Inflamm. Res. (2015) 64 (Suppl 2):S51–S248 DOI 10.1007/s00011-015-0839-4
Inflammation Research
The 12th World Congress on Inflammation
8–12 August 2015 Seaport Hotel and World Trade Center Boston, USA
The Abstracts
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List of Committees CONGRESS COMMITTEES
New Investigators Award Committee
Congress President
John Somerville, Chair, USA Ian Adcock, UK Brydon Bennett, USA Doug Morgan, USA Beverley Moore, USA Ann Welton, USA
Rod Flower, UK Congress Co-Chairs Lisa Marshall, USA Arpita Maiti, USA International Scientific Advisory Board Anthony Coyle, USA Leslie J. Crofford, USA Eugen Faist, Germany Elisabeth Peen, Denmark Fiona Powrie, UK Charles Serhan, USA Arlene H. Sharpe, USA Local Scientific Committee Lisa Schopf, Chair, USA Andrew Glasebrook, USA Liwu Li, USA Alison O’Mahony, USA Caralee Schaefer, USA Stephen Stimpson, USA Joel Tocker, USA Thomas Wynn, USA Edward Yurkow, USA
Outstanding Scientist Award Committee John Hamilton, Co-Chair, Australia Graham Wallace, Co-Chair, UK Nathalie Vergnolle, France Stephen A. Stimpson, USA Women in Inflammation Science Award Committee Ellen Berg, Chair, USA Leslie J. Crofford, USA Rod Flower, UK Graham Wallace, UK Career Development and Networking Committee Caralee Schaefer, USA Larry Burgess, USA Tineke Meijers, Canada Sharlene Velichko, USA Social Activities Committee
Abstracts and Poster Committee
Doreen Gallagher, Chair, USA
Edward Yurkow, Chair, USA Lauren Aleksunes, USA Kent Barbay, USA Alison Budelsky, USA Subba Chintalacharuvu, USA Heather Deutsch, USA Karen Duffy, USA Paul W. Fisher, USA Catherine Healy, USA Brian Jones, USA Kevin Kreutter, USA Erik Lubberts, Netherlands Ravi Malaviya, USA Carl L. Manthey, USA Christine McCauley, USA Michael McQueney, USA Beverley Moore, USA Lynne Murray, UK Tatiana Ort, USA Holly Raymond, USA Lani San Mateo, USA Pitchumani Sivakumar, USA Mary Ryan, USA Joshua Wertheimer, USA
Scientific Program Committee
Advertising Committee Alison O’Mahony, Chair, USA Heather Jones, USA Doug Morgan, USA Miranda Hanson-Baseler, USA
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Ian Adcock, UK Ellen Berg, USA Yasmine Belkaid, USA Maria Belvisi, UK Kate Blease, USA Jason Brenchley, USA Larry Burgess, USA Liudmila Buryachkovskaya, Russia Rachel Caspi, USA Ernest Choy, UK Leon Collis, USA Leslie Crofford, USA Salvatore Cuzzocrea, Italy James Ellis, USA Kate Fitzgerald, USA Rod Flower, UK Marc Gavin, USA Claire Gelfman, USA Andrew Glasebrook, USA David Glass, USA Emma Guttman-Yassky, USA Jenny Gumperz, USA Jennifer Hamilton, USA Mark Hogarth, Australia Stephen Holdsworth, Australia David Howat, UK David Huss, USA Heather Jones, USA Raj Kamath, USA
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Jeff Karp, USA Iain Kilty, USA Paul Kirkham, UK Liwu Li, USA Peter Libby, USA Nikita Lomakin, Russia Alberto Mantovani, Italy Anthony Marotta, Canada Jason McDougall, Canada Jane Mitchell, UK James Mobley, USA Kathryn Moore, USA Ikuo Morita, Japan Lynne Murray, UK Lisa Olson, USA Alison O’Mahony, USA Luke O’Neill, Ireland Tatiana Ort, USA John Parkinson, USA Robert Philibert, USA Vijay Rathinam, USA Simeon Ramsey, USA Carlo Riccardi, Italy Eric Sasso, USA Caralee Schaefer, USA Robert Schaub, USA Lisa Schopf, USA Charles Serhan, USA Arlene Sharpe, USA Gary Sims, UK Mustapha Si-Tahar, France Anthony Slavin, USA Pejman Soroosh, USA Avi Spira, USA Hergen Spits, Netherlands Thaddeus Stappenbeck, USA Chris Stevenson, UK Stephen Stimpson, USA Tetsuya Taga, Japan Mauro Teixeira, Brazil Nathalie Vergnolle, France John Wallace, Canada Cara Williams, USA Li Chun Wang, USA Thomas Wynn, USA Tim Zheng, USA
Lisa Marshall, Chair, Boston 2015 World Congress on Inflammation Ian Adcock, Chair, London 2017 World Congress on Inflammation Ian Ahnfelt-Rønne, Chair, Corporate Advisory Group
WCI 2015 Management
IRA Officers
FASEB Office of Scientific Meetings and Conferences 9650 Rockville Pike Bethesda, MD 20814 Tel: 301-634-7010 Email:
[email protected]
Joel E. Tocker, President Lisa Schopf, Vice-President Liwu Li, Treasurer Caralee Schaefer, Secretary
IAIS Steering Committee (Executive Committee plus two representatives from each member society) Brazilian Inflammation Society (BIS, Brazil) Mauro Teixeira Fernando de Queir¢z Cunha British Inflammation Research Association (BIRA, UK) Neville Punchard Matt Barnes Groupe de Recherche et d’Etude des Me´diateurs de l’Inflammation (GREMI, France) Michel Chignard Vincent Lagente Inflammation Research Association (IRA, USA) Arpita Maiti Joel E. Tocker Inflammation Research Network (IRN, Canada) John L. Wallace Jason J. McDougall Italian Inflammation Group (IIG, Italy) Salvatore Cuzzocrea Carlo Riccardi Japanese Society of Inflammation and Regeneration (JSIR, Japan) Ikuo Morita Tetsuya Taga Russian Inflammation Society (RIS, Russia) Nikita V. Lomakin Ludmila I. Buryachkovskaya Society for Cytokines, Inflammation and Leukocytes (SCIL, Australia) Stephen Holdsworth Samuel Breit
IRA Board Members IAIS EXECUTIVE COMMITTEE & IRA OFFICERS AND BOARD MEMBERS IAIS Executive Committee John Hamilton, President Stephen A. Stimpson, Vice-President Graham R. Wallace, Treasurer Nathalie Vergnolle, Secretary Kouji Matsushima, Past-President Boris Vargaftig, Chair, Brazil 2013 World Congress on Inflammation
Alison Budelsky Larry Burgess Anne M. Fourie Howard Kartstein Erik Lubberts Arpita Maiti Michael S. McQueney Alison O’Mahony Matthew A. Sleeman Edward Yurkow
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Table of Contents Sunday, August 9, 2015 Patient Perspectives: Unmet Medical Need – Osteoarthritis - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Symposium 1: Innate Memory and Programming in Acute and Chronic Inflammation - - - - - - - - - - - - - - - - - - - - - Symposium 2: Mechanisms Underlying Microbiome-Mediated Inflammation - - - - - - - - - - - - - - - - - - - - - - - - - - - Symposium 3: Disease Modification in Osteoarthritis: Myth or Reality? - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Symposium 4: The ‘‘Eicosanoid Storm’’ - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Symposium 5: Cytokines and Inflammation – Sponsored by ICIS - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Symposium 6: Novel Biologics Therapies for Immune and Inflammatory Disorders – Sponsored by SCIL-AUS - - - - - Symposium 7: New Therapeutic Strategies for Respiratory Diseases – Sponsored by ERS - - - - - - - - - - - - - - - - - - - Symposium 8: The Neutrophil: An Old Dog with New Tricks – Sponsored by GREMI - - - - - - - - - - - - - - - - - - - - - Symposium 9: Ocular Inflammation, Translation Focused – Sponsored by Ora, Inc. - - - - - - - - - - - - - - - - - - - - - - - Symposium 10: Glucocorticoids in the Regulation of Inflammatory Response – Sponsored by Italian Inflammation Group Symposium 11: A Role for Epigenetics in Inflammatory Disease: Spotlight on Monocytes and Macrophages - - - - - - - Symposium 12: Next Generation Kinase Inhibitors – Sponsored by BIRA-UK - - - - - - - - - - - - - - - - - - - - - - - - - Mini-Symposium 1: Novel Models of Inflammation - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
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Monday, August 10, 2015 Patient Perspectives: Unmet Medical Need – Inflammatory Bowel Disease- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Symposium 13: MicroRNAs (miRs) in Inflammation - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Symposium 14: Mucosal Immunity: Hot Topics in IBD - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Symposium 15: Inhibitory Receptor/Immune Checkpoints/Immunotherapeutics – Sponsored by IRA-USA - - - - - - - - - - - - - - - Symposium 16: Mechanisms of Fibrosis - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Symposium 17: Therapeutics with Localized Actions – Optimal Ways to Treat Tissue Inflammation - - - - - - - - - - - - - - - - - - - Symposium 18: Ion Channels and Inflammation – Sponsored by IRN-Canada - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Symposium 19: Harnessing Novel Resolution Mechanisms to Control Inflammation - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Symposium 20: Skeletal Muscle Inflammation - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Symposium 21: Regulation of Inflammation in Tissues by Co-Stimulatory and Co-Inhibitory Pathways - - - - - - - - - - - - - - - - - Symposium 22: Atopic Dermatitis, Psoriasis and IL-4/IL-17 Biology - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Symposium 23: Defining Sub-Phenotypes in Chronic Respiratory Diseases to Improve Drug Development and Disease Management Symposium 24: Biological and Non-Biological Therapies of Autoimmune Diseases – Sponsored by JSIR-Japan - - - - - - - - - - - Mini-Symposium 2: Cellular and Molecular Mechanisms of Fibrosis - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - New Investigator Award Winner Presentations - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
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Tuesday, August 11, 2015 Patient Perspectives: Unmet Medical Need – Lupus - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Symposium 25: Complement Targeted Therapies – Is There Light at the End of the Tunnel? - - - - - - - - - - - - Symposium 26: Regenerative Medicine and Stem Cells in Inflammation - - - - - - - - - - - - - - - - - - - - - - - - Symposium 27: iNKT Cells – Mediators at the Interface of Inflammation and Immunity - - - - - - - - - - - - - - Symposium 28: Infection and Inflammation – Sponsored by BIS-Brazil- - - - - - - - - - - - - - - - - - - - - - - - - - Symposium 29: Biomarkers in Inflammation: Focus on Rheumatoid Arthritis - - - - - - - - - - - - - - - - - - - - - - Symposium 30: SLE: Current Therapeutic Landscape and Future Promises - - - - - - - - - - - - - - - - - - - - - - Mini-Symposium 3: Mechanisms Driving Mucosal Inflammation in the Gut - - - - - - - - - - - - - - - - - - - - - - Symposium 31: New Visions in Ocular Therapy - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Symposium 32: Tregs in Human Autoimmunity - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Symposium 33: Gaseous Mediators as the Basis for Novel Anti-Inflammatory Drugs – Sponsored by IRN-Canada Symposium 34: Therapeutic Pathways in Inflammation: New Faces of Old Friends – Sponsored by RIS- - - - - - Symposium 35: Clinical Developments in Fibrosis - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Symposium 36: Novel Therapies in RA - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Mini-Symposium 4: Signaling in Inflammation - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
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Wednesday, August 12, 2015 Symposium 37: Novel Targets and/or Novel Therapies – Sponsored by Celgene Symposium 38: Cardiovascular Inflammation - - - - - - - - - - - - - - - - - - - - - Symposium 39: Predictive Toxicology - - - - - - - - - - - - - - - - - - - - - - - - - Symposium 40: Translational Medicine - - - - - - - - - - - - - - - - - - - - - - - Symposium 41: Innate Lymphoid Cells - - - - - - - - - - - - - - - - - - - - - - - -
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Posters - - - - - - - - - - - - - - - - - - - - - - - - - - - Angiogenesis - - - - - - - - - - - - - - - - - - - - - - - Apoptosis - - - - - - - - - - - - - - - - - - - - - - - - - Autoimmune Diseases—RA, Psoriasis, SLE, IBD, MS
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Inflamm. Res. Biologic Therapies for Targeting Inflammatory and Immune Mechanisms - - - - - - - - - - - - - - - Cartilage and Bone Remodeling - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Cell Adhesion and Leukocyte Migration - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Chemokines and Chemokine Receptors - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Co-Stimulatory and Co-Inhibitory Pathways - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Cytokines in Inflammatory Disease - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Epigenetics and Regulation of the Immune Response - - - - - - - - - - - - - - - - - - - - - - - - - - - Fibrosis and Tissue Remodeling - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -------GPCRs in Inflammation - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Immunotherapeutics - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Inflammasomes in Health and Disease - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Inflammation and Aging - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Inflammation and Cancer - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Inflammation and Metabolic Disorders - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Inflammatory Cell Signaling - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Inflammatory Mediators - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Inflammatory Pain and Analgesia - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Inflammatory Processes in Cardiovascular Diseases - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Inflammatory Processes in Central Nervous System Diseases - - - - - - - - - - - - - - - - - - - - - - - Inflammatory Processes in Shock and Trauma - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Inflammatory Responses, Stem Cells and Tissue Regeneration - - - - - - - - - - - - - - - - - - - - - Inflammatory Skin Disorders - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Inhibitory Receptors and Immune Checkpoints - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Innate Immunity – Macrophages, Dendritic Cells, Neutrophills, Basophils, Mast Cells, Eosinophils Innate Lymphoid Cells - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Lipids, Their Enzymes and Inflammation - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Microbiome in Health and Disease - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - microRNAs and lncRNAs in Inflammation (microRNAs=miRs; long Non-Coding RNAs=lncRNAs) Molecular Patterns and Acute Inflammation - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Mucosal Immunity - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Neutrophils, NETs and PADs - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - New Models of Inflammatory Mechanisms and Diseases - - - - - - - - - - - - - - - - - - - - - - - - - Novel and Innovative Platforms for Drug Discovery - - - - - - - - - - - - - - - - - - - - - - - - - - - Novel Targets and New Drugs in Inflammation - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Nuclear Hormone Receptors - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Ocular Inflammation - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Proteases - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Resolution of Inflammation and Tissue Repair - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Respiratory Disease and Inflammation - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Small Molecule Therapeutics for Inflammatory Diseases - - - - - - - - - - - - - - - - - - - - - - - - - T Regulatory Cells - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Tissue Damage/Repair- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Toll Receptors - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Translational Medicine and Biomarkers - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Author Index - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
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PATIENT PERSPECTIVES: UNMET MEDICAL NEED – OSTEOARTHRITIS 001 PATIENT PERSPECTIVES: UNMET MEDICAL NEED–OSTEOARTHRITIS Heather E. Jones Pfizer, Inc., Collegeville, PA, USA Objective: To increase awareness of the true unmet medical need left by current therapeutic options for osteoarthritis and understand what this means to the patient. The importance of recognizing the true unmet medical need in osteoarthritis, the most common form of arthritis affecting millions of people worldwide, is higher than ever. No drug has so far been able to cure osteoarthritis, only to can help reduce pain and maintain joint movement. To progress science and clinical development there must be a focus on understanding the pathogenesis of the disease. From a medical perspective we need to consider the patient as a whole human being and not only based on the organ(s) involved. Osteoarthritis is a degenerative disease that worsens over time and may become severe enough to make daily tasks difficult. The morning session will begin by emphasizing the unmet medical need followed by an interview with a patient suffering from osteoarthritis, with an emphasis on what they see as key objectives for further development. At the end of the session the co-chairs will summarize the signs and symptoms and the treatment algorithm for osteoarthritis, as well as the need for biomarkers to define the disease, ensure safety, guide treatment and predict treatment response.
SYMPOSIUM 1: INNATE MEMORY AND PROGRAMMING IN ACUTE AND CHRONIC INFLAMMATION 002 PTX3 AS A PARADIGM FOR THE INTERPLAY BETWEEN CELLULAR AND HUMORAL INNATE IMMUNITY IN INFLAMMATION AND CANCER Alberto Mantovani
Inflamm. Res. PTX3 plays a role in the interplay between the cellular and the humoral arm of innate immunity. PTX3 deficiency was associated with increased susceptibility to mesenchymal and epithelial carcinogenesis. PTX3 expression was epigenetically regulated in selected human tumors (e.g., leiomyosarcomas and colorectal cancer) by methylation of the promoter region and of a putative enhancer. Thus, PTX3, an effector molecule belonging to the humoral arm of innate immunity, acts as an extrinsic oncosuppressor gene in mouse and man by regulating Complement-dependent, macrophage-sustained, tumor promoting inflammation. References Sica A, Mantovani A. Macrophage plasticity and polarization: in vivo veritas. J Clin Invest. 2012, 122:787–95. Jaillon S, Moalli F, Ragnarsdottir B, Bonavita E, Riva F, et al. The humoral pattern recognition molecule PTX3 is a key component of innate immunity against urinary tract infection. Immunity, 40: 621–632, 2014. Bonavita E, Gentile S, Rubino M, Maina V. et al., PTX3 is an extrinsic oncosuppressor regulating complement-dependent inflammation in cancer. Cell 160: 700–714, 2015.
003 THE MACROPHAGE EPIGENOME AND THE CONTROL OF THE INFLAMMATORY GENE EXPRESSION PROGRAM Gioacchino Natoli European Institute of Oncology, Milan, Italy Induction of an inflammatory response requires the activation of a complex gene expression program in which hundreds of genes are activated or repressed in a kinetically complex fashion that reflects the specific functional role of their products. The activity of the transcription factors responsive to inflammatory stimuli, such as NF-kB, STAT and IRF family members, is critically influenced by the pre-existing chromatin organisation (epigenome) of the cells in which they are activated. This way inflammatory gene expression is qualitatively and quantitatively different depending on the cell type in which it is elicited. In turn, chromatin organisation in differentiated cells is controlled by lineage-determining transcription factors, such as the essential myeloid master regulator PU.1 and its binding partners (RUNX1, IRF8 and others). A wealth of genomic, biochemical and functional data accumulated in the last years has demonstrated that an important role of PU.1 is to make binding sites for inflammatory transcription factors accessible, thus enabling their recruitment to chromatin and the activation of a macrophage-specific inflammatory gene expression program.
Istituto Clinico Humanitas, Humanitas University, Rozzano, Italy Innate immunity consists of a cellular and a humoral arm. The long pentraxin PTX3 as originally cloned (cDNA and genomics, mouse and human) as an IL-1 inducible gene. We have used the long pentraxin PTX3 as a paradigm for the humoral arm of innate immunity and its interplay with cells. PTX3 is a multifunctional soluble pattern recognition receptor characterized by a C-terminal domain highly homologous to C-reactive protein and serum amyloid P component, associated to a N-terminal domain unrelated to other known proteins. PTX3 is produced upon stimulation with proinflammatory cytokines and Toll-like receptor engagement most prominently by monocytes/macrophages. The molecule binds complement components and microbial moieties. It mediates effector function via Fcg receptor and complement. Recent results suggest a function at mucosal surfaces. PTX3 plays non-redundant functions including innate immunity against selected microorganisms and regulation of inflammation. In addition
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004 EPIGENETIC REGULATION OF MACROPHAGE ACTIVATION AND FUNCTION Lionel Ivashkiv Hospital for Special Surgery, Weill Cornell Medical College, New York, NY, USA Macrophage activation phenotypes are determined by the interplay between cytokines such as IFN-gamma or TNF and acute inflammatory stimuli such as TLR ligands. Synergistic activation of inflammatory cytokine genes by IFN-gamma and TLR signaling is important for innate immunity and inflammatory disease pathogenesis. We have identified a synergy mechanism whereby IFN-gamma creates a primed
Inflamm. Res. chromatin environment to augment TLR-induced transcription of inflammatory genes such as TNF, IL6 and IL12B in human macrophages. IFN-gamma primes enhancers by inducing sustained occupancy of STAT1, IRF-1 and associated histone acetylation. In contrast to pervasive epigenomic remodeling for gene activation, IFN-gamma stably silenced only a small number of genes via chromatin regulation. These silenced genes are enriched in transcription factors that regulate aspects of alternative activation. These results suggest that M1 polarization has a stable epigenetic component that blocks reprogramming of select transcription modules by M2 stimuli, whereas the remaining (majority) of M1 genes are dynamically regulated by environmental cues. In contrast to IFN-gamma, TNF tolerizes macrophages such that inflammatory genes are not activated by TLR stimulation; this tolerization can be reversed by IFNs. Combined genome-wide analysis using RNAseq, ChIPseq and ATACseq identified chromatin-based mechanisms for reversal of tolerance under conditions where signaling is strongly attenuated. Overall the results highlight the importance of chromatin-based mechanisms in regulating transcriptional responses to acute stimuli that activate canonical inflammatory signaling pathways. Interplay between epigenetic mechanisms and signaling pathways will determine macrophage phenotypes in response to environmental cues.
005 DYNAMIC PROGRAMMING OF INNATE IMMUNITY IN HEALTH AND DISEASE
S57 Infection of gut-resident CD4+ memory T-cells during acute HIV and SIV infection is associated with rapid loss of these cells and damage to the epithelial barrier. Damage to the epithelial barrier allows translocation of microbial products from the intestinal lumen into the body. Immune activation caused by these microbial products has been associated with disease progression. Although microbial translocation has been demonstrated in SIV-infected nonhuman primates, the identity of translocating bacteria has not been determined. In this study we examined the community makeup of bacteria both within the GI tract and systemic tissues of both healthy and experimentally SIV-infected Asian macaques. While there were only modest changes in the GI tract-associated microbiome resulting from infection, there is substantial dysbiosis after administration of antiretrovirals. Analysis of bacterial DNA isolated from tissues of infected animals revealed a preference for the phylum Proteobacteria, suggesting that Proteobacter preferentially translocate. Consistent with this finding, we observed increased metabolic activity of Proteobacterial species within the colonic lumen of SIV-infected animals. Overall these data provide insights into disease progression and suggest that therapies aimed at altering the composition and metabolic activity of the GI tract microbiome could benefit chronically-HIV infected individuals particularly those on antiretroviral therapies.
Liwu Li
007 SYMBIOTIC SPHINGOLIPIDS SHAPE THE HOST IMMUNE DEVELOPMENT AND HOMEOSTASIS
Virginia Tech, Blacksburg, VA, USA
Dingding An1,2
Host innate leukocytes such as monocytes and neutrophils can be preprogrammed into distinct states depending upon the nature and quantities of external stimulants. The programming and rudimentary memory of innate immunity have significant implications in the pathogenesis both acute and chronic diseases such as sepsis and atherosclerosis. However, the responsible mechanisms are not well understood. We observed that super low dose bacterial endotoxin lipopolysaccharide (LPS) skews and programs innate leukocytes into distinct functional states, as reflected by the selective expression of inflammatory mediators in monocytes and unique modulation of neutrophil extra-cellular traps (NET). At the pathological level, we observed that mice pre-conditioned with super-low dose LPS experienced severe tissue damage, inflammation, and increased bacterial load in circulation when they were subjected to cecal-ligation and puncture (CLP). In contrast, CLP mice pre-conditioned with low dose LPS exhibited reduced tissue damage, inflammation, and reduced bacterial load in blood. In summary, our studies suggest that innate leukocytes can be dynamically programmed by varying signal strength of innate challenges, and may have far-reaching patho-physiological consequences.
SYMPOSIUM 2: MECHANISMS UNDERLYING MICROBIOME-MEDIATED INFLAMMATION 006 DYSBIOTIC MICROBES TRANSLOCATE FROM THE GI TRACT IN PROGRESSIVE SIV INFECTION Jason Brenchley, Zachary Klase LMM/NIAID/NIH, Bethesda, MD, USA
Boston Children’s Hospital, Boston, MA, USA; 2Harvard Medical School, Boston, MA, USA
1
It has been increasingly appreciated that the development of the mammalian immune system is profoundly dependent on the thousands of microbial species that the host is associated with. However, the molecular details of how microbes modulate the immune system are largely lacking. Here we report that sphingolipid-producing symbiotic bacteria regulate the host mucosal homeostasis and disease susceptibility. Many of the ubiquitous intestinal Bacteroides species possess sphingolipids, which are rare in bacteria and without much known functions. We discovered that Bacteroides fragilis produces immunomodulatory a-galactosylceramide molecules. These molecules modulate the colonic invariant killer T (iNKT) cell proliferation by competing for the limited agonist-binding space on the antigen-presenting CD1d protein. As a result, if the host is exposed to the wild-type B. fragilis or its glycosphingolipid molecules early in life when iNKT cells actively proliferate, the expansion of colonic iNKT cells in response to endogenous antigens is modulated, resulting in a lower homeostatic colonic iNKT cell level in adult life. Consequently, the host becomes more resistant to the iNKT cell-dependent colitis challenge. These results suggest an unexpected mechanism by which symbionts can help the host attain immune balance by supplementing the endogenous lipid antigen milieu with unique inhibitory glycosphingolipids. These Bacteroides glycosphingolipids provide the second known example of immunomodulatory molecules produced by a symbiont (the first being a zwitterionic polysaccharide also produced by B. fragilis). In addition, we have further discovered that bacterial sphingolipids also mediate the homeostasis of the epithelial layer in the colon and are important for the host resistance to dextran sodium sulfate-induced colitis. Our work starts to reveal the profound impacts of these unique molecules produced by bacterial symbionts on host development and homeostasis.
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SYMPOSIUM 3: DISEASE MODIFICATION IN OSTEOARTHRITIS: MYTH OR REALITY? 008 NEW TARGETS FOR JOINT DAMAGE AND ASSOCIATED PAIN IN OSTEOARTHRITIS Anne-Marie Malfait Division of Rheumatology, Rush University Medical Center, Chicago, IL, USA Osteoarthritis (OA) is the most common form of arthritis. Pain is the defining symptom of osteoarthritis (OA), yet available analgesic treatment options, of which NSAIDs are the most common, provide inadequate pain relief and are associated with serious health risks. Disease-modifying OA drugs (DMOADs) are not yet available for clinical use. It is unknown to which extent efficacious DMOADs may also have a beneficial effect on pain associated with OA. Generally, the mechanisms that generate and maintain OA-associated pain are poorly understood, and so is the relationship between joint damage and pain pathways. This gap in knowledge exists partly because modeling OA pain in laboratory animals is challenging. We employ the murine destabilization of the medial meniscus (DMM) model for the longitudinal study of OA-related pain behaviors and their molecular/neuronal pathways. Following DMM surgery, in association with slowly progressive joint pathology, mice develop pain-related behaviors in two phases: firstly, secondary mechanical allodynia in the ipsilateral hindpaw develops early on, when joint damage is minor (weeks 4–8 after surgery). The second phase (weeks 8–16) marks more severe joint pathology and is characterized by persistent mechanical allodynia and the onset of movement-provoked pain, thus likely representing a stage of chronic pain. The temporal effects of novel analgesics will be discussed. In addition, examples will be shown of how pharmaceutical modulation of joint damage affects pain pathways.
009 CELL-BASED THERAPIES FOR OSTEOARTHRITIS: FROM SYNTHETIC MATERIALS TO SYNTHETIC BIOLOGY Farshid Guilak, Jonathan M. Brunger, Ananya Zutshi, Vincent P. Willard, Katherine A. Glass, Alison K. Ross, Franklin T. Moutos, Bradley T. Estes, Charles A. Gersbach Duke University Medical Center, Durham, NC, USA The repair of articular cartilage following joint injury or degeneration remains an important challenge for the field of tissue engineering. While a number of techniques have been developed for the treatment of focal cartilage defects, there have been few attempts at tissueengineered therapies for end-stage osteoarthritis. Some of the major considerations for this approach include the design of mechanically functional scaffolds that can withstand joint loading, the identification of an abundant and accessible stem cell source, and the reprogramming of these cells for appropriate tissue differentiation within an osteoarthritic environment. Using principles of ‘‘functional tissue engineering’’, we have used techniques for three-dimensional weaving to develop large, anatomically-shaped scaffolds that can be engineered with biomimetic mechanical properties that reproduce those of native cartilage, providing the potential for complete
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Inflamm. Res. resurfacing of the entire joint surface. However, the presence of proinflammatory cytokines such as interleukin 1 or tumor necrosis factor alpha has been shown to prevent chondrogenesis and induce degradation of engineered tissues. In recent years, the advent of synthetic biology has led to the development of technologies for precisely modifying gene networks that control cell behavior. Here, we present a combination of principles from these fields to rewire cellular gene circuits in a manner that allows us to create a unique, custom-designed cell type that can sense and respond to its biochemical environment in a pre-programmed way to provide long-term signals for controlling cell differentiation or immunomodulatory response. This strategy uses recently developed methods in genome editing (CRISPR/Cas9) to reprogram cells to detect dynamic cues from the environment and to actively regulate their response with customized decision-making capabilities. By combining techniques in synthetic biology, gene therapy, and functional tissue engineering, we have developed engineered tissues with the ability for tunable, inducible, or feedback-controlled, auto-regulated immunomodulatory properties. In addition to recapitulating the biochemical and biomechanical properties of the tissue, these ‘‘smart’’ constructs can provide controlled drug delivery to the joint to enhance the success of engineered tissue replacements. Acknowledgments: Supported by grants from the NIH, NSF, Nancy Taylor Foundation for Chronic Diseases, Arthritis Foundation, and AO Foundation.
010 ADVANCES IN BIOMARKER QUALIFICATION TO FACILITATE OSTEOARTHRITIS CLINICAL TRIALS Virginia B. Kraus Duke Molecular Physiology Institute and Department of Medicine, Duke University, Durham, NC, USA Objectives: Our current reference standard for disease diagnosis and severity of osteoarthritis (OA) is often the radiograph (Hunter 2014). Moreover, the draft regulatory (FDA) guidance and current gold standard for measuring clinical efficacy in disease modifying therapy development in OA is radiographic which has a low responsiveness to change and at most moderately correlates with clinical endpoints (Hunter 2014). Biomarkers are needed to provide a method for earlier diagnosis of OA, and to inform the prognosis, monitoring and therapeutic strategies for OA (Kraus 2011 and 2015). The goal of our work has been to evaluate the ability of a set of soluble biomarkers to predict knee OA progression to provide new drug tools and surrogate outcomes in OA trials. Methods: We undertook a nested case–control study within the Osteoarthritis Initiative in knees (one knee per subject) with a Kellgren and Lawrence grade (KLG) of 1–3. Cases were defined as knees having the combination of radiographic progression (a decrease of C0.7 mm in medial tibiofemoral joint space) and pain progression (a persistent increase in WOMAC pain score of C9 on a 0–100 scale) at the 24, 36 or 48 months follow-up compared to baseline. Controls (n = 406) were eligible knees that did not meet both endpoint criteria, and included 200 with neither radiographic nor pain progression, 103 with radiographic progression only and 103 with pain progression only. A total of twelve OA-related soluble biomarkers were measured in serum and/or urine. Results: Longitudinal burden of biomarker concentrations over time (area of concentration vs time curve) was a more robust predictor of case status than change scores over time.
Inflamm. Res. Conclusions: For dynamic markers such as soluble systemic biomarkers, strategies accounting for ‘biomarker burden’ over time appear to be more appropriate for identifying progressors than strategies based on change over time.
S59 single agent alone. Collectively, these findings uncover a previously unrecognized role for PGE2 in the promotion of tumor formation and progression.
Acknowledgments: The FNIH OA Biomarkers Consortium References Hunter DJ, Nevitt M, Losina E, Kraus V. Biomarkers for osteoarthritis: current position and steps towards further validation. Best Pract Res Clin Rheumatol 2014;28(1):61–71. Kraus VB, Burnett B, Coindreau J, Cottrell S, Eyre D, Gendreau M, Gardiner J, Garnero P, Hardin J, Henrotin Y, Heinegard D, Ko A, Lohmander LS, Matthews G, Menetski J, Moskowitz R, Persiani S, Poole AR, Rousseau JC, Todman M. Application of biomarkers in the development of drugs intended for the treatment of osteoarthritis. Osteoarthritis Cartilage 2011;19(5):515–542. Kraus V, Blanco F, Englund M, Henrotin Y, Lohmander L, Losina E, ¨ nnerfjord P, S P. Guidelines for soluble biomarker assessments in O osteoarthritis clinical trials. Osteoarthritis and Cartilage 2015;in press.
SYMPOSIUM 4: THE ‘‘EICOSANOID STORM’’ 011 INFLAMMATION, INFLAMMATORY MEDIATORS AND CANCER PROGRESSION
012 INDUCTION OF EICOSANOIDS BY THE INFLAMMASOME Karsten Gronert University of California Berkeley, Berkeley, CA, USA Inflammasomes have remerged as key players in the innate immune system and have been implicated in pathogenesis of inflammatory diseases, autoimmune disorders and host defense. The inflammasome complex acts as a ‘‘last resort’’ intracellular sensor that detects intracellular microbial products or stress factors to mount a critical innate immune response. A common mechanism of most inflammasomes is activation of caspase 1 and pyroptosis. Despite the fact that this system is designed to send an immediate inflammatory signal primary effector function has been relegated to cleavage and formation of interleukin 1b (IL-1b) and IL-18, which takes hours. We recently discovered a novel early and critical effector function of inflammasome activation, namely the rapid formation of an eicosanoid storm by tissue macrophages. In view of emerging evidence that places eicosanoids as key regulators of innate as well as adaptive immune responses this novel inflammasome effector function has far reaching implications in health and disease.
Raymond N. DuBois1,2 Arizona State University, Tempe, AZ, USA; 2Mayo Clinic Arizona, Phoenix, AZ, USA 1
Although epidemiologic and experimental observations support the hypothesis that chronic inflammation and diet are risk factors for colorectal cancer, the mechanisms by which chronic inflammation and diet contribute to the development of cancer are poorly understood. Evidence for the link between inflammation and cancer comes from epidemiologic and clinical studies showing that use of nonsteroidal anti-inflammatory drugs (NSAIDs) reduce the relative risk for developing colorectal cancer (CRC) by 40–50 %. NSAIDs exert some of their anti-inflammatory and anti-tumor effects by targeting cyclooxygenase enzymes (COX1 and COX2). Metabolism of arachidonic acid, a major ingredient in animal fats, by cyclooxygenase enzymes provides one mechanism for the contribution of dietary fats and chronic inflammation to carcinogenesis. Prostaglandin E2 (PGE2) is a pro-inflammatory mediator that promotes tumor progression. We found that PGE2 exerts its effects on chemokine and cytokine expression by upregulation of the CXCR2 pathway, which increases the level of myeloid derived suppressor cells (MDSC’s) in the tumor microenvironment. MDSCs have been shown to contribute to cancer immune evasion by suppressing T cell activation, proliferation, trafficking, and viability. MDSC’s also can inhibit natural killer (NK) cells and promote activation/expansion of Foxp3 positive Treg cells. We also provided the first evidence demonstrating that MDSCs promote chronic colonic inflammation and colitis-associated carcinogenesis via suppression of colonic CD8+ T cell cytotoxicity against tumor cells. Our recent findings not only provide a rationale for developing effective therapeutic strategies to subvert inflammation- and tumor-induced immunosuppression, but also support the hypothesis that combined treatment of anti-PD-1 and anti-CXCR2 agents may provide more effective therapeutic effects than either
013 PGE2 AND THE INNATE IMMUNE RESPONSE Mariano Sanchez Crespo IBGM, CSIC, Valladolid, Spain Eicosanoids support local inflammation and exhibit immunomodulatory properties. Current views have focused on prostaglandin E2 (PGE2), because it is the most abundant and lasting eisosanoid in the inflammatory milieu due to the robust production elicited after challenge with pathogen-associated molecular patterns by the constitutive and the inducible COX isoforms. The different functions and cell distribution of E prostanoid (EP) receptors explain the difficulty so far encountered to delineate the actual role of PGE2 in the immune response. It is widely accepted that by acting in an autocrine/paracrine manner PGE2 induces a regulatory phenotype including the expression of IL-10, the inhibition of the release of IL-12 p70, and distinct effects on IL-23 production due to differential effects on the expression of the p40 and p19 (encoded by the gene il23) chains of this cytokine. Given that PGE2 may be released concomitantly with other lipid mediators, the definite assignment of a role for each mediator is not an easy task. Our studies on the regulation of the gene il23a have disclosed the cooperation of leukotriene B4, cysteinylleukotrienes, and the phospholipid mediator platelet-activating factor in the response to fungal patterns. These mediators, by acting concomitantly on their cognate G-protein coupled receptors, activate phospholipase Cß and enhance the Ca2+- and kinase-dependent routes initiated by fungal patterns involving Syk kinase and phospholipase Cc. The autocrine production of these mediators has a robust effect on the activation of transcription factors such as CREB and ATF2 that cooperate with NF-jB to establish the cytokine signature.
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B153 COLONY-STIMULATING FACTOR-1 (CSF-1) AND TUMOR NECROSIS FACTOR-A (TNF-A) IN ARTHRITIC PAIN AND DISEASE
015 TAM RECEPTOR TYROSINE KINASES REGULATE THE MAGNITUDE OF THE IMMUNE RESPONSE
See poster section for abstract.
Carla Rothlin
SYMPOSIUM 5: CYTOKINES AND INFLAMMATION—SPONSORED BY ICIS 014 RESOLVINS AND PRO-RESOLVING LIPID MEDIATORS: RESOLVING THE STORM Charles N. Serhan, Nan Chiang, Jesmond Dalli Center for Experimental Therapeutics and Reperfusion Injury Department of Anesthesia, Perioperative and Pain Medicine Harvard Institutes of Medicine, BWH and Harvard Medical School, Boston, MA, USA Local control of the acute inflammatory response by the host is critical for an ideal outcome and return of tissue function. Using a systems approach with self-limited inflammatory infectious exudates to map tissue events, cell traffic and identification of protein and chemical mediators, we identified three structurally separate families of potent novel mediators, coined resolvins, protectins and maresins. Complete structural elucidation of these new molecules demonstrated their functions in vivo in the resolution of acute inflammation. Each family member is chemically distinct functioning as pro-resolving local mediators that control both the duration and magnitude of acute inflammatory responses with actions in pico- to nano-gram concentration range. Mapping of these resolution circuits provides new avenues to probe the molecular basis of many widely occurring diseases and implicate the failure of resolution mechanism as a component of chronic inflammatory diseases (CN Serhan Nature 2014). This presentation shall focus on our recent advances on the biosynthesis and functions of specialized pro-resolving mediators (SPM) and their actions in counter-regulation of pro-inflammatory cytokines (IL-1, TNF, IL-6, etc.) that initiate leukocyte infiltration and inflammation. SPM possess potent multi-pronged anti-inflammatory, pro-resolving, and anti-microbial actions in animal models. We use LC–MS-MS lipidomics to profile SPM in human tissues and uncovered new pathways that stimulate tissue regeneration. Several SPM are in clinical development and clinical trial in humans. Identification of SPM during inflammation-resolution indicates that resolution is an active programmed process challenging the concept that resolution is a passive process where chemotactic molecules dilute and wane to resolve tissue inflammation. Together these findings indicate that endogenous resolution pathways may underlie prevalent diseases associated with uncontrolled inflammation and open the potential for resolution-based pharmacology. The authors acknowledges support of NIH GM038765 and GM P01 GM095467.
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Department of Immunobiology, School of Medicine, Yale University, New Haven, CT, USA The innate immune response of dendritic cells (DCs) and other sentinel cells functions as both the first line of defense against pathogens and also as the initiating trigger for T cell-mediated adaptive immunity. These fundamental activities notwithstanding, DC activation must be tightly regulated. While reduced DC function leads to increased susceptibility to infections, unrestrained, overactive DC responses can lead to allergy, autoimmunity, chronic inflammatory disease, and other pathological conditions. We have found that the TAM receptor tyrosine kinases, Tyro3, Axl and Mertk, are potent negative regulators of the immune response in DCs. I will present new findings that show that the TAM signaling in DCs is triggered by cells of the adaptive response with which DCs interact with. I will also discuss the specificity of different TAM receptors in the regulation of the immune response.
016 FRENEMIES IN THE GUT: INFECTIOUS AGENTS THAT DEFY CLASSIFICATION Ken Cadwell New York University School of Medicine, New York, NY, USA The gut microbiome includes trillions of commensal bacteria that provide key benefits to the host including aiding digestion, promoting the development of the immune system, and protecting the epithelial barrier. However, an inappropriate immune response directed at members of the microbiome can also lead to diseases such as inflammatory bowel disease (IBD). For these reasons, the bacterial microbiome has received much attention as critical regulators of mammalian health and disease. Less is known about the impact of other intestinal inhabitants, such as viruses that are part of the enteric virome. We present evidence that an intestinal animal virus can influence host physiology in a manner similar to commensal bacteria. Although murine norovirus (MNV) can persistently infect mice without causing obvious signs of disease, we found that this virus induces intestinal pathologies in a mouse model of IBD. Therefore, like colonization by commensal bacteria, MNV infection is typically harmless but can induce disease in a genetically susceptible host. Remarkably, we found that MNV infection reproduces beneficial functions of bacteria as well. Viral infection of germ-free mice or antibioticstreated mice reversed abnormalities in intestinal and immune development that were due to the depletion of bacteria. Moreover, MNV protected bacterially-deficient mice from chemical and infectious damage to the intestine. These finding indicate that viruses can perform functions that have been attributed to commensal bacteria, and has implications for how we view the role of non-bacterial members of the microbiome. The relationship between MNV, cytokine signaling, and other infectious entities will be discussed in the context of disease susceptibility.
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017 INNATE IMMUNE SENSING OF LPS IN THE CYTOSOL
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019 GM-CSF AND INFLAMMATORY PAIN Andrew D. Cook
Vijay Rathinam UConn Health, Farmington, CT, USA Innate immune system is central to the sensing of invading pathogens and the activation of the host immune response. Inflammasomes are multi-protein scaffolds in the cytosol containing a NLR receptor, an adapter ASC, and an effector, caspase-1. Inflammasome is an integral part of the immunosurveillance of the cytosol. Inflammasomes directly detect various ‘‘signature’’ microbial products or indirectly sense signs associated with an infection. Although lipopolysaccharide (LPS) of Gram-negative bacteria was believed to be exclusively detected at the cell surface by Toll-like receptor-4 (TLR4), it has very recently been described that the LPS is sensed in the cytosol in a TLR4-independent manner by caspase11, an inflammatory caspase. Activation of caspase-11 by intracellular LPS leads to the proteolytic activation of caspase-1, which then executes the activation of IL-1b and IL-18. Importantly, active caspase-11 triggers an inflammatory form of cell death (pyroptosis) and the release of endogenous alarmin or danger molecules that perpetuate the inflammatory reactions. The mechanistic details of this pathway regarding the molecular basis of caspase-11 activation will be presented.
SYMPOSIUM 6: NOVEL BIOLOGICS THERAPIES FOR IMMUNE AND INFLAMMATORY DISORDERS— SPONSORED BY SCIL-AUS 018 TARGETING GM-CSF IN INFLAMMATORY DISEASES Ian P. Wicks
1,2,3
1
Inflammation Division, Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia; 2Rheumatology Department, Royal Melbourne Hospital, Parkville, VIC, Australia; 3University of Melbourne, Parkville, VIC, Australia Granulocyte macrophage colony stimulating factor (GM-CSF) was originally discovered by virtue of its effects as a haemopoietic growth factor. However, GM-CSF turns out to have a variety of effects in addition to those well recognised for bone marrow progenitor cells, including on cells of the immune and inflammatory systems. As such, GM-CSF can also be considered as a pro-inflammatory cytokine. Preclinical studies established a strong rationale for targeting GM-CSF in diseases such as rheumatoid arthritis (RA). Therapeutic monoclonal antibodies that antagonise GM-CSF signalling have been developed and are being evaluated in human clinical trials. To date, the results in RA have been very promising and raise the prospect of extending this approach to other inflammatory diseases.
The University of Melbourne, Parkville, VIC, Australia Better therapies are needed for inflammatory pain. In arthritis the relationship between joint pain, inflammation and damage is unclear. Granulocyte–macrophage colony-stimulating factor (GM-CSF) is important for the progression of a number of inflammatory conditions, including arthritis, and there is encouraging data that its blockade may have clinical relevance. However, its contribution to inflammatory and arthritic pain is unknown. The aims of this study were to determine whether GM-CSF controls inflammatory and/or arthritic pain. A model of inflammatory pain (CFA footpad), as well as three arthritis models, were induced in either GM-CSF-/- mice or wildtype mice treated prophylactically or therapeutically with a monoclonal antibody to GM-CSF. Development of pain (assessment of weight distribution) and arthritic disease (histology) were assessed. Pain was further measured in a GM-CSF-driven arthritis (mBSA/GMCSF) model and the cyclooxygenase-dependence determined using indomethacin. GM-CSF was absolutely required for pain development in both the inflammatory pain and arthritis models. Therapeutic neutralization of GM-CSF not only abolished the pain within 3 days but also led to significantly reduced cartilage damage in the collagenase-induced instability model of osteoarthritis. Pain in a GM-CSF-driven arthritis model, but not the disease itself, was abolished by the cyclooxygenase inhibitor, indomethacin, indicating separate pathways downstream of GM-CSF for pain and arthritis control. GM-CSF is thus key to the development of inflammatory and arthritic pain. Importantly, GM-CSF neutralization by a therapeutic monoclonal antibody-based protocol rapidly and completely abolished existing arthritic pain and suppressed the degree of arthritis development. These results suggest that it would be worth exploring the importance clinically of GM-CSF for pain and disease in inflammatory conditions in general.
020 IL-3RA AS A THERAPEUTIC TARGET FOR LUPUS Nicholas J. Wilson1, Shereen Oon2,3,4, Eric Morand5, Ian Wicks2,3,4, Katherine Monaghan1 1 CSL Limited, Broadmeadows, VIC, Australia; 2The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia; 3The Royal Melbourne Hospital, Parkville, VIC Australia; 4The University of Melbourne, Parkville, VIC, Australia; 5Monash University, Malvern East, VIC, Australia
Systemic lupus erythematosus (SLE) is a multi-system autoimmune disease characterized by auto-reactive antibodies and elevated production of interferon-a (IFNa). SLE autoantibodies, directed against DNA and RNA components, form immune complexes that can activate toll-like receptors (TLR)-7 and -9 on plasmacytoid dendritic cells (pDCs), which produce large amounts of IFNa in response. PDCs also express high levels of interleukin-3 receptor alpha (IL-3Ra; CD123). We have developed a novel, neutralizing anti-CD123 mAb (CSL362)
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S62 with enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) against CD123-expressing cells. To study the effects of CSL362 in SLE, we recruited a cohort of SLE patients (n = 34) and matched healthy donors (n = 34). We enumerated various cell subsets and determined their CD123 expression. In addition, the in vitro effect of CSL362 on pDC depletion and subsequent IFNa production and IFNa-inducible gene expression was determined. The effect of CSL362 in vivo was also examined after subcutaneous (s.c.) administration to non-human primates (NHPs). This study shows that in healthy controls and SLE donors, pDCs have the highest average expression of CD123 (44,152 and 49,325 receptors/cell respectively), followed by basophils (28,530 and 34,094 receptors/cell respectively) compared with other cell types that have \2000 receptors/cell. SLE donors had reduced cell numbers for most cell types studied, including pDCs, basophils and NK cells; however, they had normal numbers of eosinophils, neutrophils and plasma blasts. CD123 expression was not statistically different between SLE and healthy control donors for all cell types, with the exception of pDCs, which had higher CD123 expression in SLE than healthy donors. CSL362 potently depleted pDCs in vitro in both SLE and healthy donors (EC90 = *0.01 lg/ml). CSL362 also partially depleted basophils and mDCs in vitro but did not significantly deplete other cell types. In addition, CSL362 abrogated ex vivo TLR-7 and 9-induced IFNa production in SLE and healthy donors and selectively inhibited an IFN gene signature induced by TLR-7 and -9 agonists, but not other TLR agonists. In vivo, a dose-dependent depletion of pDCs and basophils was observed in response to s.c. administration of CSL362 to cynomolgus macaques. Depletion of pDCs and basophils by CSL362 was associated with activation of NK cells both in vitro and in vivo. Subcutaneous administration of CSL362 lead to a similar inhibition of IFN-induced genes in blood stimulated with TLR-9 agonists ex vivo. This study shows that CSL362 potently and selectively depletes pDCs in vitro, in both SLE and healthy donors, and in vivo in NHPs. PDC-depletion by CSL362 was associated with decreased TLR-7/9induced IFNa production and IFNa-inducible gene expression in vitro and in vivo accompanied by increased NK cell activation. Cytoreductive therapy with CSL362 may therefore represent a novel treatment strategy in SLE. Supported by Janssen Biotech
021 TARGETING TLR-INDUCIBLE INFLAMMATORY PATHWAYS IN MACROPHAGES Matthew J. Sweet IMB, The University of Queensland, St. Lucia, QLD, Australia Toll-like receptors (TLRs) respond to both pathogen- and hostderived danger signals to drive pro-inflammatory signalling responses in innate immune cells such as macrophages. Inappropriate or dysregulated TLR activation propagates pathological inflammation in animal models of numerous inflammation-driven diseases including autoimmune diseases, atherosclerosis, neurodegeneration, liver damage and cancer metastasis. Consequently, TLR complexes and downstream signalling pathways represent attractive targets for inflammation-driven diseases. The textbook view of TLR signalling is that receptor activation results in the recruitment of toll/interleukin-1 receptor (TIR) domain-containing proteins, which in turn recruit specific serine/threonine kinases (IRAKs) to relay downstream pro-inflammatory signalling (e.g. activation of NF-
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Inflamm. Res. kappaB and MAPKs). However, there are numerous aspects of initiation and perpetuation of pro-inflammatory signalling responses downstream of TLRs that are still poorly understood, some of which may be amenable to targeting. Here we report a novel cell-surface protein that is required for agonist-induced TLR4 phosphorylation, pro-inflammatory signalling and the TLR4-inducible production of a sub-set of pro-inflammatory cytokines (IL-6, IL-12). These studies have revealed that proximal events involved in initiating TLR4 signalling impart specificity to downstream inflammatory responses, thus offering new avenues for selective manipulation of TLR-inducible cytokine production. We also describe the role of a specific histone deacetylase (HDAC7) in driving HIF-1alpha-dependent TLR4-inducible cytokine production in macrophages. HDAC7 is expressed at elevated levels in inflammatory macrophages, thus providing a mechanism for perpetuation of pathological inflammatory responses. Broad-spectrum HDAC inhibitors are efficacious in animal models of many inflammation-mediated diseases, but are also associated with a number of adverse effects. Thus, selective targeting of HDAC7 may enable the generation of new HDAC inhibitors with anti-inflammatory properties, but reduced side effects.
022 A NEW CANCER THERAPEUTIC THAT TARGETS ANGIOGENIC VESSELS Jennifer R. Gamble1,2, Yang Zhao1,2, Kaka Ting1,2, Jia Li1,2, Thorleif Moller3, Mathew A. Vadas1,2 1
Centenary Institute, Sydney, NSW, Australia; 2University of Sydney, Sydney, NSW, Australia; 3Mirrx Therapeutics, Vejle, Denmark Tumour angiogenic vessels are considered an attractive target for the development of therapeutics. One of the prime targets is VEGF, the major growth factor for blood vessels. However, the anti-VEGF therapies have shown limited efficacy resulting in only a transient effect on tumour growth but also resulting in an increase in tissue hypoxia that ultimately promotes tumour growth and limits radio- and immune-therapy. Tumour angiogenic blood vessels are characterised by both structural and functional changes. Thus, they are tortuous, lack adequate pericyte coverage and the matrix shows changes in density and composition. These changes result in increased permeability, reduced perfusion and the tissue environment is hypoxic. The concept of vessel normalisation has been proposed as an alternative to ablation of tumour angiogenic vessels. In normalisation, the vessels are converted back to a structurally more normal architecture and function, with the potential to enhance radio-, chemo- and immune-therapy. We have developed a first-in-class drug (CD5-2) that with a single intravenous injection significantly enhances, in mice, tumour vessel perfusion and pericyte coverage, decreases vascular leak and tissue hypoxia and inhibits tumour growth. CD5-2 increases the expression of the major structural endothelial cell specific junctional adhesion molecule VE-cadherin and has downstream effects on other associated pathways involved in vessel stabilisation namely the TIE-2 and the tight junction pathways. CD5-2 is a proprietary single stranded modified oligonucleotide. The profound effects of CD5-2 are being currently examined in metastatic, cytotoxic and radiotherapy models. In an age where ‘personalised’ therapy of cancers is of great interest, drugs that have potential utility across all solid cancers, and are thus ‘non-personalised’ offer a cost-effective advance.
Inflamm. Res.
023 GLUCOCORTICOID-INDUCED LEUCINE ZIPPER AS A NOVEL THERAPEUTIC IN INFLAMMATORY DISEASE Michael J. Hickey, Qiang Cheng, Eric F. Morand Department of Medicine, Centre for Inflammatory Diseases, School of Clinical Sciences at Monash Health, Monash University, Malvern East VIC, Australia Glucocorticoid-induced leucine zipper (GILZ) is an anti-inflammatory protein first identified in T lymphocytes, being highly inducible in response to dexamethasone. In previous work, we have observed antiinflammatory effects of GILZ both in vitro and in vivo. Transfection of GILZ into endothelial cells reduced their capacity to support interactions with leukocytes, via effects on expression of adhesion molecules and chemokines. Similarly, in vivo delivery of GILZ via a viral vector reduced inflammation in a mouse model of collagen-induced arthritis. As a result of these observations, we hypothesized that exogenouslydelivered GILZ has potential as a novel anti-inflammatory agent, and that it may provide a more specific anti-inflammatory effect than glucocorticoids by avoiding their well-known adverse metabolic effects. To address this, we developed a cell-permeable form of GILZ protein, termed HHph1-GILZ, via inclusion of a protein transduction domain. HHph1-GILZ, but not native GILZ, was able to enter various cell types including microvascular endothelial cells, macrophages and primary peripheral blood mononuclear cells, in vitro. Moreover, HHph1-GILZ reduced activation of endothelial cells and macrophages in response to TNF, as determined using cell lines incorporating an NFkB reporter construct. We are also assessing whether GILZ exerts harmful metabolic effects similar to those induced by glucocorticoids, using biochemical assays with hepatocyte cell lines and, to this point, there is no evidence that this is the case. In ongoing work, we are undertaking further characterisation of the molecular targets of HHph1-GILZ, with a view to assessing its anti-inflammatory capacities in in vivo models of inflammation. In conclusion, these findings support further investigation of the utility of cell-permeable GILZ as a novel therapeutic for inflammatory conditions.
SYMPOSIUM 7: NEW THERAPEUTIC STRATEGIES FOR RESPIRATORY DISEASES—SPONSORED BY ERS
S63 asthma patients do not achieve total control. The commonest reason for this is poor adherence with asthma treatment. Some patients, however, will not achieve asthma control, even with maximal doses of currently available therapy, perhaps as many as 10 %. These patients are considered to have severe refractory asthma. It has become evident that severe refractory asthma consists of a very heterogeneous population of patients. In addition, many diseases can masquerade as severe asthma. An accurate diagnosis and careful phenotyping is needed to identify which newer treatments may benefit an individual patient. A number of experimental treatments are being developed for severe refractory asthma. An example of the necessity to phenotype patients with severe refractory asthma has been the development of monoclonal antibodies (hMab) directed against interleukin (IL)-5, a cytokine produced by Th2 cells in innate lymphocytes type 2 (ILC2). Two antibodies have been developed (mepolizumab and relizumab), neither of which showed benefit in a non-selected cohort of patients with difficult-to-treat asthma; however, when studied in patients with a persisting airway eosinophilia, these treatments have been shown to reduce asthma exacerbations and improve lung function. The use of induced sputum was essential to identify these patients with a persisting airway eosinophilia, despite optimal treatment. Similarly, a hMab directed against another type-2 cytokine, IL-13 significantly improved lung function in patients with difficult-to-control asthma, but only in those with an elevated serum periostin (a protein produced by airway epithelial cells after stimulation with IL-13). Also, a hMab directed against the IL-4Ra, which is the common component of the receptor for IL-4 and IL-13, is showing promise in patients with elevated blood eosinophil counts. Thymic stromal lymphopoietin (TSLP) is produced by airway epithelial cells in response to viruses and environmental allergens. It is up-stream to the production of the type-2 cytokines, IL4, Il-5 and IL-13. Treatment with a hMab against TSLP also shows promise in asthma. It is likely that all new treatments for severe refractory asthma will require efforts at phenotyping to target therapy at the populations of patients likely to benefit, as this group of patients have such heterogeneous mechanisms causing their severe disease.
025 THERAPEUTIC STRATEGIES FOR THE TREATMENT OF CYSTIC FIBROSIS Stuart Elborn Queens University Belfast, Belfast, UK
024 ANTI-CYTOKINE THERAPY IN ASTHMA: FOCUS ON TSLP Paul M. O’Byrne McMaster University, Hamilton, ON, Canada Asthma management is focused on achieving total asthma control in two domains. First is current control, where the patient is asymptomatic all of the time, has normal lung function and no limitations in activities. The second in minimizing future risk of severe exacerbations, accelerated decline in lung function and avoiding side effects from medications. Currently available treatment, particularly with inhaled corticosteroids (ICS) with or without long-acting inhaled b2-agonists (LABA) can achieve good asthma control in many patients. However, most
Cystic fibrosis is the most common life-limiting genetic disorder affecting North Western Europeans and it occurs in all populations across the world. It is caused by reduced or abnormal function of the cystic fibrosis transmembrane regulator protein (CFTR) which is a cyclic AMP regulated chloride channel. CFTR also transports other ions particularly bicarbonate and regulates an epithelial sodium channel and interacts with inflammation related pathways within the cell. CFTR regulates hydration, pH and electrolyte concentration in epithelial lining fluid and in the lung this is mucus hydration, mucocilary clearance and innate immunity. A range of therapeutic approaches have been utilised in an attempt to restore CFTR function in people with cystic fibrosis. The aim is to restore the airways surface liquid and associated micro environment to improve mucociliary clearance and innate immune function. In a ground-breaking small molecule programme ivacaftor was identified through high throughput screening to be a potentiator of mutant CFTR. In patients with a particular mutation (G551D) where the
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026 NEW TREATMENT OPTIONS FOR PULMONARY FIBROSIS Toby M. Maher1,2 1
Imperial College, London, London, UK; 2Royal Brompton Hospital, London, UK Idiopathic pulmonary fibrosis (IPF) is an inexorably progressive disease of unknown aetiology that conveys a dismal prognosis. Until 12 months ago there were no licensed therapies in the United States for the treatment of IPF. However, a growth in pharmaceutical interest in the treatment of fibrosis coupled with the development of effective IPF clinical trials networks has led to a number of key developments in the treatment of IPF over the last decade. Both pirfenidone and nintedanib have shown efficacy signals in multicentre phase 3 trials and have now both been approved in the US and Europe for the treatment of IPF. Furthermore, advances in the understanding of the pathogenetic processes involved in the development of pulmonary fibrosis have led to the identification of a multitude of novel therapeutic targets. This in turn has contributed to a rapid expansion in the number of early phase trials in IPF with current targets including; protein kinases, cytokines, growth factors, oxidative stress and matrix turnover. These developments offer genuine hope for individuals with IPF, and for their treating physicians, that there may, in the relatively near future, be a range of therapeutic options available for the treatment of this devastating disease.
SYMPOSIUM 8: THE NEUTROPHIL: AN OLD DOG WITH NEW TRICKS— SPONSORED BY GREMI 027 NEWS ABOUT NETS Sylvie Chollet-Martin Bichat Hospital and INSERM Paris-Sud University, Orsay, France In 2004, A Zychlinsky evidenced that neutrophils were able to release the so-called neutrophil extracellular traps (NETs) in order to kill pathogens via both microbe ensnaring and protease release. The present review aims to present some of the most important new findings on NETs. Since 2004, a large number of studies have described the mechanisms leading to the formation of these web-like structures
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Inflamm. Res. composed of dsDNA, histones and various proteins from the intracellular medium. The most recent reports emphasize the implication of NOX2 and rac, but also the role of SK3 channel, the mitochondrialderived reactive oxygen species, the mTOR pathway of autophagy or the complex mechanism of citrullination and its main importance in autoimmunity. Neutrophils might even retain some of their functional capacities while expressing NETs. Netosis is one of the defense mechanisms against microbes and neutrophils are able to choose to undergo netosis or to prefer phagocytosis depending on the size of the pathogen; moreover some of them have developed strategies to overcome netosis. Beside their important antimicrobial activity, NETs have been implicated in the pathophysiology of an increasing number of human diseases. NETs components such as DNA, proteinase 3, myeloperoxidase or citrullinated proteins are considered as auto-antigens by the immune system and can thus induce autoimmunity; systemic lupus erythematosus is one of the best documented example. NETs also play a role in tissue damage in various clinical situations. During severe infections, in particular in the liver and in the lung, the majority of the injury might be induced by proteases-associated NETs rather by the microbe itself. Netosis, coagulation activation and platelets functions are linked and several lines of evidence implicate NETs in thrombosis-related diseases. Another important challenge is to better understand how NETs are involved in cancer development. Among the new clinical studies documenting the deleterious effects of NETs, the description of the NET-related primary graft dysfunction after lung transplantation is original. Interestingly, NETs can participate to the regulation of the immune response. For instance, they can downregulate dendritic cell maturation, leading to Th2 polarization. They also express some inhibitors of the inflammatory response like SLPI. Finally, given the deleterious effects of NETs on cells and tissues, several therapeutic approaches have been suggested, via the use of DNAse, the inhibition of their formation or their shedding in the circulation.
028 UNEXPECTED NEUTROPHIL CONTRIBUTIONS TO ANTIBODY-INDUCED REACTIONS Pierre Bruhns1,2 1
Antibodies in Therapy and Pathology, Institut Pasteur, Paris, France; 2INSERM U760, Paris, France
IgG antibodies are potent activators of myeloid cells that express IgG receptors (FccRs). Targeting of antibodies onto tissue-expressed antigens or onto circulating antigens can induce local or systemic inflammatory reactions, respectively. We aimed at understanding which myeloid cells are responsible for IgG antibody-induced inflammation in a local inflammatory conditions, i.e. a subcutaneous tumor targeted by a therapeutic antibody, or in a systemic inflammatory condition, i.e. circulating allergens opsonized by anti-allergen antibodies that results in allergic shock (anaphylaxis). We show that neutrophils are responsible for antibody-induced therapy of subcutaneous tumors (a syngeneic melanoma or a human breast cancer xenograft) and for the induction of allergic shock. Both antibody-induced reactions were abolished in neutropenic mice or mice lacking FccRs. But both reactions could be restored upon transfer of FccR-expressing mouse or human neutrophils. In addition, transgenic expression of human IgG receptors expressed on activated neutrophils, FccRI (CD64) or FccRIIA (CD32A), enabled both antibody-induced anti-tumor and anaphylactic reactions. Importantly, conditional knockout mice unable to perform FccR-mediated
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activation and phagocytosis specifically in neutrophils were resistant to antibody-induced cancer therapy. Our work suggests that neutrophils are necessary and sufficient for (1) mAb-induced therapy of subcutaneous tumors, and (2) anaphylactic reactions to circulating allergens that mimics drug-induced anaphylaxis. These unexpected roles of neutrophils suggest novel therapeutic avenues to improve antibody-induced anti-tumor therapy and to reduce drug-induced allergic shock.
029 DECIPHERING THE CYTOSOLIC SCAFFOLD OF THE PROLIFERATING CELL NUCLEAR ANTIGEN (PCNA) TO CONTROL NEUTROPHIL SURVIVAL IN INFLAMMATORY DISEASES 1,2,3
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Veronique Witko-Sarsat , Cle´mence Martin , Delphine Ohayon1,2,3, Pierre-Regis Burgel2,4 1 INSERM U1016, Institut Cochin, Paris, France; 2Universite´ Paris Descartes, Paris, France; 3Laboratoire d’Excellence INFLAMEX, Paris, France; 4Cochin Hospital, Paris, France
The life span of a neutrophil must be tightly regulated, as extended survival is essential for the effective elimination of pathogens and cell death necessary to prevent the release of the highly cytotoxic contents of activated neutrophils and subsequent tissue damage. We have previously shown that proliferating cell nuclear antigen (PCNA), a nuclear factor involved in DNA replication and repair of proliferating cells, is localized exclusively in the cytoplasm of neutrophils where it regulates their survival. Nuclear PCNA functions are tightly linked to its ring-shaped structure, which allows PCNA to bind to numerous partner proteins to orchestrate DNA-related processes. We have shown that only monomeric PCNA can expose its nuclear export sequence to be relocalized from nucleus to cytosol during granulocyte differentiation. When localized into the cytosol, PCNA is able to bind procaspase-3, 8, 9 and 10, which in turn precludes their activation. Another well-characterised binding partner is the cyclin dependent kinase inhibitor p21/waf1, which binds PCNA at the interdomainconnecting loop which is the preferred site of interaction for numerous PCNA partners. A small peptide corresponding to the residues 141–160 of p21/waf1 known as carboxyp21 was found to interfere with PCNA-protein interactions and in turn had a strong antiproliferative effect. Treating neutrophils with carboxyp21 peptide triggered PCNA degradation and apoptosis, while treatment with a mutated version of this peptide incapable of binding to PCNA, had no effect on apoptosis. Moreover, carboxyp21 peptide significantly inhibited G-CSF-induced neutrophil survival. We next examined if p21/waf1 expression in neutrophils can enhance their apoptosis and potentiate the resolution of inflammation. The role of p21/waf1 in neutrophil survival was also examined in a murine model of Pseudomonas aeruginosa as well as in a model of peritonitis. After 7 days of lung infection with P. aeruginosa, neutrophilic inflammation was more prominent in p21-/- mice compared to WT controls. In vitro, neutrophils isolated from p21-/- displayed enhanced survival in response to TNF-alpha and G-CSF associated with an increased in PCNA expression. Our data reveals a novel role for p21/waf1 in the resolution of inflammation after P. aeruginosa infection via its ability to promote neutrophil apoptosis via the destabilization of cytosolic scaffold of PCNA. We concluded that p21/waf1 functions as an inducible and endogenous break in the PCNA pro-survival platform, which has been previously described (Witko-Sarsat et al. J Exp Med, 2010). To our knowledge, this is the first report showing negative
regulation of the pro-survival PCNA scaffold and may have important therapeutic applications in chronic inflammatory diseases.
B232 INTRAVITAL IMAGING OF VASCULATURE REVEALS THAT NEUTROPHIL EXTRACELLULAR TRAP (NET) COMPONENTS ATTACH TO VON WILLEBRAND FACTOR IN A DNASE-INDEPENDENT MANNER See poster section for abstract.
B204 RECOGNITION OF PRODUCTS FROM NEUTROPHIL DEGRANULATION BY TOLL LIKE RECEPTORS MEDIATE KILLING OF LEISHMANIA AMAZONENSIS IN MACROPHAGES See poster section for abstract.
B282 NEUTROPHIL PROTEASES ALTER THE INTERLEUKIN-22-RECEPTOR-DEPENDENT LUNG ANTIMICROBIAL DEFENCE See poster section for abstract.
SYMPOSIUM 9: OCULAR INFLAMMATION, TRANSLATION FOCUSED—SPONSORED BY ORA, INC. 030 NK-DC CROSSTALK TRIGGERS AN INNATE IFN-c/IL-27 LOOP THAT CONTROLS THE AUTOPATHOGENIC TH17 RESPONSE Rachel R. Caspi1, Wai Po Chong1, Nicholas van Panhuys2, Jun Chen1, Phyllis B. Silver1, Yingyos (Ed) Jittayasothorn1, Ronald N. Germain2 1 Immunoregulation Section, Laboratory of Immunology, National Eye Institute, NIH, Bethesda, MD, USA; 2Lymphocyte Biology Section, Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD, USA
IFN-c is a proinflammatory cytokine. Paradoxically, its deficiency exacerbates experimental autoimmune encephalomyelitis, uveitis and arthritis. Here we demonstrate that innate production of IFN-c from NK cells is necessary and sufficient to trigger a novel endogenous regulatory circuit that limits autoimmunity. Following autoantigen challenge, DCs recruited IFN-c-producing NK cells to the draining lymph node and interacted with them in a CXCR3-dependent manner. DCs then produced IL-27, enhancing IFN-c production by NK cells
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and forming a self-amplifying positive feedback loop that was ultimately controlled by NK/DC-intrinsic IL-10. IL-27 dampened the autopathogenic Th17 response and induced a regulatory Tr1 response, which ameliorated disease in an IL-10-dependent fashion. Thus, early NK-DC crosstalk controls adaptive immunity and limits autoimmune disease though an innate IFN-c/IL-27 axis.
031 COMPLEMENT: MORE THAN A ‘‘GUARD’’ AGAINST PATHOGENS John D. Lambris Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA Nearly a century after the significance of the human complement system was recognized, we have come to realize that its functions extend far beyond the elimination of microbes. It is increasingly perceived as an intricate network of effectors, regulators and regulators that not only drives core cascade functions in health and disease but also extensively communicates with associated physiological pathways ranging from immunity and inflammation to homeostasis and development. A steady stream of experimental data reveals new fascinating connections at a rapid pace; while opening unique opportunities for research discoveries, and therapeutic interventions against various diseases and clinical conditions. I will discuss our updated view of the function, structure and dynamics of the complement network, highlight its interconnection with immunity at large and with other endogenous pathways, and illustrate its multiple roles in homeostasis and disease emphasizing its role in ocular disease. Finally, I will discuss the current status of complement therapeutics and various approaches for complement based therapies.
033 DALAZATIDE (SHK-186), A KV1.3 POTASSIUM CHANNEL BLOCKER, SIGNIFICANTLY REDUCES INFLAMMATORY CELL INFILTRATES AND DISEASE IN AN EXPERIMENTAL AUTOIMMUNE ANTERIOR UVEITIS MODEL Ernesto J. Munoz, David W. Peckham, Kayla Norton, Shawn P. Iadonato
032 ROCK-ISOFORM SPECIFIC POLARIZATION OF MACROPHAGES ASSOCIATED WITH AGERELATED MACULAR DEGENERATION Ali Hafezi-Moghadam1,2 Harvard Medical School, Department of Radiology; 2Brigham and Women’s Hospital, Boston, MA, USA
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Age is a major risk factor in age-related macular degeneration (AMD), but the underlying cause is unknown. We find increased Rhoassociated kinase (ROCK) signaling and M2 characteristics in eyes of aged mice, revealing immune changes in aging. ROCK isoforms determine macrophage polarization into M1 and M2 subtypes. M2like macrophages accumulated in AMD, but not in normal eyes, suggesting these macrophages may be linked to macular degeneration. M2 macrophages injected into the mouse eye exacerbated choroidal neovascular lesions, while M1 macrophages ameliorated them, supporting a causal role for macrophage subtypes in AMD. Selective ROCK2 inhibition with a small molecule decreased M2-like macrophages and choroidal neovascularization. ROCK2 inhibition upregulated M1 markers without affecting macrophage recruitment, underlining the plasticity of these macrophages. These results reveal age-induced innate immune imbalance as underlying AMD pathogenesis. Targeting macrophage plasticity opens up new possibilities for more effective AMD treatment.
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Kineta, Inc, Seattle, WA, USA Autoreactive effector memory T cell subsets play a major role in the etiology and chronicity of non-infectious and dry eye ocular disease by secretion of multiple inflammatory mediators. TH1 and TH17 effector memory cells infiltrate the anterior chamber during Sjogren’s syndrome and are key mediators in chronic uveitis models. Standard of care therapies for refractory uveitis and dry eye diseases are problematic, as they are broadly immunosuppressive, cause significant side effects, or exhibit poor eye penetration. Dalazatide (ShK-186) is a potent, highly specific blocker of Kv1.3, a potassium channel required for sustained intracellular calcium influx during activation of effector memory T cells. It is effective in preventing disease in preclinical models of diverse autoimmune diseases. It was well tolerated with no serious adverse events reported in phase 1 trials in healthy volunteers, and is currently being evaluated in a POC trial in psoriasis. In the current studies, topically-dosed dalazatide was evaluated for its ability to penetrate the anterior chamber, and reduce disease and Kv1.3+ and CD3+ cell infiltrates when given prophylactically in an EAAU disease model. Dose-dependent dalazatide penetration of the anterior chamber was demonstrated, with concentrations in the aqueous fluid ranging from 5 to 62 and 275 to 949 ng/mL (0.1 and 1 % dalazatide dose, respectively). No histopathology was observed in H&E-stained sections of 1 % dalazatide-treated naı¨ve eyes. In the EAAU model,
Inflamm. Res. dalazatide reduced disease penetration from 13 of 16 eyes (vehicle) to 3 of 16 (dalazatide). Dalazatide also reduced the mean composite clinical score for all eyes from 7.5 (vehicle) to 1.8 (dalazatide). Cellular infiltrates were reduced in anterior chamber fluid, as well. Histopathology confirmed that dalazatide also reduced or eliminated dilated iris vessels and cellular infiltrates. IHC demonstrated that dalazatide reduced or eliminated Kv1.3+ and CD3+ infiltrates of the anterior chamber, ciliary body, and iris. These results demonstrate that topical ocular dosing of dalazatide has potential for treating autoimmune-mediated uveitis by targeting CD3+ and Kv1.3+ inflammatory cells which mediate disease, while limiting side effects.
SYMPOSIUM 10: GLUCOCORTICOIDS IN THE REGULATION OF INFLAMMATORY RESPONSE—SPONSORED BY ITALIAN INFLAMMATION GROUP 034 INTENSE CROSS–TALK BETWEEN TNF AND GLUCOCORTICOID RECEPTOR Claude Libert Inflammation Research Center, VIB, Ghent, Belgium GC protect extremely well against acute inflammatory conditions and the cross talk between GC and cytokines is important. However, the exact mechanism of the anti-inflammatory function of GC is still unclear. Notably, large groups of patients respond insufficiently to GC, a condition called GC resistance. This problem occurs in some 5 % of asthma patients and in most of the sepsis patients. Sepsis is a form of systemic inflammatory response syndrome (SIRS) and a huge unmet medical need. Study of the mechanism of loss of GC protection, when GC aregiven after the onset of SIRS, indicate an intense cross talk between TNF and GC.
035 ROLE OF GILZ IN MEDIATING THE ANTIINFLAMMATORY ACTIONS OF GLUCOCORTICOIDS Carlo Riccardi, Monica Cimino, Tiziana Frammartino, Daniele Sorcini, Michele Biagioli, Oxana Bereshchenko, Stefano Bruscoli Department of Medicine, University of Perugia, Perugia, Italy Glucocorticoids (GC) are of extraordinary therapeutic value in a wide range of inflammatory and autoimmune diseases. Most of GC effects are receptor (GR) mediated and relates to regulation of gene transcription. Their therapeutic activity is due to effects on activation, growth and differentiation in a number of cells and tissues, including cells of the immune/inflammatory system such as T lymphocytes. We describe the GC-induced gene glucocorticoid-induced leucine zipper (GILZ), a protein rapidly induced by GC treatment. Notably, our results indicate GILZ is a mediator of GC effects. In particular, using GILZ-TG and GILZ-KO mice we demonstrate, in various disease models, GILZ is an anti-inflammatory molecule. Interestingly, GILZ regulates B and T cell activation, differentiation and survival. Moreover, it regulates cytokine production and inflammatory process
S67 development. Notably, GILZ is an essential mediator for GC-induced development of T regulatory cells (Treg), a T cell subpopulation responsible of anti-inflammatory effect. Consistent with these T cell subpopulations changes there is an increased severity of colitis, arthritis and other inflammatory disease models in GILZ-KO, while a reduction in GILZ-TG. Results indicate GILZ as a mediator of GC activity, provide new means to predict sensitivity to treatment with GC and to outline new therapeutic approaches.
036 GLUCOCORTICOID AND B CELL DEVELOPMENT: ROLE OF GLUCOCORTICOIDINDUCED LEUCINE ZIPPER (GILZ) Stefano Bruscoli, Michele Biagioli, Oxana Bereshchenko, Tiziana Frammartino, Daniele Sorcini, Monica Cimino, Carlo Riccardi Department of Medicine, Section of Pharmacology, University of Perugia, Perugia, Italy Glucocorticoids (GC) are widely used as immunosuppressive drugs and antitumor agents in some acute leukemia and multiple myeloma. Therapeutic doses of GC induce growth suppressive and cytotoxic effects on various leukocyte types including B cells. Molecular mechanisms of GC action include induction of GC target genes. Glucocorticoid-induced leucine zipper (GILZ) is a gene rapidly, potently and invariably up-regulated by GC treatment. It mediates a number of GCs effects, such as control of cell proliferation, apoptosis and differentiation. GILZ suppresses Ras/MAPK/Erk and NFkB pathways and promotes TGF-b signaling in T cells. It belongs to TSC22d family, members of which were recently found mutated in diffuse large B cell lymphoma patients. Here we address the physiologic role of GILZ in normal hematopoiesis, and evaluate its role in mediation of GC effects on various blood cells, using genetic approach. Mice deleted for gilz gene were recently generated. We have monitored white blood cell counts in wild type (wt) and in gilz knockout (KO) mice overtime. Development of lymphoid and myeloid lineages was evaluated both by peripheral blood (PB) cell counts (Hematocrit), and by flow cytometry analysis of bone marrow (BM), spleen and PB using Mac-1, B220, CD43, IgM and IgD staining. Young gilz KO mice showed normal body and lymphoid tissues weights and cell counts in PB, thymus, spleen, peripheral lymph nodes and BM. However, overtime gilz KO mice showed a 1.5- to 2-fold increase in white blood cell counts in PB. Increase in lymphocyte counts was due to accumulation of B220+ cells, while the number of Mac-1+ cells did not differ between wt and gilz KO mice. Flow cytometry analyis of B220+ cell compartment in BM revealed an increase in the frequency and number of pre-B cells (IgM loIgDlo), immature (IgMhiIgDlo) and recirculating B cells (IgMloIgDhi) already in 8-week old mice. Preliminary data suggest that the defect starts as early as at common-lymphoid progenitor (CLP) stage. Treatment of purified B220 + cell with GC in vitro resulted in different degree of apoptosis in wt and gilz KO cells, suggesting that the increase in B cells in vivo may results from decreased sensitivity to the death induced by endogenous GC. Our results show that lack of GILZ results in specific defect in B cell development, leading to the expansion of B220 + cells compartment, associated with the expansion of early B cell progenitor cells and suggest that deregulation of GILZ expression may contribute to cell survival or differentiation of early B cells and pathologies of B cell lineage.
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SYMPOSIUM 11: A ROLE FOR EPIGENETICS IN INFLAMMATORY DISEASE: SPOTLIGHT ON MONOCYTES AND MACROPHAGES 037 THE EPIGENETIC SIGNATURE OF INFLAMMATION IN WHITE BLOOD CELLS AND PULMONARY MACROPHAGES FROM SMOKERS: WHERE THERE IS SMOKE, THERE IS FIRE Robert A. Philibert Universtity of Iowa, Iowa City, IA, USA Smoking is the largest preventable cause of morbidity and mortality in the United States. Each year, over a half million people die prematurely from smoking related illnesses including coronary artery disease, stroke and chronic obstructive pulmonary disease. Peripheral white blood cells and their derivatives, most notably, alveoloar macrophages, are known to play important roles in mediating the pathogenesis of some of these smoking-related illnesses. In this presentation, we compare and contrast the whole genome DNA methylation of DNA from peripheral white blood cells and pulmonary macrophages. We show that smoking is associated with dose-dependent alteration of DNA methylation with preference for those genes mapping to immune system and inflammatory pathways in both peripheral white blood cell and alveolar macrophage DNA. These changes in DNA methylation are mirrored by corresponding changes in the expression of genes mapping to inflammatory and immune system pathways, and prior data demonstrating the role of smoking in driving the conversion of alveolar macrophages from the M1 to the M2 phenotype. Network analyses of peripheral methylation data demonstrate that smoking alters the regulation of protein networks important not only to inflammation, but to smoking associated illnesses such as stroke, heart disease (notably F2RL3) and cancer. Finally, and perhaps most importantly, the sensitivity and reversibility of DNA methylation at key loci, such as AHRR, may allow the derivation of new clinical tools for improving smoking prevention and cessation.
038 SUPPRESSION OF TYPE 1 DIABETES BY EPIGENETIC MODULATION Wenxian Fu1,2, Julia Farache1, Rab Prinjha3, Christophe Benoist1, Diane Mathis1 Harvard Medical School, Boston, MA, USA; 2University of California, San Diego, Jolla, CA, USA; 3GlaxoSmithKline, Brentford, USA
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Type 1 diabetes (T1D) is an organ-specific autoimmune disease characterized by selective destruction of the insulin-producing beta cells of the islets of Langerhans of the pancreas. Like many complex diseases, the pathogenesis of T1D is believed to be affected by both genetic associations and environmental cues. Epigenetic regulation is critical to integrate environmental signals for the cell to modulate the functional output of its genome. However, the impact of epigenetic mechanisms and regulations in T1D remains largely unexplored. Here we report the modulation of T1D in non-obese diabetic (NOD) mice by an inhibitor that specifically interferes with the recognition of acetylated lysine residues by proteins of the bromodomain and extra-
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Inflamm. Res. terminal domain (BET) family. Mice treated with a BET protein inhibitor were protected from the development of diabetes. Concordantly, insulitis was either prevented or inhibited, depending on the treatment protocol. Mechanistically, BET inhibition induced an antiinflammatory phenotype in pancreatic macrophages through the suppression of an NF-kappa-B-mediated inflammatory pathway in these cells. Strikingly, I-BET was also beneficial to pancreatic beta cells by promoting beta cell turnover in vivo. Thus, small molecule inhibition of BET protein bromodomains irreversibly blocks T1D in NOD mice via combined effects of fostering anti-inflammatory macrophages and promoting beta cell function. Together, these data provide a rationale for T1D treatment by targeting epigenetic modifications.
039 EPIGENETIC DYSREGULATION IN IMMUNE TOLERANCE: TWO SIDES TO THE SAME COINCANCER AND AUTOIMMUNITY S A. Litherland, PhD1,2,3, Michael J. Clare-Salzler, MD2, Juan Pablo Arnoletti, MD1 1
Florida Hospital Cancer Institute, Orlando, FL, USA; 2University of Florida College of Medicine, Gainesville, FL, USA; 3SanfordBurnham Medical Research Institute, La Jolla, CA, USA Immune tolerance, the ability of the immune system to distinguish self from non-self on the molecular level, varies throughout the body. A range of immune sensitive to immune privileged compartments are found, dependent on the need to balance recognition of foreign antigens mimicking self moieties with the ability to recognize and destroy altered-self antigens. Maintenance of immune tolerance is a double edged sword in disease: too little, and self-reactivity leads to autoimmune pathology; too strong and altered-self cells go undetected or are actively protected from immune responses. A proinflammatory microenvironment aides both immune-related pathologies. In autoimmunity, epigenetic dysregulation promotes the inflammatory milieu conducive to the development of immunopathology. In this pro-inflammatory environment, antigen presenting cell regulation of self-reactive T cells becomes defective, self tolerance can be lost, and auto-reactivity ensues. In autoimmune type 1 diabetes (T1D), we found that epigenetic dysregulation affects the expression of GM-CSF and its subsequent activation of COX2 expression and PGE2 production. The dysregulation is mediated by aberrant histone acetylation though dysfunctional STAT5 activation and binding to enhancer elements in both gene loci. This dysfunction seen phenotypically in T1D human monocytes, can be genetically linked in NOD bicongenic mice myeloid cells through genetic variations found in enhancer sequences affecting expression of CSF2 and PTGS2 gene loci. In cancer, promotion of immune self-tolerance can be lethal, allowing relapse and metastatic growth of treatment resistant tumors. Epigenetic dysregulation of oncogenes in tumors is emerging as a common biomarker for tumor activation and development. Pro-inflammatory products of myeloid derived suppressor cell (MDSC) promote immunosuppression of tumor recognition. These functions appear to be enhanced by the interactions of MDSC with tumor cells. In pancreatic ductal carcinoma (PDAC), we have reproducibly detected histone acetylation of chromatin promoting the activation and gene expression of mutant K-RAS gene. We use this epigenetic biomarker to identify circulating tumor cells (CTC) concentrated in the portal venous blood circulatory compartment between pancreas and liver. The level of histone acetylation-mediated activation and KRAS mutant mRNA expression is not seen in peripheral blood from the same patients or in the portal venous blood of patients without
Inflamm. Res. cancer, suggesting the immune microenvironment of the portal circulation may act as a potential metastatic cell reservoir for PDAC. Epigenetic dysregulation of inflammation and of critical gene expression in immune targets represent two faces of the same pathological coin, allowing for the development of pro-inflammatory microenvironments and promoting aberrations of self-tolerance.
040 THE EPIGENETIC-ASSOCIATED FAMILY OF BROMODOMAIN ‘‘READER’’ PROTEINS AS DRUG TARGETS TO REGULATE ACTIONS OF THE IMMUNE SYSTEM Eleonore Beurel University of Miami, Coral Gables, FL, USA Orchestration of the inflammatory response is crucial for clearing pathogens. Although the production of multiple inflammatory cytokines has been thought to be regulated by common mechanisms, recent evidence indicates that the expression of some cytokines is differentially regulated by epigenetic regulatory mechanisms. Inhibitors of bromodomain reader (BRD) proteins, including BRD2, BRD3 and BRD4, have provided promising results in a wide spectrum of therapeutic applications, especially the regulation of the immune system. BRD proteins are involved in translating histone modifications, which dramatically impacts transcription. Thus, overexpressing BRD2 leads to the development of splenic B lymphomas. BRD4 promotes NF-jB dependent gene responses after endotoxin shock. BET bromodomain protein inhibitor (I-BET) protects from otherwise lethal septic shock and blocks lipopolysaccharide (LPS) induced cytokine production. We found that IL-6 production is selectively inhibited by a low dose of I-BET151 in RAW264.7 cells stimulated with LPS, whereas I-BET151 did not alter the production of several other cytokines (TNFa, IL-1b and IL-10) at the same concentration of IBET151. I-BET151 prevented the binding of CBP to the promoter of IL-6, but I-BET151 did not affect acetylation, phosphorylation, nuclear translocation, or DNA binding of p65-NFjB. In vivo, I-BET151 treatment in the experimental autoimmune encephalomyelitis mouse model of multiple sclerosis decreased the early clinical symptoms, which are thought to be dependent on cytokine production. Altogether, these data suggest that targeting epigenetic-related proteins, such as BET proteins, may provide a strategy to reduce inflammation and the severity of inflammatory diseases, such as multiple sclerosis.
SYMPOSIUM 12: NEXT GENERATION KINASE INHIBITORS—SPONSORED BY BIRA-UK 041 TARGETING IRAK4 AS A CENTRAL REGULATOR IN INNATE IMMUNITY Iain Kilty Pfizer Inc, New York, NY, USA Interleukin 1 receptor associated kinase 4 (IRAK4) represents a key node in innate inflammatory signaling, directly downstream of the
S69 Toll-like receptors (TLR) and Interleukin-1 (IL-1) family of receptors. TLRs represent a first line of defense against pathogens such as bacteria, viruses and yeast with the IL-1 family of receptors also playing important roles in the immediate inflammatory response to invading organisms. In addition IRAK4 is expressed in T and B lymphocytes and has been reported to play an important role in cross talk between the innate and adaptive immune system. IRAK4 has both a kinase dependent signaling role as well as a scaffolding role in a larger signaling complex including proteins such as MYD88 and IRAK1. Interestingly, individuals who lack IRAK4 show impaired activation of the innate immune response but no increased susceptibility to viral or fungal infection and only increased liability to infection by a narrow range of pyogenic bacteria prior to adolescence. Therefore IRAK4 has been recognized as an interesting pharmacological target for the treatment of chronic inflammatory diseases. Using potent selective IRAK4 inhibitors we have characterized the role of IRAK4 kinase activity in mediating inflammatory signaling in primary human cells from both healthy donors and patients suffering chronic inflammatory diseases, such chronic obstructive pulmonary disease, systemic lupus erythematosus and gout. Moreover, in vivo activity of compounds has also been investigated in a range of chronic inflammatory models. The emerging rationale for IRAK4 inhibition across chronic inflammatory indications will be presented.
042 A NEW GENERATION OF INHALED KINASES INHIBITORS FOR INFLAMMATORY AIRWAY DISEASES Christopher S. Stevenson Respivert Ltd, London, UK Chronic obstructive pulmonary disease (COPD) and uncontrolled asthma are obstructive airway diseases associated with pronounced lung inflammation that cannot be attenuated with inhaled corticosteroids. Several alternative anti-inflammatory approaches are currently under clinical evaluation including orally administered molecules that target protein kinases, which transduce inflammatory signals triggered by cytokines, pathogens, and environmental stimuli (e.g., cigarette smoke). Unfortunately, the systemic adverse effects of these kinase inhibitors have limited the clinical dose range that can be assessed. In addition, the anti-inflammatory efficacy of these molecules in other inflammatory indications (e.g., rheumatoid arthritis) have been limited due to physiological escape and/or the molecular redundancy that exists for these signaling pathways. To mitigate these potential risks, Respivert have developed two novel classes of inhaled kinase inhibitors: (1) narrow spectrum kinase inhibitors (NSKIs) and (2) inhibitors of phosphatidylinositide 3-kinase isoforms. The molecules were designed for inhalation delivery to limit the systemic exposure of these molecules (in order to maximize safety margins) and to specifically target multiple kinase isoforms involved in mediating steroid resistant inflammation (to mitigate the potential for escape/redundancy). Each class of inhibitors was optimized using primary human cell phenotypic assays (including cells from patients) to assess their potential for inhibiting steroid resistant inflammatory processes. In vivo efficacy and duration of action were assessed across a number of preclinical models of steroid sensitive and insensitive inflammation. Early clinical data indicate that these molecules are well tolerated and provide an anti-inflammatory benefit. Taken together, these data indicate that inhaled NSKIs and PI3 K inhibitors hold therapeutic potential by attenuating the steroid-resistant inflammation associated with COPD and uncontrolled asthma.
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MINI-SYMPOSIUM 1: NOVEL MODELS OF INFLAMMATION B071 CIGARETTE SMOKE ENHANCES ADAPTIVE IMMUNE RESPONSE IN MURINE MODEL OF ALLERGIC AIRWAY INFLAMMATION See poster section for abstract.
B092 DISEASE RELEVANT IN VITRO AND IN VIVO MODELS FOR LUNG FIBROSIS See poster section for abstract.
B002 SYNCHROTRON MICROBEAM IRRADIATION INDUCES INFLAMMATION AND SELECTIVE VASCULAR DAMAGES IN ADULT ZEBRAFISH See poster section for abstract.
B191 DEVELOPMENT OF A MODEL OF DERMAL INFLAMMATION AND IRRITATION (URTICARIA) IN THE MINIATURE SWINE See poster section for abstract.
B237 IN VITRO MODELLING ACUTE AND CHRONIC INFLAMMATION BASED ON HUMAN MONOCYTES. ANALYSIS OF THE MODULATION OF IL-1 FAMILY MEMBERS
Inflamm. Res.
B179 MATERNAL IL17 PATHWAY PROMOTES AUTISM–LIKE PHENOTYPES IN OFFSPRING See poster section for abstract.
PATIENT PERSPECTIVES: UNMET MEDICAL NEED–INFLAMMATORY BOWEL DISEASE 043 PATIENT PERSPECTIVES: UNMET MEDICAL NEED–INFLAMMATORY BOWEL DISEASE Heather E. Jones Pfizer, Inc., Collegeville, PA, USA Objective: To increase awareness of the true unmet medical need left by current therapeutic options for Crohn’s disease and understand what this means to the patient. The importance of recognizing the true unmet medical need in autoimmune diseases is higher than ever. No drug has so far been able to cure Crohn’s disease, only to induce remission. To progress science and clinical development there must be a focus on understanding the pathogenesis of the disease. From a medical perspective we need to consider the patient as a whole human being and not only based on the organ(s) involved. Autoimmune diseases in most cases are systemic disorders that affect more than one organ. The morning session will begin by emphasizing the unmet medical need followed by an interview with a patient with Crohn’s disease, with an emphasis on what they see as key objectives for further development. At the end of the session the co-chairs will summarize the signs and symptoms and the treatment algorithm for Crohn’s disease, as well as the need for biomarkers to define the disease, ensure safety, guide treatment and predict treatment response.
SYMPOSIUM 13: microRNAs (MIRS) IN INFLAMMATION
See poster section for abstract.
B239 DYNAMIC EQUILIBRIUM OF NEUTROPHIL TRAFFICKING AT SITES OF INFLAMMATION AND INFECTIONS See poster section for abstract.
044 METABOLIC REGULATION BY MIR-33 IN MACROPHAGES CONTROLS IMMUNE EFFECTOR RESPONSES Kathryn J. Moore1, Hasini Ediriweera1, U. Mahesh Gundra1, Katey J. Rayner3, P’ng Loke1, Philip Zamore2, Gregory Steinberg4, Mireille Ouimet1 1
B235 SPONTANEOUS MUTATION IN ZFP-BINDING REGION OF TNF GENE CAUSES CHRONIC POLYARTHRITIS AND HEART VALVE DISEASE See poster section for abstract.
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New York University School of Medicine, New York, NY, USA; University of Massachusetts Medical School, Worcester, MA, USA; 3 University of Ottawa Heart Institute, Ottawa, ON, Canada; 4 McMaster University, Hamilton, ON, Canada 2
Cellular metabolism is increasingly recognized to control immune cell fate and functions. miR-33 is a regulator of cellular lipid metabolism that represses genes involved in cholesterol efflux, HDL biogenesis and fatty acid oxidation. We demonstrate that by altering
Inflamm. Res. the balance of aerobic glycolysis and mitochondrial oxidative phosphorylation, miR-33 instructs macrophage inflammatory polarization and shapes innate and adaptive immune responses. Targeted deletion of miR-33 in macrophages increases oxidative respiration, enhances spare respiratory capacity, and induces the expression of genes that define M2 macrophage polarization. We show that these changes are independent of effects on cholesterol efflux, but instead require miR-33 targeting of the energy sensor AMP-activated protein kinase. Notably, inhibition of miR-33 also increases macrophage expression of the retinoic acid-producing enzyme Aldh1a2 and retinal dehydrogenase activity both in vitro and in vivo. Consistent with the ability of retinoic acid to foster inducible regulatory T cells, anti-miR33-treated macrophages have an enhanced capacity to induce FoxP3 expression in naı¨ve CD4+ T cells. Finally, treatment of western diet-fed Ldlr-/- mice with miR-33 inhibitors for 8 weeks (conditions that do not alter HDL cholesterol levels) promoted the accumulation of inflammation suppressing M2 macrophages and FoxP3+ T regulatory cells in plaques, and reduced atherosclerosis progression by 40 %. Collectively, these results identify a novel role for miR-33 in the regulation of macrophage inflammation and show that antagonism of miR-33 reduces atherosclerotic inflammation by promoting M2 macrophage polarization and regulatory T cell induction.
045 microRNA REGULATION OF ENDOTHELIAL INFLAMMATION Mark W. Feinberg1,2 1
Brigham and Women’s Hospital, Boston, MA, USA; 2Harvard Medical School, Boston, MA, USA Endothelial cell (EC) activation and vascular inflammation occur when the endothelium is exposed to various biochemical insults such as proinflammatory cytokines, oxidative stress, hypertension, hyperglycemia, aging, and biomechanical stimuli such as shear stress. These insults lead to the pathogenesis of a range of disease states, including atherosclerosis, insulin resistance, and obesity. Several signaling pathways, especially nuclear factor jB mediated signaling, play crucial roles in these pathophysiological processes. Recently, microRNAs (miRNAs) have emerged as important regulators of EC function by fine-tuning gene expression. In this seminar, the audience will gain insights of how miRNAs regulate EC function and vascular inflammation in response to a variety of pathophysiologic stimuli. Recent studies in mice and human subjects highlight an important role for miR-181b as a suppressor of endothelial inflammatory responses in both acute (e.g., sepsis) and chronic vascular disease states (e.g., atherosclerosis, insulin resistance, and obesity). These studies have uncovered emerging roles for novel miRNA targets in a cell-specific manner. An understanding of the role of miRNAs in EC activation and dysfunction may provide novel therapeutic opportunities for controlling a range inflammatory disease states.
S71 Sepsis is a systemic inflammatory response to infection and mediated via activation of the innate immune system. Sepsis is the leading cause of death in patients in the intensive care units and opioids are the preferred analgesic in this setting. The adverse effects of chronic morphine on the immune system has been well documented over the years. Higher levels of morphine in systemic circulation reduces pathogen clearance, specifically in case of opportunistic infection, and also induces translocation of gut microbes. Interestingly, in healthy individuals, both sepsis and consequent exposure to bacterial products is characterized by an initial hyper-production of cytokines, followed by a ‘‘silencing’’ phase, where Toll Like Receptor mediated production of pro-inflammatory cytokines is suppressed. This has been variously referred to as ‘‘TLR reprogramming’’ or ‘‘endotoxin/LPS tolerance’’. The mechanisms proposed for the endotoxin tolerance range from silencing of key mediators of TLR signaling to impaired interaction between different signaling mediators. Within the past decade, while the ‘‘silencing’’ mechanism is increasingly being implicated in describing drug/endotoxin tolerance, a new class of molecules, namely the micro-RNAs (miRNA) have emerged as key players in selectively silencing the intermediaries of TLR signaling between the surface receptor and eventual NF-jB activation. Development of tolerance to endotoxin prevents sustained hyper inflammation during systemic infections. We show that chronic morphine treatment tempers endotoxin tolerance resulting in persistent inflammation, septicemia and septic shock. Morphine was found to down-regulate endotoxin/LPS induced miR-146a and 155 in macrophages. However, only miR-146a over expression, but not miR-155 abrogates morphine mediated hyper-inflammation. Conversely, antagonizing miR-146a (but not miR-155) heightened the severity of morphine-mediated hyper-inflammation. These results suggest that miR-146a acts as a molecular switch controlling hyper-inflammation in clinical and/or recreational use of morphine.
SYMPOSIUM 14: MUCOSAL IMMUNITY: HOT TOPICS IN IBD 047 MONGERSEN, AN ORAL SMAD7 ANTISENSE OLIGONUCLEOTIDE, IN ACTIVE CROHN’S DISEASE Gerald Horan1, Giovanni Monteleone2, Markus F. Neurath3, Sandro Ardizzone4, Antonio Di Sabatino5, Massimo Claudio Fantini2, Fabiana Castiglione15, Maria Lia Scribano6, Alessandro Armuzzi7, Flavio Caprioli8, Giacomo Carlo Sturniolo9, Francesca Rogai10, Maurizio Vecchi8, Raja Atreya3, Fabrizio Bossa11, Sara Onali2, Maria Fichera4, Gino Roberto Corazza5, Livia Biancone2, Vincenzo Savarino12, Roberta Pica13, Ambrogio Orlando14, Francesco Pallone2 1
046 MORPHINE INDUCED EXACERBATION OF SEPSIS IS MEDIATED BY TEMPERING ENDOTOXIN TOLERANCE THROUGH MODULATION OF MIR-146A Sabita Roy1,2, Santanu Banerjee1, Jingjing Meng1, Sundaram Ramakrishnan2 1
Department of Surgery; 2Department of Pharmacology, University of Minnesota, Minneapolis, MN, USA
Celgene Corp, Summit, NJ, USA; 2University of Tor Vergata, Rome, Italy; 3University of Erlangen-Nu¨rnberg, Erlangen, Germany; 4‘‘L. Sacco’’ University Hospital, Milan, Italy; 5University of Pavia, Pavia, Italy; 6Hospital San Camillo-Forlanini, Rome, Italy; 7Catholic University, Rome, Italy; 8University of Milan, Milano, Italy; 9 University of Padova, Padova, Italy; 10AOU Careggi University Hospital, Florence, Italy; 11‘‘Casa Sollievo Sofferenza’’ Hospital, IRCCS, San Giovanni Rotondo, Italy; 12University of Genoa, Genoa, Italy; 13Sandro Pertini Hospital, Rome, Italy; 14University of Palermo, Palermo, Italy; 15University ‘‘Federico II’’ of Naples, Italy Introduction: Crohn’s disease (CD)-related inflammation is characterized by defective activity of the immunosuppressive cytokine transforming growth factor (TGF)-b1, due to high Smad7 (an
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S72 inhibitor of TGF-b1) signalling. The effects of an oral, topically active Smad7 antisense oligonucleotide, Mongersen, were evaluated in a phase II study in patients with active CD. Aims and methods: In a double-blind, placebo-controlled trial, the efficacy of Mongersen as induction therapy was evaluated in steroiddependent or steroid-resistant patients (utilizing ECCO consensus definition) with active CD [CD activity index (CDAI) score 220–400]. Patients were randomized to Mongersen 10, 40 or 160 mg/day or placebo for 2 weeks. The primary outcomes were clinical remission (CDAI score \150 at Day 15 and maintained for C2 weeks) and safety. Secondary endpoints included clinical response (CDAI score reduction of 100 points) at Day 28. Results: Clinical remission was achieved by significantly greater proportions of patients receiving Mongersen 40 (55.0 %) and 160 mg/day (65.1 %) compared with placebo (9.5 %; p \ 0.0001 for both). No significant difference in clinical remission was seen for 10 mg/day (12.2 %) vs. placebo. The rate of clinical response was significantly greater among patients receiving 10 (36.6 %), 40 (57.5 %) or 160 mg/day (72.1 %) of Mongersen vs. placebo (16.7 %; p = 0.039, p = 0.0001 and p \ 0.0001, respectively). The rates of adverse events (AEs) and serious AEs (SAEs) were similar across groups. Nine SAEs occurred in 6 patients (placebo, n = 1; Mongersen 10 mg, n = 3; 40 mg, n = 1; 160 mg, n = 1). Most SAEs consisted of hospital admissions for CD-associated complications or symptoms, and included: pyrexia and cough (placebo); abdominal pain (n = 2), CD worsening and pyrexia (Mongersen 10 mg); seton placement for perianal fistula and surgery for hemorrhoid thrombosis (Mongersen 40 mg); and thermal burn (Mongersen 160 mg). Conclusions: Induction therapy with orally administered, topically active Mongersen for CD was well tolerated; toxicities previously reported with systemically active antisense agents were not observed. Mongersen treatment resulted in significant improvements in clinical remission and response rates within 4 weeks of initiation of treatment.
048 THE UNFOLDED PROTEIN RESPONSE AND INTESTINAL INFLAMMATION Richard Blumberg Harvard Medical School, Boston, MA, USA Protein function is fundamentally dependent on correct protein folding and post-translational modification. For proteins entering the secretory pathway, this occurs in the endoplasmic reticulum (ER) which imposes stress on the ER when misfolding occurs. The unfolded protein response (UPR) has evolved to counter this stress by adapting the cell’s protein folding capacity to the level of demand. Abnormalities in the UPR are increasingly recognized as an important contributor to disease pathogenesis. Three major branches of the UPR have evolved, each consisting of an ER transmembrane protein that serves as a proximal sensor of ER stress in conjunction with an ER resident chaperone, grp78. These three ER transmembrane proteins are inositol-requiring enzyme 1 (IRE1), with its isoforms a and ß, activating transcription factor 6 (ATF6; a and ß isoforms) and PKR (double-stranded RNAdependent protein kinase)-like ER kinase (PERK), which activate specific transcriptional programs. IRE1 acts as an endoribonuclease, excising a 26nt stretch from the mRNA encoding X-box binding protein 1 (XBP1), which results in a frame-shift and translation of the active transcription factor (XBP1s, s for spliced) that transactivates UPR target genes. ATF6 is packaged into vesicles, which are released from the ER and traverse to the Golgi, where ATF6 is cleaved by site-1 and site-2 proteases (S1P, S2P), releasing the active transcription factor fragment that enters the nucleus and also transactivates a set of
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Inflamm. Res. UPR target genes, in part by co-operating with XBP1s. XBP1 and ATF6 transactivate genes that are involved in protein translation, folding and quality control, but also in ER associated degradation (ERAD), rendering the removal of misfolded proteins via the proteasome. Finally, PERK activation results in phosphorylation and hence inactivation of elongation and initiation factor 2a (eIF2a) and consequent inhibition of mRNA transcription decreasing the global flux of proteins entering the ER. However, certain mRNAs that contain short open reading frames in their 50 untranslated regions are preferentially transcribed under conditions of limiting eIF2a. Amongst those is activating transcription factor 4 (ATF4), a further UPR transcription factor that, among other target genes, transactivates CHOP (transcription factor C/EBP homologous protein). CHOP in turn transactivates genes involved in apoptosis induction and thereby connects ER stress that has become unsustainable with cell death. A critical cell type that is affected by abnormalities of the UPR is the intestinal epithelial cell (IEC) and especially Paneth cells. Many genetic and environmental defects which act upon the elements of the UPR result in increased susceptibility to intestinal inflammation. One such factor is XBP1, which when deleted in the IEC results in spontaneous enteritis. This presentation will focus on the role played by the UPR in IECs and how these pathways interact with autophagy
049 EMERGING UTILITY OF CULTURED INTESTINAL EPITHELIAL CELLS IN PERSONALIZED MEDICINE Thaddeus Stappenbeck Washington University School of Medicine, St. Louis, MO, USA One mode of personalized medicine is to utilize cells from patients to perform in vitro assays that will in turn inform upon the treatment regimens of specific patients. Intestinal epithelial cells have the potential to be a critical cell type in such assays. In the last several years, as a field, we have developed the ability to efficiently isolate and propagate these cells from primary sources (animal models and humans). The lines creates have been the nidus for many new exciting experimental systems developed in the last several years that I will review here. I will also describe recent advances in the utility of human intestinal epithelial cell lines to show differences in the response to inflammatory stimuli that could direct treatments.
B209 ABSENCE OF DIETARY FIBRE OR THE METABOLITE SENSOR GPR43 EXACERBATES NEUTROPHIL RECRUITMENT DURING ACUTE INFLAMMATORY RESPONSES See poster section for abstract.
B032 A NOVEL IRON-MEDIATED MECHANISM FOR DEVELOPMENT OF INFLAMMATORY BOWEL DISEASE See poster section for abstract.
Inflamm. Res.
SYMPOSIUM 15: INHIBITORY RECEPTOR/IMMUNE CHECKPOINTS/ IMMUNOTHERAPEUTICS—SPONSORED BY IRA-USA 050 PREVENTION OF COLLATERAL DAMAGE BY IMMUNE INHIBITORY RECEPTORS Linde Meyaard Laboratory of Translational Immunology, UMC, Utrecht, The Netherlands The proper functioning of the immune system is based on a fine balance between activation and inhibition. Immune suppression leads to the threat of infection by pathogens and immunodeficiency, while insufficient inhibition can lead to damage to self or autoimmunity. Inhibitory immune receptors play a crucial role in this balance. Neutrophils are useful but also dangerous cells. They are essential to protect the host against invading pathogens and have the ability to quickly enter the tissues. However, their activity also causes collateral tissue damage that in ultimo can result in destruction of the tissue architecture and the formation of pus, also in non-infectious inflammatory conditions. Balancing neutrophil influx and function is essential to prevent excessive disease. Immune inhibitory receptors are potential regulators of this balance. Next to phagocytosis and degranulation, neutrophils fight bacteria through neutrophil extracellular traps (NETs), which arise from the release of the neutrophil’s nuclear content into the extracellular space and consist of decondensed DNA decorated with antimicrobial proteins. Despite the importance of NETs in host defense, NETs contribute to pathology by several mechanisms. It is suggested that the DNA and antimicrobial peptides of NETs can be recognized by B cells which leads to the production of auto-antibodies, such as in SLE. Furthermore, the antimicrobial peptides attached to the NETs have poor target specificity, which leads to the damage of the tissues surrounding the NET. Finally, NETs have recently been shown to form a platform for platelets and form a central role in thrombosis. Therefore, NET formation contributes to pathology in several diseases that involve tissue infiltration by neutrophils. We recently identified signal inhibitory receptor on leukocytes-1 (SIRL-1) as a negative regulator of human neutrophil function. SIRL1 ligation inhibits the oxidative burst and prevents the pathogenic release of NETs in SLE. SIRL-1 engagement can dampen spontaneous and anti-neutrophil antibody-induced NET formation in SLE. This opens up the interesting therapeutic opportunity of inhibiting collateral damage by engagement of inhibitory receptors.
S73 demonstrated that ITAM can also initiate inhibitory signaling toward heterologous receptors. This inhibitory signaling by ITAM-bearing receptors is coined inhibitory ITAM (ITAMi). Until now one human IgA receptor, the FcaRI (CD89) and two human low-affinity IgG receptors, the FccRIIIA (CD16A) and the FccRIIA (CD32A), that are associated with the FcRc subunit or carrying an ITAM motif on their cytoplasmic tail, have been found to act as bifunctional receptors which, depending on the type of interaction with their ligand, induce either activating or inhibitory cell signaling. While multivalent cross-linking of these FcR by immune complexes induced proinflammatory signaling, monovalent or divalent targeting of these FcR with either IgA or IgG (or with Fab/ F(ab0 )2 of anti-FcR) could trigger inhibitory signals towards a whole array of cellular functions such as phagocytosis, IgE-dependent degranulation and TLR- or cytokine-mediated responses. The ITAMi configuration is defined by weak phosphorylation of the ITAM, transient recruitment of Syk followed by stable recruitment of the tyrosine phosphatase SHP-1, whereas multivalent aggregation of these receptors promotes strong ITAMa phosphorylation with stable Syk recruitment. The ITAMi configuration promotes the actin depolymerization-dependent ‘‘trapping’’ of these FcR with the targeted activating receptors within the same lipid rafts, which is followed by the appearance of intracellular inhibisome clusters. Monomeric IgA, IgG, IVIg or anti-FcR Fab/F(ab0 )2 were able to either prevent or reverse inflammation in asthma, nephritis or arthritis mouse models through ITAMi signaling. These anti-inflammatory effects were independent of ITIM-bearing FccRIIB. These results demonstrate that circulating IgA or IgG are not functionally inert but act through continuous interaction with FcR inducing ITAMi signaling to maintain immune homeostasis. These data also support a new mechanism of action for IVIg and demonstrate the therapeutic potential of FccRIIIA targeting in inflammation. Recently, we found a new function for FccRIIA following receptor engagement by anti-FccRII F(ab0 )2 or by IVIg that reverses arthritis in mice. This mechanism has the ITAMi signaling molecular signature described for FcaRI and FccRIIIA but differs by involvement of a single tyrosine. Anti-FccRII F(ab0 )2 treatment of inflammatory synovial cells from rheumatoid arthritis patients induced inhibition of ROS production by switching their inflammatory activation state from an activated-ITAMa configuration to an ITAMi signaling. Shifting constitutive FccRIIA-mediated activation in rheumatoid arthritis to an ITAMi signaling could thus reverse inflammation, providing ground for novel treatment options in this disease.
SYMPOSIUM 16: MECHANISMS OF FIBROSIS 052 BASIC MECHANISMS OF KIDNEY FIBROSIS Jeremy S. Duffield1,2
051 ITIM AND ITAM BEARING INHIBITORY RECEPTORS ON TREATMENT OF INFLAMMATORY DISEASES Renato C. Monteiro1,2,3 Center for Research on Inflamation, Paris, France; 2Inflamex Consortium, Paris, France; 3Paris Diderot University, Paris, France
1
Immunoregulation by Fc receptors (FcR) is controlled by ITAM and ITIM motifs. This classic concept of the functional polarity of ITIM and ITAM motifs has been recently reevaluated. Several studies have
1 Biogen, Cambridge, MA, USA; 2Renal Division, University of Washington, Seattle, WA, USA
Chronic kidney diseases, represent a major burden to society, affecting up to 10 % of the adult population, and are characterized by progressive fibrosis of the glomerulus and interstitial space. Currently organ failure is managed by intervention with dialysis and for some organ transplantation. Proof of concept experiments indicate that fibrosis per se contributes to loss of function and organ demise, and that fibrosis represents an aberrant wound-repair response. Recent studies in animals have delineated cellular mechanisms by which fibrosis occurs, and the challenge now is to identify pathways that drive this process where therapeutic interventions can be safely introduced.
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S74 Fibrogenic cells undergo profound molecular, cellular and phenotypic changes from the healthy state to the pathological state, and these require changes in inflammatory signaling pathways, growth factor signaling pathways and metabolic changes, all of which contribute to the maintenance of pathological matrix forming cells This presentation will highlight several novel pathways in the area of innate immunity that play roles in the pathological process of fibrogenesis, where intervention may be safe and tractable.
053 TARGETING NOX ENZYMES IN PULMONARY FIBROSIS Victor J. Thannickal
Inflamm. Res. mAb during priming, however, displayed decreased inflammation, mucus production, and lung remodeling in the chronic phase. Together, these studies reveal redundant roles for TSLP, IL-25, and IL-33 in the maintenance of type 2 cytokine-driven pathologies and suggest that in some settings, early combined targeting of these mediators is necessary to ameliorate progressive type 2-driven disease.
SYMPOSIUM 17: THERAPEUTICS WITH LOCALIZED ACTIONS—OPTIMAL WAYS TO TREAT TISSUE INFLAMMATION 055 TARGETED POLYMERIC NANOPARTICLES: FROM DISCOVERY TO CLINICAL TRIALS
University of Alabama at Birmingham, Birmingham, AL, USA The NADPH oxidases (Nox) family of enzymes are known to mediate host defense functions in mammalian organisms. We have identified Nox4 as a highly inducible TGFb-regulated gene. Nox4 is essential to mediate TGFb-induced myofibroblast differentiation and to protect against injury-provoked pulmonary fibrosis. Recent studies support the concept that Nox4-Nrf2 imbalance regulates cellular senescence and promotes persistent fibrosis associated with aging.
054 COMBINATORIAL TARGETING OF TSLP, IL-25, AND IL-33 AND TYPE-2 CYTOKINE SIGNALING IN CHRONIC INFLAMMATION AND FIBROSIS Kevin M. Vannella1, Thirumalai R. Ramalingam1, Allen W. Cheever1,2, Lee A. Borthwick1,3, Luke Barron1, Kevin M. Hart1, Robert W. Thompson1, Kristen N. Kindrachuk1, Sandra White1, Alison L. Budelsky4, Michael R. Comeau4, Dirk E. Smith4, Thomas A. Wynn1 1 Program in Tissue Immunity and Repair, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health; Bethesda, MD, USA; 2Biomedical Research Institute; Rockville, MD, USA; 3Tissue Fibrosis and Repair Group, Institute of Cellular Medicine, Newcastle University; Newcastle upon Tyne, NE2 4HH UK; 4Department of Inflammation Research, Amgen, Amgen Court West, Seattle, WA, USA
Thymic stromal lymphopoietin (TSLP), IL-25, and IL-33 are important initiators of type 2-associated mucosal inflammation and immunity. However, their role in the maintenance of progressive type 2 inflammation and fibrosis is much less clear. Here, using chronic models of helminth infection and allergic lung inflammation, we show that collective disruption of TSLP, IL-25, and IL-33 signaling suppresses chronic and progressive type 2 cytokine driven inflammation and fibrosis. In a schistosome lung granuloma model or during chronic S. mansoni infection in the liver, individual ablation of TSLP, IL-25, or IL-33/ST2 had no impact on the development of IL-4/IL-13-dependent inflammation or fibrosis. However, significant reductions in granuloma-associated eosinophils, hepatic fibrosis, and IL-13-producing group 2 innate lymphoid cells (ILC2s) were observed when signaling of all three mediators was simultaneously disrupted. Combined blockade via mAb treatment also reduced IL-5 and IL-13 expression during primary and secondary granuloma formation in the lung. In a model of chronic house dust mite-induced allergic lung inflammation, combined mAb treatment did not decrease established inflammation or fibrosis. TSLP/IL-33 double-knockout mice treated with anti-IL-25
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Omid C. Farokhzad1,2 1
Brigham and Women’s Hospital, Boston, MA, USA; 2Harvard Medical School, Boston, MA, USA Controlled-release polymeric nanoparticles can deliver drugs in the optimum dosage over time, thus increasing the efficacy of the drug, maximizing patient compliance and enhancing the ability to use highly toxic, poorly soluble, or relatively unstable drugs, and can also be used to co-deliver two or more drugs for synergistic combination therapy. Moreover, the surface engineering of these nanoparticles may yield them ‘‘stealth’’ to prolong their residence in blood, and the functionalization of these particles with targeting ligands can differentially target their delivery or uptake by a subset of cells, further increasing their specificity and efficacy. Nevertheless, the successful clinical translation of targeted polymeric nanoparticles for drug delivery requires optimization of many distinct parameters including: variation in the composition of the carrier system, drug loading efficiency and release kinetics, surface hydrophilicity, surface charge, particle size, density of possible ligands for targeting, etc., resulting in potential variables for optimization which is impractical to achieve using a low throughput approach. Combinatorial approaches precisely engineer nanoparticles and screen multiple nanoparticle characteristics simultaneously with the goal of identifying formulations with the desired physical and biochemical properties for each specific application. In this talk, I will present our efforts in the design and optimization of targeted polymeric nanoparticles for medical applications, which formed the foundation for the clinical translation of the first-in-human targeted and controlled-release nanoparticles, BIND-014 and SEL-068.
056 IMMUNE TARGETING NANOPARTICLES CONTAINING siRNAs TO CURTAIL LEUKOCYTEIMPLICATED DISEASES Dan Peer1 1
Tel Aviv University, Tel Aviv, Israel
RNA interference (RNAi)-based approaches have greatly contributed to better understanding of gene expression and function in vitro. The capability to apply these strategies in vivo in order to validate the role of specific genes in normal or pathological conditions, and to induce therapeutic gene silencing, opened new avenues for utilizing RNAi as a novel therapeutic modality. However, the translation of RNAi from an effective genomic tool into a novel therapeutic modality has been
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hindered by the difficulty to deliver RNAi molecules into their target tissues by systemic administration, especially to hematopoietic cells. In this presentation, I will describe some of the challenges and opportunities in modulating leukocytes response using RNAi and discuss adverse effects such as immuno-toxicity. Several examples will be discussed among them the discovery of cyclin D1 as a potential anti inflammatory targeted in Inflammatory bowel disease (IBD). Cyclin D1 (CyD1) is a pivotal cell cycle–regulatory molecule and a well-studied therapeutic target for cancer. Although CyD1 is also up regulated at sites of inflammation, its exact roles in this context remain less understood. To address this question, we developed a strategy to target gut mononuclear leukocytes and selectively silence CyD1 in leukocytes in vivo. Targeted stabilized nanoparticles (tsNPs) were loaded with CyD1-small interfering RNA (siRNA). Antibodies to b7 integrin (b7 I) were then used to target specific leukocyte subsets involved in gut inflammation. Using PET/CT scan we were able to detect 64Cu-labeled -b7 I-tsNPs accumulation in the gut of mice with DSS-induced colitis. Intravenous administration of b7 I-tsNPs silenced CyD1 in leukocytes and reversed colitis by suppressing leukocyte proliferation and TH1 cytokine expression. This study reveals CyD1 to be a potential anti-inflammatory target, and suggests that the application of similar modes of targeting by siRNA may be feasible in other therapeutic settings. Examples of our current research in blood cancer (mantle cell lymphoma) and from naı¨ve CD4 T cells will also be discussed.
057 AN INFLAMMATION RESPONSIVE PLATFORM FOR CONTROLLED DRUG DELIVERY 1,2,3
Jeffrey M. Karp 1
Brigham and Women’s Hospital, Boston, MA, USA; 2Harvard Medical School, Boston, MA, USA; 3Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA, USA; 4Harvard Stem Cell Institute, Cambridge, MA, USA We have developed an autonomous delivery system that titrates the amount of drug released in response to the level of inflammation, ensuring the drug is released only when needed at a therapeutically relevant concentration. Translation of such technology would represent a paradigm shift in the treatment of inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease which has characteristic flares followed by periods of lower disease activity. We have evaluated its ability to release drugs in response to enzymes that are abundant in human synovial fluid and ulcerated gut tissue using a series of in vitro and in vivo experiments.
SYMPOSIUM 18: ION CHANNELS AND INFLAMMATION—SPONSORED BY IRNCANADA 058 BACTERIAL SIGNALS TO SENSORY NEURONS THAT MODULATE INFLAMMATION AND PAIN Isaac M. Chiu1, Clifford J. Woolf2 1 Harvard Medical School, Department of Microbiology and Immunobiology, Boston, MA, USA; 2Boston Children’s Hospital, Kirby Neurobiology Center, Boston, MA, USA
Primary somatosensory neurons densely innervate barrier tissues that are often exposed to pathogens, including the skin, respiratory, and gastrointestinal tract. Pain, which is a hallmark of many infectious diseases, is mediated by nociceptor sensory neurons as a protective mechanism against damaging stimuli. However, the molecular mechanisms leading to nociceptor neuron activation during bacterial infection and the role of the nervous system in host defense are not well understood. We find that nociceptor neurons directly detect two classes of bacterial ligands: N-formulated peptides and pore-forming toxins. Detection of these bacterial signals occurs through distinct mechanisms, producing differential mechanical and thermal hyperalgesia. Using a mouse model of skin infection, we find that Staphylococcus aureus-induced pain does not depend on critical host defense pathways or immune cell-types. Hyperalgesia directly correlates with live bacterial load rather than tissue swelling or immune activation. Targeted ablation of nociceptor neurons leads to significant increases in immune cell recruitment and lymph node hypertrophy during infection. Therefore, sensory neurons directly detect bacterial ligands to produce pain and to modulate inflammation. We and others are working to further elucidate the molecular interactions between bacterial pathogens and the somatosensory nervous system that could be key mediators of pain, inflammation, and host defense.
059 TRPA1 CHANNELS ARE EARLY SENSORS OF GRAM-NEGATIVE BACTERIAL ENDOTOXINS Felix Viana1, Victor Meseguer1, Yeranddy A. Alpizar2, Enoch Luis1, Sendoa Tajada3, Bristol Denlinger1, Jan-Albert Manenschijn1, Otto Fajardo1, Carlos Ferna´ndez-Pen˜a1, Arturo Talavera2, Tatiana Kichko4, Bele´n Navia5, Alicia Sa´nchez2, Rosa Sen˜arı´s5, Peter Reeh4, Marı´a Teresa Pe´rez-Garcı´a3, Jose´ Ramo´n Lo´pez-Lo´pez3, Thomas Voets2, Carlos Belmonte1 1
Instituto de Neurociencias de Alicante, UMH-CSIC, Alicante, Spain; Laboratory of Ion Channel Research, KULeuven, Leuven, Belgium; 3 Instituto de Biologı´a y Gene´tica Molecular, UV-CSIC, Valladolid, Spain; 4Institute of Physiology and Pathophysiology, University of Erlangen-Nuremberg, Erlangen, Germany; 5Department of Physiology, University of Santiago de Compostela, Santiago de Compostela, Spain 2
TRPA1 are calcium permeable, non-selective cation channels expressed in sensory endings of somatic and visceral nociceptors and many non-neuronal cells, including fibroblasts, endothelial and glial cells. These ion channels are critically involved in the biological response to physical stimuli (temperature and pressure) and natural and synthetic environmental irritants, including many electrophiles, reactive oxygen species and endogenous inflammatory mediators produced following tissue damage. Abnormal activation of TRPA1 has been linked to the pathogenesis of neuropathic pain and itch, and a number of systemic inflammatory diseases, including atopic dermatitis, irritable bowel syndrome and asthma. Gram-negative bacterial infections are accompanied by inflammation and somatic or visceral pain. These symptoms are generally attributed to sensitization of nociceptors by inflammatory mediators released by immune cells. Nociceptor sensitization during inflammation by lipopolysaccharide (LPS), a toxic byproduct of gramnegative bacterial lysis, is thought to occur through activation of pattern-recognition receptors, in particular the Toll-like-receptor 4 (TLR4) signaling pathway. Using a combination of experimental techniques, including electrophysiological recordings and cellular calcium imaging, we found
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S76 that LPS exerts fast (seconds) activation of mouse somatic and visceral nociceptors. These actions were mediated by opening of TRPA1 channels leading to neuronal depolarization, firing of action potentials and rapid elevation of intracellular calcium levels. Human TRPA1 channels showed a similar sensitivity to LPS. LPS, injected intradermally, produced a rapid (minutes) inflammatory response that was accompanied by pain and acute vascular reactions, including neurogenic inflammation and CGRP release (measured by enzyme immunoassay). These responses were severely blunted in TRPA1 KO mice and developed independently of TLR4 activation. Moreover, the capacity of various forms of LPS, purified from different pathogens, to activate TRPA1 in vitro correlated with their ability to induce inflammatory responses in vivo. In summary, we identified TRPA1 channels as key molecular determinants of rapid LPS effects on sensory neurons and their terminals, leading to acute neurogenic inflammation and pain. These results suggest that TRPA1 channels may play a previously unrecognized role in the first line of immune defense against microbial pathogens. Pharmacological targeting of TPA1 channels could represent a novel therapeutic avenue for treatment of complications derived from gram negative bacterial infections, including septic shock. Acknowledgements: Financial support provided by project SAF201345608-R of MINECO.
060 A ROLE OF THE CATION CHANNEL TRPV4 IN LUNG INFLAMMATION Mustapha Si-Tahar INSERM U1100, Centre d’e´tude des pathologies respiratoires (CEPR), Tours, France Cystic fibrosis (CF) is an inherited disease associated to a severe and chronic lung inflammation, causing an early death. To improve patients’ outcome, there is an urgent need to develop safe and efficient anti-inflammatory treatments. To do so, we need to characterize new cellular and molecular components that could contribute to mechanisms of lung inflammation. Here, we focused on the potential role of ‘‘Transient Receptor Potential Vanilloid-400 (TRPV4), a non-selective, calcium channel in respiratory epithelial cells-mediated inflammatory signaling. We first showed that 5,6-, 8,9-, 11,12- and 14,15-epoxyeicosatrienoic acids, i.e. four natural lipid-based TRPV4 agonists are present in sputum of CF patients. Then, we used in vitro and in vivo approaches to demonstrate that epithelial TRPV4 triggers a secretion of pro-inflammatory mediators (cytokines, lipids) as well as a neutrophil recruitment. We also found an alteration of TRPV4-dependent signaling in the CF context, suggesting that TRPV4 could constitute a promising target for the development of new anti-inflammatory treatments in diseases such as CF.
061 SENSITIZATION OF THE TRANSIENT RECEPTOR POTENTIAL VANILLOID 1 (TRPV1) CHANNEL IN NEUROGENIC INFLAMMATION AND PAIN Christophe Altier University of Calgary, Calgary, AB, Canada
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Inflamm. Res. Inflammatory pain associated with tissue damage, infection or auto immune disease, results from sequential events initiated during immune responses. Our lab investigates the plasticity of specialized pain-sensing dorsal root ganglion (DRG) neurons that participate in peripheral sensitization of the nociceptive system during inflammation. These neurons (nociceptors) exposed to inflammatory molecules undergo molecular changes that drive their hyperexcitability by lowering their threshold of activation. The hyperexcitability of nociceptors is mediated by altered expression, activity and trafficking of ion channels. Among a large variety of ion channels, DRG neurons express TRP channels—a family of cationic ion channels that participate in the regulation of Ca2+ homeostasis. TRP channels contribute to changes in cytosolic Ca2+ concentration [Ca2+]i by regulating Ca2+ influx at the membrane or Ca2+ release from internal Ca2+ stores. The Transient receptor potential vanilloid 1 (TRPV1) channel is a non-selective cation channel that transduces inflammatory signals into electrical signals leading to action potential propagation in sensory neurons. In this presentation I will summarize our recent work that pertains to targeting TRPV1 subunit association to reduce inflammatory pain. In this study, we have identified the molecular determinants of TRPV1 channel subunit assembly and were able to abolish TRPV1 function and thus inflammatory pain by targeting this specific motif. In a second part of my talk, I will present our findings on the regulation of TRPV1 signaling in the context of inflammation. Here, using the dextran sulfate sodium (DSS)-induced colitis model, we demonstrated a critical role of substance P-mediated sensitization of TRPV1 channel in the development of post-inflammatory pain.
SYMPOSIUM 19: HARNESSING NOVEL RESOLUTION MECHANISMS TO CONTROL INFLAMMATION 062 RESOLVINS AND SPM IN RESOLUTION AND THEIR RELATIONSHIP TO INFECTION AND TISSUE REGENERATION Charles N. Serhan, Jesmond Dalli, Nan Chiang, Paul Norris, Sesquile Ramon, Romain Colas Center for Experimental Therapeutics and Reperfusion Injury Department of Anesthesia, Perioperative and Pain Medicine Harvard Institutes of Medicine, BWH and Harvard Medical School, Boston, MA, USA In the resolution of inflammation novel chemical mediators are produced in resolving exudates that limit inflammation, stimulate resolution and tissue regeneration. Identification of novel signals that regulate these processes and their pathways is of general interest. This presentation shall review recent results from regenerating planaria, infectious murine exudates, human milk and macrophages, where we identified novel potent molecules that stimulate tissue regeneration in planaria and promote tissue repair in mice. These molecules proved to be peptide-conjugated maresins and are coined maresins conjugates in tissue regeneration (MCTR). They also displayed potent anti-inflammatory and proresolving actions i.e. limiting neutrophil infiltration and promoting the containment and clearance of infections in vivo. At the cellular level, they stimulate bacterial phagocytosis by macrophages and efferocytosis of apoptotic cells both with mice in vivo and human cells in vitro. MCTR1 and MCTR2 are each biosynthesized from docosahexaneoic acid and both carried sulfido-conjugated triene
Inflamm. Res. double bond systems. These are joined by two other recently uncovered novel series of bioactive conjugates from the resolvin and protectin families denoted RCTR, PCTR, respectively. Together, these results identify new, conserved resolution moduli present in planaria, mice and humans that each regulate host responses to control infection, tissue regeneration and organ protection.
063 THERAPEUTIC INNOVATION IN INFLAMMATION: TARGETING PRO-RESOLVING RECEPTOR SIGNALING Mauro Perretti Queen Mary University of London, London, UK The resolution of acute inflammation is enabled by counter-regulatory checkpoints to terminate the host reaction, therefore, in line with the onset phase of inflammation, pro-resolving mediators encompass bioactive lipids, proteins and peptides, autacoids and gases, that act on target cells to extinguish this response. When fully operative, the inflammatory reaction is controlled in time and space, ensuring tissue repair and regain of function. Importantly, disruption of the key processes involved in resolution phase of inflammation could result in delayed restoration of tissue homeostasis, fibrosis and persistent inflammation. The large majority of current therapeutics for the clinical management of inflammatory diseases act by blocking specific enzymes or antagonising receptor targets. Using the endogenous protective pathway centred on a receptor termed FPR2/ALX, which is activated by Annexin A1, Lipoxin A4 and Resolvin D1, strategies that could lead to innovation for next generation anti-inflammatory medicines will be discussed. In particular, we step-change derives from the notion that it is not only important to select the appropriate ‘receptor target’ for the drug discovery programmes, but also identify the pro-resolving signalling signature as guidance for predicting the wanted biological properties. Pro-resolving based medicines—identified and developed on the rationale of resolution, using targets and signalling validated in physio-pathological settings—will be modulatory in their function and devoid of major side effects, affording a therapeutic approach based on the enhancement of the patient’s own mechanisms of repair.
064 MECHANISMS OF INFLAMMATORY RESOLUTION IN HUMANS
S77 ‘‘inflammaging’’ and that rectifying these defects may improve vaccine efficacy in the elderly. To understand these processes better as they pertain to humans and human aging, we have developed models of acute, self-limiting inflammation in healthy volunteers. As a result of these studies we are mapping pathways of inflammation with emphasis on mononuclear phagocytes and lipid mediators and are finding clear differences between young and older individuals with regard to proresolution processes. In my presentation, I will present these new data and speculate on their potential contribution to the aetiology of chronic inflammation and the role aging plays in this process.
SYMPOSIUM 20: SKELETAL MUSCLE INFLAMMATION 065 DIFFERENT MACROPHAGE SUBSETS CONTROL MATRIX REMODELING AND FIBROSIS DURING MUSCLE REGENERATION VERSUS CHRONIC DEGENERATIVE MYOPATHIES Be´ne´dicte Chazaud1,2, Marielle Saclier2, Gae¨tan Juban1, Lin Yang3, Basil J. Petrof4, Re´mi Mounier1 1 Universite´ Claude Bernard Lyon 1, Villeurbanne, France; 2Institut Cochin, INSERM, Paris, France; 3University of Florida, Gainesville, FL, USA; 4McGill University, Montrel, QC, Canada
Macrophages play beneficial roles during skeletal muscle regeneration. Ly6Cpos pro-inflammatory macrophages entry the damaged muscle where that sustain myoblast proliferation, then switch into Ly6Cneg anti-inflammatory macrophages that stimulate myogenesis. We characterized phenotypes and functions of macrophage subsets in myopathies characterized by chronic inflammation and fibrosis. We showed that pro-inflammatory marker-expressing macrophages were associated with fibrosis in both mouse and human Duchenne muscle. Functional experiments showed that in regenerating muscle, sorted Ly6Cneg macrophages were involved in matrix remodeling while Ly6Cpos macrophages induced fibroblast apoptosis. On the contrary, in dystrophic muscle, Ly6Cpos stimulated fibroblasts to produce collagen, and sustained their persistence, triggering fibrosis, through the action of TGFb. In vivo, prevention of Ly6Cpos macrophage entry into mdx muscle, as well as anti-inflammatory (NaHS) or Metformin treatments, improved the dystrophic phenotype, i.e. induced a reduction of necrosis and fibrosis, which was associated with a decrease of proinflammatory macrophages.
Derek W. Gilroy University College London, London, UK Investigate the cells, soluble mediators and receptors that collectively help switch inflammation off, so-called inflammatory resolution. My overall hypothesis is that understanding how acute inflammation resolves will provide insight into the aetiology of chronic inflammatory diseases. In addition, identifying mediators and receptors essential for resolution will help develop drugs that will drive ongoing inflammation down a pro-resolution pathway. However, recent work suggests that resolution of acute inflammation is not the end of immune responses to infection/injury but, that through cells of the mononuclear phagocyte system expressing arachidonic acid, docosahexaenoic acid and eicosapentaenoic acid metabolising enzymes resolution acts as a bridge between innate and adaptive immunity. We believe that defects in these pathways may contribute to the aetiology of, for instance,
066 CYTOKINE-MODULATED CELLULAR RESPONSES IN SKELETAL MUSCLE DAMAGE AND REPAIR Andrew N. Billin GlaxoSmithKline, Brentford, UK Regeneration of damaged skeletal muscle throughout life depends on a population of adult stem cells (satellite cells) that both self-renew and expand as myogenic precursors in response to injury. Satellite cell regenerative functions are modulated by extracellular signals and these
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S78 signals are likely to play a pivotal role in the process of muscle repair. In order to identify key extracellular regulators of satellite cell activity we performed a high-throughput in vitro screen using murine satellite cells and a library containing a comprehensive set of recombinant secreted proteins and the extracellular domains of transmembrane proteins. We identified proteins that increased or decreased satellite cell proliferation and a significant fraction of the identified proteins are inflammatory cytokines. The enrichment of this class of proteins as active hits in the screen suggests that inflammatory cytokines as a class play a major role in determining the activity of satellite cells during muscle repair. The implications of these findings will be discussed in the context of normal muscle repair and in the background of disease or aging.
067 MODULATION OF FIBROSIS BY INFLAMMATORY CELLS DISRUPTED IN CHRONIC MUSCLE INJURY Fabio Rossi1, Dario Lemos1, Farshad Babaeijandaghi1, Marcela Low1, Chih–Kai Chang1, Daniela Fiore2, Regan–Heng Zhang1, Sergei Nedospasov3 A. Nedospasov3,4
Inflamm. Res. Co-inhibitory or immune checkpoint receptors play a key role in resolving tissue inflammation and restoring immune homeostasis. This reflects their function in terminating effector T cell responses. In cancer, the heightened expression and function of co-inhibitory receptors on tumor-infiltrating lymphocytes results in dampened antitumor immunity. Currently, therapies that block the co-inhibitory receptors CTLA-4 and PD-1 are demonstrating unprecedented efficacy in restoring productive anti-tumor immunity in certain cancers. While these therapies continue to be very promising in the clinic, a significant fraction of treated patients remain unresponsive to these therapies and some cancers have proven refractory. This has spurred investigation into targeting other co-inhibitory receptor pathways. The growing landscape of co-inhibitory receptors raises new opportunities but also important questions regarding mechanisms of action and therapeutic application. Data on emerging co-inhibitory receptor targets will be discussed in the context of current therapies.
069 CONTROL OF FIBROTIC ACTIVITY IN THE LUNG AND SKIN BY LIGHT (TNFSF14) Michael Croft
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The Biomedical Research Centre, University of British Columbia, Vancouver BC, Canada; 2Department of Experimental Medicine, Section of Medical Pathophysiology Food Science and Endocrinology, Sapienza University of Rome, Rome, Italy; 3 Engelhardt Institute of Molecular Biology and Lomonosov Moscow State University, Moscow, Russia; 4German Rheumatism Research Center, Berlin, Germany Depending on the inflammatory milieu, injury can either result in a tissue’s complete regeneration or in its degeneration and fibrosis, potentially leading to permanent organ failure. Our data indicates that in acutely damaged skeletal muscle, sequential interactions between multipotent mesenchymal progenitors and infiltrating inflammatory cells determine the outcome of the reparative process. Infiltrating inflammatory macrophages, through their expression of Tnf, directly induce apoptosis of fibro/adipogenic progenitors (FAPs). In states of chronic damage, however, such as in mdx mice macrophages lost their ability to induce apoptosis and FAPs differentiate into fiborgenic cells. Treatment with nilotinib, a kinase inhibitor with proposed anti-fibrotic activity, can restore FAP apoptosis and reduce muscle fibrosis in mdx mice. Our findings suggest that disruption of the precisely timed inflammatory phases that characterize the response to acute damage favors fibrotic degeneration of the muscle during chronic injury.
SYMPOSIUM 21: REGULATION OF INFLAMMATION IN TISSUES BY CO-STIMULATORY AND COINHIBITORY PATHWAYS 068 TARGETING CO-INHIBITORY RECEPTORS IN CANCER Ana C. Anderson1,2 1
Harvard Medical School, Boston, MA, USA; 2Brigham and Women’s Hospital, Boston, MA, USA
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La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA The tumor necrosis factor receptor (TNFR) superfamily consists of 29 membrane and soluble proteins with similar structural features. Many of these molecules are expressed on cells of the immune system and are potential targets of therapeutic intervention in a number of immune based diseases. They can play strong roles as stimulatory receptors for effector CD4 and CD8 T cells, as well as regulating activity of dendritic cells and B cells and functioning to organize lymphoid structures. Recent studies have suggested that certain molecules in this family also may be important in regulating non-lymphoid cells controlling the cross-talk that results from innate and adaptive immunity. One consequence of this cross-talk between the immune system and non-lymphoid cells that occurs in chronic diseases is fibrosis and tissue remodeling. Fibrosis is a common feature of asthma, GvHD, IPF, and several autoimmune diseases such as systemic sclerosis and RA. It largely involves the deposition of extracellular matrix proteins such as collagen in the lungs, skin, and other tissues, and often accompanying this is differentiation of epithelial cells or fibroblasts such that they gain characteristics of smooth muscle, which further impairs normal tissue function. Current treatment involves global immunosuppression and therefore defining specific molecules that promote fibrosis may be important therapeutically. In a model of severe asthma, we found that LIGHT (CD258/TNFSF14) blockade reduced lung collagen production and smooth muscle hyperplasia, associated with impaired TGFbeta and IL-13 expression, two cytokines previously implicated in driving fibrosis. We have extended this data to different inflammatory conditions assessing fibrosis driven by rhinovirus infection, and by the antibiotic bleomycin that induces a response similar to that in human systemic sclerosis and idiopathic pulmonary fibrosis. Data will be shown on the importance of LIGHT to these syndromes in studies of mice lacking LIGHT that show decreased collagen deposition and smooth muscle mass in their lungs and skin and overall less fibrosis. LIGHT has two receptors in the TNFR superfamily, namely the herpes virus entry mediator (HVEM) and the lymphotoxin beta receptor (LTbetaR). Both receptors are broadly expressed on cells implicated in driving fibrotic activity, including epithelial cells in the lung, keratinocytes in the skin, and fibroblasts. Results will also be discussed assessing the range of activities of LIGHT on these cell types and how this may contribute to the inflammatory milieu that drives both early and late stages of fibrotic disease.
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070 IMMUNOREGULATORY MECHANISMS THAT LIMIT AUTOIMMUNITY AND CANCER Dario AA Vignali University of Pittsburgh, Pittsburgh, PA, USA Regulatory T cells (Tregs) inhibit beneficial anti-tumor responses. Treg depletion enhances tumor rejection in animal models and the clinic but also leads to substantial adverse events. Thus approaches have been sought to target Tregs in tumors while limiting systemic autoimmune and inflammatory manifestations. First, the signals that maintain Treg stability and potentiate their function remain obscure. We have shown that the immune cell surface ligand semaphorin-4a (Sema4a) on conventional T cells and DCs, and the Treg-restricted receptor neuropilin-1 (Nrp1) interact to potentiate Treg function. Mice with a Tregrestricted deletion of Nrp1 exhibit limited tumor-induced tolerance and thus substantial resistance to tumors, yet do not develop any autoimmune manifestations. Thus, Nrp1 ligation maintains Treg stability and function in highly inflammatory sites but is dispensable for the maintenance of immune homeostasis, highlighting Nrp1 as a potential immunotherapeutic target in cancer. Second, the relative importance of different Treg suppressive mechanisms remains contentious. Interleukin-35 (IL35) is a Treg-secreted cytokine known to inhibit effector T cell proliferation and mediate infectious tolerance via induction of suppressive IL35-producing induced Tregs, iTr35. Using antibody-mediated neutralization, Treg-restricted deletion of Ebi3 and novel reporter mice, we have shown that IL35 facilitated tumor growth by limiting anti-tumor immunity in transplantable and genetically-induced murine models of melanoma and lung carcinoma. These findings reveal the previously unappreciated importance of IL35 in limiting anti-tumor immunity and present IL35 as a potential therapeutic target in cancer. The inhibitory receptor LAG-3 regulates T cell function and is particularly evident in inflammatory sites. Indeed, we have observed extensive expression of LAG-3 on tumor-infiltrating CD4+ and CD8+ T cells in three distinct transplantable tumors. We will discuss the function of LAG3 on effector T cells versus Tregs in autoimmunity and cancer. Our recent observations suggest that LAG3 may be differentially utilized by different cell types.
S79 responses) in 80–90 % of patients using a single drug. This success has been enabled by a ‘‘fine mapping’’ of disease-related genes and pathways using genomic methods to identify disease-associated genes and associated responses to progressively more ‘‘narrow’’ immune or cytokine inhibitors. While psoriasis displays activation of Th1, Th17, and Th22 T-cells (as well as corresponding CD8+ subsets, cd subsets, and innate lymphoid subsets), the central axis of disease pathogenesis is now understood as the IL-23/T17 Pathway. This axis begins with excess IL-23 production by a group of myeloid (CD11c+) dentritic cells after these cells are stimulated in vivo by TNF, possibly from an autocrine activation loop (these DCs have been defined as TNF- and iNOS-producing or TIP-DCs). The excess IL-23 drives T-cell activation as Th17, and more broadly as T17, with apparent co-activation of Th22 T-cells. T17 T-cells are activated within skin lesions to produce excess IL-17A and IL-17F. In turn, IL-17 has major actions to alter gene transcription within epidermal keratinocytes and other skin-resident cells, leading to altered production of several-hundred gene products, many of which define the psoriasis vulgaris disease phenotype. Induced genes include chemokines, cytokines, antimicrobial peptides, and S100 proteins that create ‘‘feed forward’’ activation loops for dendritic cells, T17 T-cells, and epidermal perplasia/altered differentiation. Th22 T-cells produce high levels of IL22, which in combination with other ‘‘IL-20-family’’ cytokines (IL19, IL-20, IL-24), directly induce hyperplasia in epidermal keratinocytes. Clinical studies have shown that targeting the Th1 axis with antibodies to gamma-interferon is highly ineffective, whereas targeting the IL-23/T17 axis with antibodies to IL-23, IL-17A, or the IL-17 receptor induces PASI75 responses ([75 % disease improvement) in 80–90 % + of treated patients and virtual disease remission (PASI100) in a high fraction of treated patients. Molecular profiling of the treatment response to these antagonists has shown ability to restore pathologic and disease-defining gene transcription changes to levels that are close to background skin.
072 THE TRANSLATIONAL REVOLUTION IN ATOPIC DERMATITIS CENTERED ON TH2/TH22 CYTOKINES Emma Guttman-Yassky
SYMPOSIUM 22: ATOPIC DERMATITIS, PSORIASIS AND IL-4/IL-17 BIOLOGY
Center for Excellence in Eczema and Laboratory for Inflammatory Skin Diseases, Icahn School of Medicine at Mount Sinai Medical Center, New York, NY, USA
071 PSORIASIS: AN IL-23/TH17-DRIVEN DISEASE
Although originally two competing hypotheses were considered for atopic dermatitis (AD), the disease is emerging as a T-cell mediated disease, with cytokine-driven epidermal responses. The predominant inflammatory pathways in AD are T helper type 2 (Th2) and Th22, with input from other T cell subsets such as Th1 and Th17. The cytokines interleukin (IL)-4 and IL-13 are key drivers of Th2 inflammation, and their expression is associated with features of skin barrier dysfunction related to decreases in expression of filaggrin, increases in epidermal hyperplasia, and spongiosis. The pathogenic role of IL-4/IL-13 signaling and reversibility of the epidermal pathology in AD have been confirmed in studies of dupilumab in patients suffering from moderateto-severe disease. Dupilumab is a fully-human monoclonal antibody directed against the IL-4 receptor alpha subunit that blocks both IL-4 and IL-13 signaling. In these early phase clinical trials, dupilumab significantly improved clinical signs and symptoms of AD, and was well-tolerated with a favorable safety profile1–3. Four weeks of dupilumab treatment resulted in a dose-dependent shift from lesional to more non-lesional transcriptional phenotypes. Modulated genes included those related to epidermal proliferation, keratin, and
James Krueger The Rockefeller University, New York, NY, USA Psoriasis vulgaris is one of the most common T-cell mediated inflammatory diseases in humans, affecting 2–3 % of the population. Psoriasis vulgaris is primarily a skin disease in which growth and differentiation of resident skin cells is altered in response to infiltration of skin by T-cells and CD11c+ dendritic cells, with attendant production of inflammatory cytokines. Psoriasis is similar to other inflammatory diseases of peripheral organs in that it displays complex genetics and genomics with [30 gene variants contributing disease risk and with alterations in expression of [4000 mRNAs in skin lesions. However, psoriasis vulgaris is now the most successfully treated human inflammatory disease with a series of immune-targeted therapeutics able to produce high grade improvement (PASI75
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S80 inflammatory mediators4. Of note, dupilumab did not reduce mRNA expression of IL-22 in the skin; however, expression of IL-22-modulated genes was significantly suppressed, suggesting a modulation of Th22 activity. Translational medicine offers insight into Th2- and Th22-related mechanisms underlying AD pathogenesis. (Clinicaltrials.gov identifiers: NCT01259323, NCT01385657, NCT01548404, NCT01639040, and NCT01859988) References: 1. Beck LA, Thac¸i D, Hamilton JD, et al. Dupilumab treatment in adults with moderate-to-severe atopic dermatitis. N Engl J Med. 2014;37:130–139. 2. Thac¸i D, Simpson E, Bieber T, et al. Efficacy and Safety of Dupilumab in adults with moderate-to-severe atopic dermatitis (AD) inadequately controlled by topical therapies: final results of a phase 2b study. American Academy of Dermatology—73rd Annual Meeting. 2015; Abstract F037. https://www.aad.org/Symposium/LBAM 2015/. Accessed on April 30, 2015. 3. Simpson E, Worm M, Soong W, et al. Dupilumab Improves Patient-Reported Outcomes (PROs) in a Phase 2 Study in Adults with Moderate-to-Severe Atopic Dermatitis. J Allergy Clin Immunol. 2015;135:AB167. 4. Hamilton JD, Sua´rez-Farin˜as M, Dhingra N, et al. Dupilumab improves the molecular signature in skin of patients with moderate-tosevere atopic dermatitis. J Allergy Clin Immunol. 2014;134:1293–1300.
073 FROM PATHWAY TO TARGETED THERAPY: IL-4 AND IL-13 IN ALLERGIC DISEASES Jennifer D. Hamilton Regeneron Pharmaceuticals, Inc, Tarrytown, NY, USA Interleukin (IL)-4 and IL-13 are key cytokines driving allergic and T helper cell type 2 (Th2)-polarized inflammatory processes. These cytokines are now known to originate from several cell types, including Th2 cells, innate lymphoid Type 2 cells (ILC2), mast cells, eosinophils and basophils. IL-4 and IL-13 are upstream drivers of Th2 inflammatory processes, such as recruitment of T cells, mast cells, eosinophils and monocytes to tissues; mucus production; B cell activation; and immunoglobulin E production. Different disease endotypes may exist among patient subpopulations. Biologics targeting Th2-associated cytokine pathways are in development for several allergic diseases, including atopic dermatitis, asthma, chronic sinusitis with nasal polyposis, and eosinophilic esophagitis. Data from early phase trials of these targeted therapies provide insight into Th2-mediated inflammatory processes and provide further insight into the role of IL-4 and IL-13 in human allergic diseases. In addition to the potential for providing new treatment options for patients, targeted therapeutics also provide a unique opportunity to advance the understanding of disease pathogenesis and endotypes in atopic disorders.
074 PREDICTION OF CLINICAL THERAPEUTIC RESPONSES IN INFLAMMATORY SKIN DISEASES BASED ON EARLY TIME POINT GENE EXPRESSION DATA Mayte Suarez-Farinas1,2, Joel Correa da Rosa2 1
Mount Sinai School of Medicine, New York, NY, USA; 2Rockefeller University, New York, NY, USA
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Inflamm. Res. The development of drugs targeting key modulators of immune function has brought about a revolution in the treatment of many diseases. However, such drugs pose unique and novel challenges for drug development: as they target specific human immune components it is often harder to extrapolate from in vitro or animal models what their clinical efficacy may be, and the clinical response can require months to manifest. Thus longer trials with human patients are necessary, adding considerable cost, complexity and development time to the current cost of four billion dollars and 8 years in development for each successful drug brought to market. In particular, Psoriasis has become the ‘‘go to’’ immune disease for proof-of-concept studies of new immune targeted compounds, with more than 34 compounds in different phases of development spanning from biological agents to small molecules. As a result, there is abundant genomic data for model development, much of which has been generated by our laboratory over the past decade. Although psoriasis is highly responsive to pathway-specific immune antagonists it often takes 12–16 weeks for clinical response measures to be meaningful, and efficacy ranges from 30 to 80 % success rate. We used longitudinal skin gene expression profiles to build a genomic classifier that use early time-points (baseline, 1st, 2nd and 4th weeks) to predict clinical response in psoriatic patients after 12 weeks of treatment. Clinical response was defined as PASI75 at 12 weeks: i.e. the percentage of patients with at least 75 % reduction of the Psoriasis Area and Severity Index (PASI) after 12 weeks of treatment. Two classifiers are developed that predicts response regardless of the agent the universal classifier, and a treatmentspecific classifier. For the treatment specific classifier, baseline and week 1 after treatment can predict treatment response with [97 % accuracy for etanercept, metrotexate, adalimumab and ustekinumab. The universal classifier achieved 95 % accuracy with data up to 2 weeks and [97 % accuracy when data from week 4 is added. The universal classifier was evaluated in unseen data from 24 patients, 16 of whom were treated with ustekinumab/45 mg with profiles available for baseline and week 1 only and 8 who were treated with placebos with profiles at baseline and week 2. The exciting results observed in predicting treatment response after a short clinical trial in psoriasis would allow us to use this methodology in other diseases and conditions, like Atopic Dermatitis.
SYMPOSIUM 23: DEFINING SUBPHENOTYPES IN CHRONIC RESPIRATORY DISEASES TO IMPROVE DRUG DEVELOPMENT AND DISEASE MANAGEMENT 075 BIOMARKER DISCOVERY AND COMPANION DIAGNOSTIC DEVELOPMENT IN ASTHMA Joseph R. Arron Genentech, Inc, South San Francisco, CA, USA Asthma has been regarded as an allergic disorder characterized by type 2 cytokine expression and eosinophilic inflammation in the airways. However, it is clearly heterogeneous on molecular, pathological, and clinical levels. Genomic approaches have identified heterogeneous gene expression patterns in asthmatic airways corresponding to the degree of type 2 cytokine expression and eosinophilic inflammation. These gene expression patterns have led to the identification of candidate biomarkers of eosinophilic airway inflammation that do not require
Inflamm. Res. bronchoscopy or sputum induction. Candidate biologic therapies targeting mediators of type 2 airway inflammation have progressed through clinical studies in moderate-severe asthma patients in recent years. Serum periostin, fractional exhaled nitric oxide (FeNO), and blood eosinophil counts have emerged as predictive and pharmacodynamic biomarkers that may enrich for clinical benefit in clinical studies of biologic therapies targeting IL13, IL5, and IgE. These clinical studies have shown that asthma heterogeneity impacts target, patient, and outcome selection, and biomarker-guided clinical development will help to: (1) deliver targeted therapeutics to patients most likely to benefit, (2) redefine the unmet medical need in asthma to inform future target discovery research efforts.
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SYMPOSIUM 24: BIOLOGICAL AND NONBIOLOGICAL THERAPIES OF AUTOIMMUNE DISEASES—SPONSORED BY JSIR-JAPAN 077 MOLECULAR UNDERSTANDING OF TARGETED THERAPIES IN PATIENTS WITH RHEUMATOID ARTHRITIS Tsutomu Takeuchi
076 INTEGRATING CLINICAL AND MOLECULAR FEATURES TO IDENTIFY DISTINCT ASTHMA SUB-PHENOTYPES Frederic Baribaud Janssen R&D, Los Angeles, CA, USA Rational: Asthma is a heterogeneous disease that includes clinically relevant subgroups. Objectives: To identify subgroups of asthma in the airways disease endotyping for personalized therapeutics (ADEPT) study using cluster analyses. Materials and Methods: Clinical features and accessible biomarkers with molecular characteristics were collected by profiling asthmatic subjects and healthy non-atopic volunteers. Assessments included questionnaires, pulmonary function tests, airway hyper responsiveness, FENO, and biomarkers from induced sputum, endobronchial biopsy and brushings. Partition-around-medoid clustering was performed on the ADEPT dataset using pre-specified clinical variables or using the gene set variation analysis (GSVA) enrichment scores (ES) using an in vitro generated glucocorticoid (GC) signature. Models for cluster classification probabilities were derived and applied to the U-BIOPRED adult asthma dataset. CCL26 (eotaxin-3) expression or GSVA ES of an IL13 IVS were used for evaluation of Th2high versus Th2low classification within clusters. Results: Four clinical clusters were identified in the ADEPT-asthma study population with unique clinical and biomarker profiles. Group 1 represents well-controlled asthma. Group 2 represents controlled, albeit highly hyper-reactive patients mostly with a Th2high phenotype. In contrast, Group 3 has a less controlled, restrictive, Th2low phenotype. Group 4 consists of the most poorly controlled patients, a mixed Th2high/low phenotype. U-BIOPRED asthma patients were classified to the ADEPT clusters with similar probability distributions as compared to ADEPT. Importantly, the distributions for most clustering and non-clustering (e.g., demographics and biomarkers) variables were similar to those in ADEPT, rather than being biased by only a minority of variables. Three GC molecular response clusters were identified in ADEPT, a GC responsive (R), a GC non-responsive (NR) and a GC partially-responsive (PR) cluster. In clinical clusters 2 and 4 about 30 % of patients were GC NR, 25 % were GC R and about 20 and 38 % were GC PR in cluster 2 and 4 respectively. In clinical cluster 3 however, about 10 % were GC NR and PR respectively and about 70 % were GC R. Conclusions: Four discrete clinical clusters were identified, and confirmed, that have unique clinical and biomarker profiles. Clusters 2 and 4 represent well-controlled, albeit highly hyper-reactive and poorly controlled patient groups with a mixed GC responsive profile, respectively, that may benefit from Th2high-targeted intervention. In contrast, cluster 3 has a Th2low, restrictive phenotype with a largely GC NR profile that would likely require different interventions.
Division of rheumatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan Recent understanding of pathogenesis of the autoimmune diseases has been enormously facilitated by clinical studies and careful observation with targeted therapies on the molecules involved in the disease process. One example can be rheumatoid arthritis (RA), which is characterized by persistent destructive polyarthritis with autoantibodies against citrullinated proteins. The appropriate targets in this autoimmune disease, RA, are TNFa and IL-6, while the potential targets proposed by animal models such as IL-1ß and IL-17 did not exhibit the comparable clinical efficacy in human RA. In addition, it has been shown the rapid and excellent clinical response in placebo-controlled dose finding study for fully human monoclonal antibody against GMCSF receptor. Given such robust evidences of the appropriate targets, we further need to fully understand the role of these targets in the pathogenesis of the autoimmune disease. In this symposium, I will focus on the in vivo change of a variety of biomarkers including cytokines during anti-TNF or anti-IL-6 receptor therapies in patients with RA in order to explore the relationship between TNFa and IL-6 as the appropriate targets. Then, we examined the in vivo change of immune cells during the targeted therapies by modern standardized immune-phenotyping to test their potential effects on B cells, T cell subsets and monocyte subsets. Finally, I will discuss the molecular distinction among the targeted therapies with comparable clinical efficacy and the basis for personalized strategy of the targeted therapies.
078 LOW MOLECULAR WEIGHT IMMUNOSUPPRESSANT THERAPY OF AUTOIMMUNE DISEASES Shinichi Kawai Toho University School of Medicine, Tokyo, Japan Autoimmune diseases are chronic inflammatory diseases characterized by immunological abnormalities and also by the specific organ disturbances. Various low molecular weight immunosuppressants such as azathioprine, cyclophosphamide, methotrexate, mycophenolate mofetil, cyclosporine A, and tacrolimus are widely used for treatment of autoimmune diseases. The primary lesion of rheumatoid arthritis (RA), an autoimmune disease is considered to be in synovial membranes. Synovial cell proliferation gradually affects surrounding cartilage and bones, frequently leading to the destruction and deformation of joints. Methotrexate and some other immunosuppressants are used in patients with RA for not only improvement of inflammation and immunological abnormalities but also prevention of joint destruction. I will introduce the intracellular mechanisms of action of methotrexate in RA patients. I also introduce the basic
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S82 and clinical data of tacrolimus which is approved in Japan for treatment of RA. Systemic lupus erythematosus (SLE) is an autoimmune disease that causes various symptoms and immunological abnormalities. The standard treatment is corticosteroid therapy, but a combination of corticosteroid with a low molecular weight immunosuppressant is used for SLE especially with severe organ involvement. In Japan, tacrolimus is approved for treatment of lupus nephritis as maintenance therapy. I will then introduce the data of some clinical studies of tacrolimus for SLE patients. In this presentation, some basic studies and clinical data of efficacy and safety of several low molecular weight immunosuppressants especially for the Japanese patients with RA and SLE will be discussed.
Inflamm. Res.
B095 POLYCYSTIC LIVER DISEASE IS SUPPRESSED BY IL-4/IL-13 AND TGF-ß SIGNALING See poster section for abstract.
B096 EVALUATING IL-13RA2 AS A THERAPEUTIC TARGET FOR PULMONARY FIBROSIS See poster section for abstract.
B034 WONDERBUMIN: A FULLY SYNTHETIC ALTERNATIVE TO IGT FOR AUTOIMMUNE DISEASE See poster section for abstract.
B047 NEUTRALIZING THE PRO-METASTATIC IMMUNE RESPONSES OF MDSC AND T CELLS THROUGH B CELL TARGETING See poster section for abstract.
MINI-SYMPOSIUM 2: CELLULAR AND MOLECULAR MECHANISMS OF FIBROSIS
B091 IDENTIFICATION OF BOTH IMMUNOMODULATORY AND ANTI-FIBROTIC ACTIONS OF NINTEDANIB AND PIRFENIDONE USING BIOMAPÒ HUMAN PRIMARY CELLBASED DISEASE MODELS See poster section for abstract.
B025 THE SYK INHIBITOR FOSTAMATINIB LIMITS TISSUE DAMAGE AND FIBROSIS IN A BLEOMYCIN-INDUCED SCLERODERMA MOUSE MODEL: EFFICACY IN SYK INHIBITION IN ANIMAL SCLERODERMA See poster section for abstract.
B093 LINEAGE TRACING OF RESIDENT FIBROBLASTS BY THE INTRATRACHEAL CELL TRANSFER ELUCIDATES THE CELLULAR ORIGIN OF ACTIVATED FIBROBLASTS IN PULMONARY FIBROSIS See poster section for abstract.
NEW INVESTIGATOR AWARD WINNER PRESENTATIONS B061 COMBATING SKIN INFLAMMATION WITH CXCL12 CHEMOKINE NEUTRALIGANDS See poster section for abstract.
B097 CROSSTALK BETWEEN MACROPHAGES AND HUMAN HEPATIC FIBROBLASTS SHOW THAT INFLAMMATORY RESPONSE ATTENUATE THE ACTIVATION OF FIBROBLASTS IN THE FIBROSIS RESPONSE
B050 THE ROLE OF RESOLVIN D1 IN THE REGULATION OF INFLAMMATORY AND CATABOLIC FACTORS IN OSTEOARTHRITIS
See poster section for abstract.
See poster section for abstract.
B094 PERICYTE MYD88 CONTROLS INFLAMMATORY AND FIBROTIC RESPONSES TO TISSUE INJURY
B099 ACETATE PROTECTS THE ISCHEMIC INTESTINE FROM REPERFUSION INJURY
See poster section for abstract.
See poster section for abstract.
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B246 IDENTIFICATION OF CD44BRIGHT, A NOVEL SUBSET OF UTERINE NK CELLS CORRELATED TO ABORTION IN RECURRENT PREGNANCY LOSS MODEL MICE See poster section for abstract.
B266 A NOVEL ROLE FOR BK CHANNEL IN REGULATING METALLOPROTEASE ACTIVITY See poster section for abstract.
PATIENT PERSPECTIVES: UNMET MEDICAL NEED–LUPUS 079 PATIENT PERSPECTIVES: UNMET MEDICAL NEED–LUPUS Heather E. Jones Pfizer, Inc., Collegeville, PA, USA Objective: To increase awareness of the true unmet medical need left by current therapeutic options for SLE and understand what this means to the patient. The importance of recognizing the true unmet medical need in autoimmune diseases is higher than ever. No drug has so far been able to cure the SLE, only to induce remission. To progress science and clinical development there must be focus on understanding the pathogenesis of the disease. From a medical perspective we need to consider the patient as a whole human being and not only based on the organ(s) involved. Autoimmune diseases are systemic, in most cases affecting more than one organ. The morning session will begin by emphasizing the unmet medical need followed up by an interview of a patient with SLE, telling their story, with an emphasis on what they see as key objectives for further development. At the end of the session the co-chairs will summarize the signs and symptoms and the treatment algorithm for SLE, as well as the need for biomarkers to define the disease, ensure safety, guide treatment and predict treatment response.
SYMPOSIUM 25: COMPLEMENT TARGETED THERAPIES—IS THERE LIGHT AT THE END OF THE TUNNEL? 080 LINKAGES BETWEEN COMPLEMENT, HISTONES AND ACUTE LUNG INJURY Peter A. Ward University of Michigan Medical School, Ann Arbor, MI, USA
S83 There is increasing evidence that there is a linkage between development of acute lung injury (ALI), complement and histones, both in humans and in mice with ALI. In C57BL/6 young male mice, ALI has been induced by airway instillation of LPS or recombinant murine C5a. This induces an intense neutrophil (PMN)-rich inflammatory response in alveoli, accompanied by edema, fibrin deposition and extracellular histones. The requirement for complement (C5a) has been demonstrated by the greatly reduced intensity of ALI as quantitated by albumin leak and buildup of cytokines, chemokines and histones in bronchoalveolar fluid (BALF). The absence of receptors for C5a greatly reduces this inflammatory response including sharply reduced BALF levels of histones. PMN depletion also is protective and greatly reduces all parameters of injury (including BALF histones). It appears that generation of C5a with its reactions with C5a receptors on PMNs results in formation of neutrophil extracellular traps (NETs), which are associated with appearance of histones. Histones are extremely phlogistic and prothrombotic. ALI can be sharply reduced by the presence of neutralizing antibodies to extracellular histones. These studies provide evidence for close interactions between complement (C5a and its receptors) and histones in the setting of ALI.
081 FINE-TUNING INFLAMMATION: ‘DESIGNER’ LIGANDS FOR THE COMPLEMENT PEPTIDE RECEPTORS Peter N. Monk University of Sheffield, South Yorkshire, UK Complement activation is a factor in most inflammatory diseases and is also known to occur in many conditions not usually thought of as having an inflammatory element, such as cancer. Small, biologically active protein fragments, the complement peptides C5a and C3a, are generated which can act on many aspects of the immune response. The receptors for these peptides are widely expressed on immune and non-immune cells and have long been important drug targets. C5a is generally pro-inflammatory and excessive production can lead to tissue damage in rheumatoid arthritis, for example. In contrast, C3a often has an anti-inflammatory role, sometimes working in opposition to C5a. However, in asthma, C5a has been shown to be protective in the early sensitisation phase whereas both C5a and C3a are destructive in the late, effector, phase. Both C5a and C3a can also be generated without activation of the complement cascade, through the extrinsic pathway. Extra- and intra-cellular extrinsic pathways have been described that enable local or autocrine production; such pathways are now thought to be involved in T cell differentiation. In addition, some pathogens can modulate extrinsic generation of C5a and C3a to produce a less hostile microenvironment. This complex biology might limit the possibilities for therapeutic intervention in complement peptide-mediated inflammation. Our understanding of how the complement peptide receptors (C5aR1, C5aR2 and C3aR) function is now leading to the generation of new ligands which modulate receptor activity rather than acting simply as antagonists. Biased agonists that can select the downstream signalling pathways, partial agonists that diminish maximum responses, and strategies to control of receptor homo- and hetero-dimerisation will be discussed as promising strategies to control pathology associated with complement peptide production. The first selective ligand for C5aR2 has recently been discovered and its ability to modify a subset of C5a responses will be described. The potential of this novel ligand for a complement peptide receptor will be illustrated by a newly-discovered pathway for the control of human T cell differentiation.
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082 COMPSTATIN CP40: AN IMMUNE MODULATOR ON A JOURNEY FROM DISEASE MODELS TO CLINICAL DEVELOPMENT
Princeton, NJ, USA; 4Renal Section, Boston University Medical School, Boston, MA, USA; 5Division of Rheumatology, University of Colorado Medical School, Aurora, CO, USA; 6Departments of Neurology and Pharmacology and Physiology, George Washington School of Medicine, Washington, DC, USA
John D. Lambris1, Alexandra Primikyri1, Sophia Koutsogiannaki1, Malvina Papanastasiou1, Jihyoung Seong1, HongBin Wang1, Gang Chen2, Markus Huber-Lang3, Martijn van Griensven4, Bo Nilsson5, Kristina Nilsson-Ekdahl5, Alireza Biglarnia5, Richard J. Smith6, George Hajishengallis7, Despina Yancopoulou8, Antonio M. Risitano9, Daniel Ricklin1
Introduction: Uncontrolled complement activation plays a pivotal role in a variety of disorders such as PNH and aHUS. Aims: ALN-CC5 is a subcutaneous (S.C.) investigational RNAi therapeutic targeting complement C5 (C5) with the purpose of decreasing terminal complement activity and thereby protects against intravascular hemolysis and complement mediated tissue damage. Materials and methods: Preclinical studies in mouse, rat and nonhuman primate (NHP) models were used to investigate the ability of ALN-CC5 to reduce complement C5, inhibit complement-mediated hemolytic activity and complement alternative- and complement classic-pathway (CAP and CCP). Furthermore, ALN-CC5 was investigated in a rat membranous nephropathy model, the mouse anticollagen antibody induced arthritis model and in a rat passive myasthenia gravis model. A placebo controlled double blind phase 1 clinical study in healthy volunteers and patients with PNH is ongoing. Several cohorts in part A, a single ascending dose study have been completed and Part B, a multiple ascending dose study is currently ongoing. Part C will be a multiple dose study in patients with PNH. Primary endpoints are safety and tolerability. Secondary endpoints are pharmacokinetics, reduction of circulating C5, reduction in hemolytic and CAP as well as CCP activity. Results: Pre-clinical studies demonstrated that ALN-CC5 resulted in mean 98.4 ± 0.7 % reduction of C5 levels in NHPs, mean 88 ± 6.1 % reduction in hemolysis and mean 95.1 ± 0.93 % reduction in CAP with every-other week or monthly S.C. dosing. Furthermore, C5 silencing significantly reduced proteinuria in a rat membranous nephropathy model, reduced disease activity in the mouse arthritis model to a level comparable to anti-C5 monoclonal antibody; and reduced clinical disease severity in the myasthenia gravis model. In Part A of the phase 1 study, human volunteer subjects were randomized (3:1) to placebo or a starting single subcutaneous dose of 50 mg ALN-CC5 and followed for a t least 70 days. Following safety review, additional single ascending dose cohorts were authorized. Part B multiple ascending cohorts are planned to start in parallel to Part A. Up-to-date results on safety, tolerability and C5 knockdown, changes in CAP, CCP and hemolytic activity from study will be presented. Conclusion: Collectively, these data suggest that the use of a novel RNAi therapeutic targeting C5 is a promising approach for inhibiting complement in PNH, aHUS and other complement mediated diseases. The subcutaneous route of administration and infrequent dosing make this a particularly encouraging potential therapy.
1
University of Pennsylvania, Perelman School of Medicine, Philadelphia, USA; 2Huazhong University of Science and Technology, Wuhan, China; 3Ulm University, Ulm, Germany; 4 Technical University Munich, Munich, Germany; 5Uppsala University, Uppsala, Sweden; 6University of Iowa, USA; 7University of Pennsylvania, School of Dentistry, Philadelphia, USA; 8Amyndas Pharmaceuticals, S.A., Glyfada, Greece; 9Federico II University, Naples, Italy As a prime sensor of inflamed and damaged tissue, cellular debris and foreign cells/materials, the complement system critically contributes to host defense and homeostasis, yet its formidable immune surveillance capabilities can turn awry and inflict damage to self-cells. Therapeutic complement inhibition has therefore been emerging as a promising strategy in broad spectrum of immune, inflammatory, and biomaterial-induced disorders. By blocking the central complement component C3, the peptidic inhibitor compstatin and its analogs have early shown promise in many disease models. Recent years have provided insight into the mode of action and structural determinants of this inhibitor class and allowed for the design of compstatin derivatives with largely improved pharmacodynamic and kinetic properties. Among them, analog Cp40 has shown particular promise and has been successfully evaluated in a variety of clinically relevant models, ranging from C3 glomerulopathies, paroxysmal nocturnal hemoglobinuria and periodontal disease to hemodialysis-induced inflammation, hemorrhagic shock and solid organ transplantation. Building on these strong preclinical results, a Cp40-based drug (AMY-101, Amyndas) has meanwhile entered clinical development. The combined preclinical and clinical studies already revealed a unique pharmacokinetic behavior that is expected to facilitate longterm administration, and indicated a beneficial safety profile. This presentation highlights milestones in the recent development of compstatin Cp40, showcases data from disease-related studies, and discusses general implications on therapeutic complement inhibition at the level of C3.
083 A SUBCUTANEOUSLY ADMINISTERED RNAI THERAPEUTIC (ALN-CC5) TARGETING COMPLEMENT C5 FOR TREATMENT OF PNH AND COMPLEMENT MEDIATED DISEASES Anna Borodovsky1, Benny Sorensen1, Jorg Taubel2, James Bush3, Noriyuki Kawahata1, Lauren Melton1, Jeffrey Cehelsky1, Christine Powell1, Kristina Yucius1, Prasoon Chaturvedi1, Garvin Warner1, David Salant4, V. Michael Holers5, Nirmal Banda5, Linda Kusner6, Henry Kaminski6, Akshay Vaishnaw1, Pushkal Garg1 1
2
Alnylam Pharmaceuticals, Cambridge, MA, USA; Richmond Pharmacology, London, UK; 3Covance Clinical Research Unit,
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SYMPOSIUM 26: REGENERATIVE MEDICINE AND STEM CELLS IN INFLAMMATION 084 THE ROLE OF STEM CELL EXHAUSTION IN AGING AND DISEASE Johnny Huard University of Pittsburgh, Pittsburgh, PA, USA Aging is characterized by the progressive erosion of tissue homeostasis and functional reserve in all organ systems. There is evidence
Inflamm. Res. that the accumulation of damage in stem cells renders them defective for self-renewing and regenerating damaged tissues. We have demonstrated that a population of muscle progenitor cells (MPCs) isolated from the ERCC1-deficient mouse model of accelerated aging, are defective in their proliferation abilities, differentiation capacity and resistance to oxidative stress. We have observed that intraperitoneal (IP) injections of wild-type (WT)-MPCs into Ercc1 knockout (Ercc1-/-) mice resulted in an improvement in age related pathologies. In an attempt to determine whether the defect observed in ERCC deficient MPCs was not exclusive to this progeria model, we have isolated and characterized MPCs from another progeroid mouse models, the zinc metalloproteinase (Zmpste24) knock-out mouse, an animal model of the Hutchinson-Gilford progeria syndrome (HGPS). Similar to ERCC deficient MPCs, we have observed that Zmpste24-/- MPCs have proliferation and differentiation defects, characteristics also observed in MPCs isolated from naturally aged mice. These results suggest that the defect in MPCs is not specific to a particular model of progeria and can also be observed in naturally aged animals. Finally, we have investigated whether a defect in MPCs can also be observed in skeletal muscle disease such as Duchenne muscular dystrophy (DMD), which is a degenerative muscle disorder characterized by the lack of dystrophin expression at the sarcolemma of muscle fibers. Interestingly, DMD patients lack dystrophin from the time of birth; however, the onset of muscle weakness only becomes apparent at 4–7 years of age, which happens to coincide with the exhaustion of the MPC pool. There are several lines of evidence that support this concept including the gradual impairment of the myogenic potential of MPCs isolated from patients during aging, which results in a reduction of muscle regeneration in older patients. In addition to muscle weakness, patients acquire osteopenia, fragility fractures, and scoliosis indicating that DMD may represent a model of premature musculoskeletal aging with a potential dysfunction in MPCs. Here, we report that dystrophin–utrophin double knockout (dko) mice exhibit a spectrum of degenerative changes in various musculoskeletal tissues including skeletal muscle and bone. In contrast to that observed with MPCs isolated from the mdx mice (dystrophin deficient and mild phenotype), we have recently shown a defect in the MPCs isolated from dKO mouse. We have observed that the MPC defect from the dKO mouse model appears to be age dependent and not specific to MPC since other stem cell population also appears to be affected.
085 INHIBITING CHRONIC SKELETAL MUSCLE INFLAMMATION IN AGING RESTORES THE SATELLITE CELL NICHE AND IMPROVES MUSCLE REGENERATION Amy J. Wagers Harvard University, Cambridge, MA, USA It has been known for some time that muscle repair potential becomes increasingly compromised with advancing age, and that these agerelated defects are associated with reduced activity of muscle satellite cells and with the presence of chronic, low grade inflammation in the muscle. Working from the hypothesis that a heightened inflammatory tone could contribute to poor regenerative capacity in aged muscle, we developed genetic systems to inducibly modulate inflammatory gene expression in muscle satellite cells or muscle fibers by modulation of the activity of nuclear factor kB (NF-kB), a master transcriptional regulator of inflammation in many tissues whose activity is dramatically upregulated in aged skeletal muscle and
S85 muscle satellite cells. Surprisingly, in vivo studies revealed that activation of NF-kB activity in muscle fibers, but not in satellite cells, drives muscle dysfunction and that life-long inhibition of NF-kB activity in myofibers preserves muscle regenerative potential with aging via cell-non-autonomous effects on satellite cell function. Building on this unexpected discovery, we analyzed differential gene expression in muscle with varying NF-kB activity, and identified a secreted phospholipase (PLA2G5) as a myofiber-expressed NF-kB regulated gene that governs muscle regenerative capacity with age. Together, our data are consistent with a model in which NF-kB activation in muscle fibers increases Phospholipase 2 expression and subsequently drives the impairment in regenerative function characteristic of aged muscle. Importantly, inhibition of NF-kB function reverses this deficit in repair, suggesting that FDA-approved drugs, like salsalate, a prodrug form of sodium salicylate, may provide new therapeutic avenues for elderly patients with reduced capacity to recover effectively from muscle injury. Together, these studies thus identify a novel NF-kB regulated, non-cell autonomous mechanism by which stem cell function is linked to lipid signaling and homeostasis, and provide important new targets for therapeutic intervention to stimulate muscle repair in aged individuals.
B121 DYSREGULATION OF RESOLUTION OF ACUTE INFLAMMATION IN AGED HUMANS See poster section for abstract.
B190 PROTECTIVE EFFECT OF HEMATOPOIETIC STEM CELLS IN STROKE See poster section for abstract.
SYMPOSIUM 27: INKT CELLS— MEDIATORS AT THE INTERFACE OF INFLAMMATION AND IMMUNITY 086 HUMAN INVARIANT NATURAL KILLER T CELLS ACTIVATE MONOCYTE-DERIVED DCS TO INITIATE A PATHWAY OF STERILE INFLAMMATION THAT PROMOTES HOST DEFENSE Jenny E. Gumperz1,2 1
University of Wisconsin School of Medicine and Public Health; Department of Medical Microbiology and Immunology, Madison, WI, USA
2
Invariant natural killer T (iNKT) cells are innate T lymphocytes that recognize lipids as antigens presented by CD1d glycoproteins. CD1d molecules are non-classical antigen presenting molecules expressed by most myeloid cell types (monocytes, macrophages, and DCs), as well as by B lymphocytes. iNKT cells are known to be able to potently affect the outcome of immune responses through cytokine
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S86 production and by interacting with and affecting the functions of CD1d+ antigen presenting cells. iNKT cells and CD1d+ myeloid cell types have been found in a variety of inflamed human epithelial and endothelial tissues, and studies in murine model systems have suggested that both of these subsets are often early recruits to sites of tissue inflammation. To gain a better understanding of how iNKT cells contribute during acute inflammation, we have investigated how they influence the functions of human monocyte-derived DCs (a type of DC that is often recruited to sites of inflammation). We show that iNKT cells induce sustained cytoplasmic calcium signaling in DCs that is associated with the induction of eicosanoid lipid mediator biosynthesis, and that does not require stimulation by microbial compounds. This sterile inflammatory pathway induces vascular permeabilization and neutrophil recruitment and activation in vivo, and appeared to facilitate host defense against challenge by the opportunistic pathogen Candida albicans.
087 IMAGING iNKT CELLS IN VIVO-DIFFERENT FUNCTIONS IN DIFFERENT ORGANS Paul Kubes Snyder Institute for Chronic Diseases University of Calgary, Calgary, AB, Canada Using spinning disk microscopy allows us to track iNKT cells in various organs including the liver, joints, spleen and lung. In the liver iNKT cells crawl randomly in the sinusoids of the liver. During an infection Kupffer cells catch bacteria from the vasculature and call iNKT cells to their surface where they present pathogen-derived glycolipids on CD1d to the iNKT cells prompting them to make interferons. During non-infectious insults, the iNKT cells can make cytokines to help in repair. iNKT cells can even respond to neurotransmitters that help to immunosuppress the host and reduce inflammation. It appears that each organ has its own particular iNKT cells with organ specific localization. It is well known that iNKT cell deficient mice get a huge burden of Borreliosis into joints and when joints were visualized it became apparent that iNKT cells surround the vasculature of joints and prevent entry of Borrelia into this tissue. They do this independent of CD1d and via direct granzyme killing. They may also limit exit from joints of bacteria such as S. aureus. iNKT cells are also found in the vasculature of the lungs where they reside in capillaries but remain stationary. Once a pathogen is detected, they begin to crawl slowly migrating out of the vasculature where they receive stimulation via glycolipids found on dendritic cell CD1d. However, their ability to emigrate appears to be closely associated with emigration of neutrophils. Clearly each organ has its own characteristic iNKT cell localized either within the vasculature, surrounding the vasculature or in the case of lung in the vasculature with the capacity to emigrate out upon infection.
088 HARNESSING THE IMMUNOSTIMULATORY PROPERTIES OF INVARIANT NATURAL KILLER T (iNKT) CELLS IN NOVEL iNKT CELL-BASED THERAPIES FOR CANCER Rupali Das1, Peng Guan1, Elizabeth Evans2, Felix Scheuplein3, Maurice Zauderer2, Robert Schaub3, Kim E. Nichols4
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Division of Oncology, Children’s Hospital of Philadelphia, Philadelphia PA, USA; 2Vaccinex, Inc. Rochester, NY, USA; 3 Research and Development, NKT Therapeutics, Waltham, MA, USA; 4 Department of Oncology, St. Jude Children’s Research Hospital, Memphis, TN, USA
Invariant natural killer T (iNKT) cells comprise a unique lineage of CD1d-restricted lipid-reactive T lymphocytes that kill tumor cells and exhibit robust capacity to transactivate the tumor-directed functions of dendritic (DC), NK, T and B cells. In most cases, optimal iNKT cell killing of tumors is T cell receptor (TCR)-dependent, requires tumor cell expression of the antigen-presenting molecule CD1d and the pre-loading of tumor cells with stimulatory lipid antigens that induce iNKT cell activation. To capitalize on the anti-tumor properties of iNKT cells, our laboratory is developing novel iNKT cell based cancer therapies. As our first approach, we have engineered a recombinant fusion protein in which the human CD1d molecule is joined to a single chain antibody fragment (scFV) specific for human CD19, a tumor antigen expressed on most B cell cancers. We have produced and purified the fusion and demonstrated that it binds with specificity to human CD19+ targets. Once loaded with a stimulatory lipid antigen such as the prototypical iNKT cell agonist a-galactosyl ceramide (aGC), the fusion induces robust in vitro iNKT cell activation, cytokine production and lysis of immortalized CD19+ CD1dB cell lines. Currently, we are testing whether the fusion will activate iNKT cells in situ and induce iNKT cell-dependent mechanisms of tumor clearance. As our second approach, we are characterizing the iNKT cell stimulatory properties of a recombinant humanized monoclonal antibody NKT 320, which binds selectively and with high affinity to the human invariant TCR. Soluble and immobilized NKT 320 induces a dose-dependent iNKT cell activation, proliferation, degranulation and activation of bystander immune cells. iNKT cells stimulated by plate-bound NKT320 also robustly secrete Th1 and Th2-type cytokines and chemokines. Consistent with these in vitro data, the treatment of Va24 transgenic mice, which express the human iTCR alpha chain, leads to up-regulation of activation markers and intracellular IFN-g production by iNKT cells, and incorporation of BRDU, indicating iNKT cell proliferation. We are now developing animal models with which to test the anti-tumor effects of NKT 320 or the related antibody NKT 14 m, which binds to murine iNKT cells and induces their activation in vitro and in vivo.
089 iNKT CELLS: MEDIATORS AT THE INTERFACE OF INFLAMMATION AND IMMUNITY Richard S. Blumberg1, Torsten Olszak1, Dingding An1, Joana F. Neves1, Marie Dowds1, Sungwhan Oh1, Arthur Kaser1, Dennis L. Kasper1, Sebastian Zeissig1 1
Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA; 2Department of Internal Medicine I, University Medical Center Schleswig–Holstein, Kiel, Germany The mechanisms by which homeostasis at mucosal surfaces is maintained is of central importance to the pathogenesis of inflammatory bowel disease (IBD) and intestinal inflammation in general. Critical to these processes is the intestinal epithelial cell (IEC), which regulates the responses of immune cells to environmental factors. CD1d has been demonstrated to function in IECs based upon studies in in vitro models. These studies have shown that IEC lines can express CD1d and present alpha-galactosylceramide to invariant Natural Killer T (iNKT) cells. CD1d-iNKT restricted pathways play an important role in antimicrobial responses in mucosal tissues for
Inflamm. Res. both pathogens and management of microbial commensalism. CD1d has also been implicated in the pathogenesis of IBD using oxazoloneinduced colitis which is dependent upon CD1d and iNKT cells and evidence of increased NKT cell activity in the human condition based upon evidence for increased production of IL-13 upon stimulation with CD1d agonists. CD1d-restricted iNKT cells are also regulated by the microbiota. Under GF conditions, oxazolone colitis is increased and can only be normalized if recolonization of the GF state occurs during the first 2 weeks of life; indicating an important window of opportunity in immune education of the iNKT cells in the colon. This is due to microbial induced signals that are yet to be defined which regulate CXCL16 production of the intestinal epithelium and bacterial derived sphingolipids from specific microorganisms such as Bacteroides fragilis which impede the proliferation of the infiltrating colonic iNKT cells. In addition, CD1d expression on IECs is decreased in human IBD suggesting a protective function for CD1d in the locale consistent with CD1d cross-linking studies on IEC lines which induces IL-10 production. The production of IL-10 is due to a novel pathway of IEC-dependent regulation of mucosal homeostasis that critically depends upon epithelial cell expression of microsomal triglyceride transfer protein (MTP), which we have previously shown is responsible for CD1d lipidation in the secretory pathway, CD1d itself and production of HSP110 and IL-10 in a pathway that depends upon STAT3 signaling. Further, our studies implicate the hematopoietic system as the source of CD1d-mediated intestinal inflammation when activated. These studies reveal a critical role for CD1d-iNKT cells in regulating the IEC-microbial interface.
SYMPOSIUM 28: INFECTION AND INFLAMMATION – SPONSORED BY BISBRAZIL 090 MECHANISMS INVOLVED IN THE IMMUNEDYSFUNCTIONS INDUCED BY SEVERE SEPSIS Fernando Q. Cunha School of Medicine Ribeirao Preto, University of Sao Paulo, Sao Paulo, Brazil Mechanisms involved in the immune-dysfunctions induced by severe sepsis FERNANDO Q. CUNHA, Department of Pharmacology of School of Medicine of Ribeira˜o Preto, USP Clearance of pathogens during infections depends on efficient neutrophil migration. Using animal models and neutrophil from patients, we are showing a defect in neutrophil recruitment into infectious focus during severe sepsis, which is followed by a reduced bacterial clearance. In fact, neutrophil from septic animals or patients present reduced chemotactic activity to CXCR1/2 agonists. This impairment of neutrophil migration associated with the down-regulation of CXCR1/2 in this cell type. Investigating the mechanism involved in the neutrophil migration to infection focus during sepsis, we are demonstrating that in parallel with CXCR1/2 internalization, the CCR5 is expressed in the neutrophil surface. It seems that the CCR5 expression is a compensatory mechanism in an attempt to reestablish the neutrophil migration to infection focus.
S87 The CCR5-/- mice have a more pronounced reduction of neutrophil migration into the infection focus, compared with WT mice. Both events (CXCR1/2 down-regulation and CCR5 expression) are due to activation of Toll-like receptors (TLRs). Neutrophils harvested from TLR-2 or -4 deficient mice submitted to polymicrobial (CLP) sepsis do not present chemotactic response to CXCR2 agonists, but become responsive to CCR5 agonists. Although TLR activation induces both events, the signaling mechanisms are different. The CXCR1/2 internalization in neutrophils harvested form septic mice or patients or from naı¨ve mice and stimulated in vitro with LPS associates with the increase of the expression and the activity of G-protein-coupled receptor kinases (GRK-2/-5). On the other hand, the CCR2 expression in the same cells depends on TNF-a production. The expression of GRKs and down-regulation of CXCR1/2 were prevented by pharmacological inhibition of iNOS (1400 W)/soluble guanilate cyclase (ODQ)/PKG (KT5823) pathway. In conclusion, these results, by a side, emphasize the harmful role of TLRs/iNOS/NO-GC-cGMP-PKG pathway on down-regulation of CXCR1/2 receptors expression in neutrophils via induction of GRKs expression, and by other side, highlight a compensatory pathway (TLRs/TNF-a production) in an attempt to reestablish the neutrophil migration. Financial support CNPq, FAPESP and Timer-EC.
091 SPECIALIZED PRO-RESOLVING MEDIATORS IN LUNG INFLAMMATION AND INJURY Bruce D. Levy1,2 Brigham and Women’s Hospital, Boston, MA, USA; 2Harvard Medical School, Boston, MA, USA
1
Acute lung inflammation is fundamentally important to host defense, but chronic or excessive inflammation can lead to several important diseases. The resolution of inflammation is an active process that is directed, in part, by specialized pro-resolving mediators that are enzymatically derived from polyunsaturated fatty acids. In health, cell–cell interactions at the onset of acute inflammation establish biosynthetic circuits for these pro-resolving mediators, including the omega-3 fatty acid-derived resolvins, protectins and maresins, which serve as agonists to orchestrate a return of the inflamed tissue to homeostasis. Understanding the cellular and molecular mechanisms for pro-resolving mediators in catabasis is providing new insights into lung tissue responses for resolution of inflammation in health and the pathophysiology of disease, including the host response to infections; as well as opportunities for therapeutic intervention. E-series and D-series resolvins are enzymatically derived from the essential omega-3 fatty acids eicosapentaenoic acid and docosahexaenoic acid, respectively. Protectin D1 and maresin 1 (MaR1) are also derived from DHA. Relevant to lung inflammation, lipoxin A4 (LXA4), Resolvin E1 (RvE1), resolvin D1 (RvD1) and protectin D1 (PD1) are generated in murine lung. LXA4 and PD1 are generated in human lung. Receptors for LXA4 and RvD1 (ALX/FPR2) and for RvE1 (CMKLR1) are expressed in lung and are dynamically regulated with lung inflammation. Evidence will be presented for cellular and molecular mechanisms for representative pro-resolving mediators in their protective actions in the regulation of airway inflammation during innate and adaptive immune responses.
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092 CHIKUNGUNYA VIRUS-INDUCED INFLAMMATORY DISEASE Suresh Mahalingam Griffith University, Nathan, QLD, Australia Chikungunya virus (CHIKV)-induced inflammatory disease is a significant cause of human morbidity worldwide. CHIKV has caused explosive outbreaks in Africa, Indian Ocean island territories, Asia, Europe and now the Americas. The recent CHIKV outbreak in the Caribbean and subsequent spread within the Americas highlights the potential for a pandemic in neighboring North and South American countries. The total number of cases in the Americas has now approached more than 1.2 million cases. There is a clear need to improve our understanding of the pathogenesis of this disease. In recent years my laboratory has unravelled the mechanisms of how CHIKV cause musculoskeletal disease (arthritis, arthralgia, myositis). We have carried out extensive work in understanding the pathogenesis of CHIKV disease and identifying new mechanisms, which has led to the identification of new candidate therapies for viral arthritides. These mechanisms and recent human studies will be discussed.
093 MECHANISMS OF DISEASE AND PROTECTION DURING VIRAL INFECTIONS Mauro M. Teixeira1, Luciana Padua1, Cristiana C. Garcia2, Daniele G. Souza1 1
Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; Instituto de Oswaldo Cruz, Fiocruz, Rio de Janeiro, Brazil
Inflamm. Res. deal with infection. In the context of Influenza A/WSN/33 H1N1 infection, there is much more neutrophil influx and damage in the lungs of PI3Kc-deficient mice. This is secondary to the decreased transmigration of T CD8+ lymphocytes, NK cells and resolving macrophages to the airways of PI3Kc deficient mice. Type I IFN responses induced by flu infection were abolished in lungs of KO mice leading to increased viral loads. Therefore, PI3Kc activation during Influenza A infection is necessary for an early antiviral response that promotes recruitment of effector cells against the infected cells and contributes to viral clearance. Therefore, our studies suggest that the role of PI3Kc during sterile inflammation and infection is context-dependent. Overall, inhibition or absence of PI3Kc tend to decrease sterile inflammation by preventing excessive leukocyte activation and tissue damage. During infection, these strategies tend to decrease infections with a strong systemic component but may affect negatively the capacity of the host to deal with focal infection, as in the model of pulmonary infection with influenza A. The concept of anti-inflammatory drugs for infectious disease is a valid principle but needs to be tested in individual infections to avoid unwanted increase in infection load and tissue damage.
SYMPOSIUM 29: BIOMARKERS IN INFLAMMATION: FOCUS ON RHEUMATOID ARTHRITIS 094 TOWARDS A MOLECULAR TAXONOMY IN RHEUMATOID ARTHRITIS: CAN WE DERIVE CLINICALLY USEFUL PROGNOSTICS AND THERAPEUTIC PREDICTORS?
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The inflammatory response that accompanies infectious diseases is central for the host ability to contain the growth of the pathogen and prime an adaptive immune response. However, excessive, misplaced or altered inflammatory responses may cause disease and even death of the host. It is possible that drugs which prevent the unwanted inflammatory response to infection may be useful as co-adjuvant treatment for infectious disease. We will discuss the potential effects of inhibitors of PI3Kc in models of sterile inflammation and infection. PI3Ks are central signaling enzymes, involved in cell growth, proliferation, survival and migration. Class IB PI3K or PI3Kc is mainly expressed on leukocytes and activated by GPCR coupling, and involved in cell migration under inflammatory conditions. Blockade or absence of PI3Kc is in general associated with decreased neutrophil activation but only partial inhibition of the influx of these cells in vivo. Decrease of the accumulation of neutrophils occurs only when PI3kc and PI3Kdelta are inhibited together. In the context of sepsis, absence or inhibition of PI3Kc is accompanied by an unexpected enhancement of neutrophil influx and consequent greater ability to deal with the septic insult. This is secondary to the decreased activation of the systemic activation of neutrophils. In the context of dengue infection, absence of PI3Kc decreases systemic disease without altering the ability of the host to
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Iain B. McInnes University of Glasgow, Glasgow, UK Rheumatoid arthritis (RA) treatment has been transformed by the advent of biologic therapeutics and by the application of formal strategies to align and enhance the use of such therapeutics. Current unmet clinical needs include the predominance of partial responses to most monotherapies, and the relatively rare occurrence of remission in the context of general treatment. Much work is ongoing to elucidate mechanisms that underlie the emergence of autoimmunity in RA and factors that mediate transition to, and maintain chronicity of synovial inflammation thereafter, representing failure of homeostatic inflammation resolution. Complex interplay exists between ‘professional’ immune cells and stromal cellular pathways that together contrive to perpetuate, or resolve articular inflammation. In this presentation I shall discuss the recent advances made in defining the biosignatures that are proposed to predict natural history and therapeutic response characteristics in RA. Unresolved issues surround the appropriate modality to define a molecular profile e.g. transcriptomics, proteomics and metabolomics, and even the utility of peripheral blood derived signals versus those form synovial tissues obtained at biopsy.
Inflamm. Res.
095 14-3-3 g: A JOINT-DERIVED MARKER WITH AUTOIMMUNE AND PERSONALIZED MEDICINE APPLICATIONS Anthony Marotta Augurex Life Sciences Corp, Vancouver, BC, Canada Background: RA is a chronic autoimmune disease that if left untreated results in severe joint destruction. Comorbid joint manifestation occur in approximately 15–20 % of patients with other autoimmune conditions, such as SLE, psoriasis and inflammatory bowel disease. It is estimated that 30–50 % of RA patients end up on workplace disability within 5 years of diagnosis. Early identification of RA, assessment of disease severity at presentation, together with a prompt and effective treatment strategy can significantly improve clinical outcomes and reduce the personal and economic burden of this disease. Need for New Markers: To enable this, new markers are needed to (1) improve the diagnostic capture rate of early RA; (2) inform joint damage prognosis to optimize treat-to-target strategies; and (3) monitor the disease process along the uncoupling of inflammation and joint damage aligning clinical response measures with damage progression risk. The goal of personalized medicine for RA is an ambitious but worthy one because of the heterogeneity of disease amongst patients and within individuals along the disease course. In that regard, if a biomarker target also serves as a drug target, companion biomarker levels can directly guide the selection of likely responders and allow for tailored dosing and administration schedules. 14-3-3g Markers and Personalized Medicine Potential: 14-3-3 proteins are an evolutionary conserved family of molecular chaperones that play a critical role in the regulation of intracellular functions. In RA, externalization of 14-3-3g is believed to couple with the release of lipid microvesicles or exosomes in a similar manner to other endokines. In vitro studies demonstrate that stimulation of cells with 14-3-3g lead to the induction of inflammatory transcripts, such as TNFa, and factors linked to the joint damage process, such as receptor activation of nuclear factor jB ligand (RANKL) and MMP-1. Since 14-3-3g is not normally present extracellularly, the immune system sees soluble 14-3-3g as foreign resulting in a specific auto-antibody response. Serum 14-3-3g and its auto-antibodies together identify more than 90 % of early RA patients. Those who are 14-3-3g protein positive have worse disease outcomes over 5 years, and in those patients who achieve DAS remission, a positive 14-3-3g test predicts a higher risk of joint damage progression, despite a good clinical response. 14-3-3g mechanistic findings converge strongly with in vivo data, wherein treatment of CIA mice with anti-14-3-3g monoclonal antibodies delays the disease onset and reduces its severity, providing a rational approach to personalized medicine with this novel factor. Learning Objectives 1. List the unmet needs for biomarkers based on RA clinical management objectives. 2. Describe 14-3-3g’s mechanistic role in RA pathogenesis and how its serological measurements assist with patient management.
S89 3. Explain how and why 14-3-3g serves as a personalized medicine target.
096 VECTRA DA, A MULTI-BIOMARKER DISEASE ACTIVITY BLOOD TEST THAT PREDICTS RISK FOR JOINT DAMAGE IN PATIENTS WITH RHEUMATOID ARTHRITIS Eric H. Sasso Crescendo Bioscience, South San Francisco, CA, USA Rheumatoid arthritis (RA) is a chronic disabling disease that requires ongoing monitoring of disease activity and adjustment of therapy to prevent irreversible damage to joints. The Vectra DA blood test measures 12 serum protein biomarkers and uses a validated algorithm to provide a score, on a scale of 1–100, that represents the level of RA disease activity. The Vectra DA score was developed to correlate with DAS28-CRP, a conventional, clinically based composite tool for assessing RA disease activity. Changes in Vectra DA score correlate with changes in DAS28-CRP. In addition, the Vectra DA score has been shown to be associated with risk for new joint damage, or radiographic progression, and is a better predictor of joint damage risk than clinically based measures or the CRP blood test. As such, Vectra DA can detect potentially damaging disease activity in patients with low levels of disease according to conventional measures and, conversely, it can identify patients with limited risk for progression despite a clinical appearance of active disease. This presentation will review studies demonstrating that, as a predictor of joint damage risk, Vectra DA measures pathologically meaningful disease activity in patients with RA and can supplement conventional assessment tools for therapeutic decision-making.
097 MRI BIOMARKERS FOR DISEASE ACTIVITY AND STRUCTURAL DAMAGE IN RHEUMATOID ARTHRITIS Charles Peterfy Spire Sciences, Inc, Boca Raton, FL, USA The past two decades have seen unprecedented advances in therapy for rheumatoid arthritis (RA), with several effective biologics introduced since 1998. Imaging played a central role in validating these novel treatments; however, a number of factors have recently made using radiography in clinical trials increasingly impractical if not infeasible. Ironically, the most important of these has been the success of the biologics revolution itself, as the availability of effective therapy has made extended placebo control unethical. Current guidelines call for
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S90 withdrawal or rescue therapy for non-responders within only 12–14 weeks. Unfortunately, traditional radiographic endpoints, such as Sharp scoring, are unable to discriminate treatment effects that quickly, even with large numbers of patients per arm. Magnetic resonance imaging (MRI) has been shown to resolve structural joint damage more sensitively than radiography can, while also assessing the upstream inflammatory drivers of erosions and cartilage loss, namely synovitis and osteitis. An increasing number of randomized clinical trials using MRI markers of these RA features have demonstrated that MRI can discriminate progression and treatment effect with respect to structural damage in only 12 weeks and with respect to inflammation in as little as 2 weeks, using fewer than 50 patients per arm. Further, composite clinical measures of disease activity, such as DAS28, SDAI and CDAI are responsive to treatment, but often fail to identify patients who continue to progress structurally despite satisfying clinical criteria for remission. MRI can thus serve as a useful adjunct to clinical measures in treat-to-target patient management as well. This presentation will review the performance of MRI as an imaging biomarker for assessing inflammation and structural damage in patients with RA from the perspectives of validity, discrimination and feasibility, and provide recommendations for its use.
Inflamm. Res.
MINI-SYMPOSIUM 3: MECHANISMS DRIVING MUCOSAL INFLAMMATION IN THE GUT B031 MULTIPLE MESENTERIC LYMPHATIC ANOMALIES IN MURINE CROHN’S DISEASE See poster section for abstract.
B028 ENHANCEMENTS TO THE PREDICTABILITY AND CLINICAL RELEVANCE OF TWO MURINE MODELS OF CHRONIC INFLAMMATION: ADOPTIVE TRANSFER-INDUCED COLITIS AND GVHD See poster section for abstract.
SYMPOSIUM 30: SLE: CURRENT THERAPEUTIC LANDSCAPE AND FUTURE PROMISES 098 MODULATION OF PLASMACYTOID DENDRITIC CELLS FOR TREATING AUTOIMMUNE DISEASES LIKE LUPUS Jo Viney Biogen, Cambridge, MA, USA Systemic lupus erythematosus (SLE) is a debilitating autoimmune disease with potentially devastating consequences. Gene expression studies have revealed characteristic Type I interferon (IFN-I) signatures in SLE. The immune complexes that are prevalent in SLE bind to FcgammaRIIa on plasmacytoid dendritic cells (pDC), driving the production of IFN-I (as well as other proinflammatory cytokines and chemokines). Plasmacytoid DC exclusively express BDCA2, a receptor that functions to inhibit the production of IFN-I. We have developed 24F4A, a humanized monoclonal antibody that binds to human and cynomolgus monkey BDCA2 with similar potency. In vitro human whole blood and PBMC assays demonstrate the ability of 24F4A to potently inhibit IFN-I production and other proinflammatory mediators following stimulation with TLR agonists or SLE-derived immune complexes. The inhibition of IFN-I is comparable in healthy and SLE donor assays. In vivo studies in cynomolgus monkeys established a PK/PD correlation with strong concordance between drug levels, BDCA2 receptor internalization, and inhibition of IFN-I production. Mechanism of action studies have revealed the inhibitory effect of 24F4A on immune-complex-induced IFN-I production by pDC is mediated via more than one mechanism. This presentation will highlight the novel therapeutic potential of an effector competent antiBDCA2 mAb that has a dual mechanism to dampen pDC responses in SLE and other autoimmune diseases. The author is an employee of Biogen.
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B215 CRITICAL ROLE OF PLASMACYTOID DENDRITIC CELLS IN IMMUNOREGULATION INITIATED BY A COMMENSAL MICROBIAL POLYSACCHARIDE See poster section for abstract.
B013 SUPPRESSING INFLAMMATORY BOWEL DISEASE BY MODULATING PROINFLAMMATORY GENE EXPRESSION AND IMMUNE CELL POPULATIONS See poster section for abstract.
B020 AMELIORATION OF CHRONIC COLITIS IN MICE BY GENETIC DELETION OF THE IL-13 DECOY RECEPTOR See poster section for abstract.
B252 FILGOTINIB, JAK1-SELECTIVE INHIBITOR, REPRESSES SIMILARLY JAK1/STAT3 PATHWAY IN THE COLON OF MICE WITH DSS-INDUCED COLITIS AND IN CULTURES OF COLON BIOPSIES FROM INFLAMMATORY BOWEL DISEASE PATIENTS See poster section for abstract.
Inflamm. Res.
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SYMPOSIUM 31: NEW VISIONS IN OCULAR THERAPY
SYMPOSIUM 32: TREGS IN HUMAN AUTOIMMUNITY
099 TOPICAL DELIVERY OF NOVEL MUCUSPENETRATING RTKIS FOR SUCCESSFUL ANGIOGENESIS BLOCKADE
100 ARE miRNA RESPONSIBLE FOR THE TREG DEFECTS OBSERVED IN MULTIPLE SCLEROSIS?
Lisa R. Schopf2, Elizabeth M. Enlow2, Pawel Nowak2, Winston Ong2, Jim Bourassa2, Kim Brazzell2, Hongming Chen2
Amy Lovett-Racke, Mary Severin, Mireia Guerau-de-Arellano, Michael Racke Ohio State University, Columbus, OH, USA
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Topical Delivery of Novel Mucus-penetrating RTKis for Successful Angiogenesis Blockade; 2Kala Pharmaceuticals, Waltham, USA Inflammation leading to neovascularization at the back of the eye is a key contributor to vision loss in diseases such as age-related macular degeneration (AMD). Vascular endothelial growth factor (VEGF) ligand blockade is a clinically proven treatment for AMD currently achieved through repeated intravitreal (IVT) injections of a biologic agent. A significant challenge in ophthalmology continues to be delivering sufficient levels of therapeutic agent to the back of the eye by non-invasive means. The objective of this work was to develop a novel RTKi that selectively blocks the VEGF pathway and can be delivered topically. While nanoparticles may have the potential to improve ocular tissue exposure from topical administration, this potential has been undermined by the adhesive nature of the ocular mucus layer, which serves to protect the eye from allergens, pathogens, and debris by effectively trapping and rapidly clearing foreign particles from the ocular surface. The mucus-penetrating particle (MPP) technology is a novel drug delivery platform that can be used to design nanoparticles that effectively penetrate mucus secretions. MPPs of certain compounds tested in the vaginal tract, lung, and gastrointestinal tracts of animals showed even distribution, and provided prolonged duration on these mucosal surfaces. Recently we have demonstrated that MPPs of certain compounds can provide a prolonged residence time at the ocular surface, which in turn translated into enhanced drug delivery to ocular tissues, superior pharmacokinetics (PK) and improved efficacy in animals. We have designed a next generation RTKi with nanomolar potency against VEGFR (KDR) and improved selectively over undesirable targets such as EGFR, FGFR, CDK and RET. Formulated as an MPP the inhibitor has the ability to penetrate mucus, residing longer at the ocular surface in animals and allowing for biologically active concentrations to be delivered to the back of the eye. Tissue distribution studies in rabbits and mini-pigs revealed retinal and choroid levels well above the cellular IC50 and higher compared to systemic compartments (i.e., plasma, heart, kidney and brain). In a rabbit model of vascular leakage, the topically applied RTKi was statistically equivalent to treatment with Avastin. In summary, there is a clear unmet medical need for non-invasive, topical drug delivery to both the anterior and especially to the posterior segment of the eye. We have demonstrated here that in animal models, an RTKi formulated as an MPP provided enhanced drug penetration not only to the ocular surface but also to the posterior segment. These data support the potential for a non-invasive therapy for VEGF pathway blockade which would be a breakthrough in the treatment of neovascular ophthalmic diseases.
Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system with an unknown etiology. Several studies have demonstrated a defect in CD4 regulatory T cells (Tregs) in MS patients but the cause of this defect lacks explanation. We performed a comprehensive miRNA profiling study on CD4 T cells of untreated MS patients and controls. Numerous miRNA were differentially expressed in subpopulations of CD4 T cells of MS patients. Notably, naı¨ve CD4 T cells had altered expression of several miRNAs that were predicted to target components of the TGFbeta, a pathway critical to the development and function of Tregs. Analysis of expression levels of various components of the TGFbeta signaling pathway found that TGFbeta receptor 1 and Smad4 expression was reduced in CD4 T cells of MS patients. When these miRNAs were over-expressed in naı¨ve T cells from control subjects, Treg development was impaired. Administration of these miRNAs to neonatal mice at the time when Treg development is peaking in the thymus resulted in suppressed Treg development. Furthermore, adult-onset EAE was exacerbated in mice which receive these miRNAs during early life. This study suggests that miRNA dysregulation in naı¨ve CD4 T cells may underlie the Treg deficit observed in MS patients and may be a risk factor for the development of MS.
101 DISENTANGLING THE ROLES OF IL-2 IN IMMUNE ACTIVATION AND SUPPRESSION Jason D. Fontenot Biogen, Cambridge, MA, USA IL-2 is a key nodal regulator of immune activation and suppression. There is significant interest in therapeutic modulation of the IL-2 pathway to potentiate cancer immunotherapy, facilitate transplant tolerance and treat autoimmune and inflammatory diseases. Given the ability of IL-2 to both promote effector T cell responses and limit immune activation through maintenance of regulatory T cells, extrapolating known effects on cell-type specific biologies to an aggregate therapeutic impact is difficult to predict, but critical to understand. Focusing on the role of CD25, the obligate component of the high affinity IL-2 receptor, we examine the impact of targeting IL2 biology during immune homeostasis and in the context of autoimmune pathology.
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102 IN VIVO MAINTENANCE OF HUMAN REGULATORY T CELLS DURING CD25 BLOCKADE David J. Huss1, Devangi S. Mehta1, Akanksha Sharma1, Xiaojun You1, Katherine A. Riester1, James P. Sheridan2, Lakshmi S. Amaravadi1, Jacob S. Elkins1, Jason D. Fontenot1
Inflamm. Res. proinflammatory mediators and elevated body temperature were not induced with Fc.muteins. Unexpectedly, certain Fc.muteins were more effective than Fc.WT or Proleukin at increasing Treg frequency and upregulating FOXP3. This property was found to correlate with an ability to stably associate with cell surface CD25 and stimulate low levels of IL-2R signaling for extended periods of time. Thus, optimal Treg-selectivity resulted from a combination of reduced activation of effector cells and better agonism of Treg. Our results demonstrate that a high degree of Treg-selectivity can be achieved through subtle changes in IL-2/IL-2R interactions.
1 Biogen, Cambridge, MA, USA; 2AbbVie Biotherapeutics, Redwood City, CA, USA
Regulatory T (Treg) cells mediate immune tolerance to self and depend on IL-2 for homeostasis. Treg deficiency, dysfunction and instability are implicated in the pathogenesis of numerous autoimmune diseases, including relapsing-remitting multiple sclerosis (RRMS). Daclizumab high yield process (DAC HYP) is a humanized monoclonal antibody that binds the IL-2 receptor alpha subunit (IL2Ra or CD25) and prevents IL-2 binding. DAC HYP has demonstrated clinical efficacy in patients with RRMS, despite causing a reduction in circulating Treg cell numbers. Here we investigate the impact of DAC HYP-mediated CD25 blockade on Treg cell homeostasis in RRMS patients. Based on analysis of a large clinical sample set, we report that DAC HYP treatment caused an *50 % decrease in Treg cells by week 8 of treatment that was sustained over a 52-week period. Remaining Treg cells retained a demethylated TSDRin the FOXP3 promoter, maintained active cell cycling and had minimal production of IL-2, IFN-gamma, and IL-17. In the presence of DAC HYP, IL-2 serum concentrations increased and CD25-independent intermediate affinity IL-2R-signaling induced STAT5 phosphorylation and sustained FoxP3 expression. Our results demonstrate that Treg cell phenotype and lineage stability can be maintained in the face of CD25 blockade.
103 MODULATING IL-2/IL-2R INTERACTIONS TO THERAPEUTICALLY SHIFT THE BALANCE BETWEEN REGULATORY T CELLS AND EFFECTOR LYMPHOCYTES Marc A. Gavin2,1, Li Li1, Dina Alcorn1, Margaret Karow1, Jacqueline Kirchner1, Kevin Gorski1, David Martin1, Katsu Ishida1, Guna Kannan1, Joshua Pearson1 1 Amgen, Inc., Thousand Oaks, CA, USA; 2Benaroya Research Institute, Seattle, WA, USA
IL-2-based therapeutics that enhance CD4+ regulatory T cells (Treg) represent a promising new modality for the treatment of inflammatory disease. IL-2-mediated Treg enrichment inhibits disease progression in multiple mouse models of autoimmunity, and in initial clinical studies, low dose IL-2 (Proleukin) enriches Treg and alleviates symptoms in patients suffering from various inflammatory conditions. One concern with this approach, however, is that efficacy will be compromised in certain diseases or patients due to IL-2 activity on autoreactive T cells, NK cells, or innate lymphoid cells, or that IL-2mediated lymphocyte activation will exacerbate pathology. To explore ways to increase Treg-selectivity, we have generated human IL-2 muteins with reduced potency and increased dependence on high IL-2Ra/CD25 expression. In humanized mice and cynomolgus monkeys, Fc-fused IL-2 muteins (Fc.muteins) were highly effective at stimulating Treg growth, but were poor agonists of conventional T cells and NK cells relative to Fc-fused wild-type IL-2 (Fc.WT) or Proleukin. Furthermore, unlike with Fc.WT or Proleukin,
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SYMPOSIUM 33: GASEOUS MEDIATORS AS THE BASIS FOR NOVEL ANTIINFLAMMATORY DRUGS—SPONSORED BY IRN-CANADA 104 IT’S JUST THE BEGINNING: NO, CO, H2S AND OTHER GASOTRANSMITTERS Rui Wang Laurentian University, Sudbury, Ontario, Canada The revolution in our understanding of the cellular signaling mechanisms started from the discovery of nitric oxide (NO) as a novel signaling molecule of gas. Distinctive from conventional neurotransmitters, NO transmits and amplifies biological signals intracellularly and intercellularly without the requirement and restrictions of the existence of secretory vesicles, specific membrane receptors, or intracellular second messengers. Not alone in utilizing this unique signal transduction strategy, NO has been joined by carbon monoxide (CO) and hydrogen sulfide (H2S) in the family of gasotransmitters. Being endogenously generated molecules of gas, gasotransmitters are crucially important for the regulation of a wide spectrum of cellular and molecular events. Altered bioavailability of gasotransmitters and their abnormal interactions with the target molecules have direct correlation with numerous diseases. Among the mostly studied molecular mechanisms for biological effects of gasotransmitters are the post-translational modifications of proteins. While NO can induce S-nitrosylation of a protein to decrease its function, H2Sinduced protein S-sulfhydration usually increases the protein function. Gasotransmitters can act on the same molecular target with different outcomes. They can also act on different molecular targets to achieve the same functional outcome. The interactions among gasotransmitters may potentiate or cancel each other’s function. If two’s company and three’s a crowd (Wang, FASEB J, 16: 1792–1798, 2002), what is four? It is ammonium (NH3) (Wang, Trends Biochem Sci, 39 (5):227–232, 2014). Against all criteria for gasotransmitters, ammonia certainly is qualified and its biological importance has been gaining more recognition. Gasotransmitter research has flourished in recent decades but it is still just the beginning of the revolution. (Supported by Canadian Institutes of Health Research).
105 HYDROGEN SULFIDE-BASED ANTIINFLAMMATORY THERAPEUTICS John L. Wallace University of Calgary, Calgary, AB, Canada
Inflamm. Res. Hydrogen sulfide is a gaseous mediator that is produced throughout the body, as well as by some bacteria residing in the gastrointestinal tract. In recent years, it has become clear that H2S mediates many physiological processes, including regulation of blood flow, adherence of leukocytes to the vascular endothelium, mitochondrial respiration, wound healing and regulation of blood pressure. The ability of H2S to increase the resistance of the gastrointestinal (GI) tract to injury, to accelerate the healing of preexisting damage, and to reduce inflammation, led us to design a series of drugs that release H2S. Specifically, we focused on existing anti-inflammatory drugs such as nonsteroidal anti-inflammatory drugs (NSAIDs) and mesalamine (for treatment of inflammatory bowel disease). Several H2S-releasing NSAIDs were developed and tested in a range of animals models. One of these, ATB-346, is a derivative of naproxen, one of the most commonly used NSAIDs for treatment of arthritis. In animal studies, ATB-346 exhibited comparable anti-inflammatory activity to the parent drug, but produced markedly less GI damage. Even when given to animals with severely compromised mucosal defence, ATB-346 was found to be safe. Indeed, this compound significantly accelerated healing of pre-existing ulcers in the GI tract. In human trials, ATB-346 exhibited enhanced anti-inflammatory activity as compared to the parent drug, presumably attributable to the anti-inflammatory effects of the H2S-releasing moiety, but also likely due to altered pharmacokinetics (including a much longer half-life than that of naproxen). Clinical trials of this compound are ongoing. H2S is a promising candidate for enhancing the activity of existing drugs and for a wide range of indications.
106 SWITCHING ‘‘OFF’’ CHRONIC NEUROPATHIC PAIN BY SWITCHING ‘‘ON’’ THE A3 ADENOSINE RECEPTOR SUBTYPE (A3AR) Daniela Salvemini Saint Louis University School of Medicine, St. Louis, MO, USA Chronic pain is a global burden that promotes disability and unnecessary suffering. To date, efficacious treatment of chronic pain has not been achieved. Thus, new therapeutic targets are needed. Our studies demonstrate that increasing endogenous adenosine levels through selective adenosine kinase inhibition produces powerful analgesic effects in rodent models of experimental neuropathic pain through the A3 adenosine receptor (A3AR) signalling pathway. Similar results were obtained by the administration of novel and highly selective A3AR agonists. These effects were prevented by blockade of spinal and supraspinal A3AR, lost in A3AR knock-out mice, and independent of opioid and endocannabinoid mechanisms. A3AR activation also relieved non-evoked spontaneous pain behaviours without promoting analgesic tolerance or inherent reward. Further examination revealed that A3AR activation reduced spinal cord pain processing by decreasing the excitability of spinal wide dynamic range neurons and producing supraspinal inhibition of spinal nociception through activation of serotonergic and noradrenergic bulbospinal circuits. Critically, engaging the A3AR mechanism did not alter nociceptive thresholds in non-neuropathy animals and therefore produced selective alleviation of persistent neuropathic pain states. These studies reveal A3AR activation by adenosine as an endogenous anti-nociceptive pathway and support the development of A3AR agonists as novel therapeutics to treat chronic pain.
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SYMPOSIUM 34: THERAPEUTIC PATHWAYS IN INFLAMMATION: NEW FACES OF OLD FRIENDS—SPONSORED BY RIS 107 THE NEW METHOD OF EVALUATION OF ß-ADRENORECEPTOR’S ACTIVITY Kirill A. Zykov1, Olga Y. Agapova1, Yuri S. Skoblov2, Valery P. Masenko3, Irina E. Chazova3 1 Laboratory of Pulmonology, Moscow State University of Medicine and Dentistry named after A.I. Evdokimov, Moscow, Russian Federation; 2Laboratory of Isotope Analysis, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, RAS (IBCh RAS), Moscow, Russian Federation; 3Department of Arterial Hypertension, Russian Cardiology Research And Production Complex, Moscow, Moscow, Russian Federation
Body: ß-adrenoreceptor active drugs are often used in therapy of bronchoobstructive and cardiovascular diseases. These drugs can change the expression and affinity of ß-receptors. Receptor’s affinity and expression can be assessed by a radioligand method on lymphocytes and extrapolated to bronchial smooth muscle, but this method is not suitable for clinic because it needs more than 20 million cells (approximately 100 mL of blood). Objective: To evaluate the change of ß-adrenoreceptors relative activity in patients treated with ß-agonists using a new optimized radioligand method. Methods: Evaluation of relative activity of ß-adrenoreceptor on T-lymphocytes was performed using an optimized method based on the radioligand assay and detection of the change of the amount of 125I-cyanopindolol in presence of specific ß2 ligand. We defined the relative receptor activity as the availability of the receptor on the cell surface for specific ligand under the given conditions. Optimal conditions for the specific binding to assess ß2-adrenergic 0.5 fmol per 1 million T-lymphocytes using (125I)-cyanopindolol were found. Healthy volunteers and patients with bronchial asthma (BA) and arterial hypertension (AH) were enrolled. Patients with BA had monotherapy with inhaled corticosteroids. Results: 6 healthy persons aged 35.0 ± 7.8 initially had specific ß2receptor binding 4.2 ± 2.3 fmol/109; after 30 min of inhalation of a short-ß2-agonist specific ß2-receptor binding decreased (3.6 ± 2.3). 3 patients examined after 2 h. Specific ß2-receptor binding returns to the initial level (3.5 ± 1.6; 3.0 ± 1.9; 3.3 ± 1.7 fmol/109). Five patients with AH and BA aged 59.6 ± 11.7 initially had specific ß2-receptor binding 2.5 ± 2.2, than 4.0 ± 2.1 and 1.9 ± 1.3 fmol/109 after 2 h. Conclusion: This method allows to assess relative activity of T-cells’ ß-adrenoreceptors. Different type of reaction of specific ß2adrenoreceptor binding were detected in healthy volunteers and patients with BA and AH. Further research is needed to establish clinical relevance of different dynamics of relative activity of ß2adrenoreceptors of blood cells.
108 CELLULAR SOURCES OF PROINFLAMMATORY CYTOKINES IN DISEASE AND CELL TYPERESTRICTED CYTOKINE TARGETING Sergei A. Nedospasov1,2,3
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Engelhardt Institute of Molecular Biology, and Lomonosov Moscow State University, Moscow, Russia; 2Lobachevsky University, Nizhni Novgorod, Russia; 3German Rheumatism Research Center, a Leibniz Institute, Berlin, Germany Proinflammatory cytokines TNF, IL-6 and IL-1 may contribute to pathogenesis of autoimmune diseases and therefore systemic inhibition of these cytokines became an essential part of the therapy. Our recent studies in experimental arthritis suggest that TNF from one particular cellular source may play an anti-inflammatory role. If so, systemic cytokine inhibition may be like a double-edged sword disrupting both pathogenic and protective signaling. Based on these findings we are developing an approach to cell type-restricted cytokine neutralization by utilizing bispecific antibodies that would attach to the cell surface of a particular type of immune cells, and capture the cytokine released by these cells, preventing its dissemination. Our constructs are based on single domain antibodies (VHH) specific for human or mouse TNF, human IL6 and for cell type-specific markers, in particular, for F4/80 surface molecule expressed on macrophages. We find that such antibodies can effectively attach to the cell surface, capture and retain released cytokines. Using mice humanized for the TNF system and with macrophages isolated from such mice we assessed activity of these constructs in vitro and in vivo. Our findings may serve as a basis for bioengineering of new type of cytokine inhibitors. Supported by Russian Scientific Fund grant 14-25-00160 (for IL-6 related studies) and Russian Ministry of Science and Education grant 14.Z50.31.0008.
109 COXSACKIE: ADENOVIRUS RECEPTOR EXPRESSION ON CARDIOMYCYTES AND PLATELETS IN PATIENTS WITH INFLAMMATORY CARDIAC PATHOLOGY Elena M. Gupalo, Liudmila I. Buryachkovskaya, Irina A. Uchitel, Peter V. Chumachenko, Natalia A. Mironova Russian Cardiology Research Complex, Moscow, Russian Federation Background: Coxsackie-adenovirus receptor is a protein of tight junction, which persists of the different cell types. Recently it was shown that CAR can be expressed on the platelets surface, the cells which are known to play a crucial role in inflammation processes and which can directly reflect systemic inflammation. It remains unknown how CAR distributed in heart tissue and peripheral blood platelets in patient with inflammatory cardiac pathology Methods: There were included 23 patients with inflammatory dilated cardiomyopathy (DCMi) and 12 healthy subjects (HS). Diagnosis of DCMi was confirmed according endomyocardial biopsy (EMB) data. CAR persistence on cardiomyocytes was analyzed by immunohistochemistry in EMB samples. Platelet morphology of pts and HS was assessed in whole blood by scanning electron microscopy. CAR persistence on platelets was analyzed using immunofluorescent method. Spontaneous and 1.0 lM ADP-induced aggregation was estimated using light transmission aggregation analyzer BIOLA (Russia). In all pts additionally the level of IL-6 and TNF-alpha was analyzed be ELISA. Results: According to EMB evaluation analyses there was three grades of CAR expression: (1) intensive staining of intercalated disc and whole membrane, (2) spread along all the cells surface, (3) weak staining of lateral cell membranes. There was no relation between the level of CAR expression on platelets and cardiomyocytes. In patients with high IL-6 level the persistence of CAR on platelets was maximal
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Inflamm. Res. (2.5 ± 0.55 vs. 2.0 ± 0.43, p = 0,036). Only 0.2–6.4 % platelets were CAR positive in healthy subjects. CAR was redistributed among all the platelet membrane. In DCMi pts the number of CAR positive platelets varied from 1 to 57 % with mean level of 8.0 percent that was significantly higher than in HS. In all DCMi patients CAR was localized at the cites of intercellular communications in the small platelet aggregates. The level of CAR expression correlated with increased level of IL6 and TNF alpha (r = 0.51, 0.48). In pts spontaneous aggregation was 3.7 ± 1.8 % at the first 5 min, but to the 9–14 min its level grew up to 37 ± 17.3 % similar to 1.0-ADP induced aggregation as a result of platelet release reaction that was never observed in HS (p = 0.0001). In whole blood pts were detected circulating platelet microaggregates leukocyte-platelet and erythrocyte-platelet aggregates that are typical for inflammation Summary: The level of CAR expression on cardiomyocytes is related to increased level of IL6. There is no relation between CAR expression on cardiomyocyte and platelets in DCMi pts. Increased CAR expression on platelets of pts is associated with increased level of IL6 and TNF alpha. The high level of CAR expression, its appearing in the sites of intracellular connection and relation to increased platelet aggregation may indicate the role of this receptor in platelets changes during inflammation. Financial support We acknowledge for financial support Rfbr: grant 14-04-01754
B117 P38DELTA MAPK: A NOVEL EFFECTOR IN NLRP3 INFLAMMASOME ACTIVATION UPREGULATED IN HUMAN CORONARY ATHEROSCLEROTIC LESIONS See poster section for abstract.
B125 CONTROL OF LUNG CANCER WITH ASPIRINTRIGGERED STIMULATION OF RESOLUTION See poster section for abstract.
SYMPOSIUM 35: CLINICAL DEVELOPMENTS IN FIBROSIS 110 TARGETING IL13 IN IDIOPATHIC PULMONARY FIBROSIS Joseph R. Arron Genentech, Inc, South San Francisco, CA, USA Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal parenchymal lung disease of unknown etiology. Preclinical models of fibrotic diseases have implicated interleukin-13 (IL13) activity on multiple cell types, including macrophages and fibroblasts, in initiating and perpetuating pathological fibrosis, which can be ameliorated by therapeutic inhibition of IL13 with monoclonal antibodies. IL13 and IL13-inducible target genes are expressed at significantly elevated levels in lung tissue from patients with IPF compared to control
Inflamm. Res. lung tissue. Systemic biomarkers related to IL13 activity are elevated in subsets of IPF patients and have been associated with disease severity and prognosis. Monoclonal antibody therapies targeting IL13 have demonstrated clinical efficacy and are generally well tolerated and safe in asthma patients, and are currently under investigation in clinical studies of IPF patients. Given the molecular, pathological, and clinical heterogeneity of IPF, biomarker-guided clinical development strategies may be important in determining the success of targeted therapeutic approaches.
111 SMALL MOLECULE INHIBITORS OF LOXL2 FOR FIBROSIS Wolfgang Jarolimek Pharmaxis, Ltd, Frenchs Forest, NSW, Australia Fibrosis is the result of excessive accumulation of matrix proteins like collagen and elastin. Degradation of these proteins is hindered by increased activity of lysyl oxidases which cross link matrix protein to form fibrotic areas. Therefore, inhibition of lysyl oxidase activity seems to be a promising mechanism to treat fibrosis. Lysyl oxidases are a five membered family (Lox, Loxl1, Loxl2, Loxl3, Loxl4) of which Loxl2 seemed to be particularly important in diseases and a functional antibody diminished fibrosis is various animal models (Nat Med 16:1009–1017, 2010). However, similar results can be obtained with a functional Lox antibody (Cancer Res 73:1721–1732, 2013) suggesting that the role of lysyl oxidases can be complimentary in diseases. To discover small molecules the only known small molecule inhibitor, b-aminoproprionitrile (BAPN), is not a good template as it is non-selective and does not have good developability properties. Based on the successful development of a mechanism-based semicarbazide-sensitive amine oxidase inhibitor (SSAO/VAP-1) we attempted to develop Loxl2 selective small molecules. Through iterative medicinal chemistry efforts we could identify low molecular weight mechanism-based inhibitors that have various selectivity ratios and some are very selective for Loxl2 over Lox in biochemical assays. These compounds show all characteristics of mechanismbased inhibition and have good developability properties. The CCl4-induced liver fibrosis model has been used to assess the efficacy of our small molecule Loxl2 inhibitors to reduce fibrosis. Consistently, the area of fibrosis and a-smooth muscle actin were significantly reduced by therapeutic applications of Loxl2 inhibitors. In summary, small molecule, selective inhibitors of Loxl2 can be developed. These compounds are very efficacious anti-fibrotic drugs and may be useful for the treatment of various diseases.
112 TARGETING LPA AS A CORE FIBROTIC PATHWAY: SIMILARITIES AND DIFFERENCES ACROSS ORGANS Andrew M. Tager1,2,3 Division of Pulmonary and Critical Care Medicine; 2Massachusetts General Hospital, Boston, MA, USA; 3Harvard Medical School, Boston, MA, USA
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Fibrosis—the pathologic accumulation of fibroblasts and extracellular matrix—can affect essentially any tissue or organ. Although fibrotic diseases present clinically with organ-specific manifestations, there
S95 has been a growing appreciation of common mechanisms in their pathogenesis. The discovery of core fibrotic pathways shared by these diseases suggests that therapeutics developed to target such pathways could have broad clinical applicability. We have identified important roles for lysophosphatidic acid (LPA) in multiple mouse models of fibrotic diseases, each affecting a different organ, including the lung, skin, peritoneum and kidney. Although LPA signals through at least six different receptors, identified as LPA1–6, we have found important roles for LPA signaling specifically through LPA1 in all organs we have investigated. In the lung, we have found that LPA-LPA1 signaling promotes epithelial cell apoptosis, vascular leak and fibroblast recruitment and persistence. In the skin, we have found that LPALPA1 signaling is required for fibroblast differentiation into myofibroblasts. In the peritoneum and the kidney, we have identified a pathway through which LPA-LPA1 signaling promotes expression of pro-fibrotic genes, such as CTGF, which drive fibroblast proliferation. This pathway involves Ga12/13, RhoA and ROCK activation, actin polymerization, the transcriptional co-activators MRTF-A and MRTF-B, and the transcription factor SRF. In contrast to similarities we have found in pro-fibrotic LPA signaling being mediated by LPA1 across organs, we have found important cross-organ differences in the generation of LPA during the development of fibrosis. Two major enzymatic pathways have been described for LPA production. LPA can be produced from phosphatidic acid by phospholipase A1 (PLA1) and phospholipase A2 (PLA2) family members, including Lipase H. Alternatively, PLA1 and PLA2 can first convert phospholipids to lysophospholipids such as lysophosphatidylcholine (LPC), which can then be converted to LPA by autotaxin (ATX). We have found that ATX expression is elevated in, and its activity is required for, the development of dermal fibrosis. In contrast, we have found that ATX activity is not required for the development of lung fibrosis. In keeping with LPA production not being ATX-dependent in lung fibrosis, mass spectroscopy analyses of lysophospholipid species during the development of lung fibrosis do not support a substrateproduct relationship between LPC and LPA. It consequently appears that the LPA pathway can be inhibited more broadly across organs by targeting the LPA1 receptor, rather than by targeting the enzymes that are involved in LPA generation. The strategy of targeting LPA1 is currently being investigated in two fibrotic diseases, with antagonists of this receptor being evaluated in clinical trials in idiopathic pulmonary fibrosis and scleroderma.
SYMPOSIUM 36: NOVEL THERAPIES IN RA 113 RHEUMATOID ARTHRITIS: WHERE WE ARE AND WHAT IS THE FUTURE Ernest H. Choy Cardiff University, Cardiff, UK Rheumatoid arthritis (RA) is a chronic inflammatory arthritis, which result in pain, disability and reduced quality of life. Chronic synovial inflammation leads to irreversible joint damage. Biologics have transformed the management of RA, reducing disability and improving prognosis. In RA, ‘‘treat-to-target’’ to maintain suppression of the disease activity is essential to halt joint destruction and is the current standard of care. However, \30 % of patients in routine clinical practice. Therefore, there is a continued need to develop novel treatments that achieve higher rate of disease remission.
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S96 Over the two decades, five classes of biologic agents have been licensed for the treatment of RA: tumor necrosis factor alpha (TNFa) inhibitors; anti-interleukin-6 (IL-6) receptor monoclonal antibody (mAb); the anti-CD20 B-cell–depleting mAb; T-lymphocyte costimulation inhibitor (abatacept); and the IL-1 receptor antagonist anakinra. More recently, a selective small molecule inhibitor of Janus-activated kinase (JAK), tofacitinib, has been approved in the US and other countries in the world for the treatment of RA1. Optimizing treatment and predicting response to treatment are important to maximise health economic value and minimise the risk of side effects. Currently, there are many research programmes trying to develop biomarkers that can predict response to biologic treatment, in particular, one study suggested that synovial histology obtained by biopsies before treatment may predict response to adalimuamb and tocilizumab2. It found that patients with high ICAM expression and low CXCL13 were more likely to respond to adalimumab conversely patients with low ICAM-1 and high CXCL13 were more likely to respond to tocilizumab. IL-6 differs from TNFa in that it has a major role in the adaptive immune response through effects on T and B cells. CXCL13 is a B cell chemoattractant. High level of CXCL13 suggest active adaptive immune response. Recent studies using BioMap have lent further support this hypothesis by demonstrating that methotrexate, adalimumab and tocilizumab have distinct immunologic phenotypic profiles consistent with their mode of action3. Interestingly methotrexate has a significant inhibitory effect on B cell function which is reflected in its ability to reduce immunogenicity of therapeutic mAb. Methotrexate and adalimumab when added together had more pharmacodynamics interaction than combining methotrexate to tocilizumab4. This may explain why the magnitude of benefit when methotrexate is combined with tocilizumab is less than those observed with methotrexate and TNFa inhibitors5–7. References 1. Choy EH et al. Nat Rev Rheumatol. 2013 Mar;9(3):154–63 2. Dennis et al. Arthritis Research & Therapy 2014, 16:R90 3. Tan SL et al. ACR 2013. 4. O’Mahony A et al. EULAR 2014 Abs no THU0526. 5. Dougados et al. Ann Rheum Dis 2013;72:43–50. 6. Breedveld FC, et al. Arthritis Rheum 2006; 54:26–37. 7. Klareskog L et al. Lancet 2004; 363(9410): 675–681.
114 GM-CSF: A NEW TARGET FOR INTERVENTION IN RHEUMATOID ARTHRITIS Michael E. Weinblatt Brigham and Women’s Hospital, Boston, MA, USA Granulocyte macrophage colony stimulating factor (GM-CSF) is a pro-inflammatory cytokine involved in the activation, differentiation and survival of cells of the myeloid compartment, notably neutrophils, macrophages and dendritic cells. Over the last two decades significant evidence points to this cytokine as playing a key role in rheumatoid arthritis (RA). Synovial fluids isolated from patients with RA have elevated GM-CSF levels (Bell et al. 1995), GM-CSF receptor positive cells are increased in the synovial tissue (Greven et al. 2014) and chromosome 5q31, in the region of IL-3 and GM-CSF, was recently identified as a risk loci for RA (Okada et al. 2014). In addition the use of recombinant GM-CSF to treat neutropenia has been shown to promote arthritis flares in patients with felty syndrome (Hazenberg et al. 1989). These clinical observations are supported by arthritis studies in vivo demonstrating that either mice deficient in GM-CSF (Campbell et al. 1998) or treated with neutralising monoclonal antibodies to either the ligand (Cook et al. 2001) or receptor (Greven et al.
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Inflamm. Res. 2014) are protected from disease. Mavrilimumab is an IgG4 human monoclonal antibody that binds to the GMCSFR chain and prevents GM-CSF binding to its receptor. Recently this antibody has demonstrated efficacy in a 24 week Phase IIb double blind randomised clinical trial in RA patients that have had an inadequate response to traditional disease modifying anti-rheumatic drugs (DMARDS). The role of the GM-CSF pathway in RA will be introduced and the results of the clinical trials in rheumatoid arthritis will be presented. References Bell et al (1995). Measurement of colony-stimulating factors in synovial fluid: potential clinical value. Rheumatol Int. 14(5):177–82. Greven et al (2014). Preclinical characterisation of the GM-CSF receptor as a therapeutic target in rheumatoid arthritis. Ann Rheum Dis. (Epub 16 June) Hazenberg et al (1989). Correction of granulocytopenia in feltys syndrome by GM-CSF. Simultaneous induction of IL-6 release and flare-up of the arthritis. Blood. 74(8): 2769–70. Campbell et al (1998) Protection from collagen-induced arthritis in GM-CSF deficient mice. J. Immunol. 161(7): 3639–44. Cook et al. (2001). Blockade of collagen induced arthritis post-onset by antibody to GM-CSF: requirement for GM-CSF in the effector phase of the disease. Arthritis Res. 3(5): 293–8.
115 A NEW THERAPEUTIC APPROACH FOR RA TREATMENT USING AN ANTI-TLR4 MONOCLONAL ANTIBODY INHIBITING TLR4 AND FCGAMMAR SIGNALING AND EXPLOITING CITRULLINATED PROTEIN IMMUNE COMPLEXES AS POTENTIAL BIOMARKERS FOR PERSONALIZED MEDICINE Emmanuel Monnet1, Limin Shang1, Genevie`ve Lapeyre1, Eric Hatterer1, Kathy deGraaf1, Venassa Buatois1, Greg Elson1, Walter Ferlin1, Cem Gabay2, Jeremy Sokolove3, Simon Jones4, Ernest H. Choy4, Iain B. McInnes5, Marie Kosco-Vilbois1, Cristina de Min1 1
Novimmune SA; 2Geneva University Hospitals,Geneva, Switzerland; Stanford University, Stanford, CA, USA; 4Cardiff Institute for Infection and Immunity, Cardiff , UK; 5Glasgow Biomedical Research Center, Institute of Infection, Immunity and Inflammation, Glasgow, UK
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Background: Innate immunity is implicated in rheumatoid arthritis (RA) pathogenesis and is likely initiated via TLR pathways. NI-0101 is the first humanized monoclonal antibody (mAb) that blocks TLR4 signaling independently of ligand type (i.e., exogenous/endogenous) and concentration. Objectives: To determine preliminary tolerability, safety, pharmacokinetic (PK)/pharmacodynamic (PD) profiles after single administrations of different NI-0101 doses to healthy volunteers (HV), as well as to investigate NI-0101 ability to block TLR4-mediated inflammatory cytokine production induced by endogenous TLR4 ligands. Methods: A PK/PD guided single ascending dose Phase 1 study was conducted in 73 HV, in the presence of in vivo and ex vivo challenges with the exogenous TLR4 ligand, lipopolysaccharide (LPS). In parallel, monocytes obtained from RA patients were stimulated either with endogenous TLR4 ligands or synovial fluids (SF) of RA patients in presence and absence of NI-0101. The correlation of TLR4 blockade with level of endogenous TLR4 ligands in SF was assessed. Finally, the anti-mouse TLR4 surrogate mAb, 5E3, was tested in murine models of RA (IL-1Rn-/- mice and collagen induced arthritis).
Inflamm. Res. Results: NI-0101 was administered up to a single dose of 15 mg/kg in the Phase 1 study in HV and showed no safety concerns. The predictable PK profile was biphasic, similar to other therapeutic IgG targeting cell surface receptors. NI-0101 administration completely inhibited ex vivo and in vivo LPS-induced cytokine release from a dose of 1 mg/kg, as well as prevented all laboratory and clinical changes expected following LPS administration to HV. NI-0101 PK/ PD profiles allowed, through modeling and simulation of multiple administration of NI-0101, to identify an appropriate dose range to be tested in Phase 2. In vitro interaction of NI-0101 with TLR4 and Fcc Receptor efficiently blocked TLR4 activation by citrullinated protein immune complexes. NI-0101 blocked the activation of monocytes stimulated with SF from a sub-population of RA patients. This inhibition correlated with the presence of anti citrullinated protein antibodies in synovial fluids and their matching sera. Therapeutic administration of 5E3 ameliorated disease progression in both mouse models of arthritis. Conclusions: Taken together, these data strongly support TLR4 as a valid therapeutic target in RA, and provide an opportunity to evaluate antibodies against citrullinated proteins as biomarkers predictive of treatment response in Phase 2.
116 CELL THERAPIES IN RHEUMATOID ARTHRITIS John D. Isaacs Newcastle University, Newcastle upon Tyne, UK Rheumatoid arthritis (RA) is an autoimmune disease characterised by joint inflammation as well as by extra-articular features such as interstitial pneumonitis, and comorbidities such as cardiovascular disease. Current therapies for RA are anti-inflammatory and/or immunosuppressive, and the vast majority of patients require chronic treatment with the risk of adverse effects such as severe infections and possibly malignancy. Furthermore, even initially effective therapies often lose their benefit with time. The ideal therapy for RA would switch off the autoimmune process and restore self-tolerance, providing long-term benefit from a shortterm intervention. So-called therapeutic tolerance has been achieved in a number of animal models of autoimmunity and transplantation but translation to the clinic has been slow. Trials of tolerogenic antibodies such as anti-CD4 have recently given way to tolerogenic cellular therapies. These include regulatory T-cells, tolerogenic dendritic cells and mesenchymal stem cells. Each of these is based around a natural immunoregulatory cell type and each has its own relative advantages and disadvantages. Treatment generally involves the isolation of an appropriate cellular subset; its ex vivo manipulation, usually involving expansion and/or differentiation, in a laboratory that conforms to current Good Manufacturing Practice regulations; and its subsequent administration following assessment against specific quality control or ‘release’ criteria. Some cellular therapies are autologous, others allogeneic; and they can be administered fresh or following cryopresevation. Most work in RA has focussed on tolerogenic dendritic cells although only three trials have been reported to date, and only in abstract form at the time of writing. My own research group has recently completed the first ever trial of intra-articular tolerogenic dendritic cells in rheumatoid and inflammatory arthritis patients (the AuToDeCRA trial). As with any phase I study it was designed primarily to study safety but potential efficacy was also explored. My lecture will use AuToDeCRA to illustrate the points to consider and understand before embarking on a tolerogenic therapy in humans, including how to measure ‘success’. The other cellular therapies that are in development for RA will also be discussed briefly.
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B259 14-3-3 ETA AS A NOVEL RA DRUG TARGET: ANTI-14-3-3 ETA MONOCLONAL ANTIBODY DELAYS THE ONSET AND MITIGATES THE SEVERITY OF ARTHRITIS IN CIA MICE See poster section for abstract.
B316 RESTORATION OF TREG CELL FUNCTION WITH PD-L1 FC PROTEIN IN RHEUMATOID ARTHRITIS See poster section for abstract.
MINI-SYMPOSIUM 4: SIGNALING IN INFLAMMATION B139 SALT-INDUCIBLE KINASES (SIK) INHIBITION IN HUMAN MYELOID CELLS MODULATES TLR AND IL-1R SIGNALING AND INDUCES AN ANTIINFLAMMATORY PHENOTYPE See poster section for abstract.
B197 RIP KINASE SIGNALING IN KERATINOCYTE CONTROLS NECROPTOSIS: MEDIATED SKIN INFLAMMATION See poster section for abstract.
B281 BLOCKING LTßR SIGNALING IN VIVO SUPPRESSES THE DEVELOPMENT OF SMOOTH MUSCLE HYPERTROPHY IN A CHRONIC MURINE MODEL OF ASTHMA See poster section for abstract.
B069 OPPOSITE ROLE OF SOLUBLE FORMS OF IL-6 RECEPTOR, SIL-6R AND SGP130, ON THE REGULATION OF INFLAMMATORY STATUS IN RHEUMATOID ARTHRITIS See poster section for abstract.
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B082 COMBINATORIAL TARGETING OF TSLP, IL-25, AND IL-33 IN TYPE 2 CYTOKINE-DRIVEN INFLAMMATION AND FIBROSIS
Oklahoma City, OK, USA; 5Allergy and Asthma Assoc. Santa Clara County Research Center, San Jose, CA, USA; 6California Allergy and Asthma Medical Group, Los Angeles, CA, USA; 7Pennisula Research Assoc., Rolling Hills Estate, CA, USA; 8Clinical Research Institute of Southern Oregon, Medford, OR, USA; 9University of Pittsburgh Asthma Institute, Pittsburgh, PA, USA
See poster section for abstract.
Asthma and other allergic diseases are associated with mast cell activation and prostaglandin D2 (PGD2) generation. PGD2 exerts pro-inflammatory activity via chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTh2). ARRY-502 is a potent and selective CRTh2 antagonist which was studied in mild atopic asthmatic adults in a double-blind, placebo-controlled 4 week phase 2a study. Enrolled patients were free of inhaled corticosteroids with an FEV1percent predicted of 60–85 %. Potential participants were screened and randomized to receive 200 mg BID ARRY-502 (n = 93) or matching placebo (n = 91). The primary endpoint was change from baseline FEV1 compared to placebo. Secondary endpoints included additional spirometry evaluations, measures of asthma control and two quality of life (QOL) assessments. Safety was evaluated by incidence and severity of adverse events, vital signs, hematology and EKG parameters. FEV1 improved 3.9 % compared to placebo (Day 29, p = 0.02). Asthma Control Questionnaire-7, ß-agonist use and symptom free days improved compared to placebo (p \ 0.001, p \ 0.001 and p = 0.07 respectively). Asthma and Rhinitis QOL improved compared to placebo (p = 0.012, p = 0.007). ARRY-502 was well tolerated with less adverse events than placebo. In patients with elevated Th2 associated biomarkers at baseline, activity outcomes between ARRY-502 and placebo were numerically and statistically greater in Th2 high (baseline) participants (for example: DFEV1 = 6.7 %; p = 0.008). In addition, there was a significant reduction in markers of Th2 driven inflammation in these patients. These results support the activity of ARRY-502 in mild asthma patients, suggest that patients that suffer from other Th2-driven allergic diseases like atopic dermatitis could benefit and warrant further development of ARRY502
B083 DECIPHERING THE DELETERIOUS EFFECT OF IFN-C IN A NEW RODENT MODEL FOR EXPERIMENTAL SEVERE MALARIA See poster section for abstract.
B325 NEURONAL TLR SIGNALING CONTROLS NEURONAL EXCITABILITY, NEUROINFLAMMATION, PAIN, AND ITCH See poster section for abstract.
SYMPOSIUM 37: NOVEL TARGETS AND/ OR NOVEL THERAPIES—SPONSORED BY CELGENE 117 DEVELOPING CAR T CELLS FOR CANCER Marcela Maus University of Pennsylvania, Philadelphia, PA, USA Chimeric antigen receptor (CAR) T cell immunotherapy has achieved unprecedented responses in hematologic B cell malignancies, and has been granted Breakthrough Therapy designation by the FDA. CAR T cells for solid tumors have shown great promise in pre-clinical models, and is in the early phases of clinical development. Despite having been developed in the academic setting, many T cell therapies are now entering an industry setting to be developed into commercial therapies to treat cancer. We will discuss the components of chimeric antigen receptors, the technologies used in making a T cell product, some of the factors considered to be important for efficacy, and recent results in hematologic and solid tumors.
118 ARRY-502: A POTENT, SELECTIVE CRTH2 ANTAGONIST FOR ASTHMA Laurence Burgess1, David Chantry1, Christine Eberhardt1, Robert Hopkins1, Michael Saunders1, Lisa Anderson1, Roger Aitchison1, Stacie Bell1, Kenji Izuhara2, Junya Ono3, Jeremy Cole4, James D. Wolfe5, Sheldon Spector6, Lawrence Sher7, Edward Kerwin8, Sally Wenzel9 1
Array BioPharma, Boulder, CO, USA; 2Saga Medical School, Saga, Japan; 3Shino Test Corp., Tokyo, Japan; 4IPS Research Co.,
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119 MECHANISMS OF DUAL INHIBITION OF TNF AND IL-17: TRANSLATION TO CLINICAL STUDIES WITH ABT-122 Carolyn Cuff, Melanie Ruzek, Robert Padley, Heikki Mansikka, Jeffrey Voss, Margaret Hugunin, Alexander Ivanov, Chung-Ming Hsieh AbbVie, North Chicago, IL, USA ABT-122 is an anti-TNF/IL-17 dual variable domain immunoglobulin (DVD-IgTM) currently in Phase II clinical trials for Rheumatoid Arthritis (RA) and Psoriatic Arthritis (PsA). This molecule is capable of simultaneously binding to both TNF and IL17. ABT-122 neutralized both TNF and IL-17 induced IL-6 production by fibroblast-like synoviocytes in vitro and similar results were demonstrated with the sera from ABT-122-dosed healthy volunteers. Single dose Phase I studies have shown acceptable safety and tolerability of ABT-122 that have enabled progression of clinical testing to Phase 2. In an effort to understand the mechanism of action of ABT-122 as well as identify potential biomarkers for clinical studies, gene arrays were evaluated in the collagen-induced arthritis mouse model, which demonstrated greater efficacy with blockade of TNF and IL-17 compared to blockade of either cytokine alone. This analysis revealed several genes and pathways
Inflamm. Res. affected only under conditions of dual cytokine neutralization, which identified multiple chemokines as potential soluble biomarkers that were robustly modulated. Consistent with modulation of chemokine pathways, we have also found certain chemokine receptors, CXCR4 and CXCR5, modulated on leukocyte subsets in healthy volunteers following a single dose of ABT-122. In addition, increased IL-10 and decreased GM-CSF responses were observed in PBMC with LPS stimulation ex vivo. As these chemokine receptors and cytokine responses have been suggested to play a role in disease pathology or its resolution, these data suggest that dual blockade of TNF and IL-17 by ABT-122 could provide a new therapeutic approach to patients with RA and immune mediated inflammatory diseases.
120 RESULTS OF A SINGLE DOSE ASCENDING FIRSTIN-MAN TRIAL OF A NOVEL BIOLOGIC, HUMAN STRESS PROTEIN RASOLVIR (BIP), IN RHEUMATOID ARTHRITIS (RA): THE RAGULO TRIAL Gabriel S. Panayi2,1, Bruce Kirkham1, Khaldoun Chaabo1, Christopher Hall2, Angela Vincent1, Joana Vasconcelos3, Toby Prevost3, Toby Garrood1, Valerie Corrigall2 1
Department of Rheumatology, Guys & St Thomas’s NHS Hospital Trust (GSTT), London UK; 2Academic Department of Rheumatology, Centre for Molecular and Cellular Biology of Inflammation, Kings College London (KCL), London, UK; 3Department of Primary Care and Public Health Sciences, KCL, London UK Background: RasolvirTM (BiP) is a human endoplasmic reticulumresident stress protein. In pre-clinical studies it has prolonged antiinflammatory properties by the induction of regulatory cells. A single intravenous infusion suppresses ongoing collagen-induced arthritis in the mouse; ameliorates arthritis and inhibits bone loss in the TNFa transgenic mouse; and suppresses inflammation in the RA synovial membrane/SCID transplantation model. It is thus a potential new therapy for RA acting by a novel mode of action. The objectives of this randomised placebo-controlled, dose ascending double blind Phase I/IIA trial of RasolvirTM were safety (Primary Objective) and efficacy as measured by the DAS28-ESR (Secondary Objective). Methods: 24 patients with active RA who had failed one or more DMARDs were sequentially assigned to three cohorts each of eight patients randomly allocated to receive placebo (two patients) or RasolvirTM (six patients). Patients received a single intravenous infusion over 1 h and were observed as inpatients overnight. No patients were re-treated. Clinical, rheumatological and laboratory assessments for safety and efficacy (DAS28-ESR) were carried out at stated intervals for 12 weeks. Findings: No infusion reactions or serious adverse reactions were noted. Adverse events were evenly distributed between placebo and RasolvirTM groups. There were no RasolvirTM-related toxicities. Haematological, renal and metabolic parameters remained within the normal range. No placebo patient and no patient who had received 1 mg Rasolvir TM went into remission. At 12 weeks, one patient who had received 5 mg and two patients who had received 15 mg RasolvirTM were in remission (DAS28 = \2.6). Interpretation: RasolvirTM is safe when administered up to a dose of 15 mg in patients with active RA. RasolvirTM induces remission after a single dose. It merits further study as a treatment for RA and other immuno-inflammatory diseases.
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SYMPOSIUM 38: CARDIOVASCULAR INFLAMMATION 121 MYELOID CELLS IN ISCHEMIC HEART DISEASE Matthias Nahrendorf Massachusetts General Hospital, Boston, MA, USA Monocytes and macrophages are innate immune cells that reside and accumulate in atherosclerotic lesions but also in the healthy and injured heart and brain. The cells and their subsets pursue distinct functions in steady state and disease, and their tenure may range between hours to months. Some subsets are highly inflammatory, while others support tissue repair. The talk discusses current concepts of cell supply by the hematopoietic system, lineage relationships and systems’ cross talk, highlights open questions, and describes imaging tools for studying monocytes, macrophages and their progenitors.
122 NETs FUELING DISEASE AND VICE VERSA Denisa D. Wagner Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA For many years, my lab has been studying the interplay between inflammation and thrombosis. These processes occur together, stimulate each other and share cellular and molecular components. The latest example of a common functional component is neutrophil extracellular traps (NETs). NETs are chromatin released together with toxic granular components from highly stimulated neutrophils. Originally found to trap/sequester invading pathogens, they were soon also seen to be part of sterile inflammatory and thrombotic processes. They interact with VWF, which is also involved in platelet and leukocyte recruitment and is crucial for venous thrombus development after inferior vena cava stenosis. This will be discussed together with the role of NETs and the enzyme that generates them (PAD4) in animal models of deep vein thrombosis and myocardial infarction. We observed that various cancers in mice prime neutrophils for NETosis. This causes cancer-associated thrombosis, and the production of NETs may affect tumor biology. Diabetes both in mice and man also promotes NETosis. NETs hinder wound healing and this is exacerbated in diabetes. Healing of skin wound in PAD4-/- mice is faster than in wild type and it is not affected by diabetes. Surprisingly deficiency of PAD4 does not make mice immunodeficient in a sepsis model of cecal ligation and puncture. Thus inhibition of PAD4 to prevent NETosis or NET destruction by DNase could be beneficial in diabetic wound healing and in many inflammatory and thrombotic conditions where NETs contribute to the pathology.
123 DEFECTIVE INFLAMMATION RESOLUTION IN ATHEROSCLEROSIS: MECHANISMS AND THERAPEUTIC OPPORTUNITIES Gabrielle Fredman1, Nazila Kamaly2, Omid Farokhzad2, Ira Tabas1 1 Columbia University Medical Center, New York, NY, USA; 2Harvard Medical School, Boston, MA, USA
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S100 The inflammatory response to acute and reversible infection or tissue damage, mediated by PAMPs and DAMPs, respectively, triggers an essential resolution response that curtails inflammation and restores tissue homeostasis. This resolution response is mediated by a panoply of lipid and protein mediators that activate specific cell surface receptors and signal transduction pathways to trigger resolution endpoints. However, when the inflammatory response is indolent and persistent, the resolution phase is often not engaged, leading to a chronic, low-grade inflammatory response that causes clinically serious tissue damage. We have proposed that the maladaptive inflammatory response in atherosclerosis is, in essence, a classic example of failed resolution. The initial inflammatory response is triggered by the subendothelial retention of apoB-containing lipoproteins, and resolution fails because this trigger is not only persistent but is actually amplified. This scenario leads to a vicious cycle of failed resolution, tissue damage-mediated DAMP formation, and amplified DAMP-mediated inflammation. This talk will review the features of atherosclerosis progression that are consistent with failed resolution; reveal new molecular-cellular mechanisms of how certain resolution mediators activate signaling pathways in macrophages, a key inflammatory cell type in atherosclerosis; and discuss and show data supporting the contention that inflammation resolution mediator therapy may be an ideal way to prevent atherosclerosis progression in a manner that would not compromise in host defense.
SYMPOSIUM 39: PREDICTIVE TOXICOLOGY
Inflamm. Res. Access BIO, Boyce, VA, USA The purpose of preclinical safety assessment is to understand the potential risks of a new drug or biologic in order to aid clinical decision-making. No drug or biologic is 100 % safe. A drug or biologic is less safe if it is used in a way that decreases foreseeable benefit, or increases the risk, or if the actual risks are greater than the predicted risks. In vitro and ex vivo studies using cells or tissues from patients have been used to screen for compounds, understand mechanism of action and establish proof of concept. Animal models of human disease are also commonly utilized to gain insight into the pathogenesis of disease and evaluate the efficacy but they are less frequently utilized in safety assessment. While animal models of human disease may not mimic all aspects of disease, understanding the limitation of the animal models together with understanding the human disease and the potential mechanisms of toxicity should allow for a better prediction of risk in the intended disease populations. Importantly, regulatory authorities are a becoming more willing to accept and even recommend data from experimental animal disease models that combine efficacy and safety.
126 TRANSLATIONAL SAFETY IN DRUG DEVELOPMENT: THE ROLE OF SAFETY BIOMARKERS IN PREDICTIVE TOXICOLOGY John Michael Sauer
124 TRANSLATIONAL IMPACT OF SECONDARY PHARMACOLOGY IN DRUG DISCOVERY Laszlo A. Urban Preclinical Safety Profiling (PSP) at the Novartis Institutes for Biomedical Research (NIBR), Cambridge, MA, USA The safety bar for medicines has been raised by regulatory authorities and became a determinant factor in successful product launch. Accordingly, the pharmaceutical industry introduced broader and earlier risk assessment for better understanding of molecular bases of toxic reactions which are often shared between drugs. Safety conscious product development requires parallel, integrated optimization of efficacy and safety. For early risk assessment a powerhouse of predictive in vitro and in silico tools have been developed which enable the inexpensive de-prioritization of chemical structures associated with ADRs, and prevents compounds with ‘‘toxic’’ off-target effects entering clinical trials. Learning from clinical experience led to the development of the concept of ‘‘reverse pharmacology’’ which further improved the predictive value of early risk assessment. These should result in diminished attrition, better side effect profiles, improved compliance and significant savings for pharmaceutical drugs. In this presentation, I will review the strategy of early safety risk assessment/mitigation of drug candidates based on identification of molecular targets, pathways associated with pathophysiological clinical consequences.
Critical Path Institute, San Diego, CA, USA In nonclinical safety studies applied during drug development, drug induced organ injury can be identified by histopathological changes. However, histopathological analysis is rarely an option in clinical drug development studies. Therefore, fluid based biomarkers are used to predict drug induced tissue injury in humans. Although many of these traditional safety biomarkers have acceptable predictive accuracy under controlled conditions, many also have deficits in their selectivity and/or sensitivity. Furthermore, many of these biomarkers have not undergone programmatic evaluation, but instead have been deemed acceptable based on clinical experience. The Predictive Safety Testing Consortium (PSTC) is a unique public private partnership that brings together pharmaceutical companies to share and validate each other’s safety testing methods in cooperation with the U.S. Food and Drug Administration (FDA), the European Medicines Agency (EMA), and the Japanese Pharmaceuticals and Medical Devices Agency (PMDA). The PSTC is currently involved in the rigorous evaluation of novel safety biomarkers for use in clinical trials with the ultimate goal of obtaining regulatory endorsement and qualification of these biomarkers. PSTC is also working towards a translational safety strategy for use in drug development that would integrate human biology-based in vitro models, novel safety biomarkers, computational tools, and non-safety data under the umbrella of systems toxicology. The objective of this presentation will be to describe the ongoing research across the PSTC working groups, and outline how these applied regulatory science outcomes fit into our vision of translational safety.
125 OPTIMIZING CLINICAL DEVELOPMENT: LEVERAGING PRECLINICAL DISEASE MODELS FOR ASSESSING SAFETY AND EFFICACY
127 LARGE MOLECULE PREDICTIVE SAFETY: EXPERIENCES AND POINTS TO CONSIDER
Joy Cavagnaro
Amgen, Thousand Oaks, CA, USA
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Michelle J. Horner
Inflamm. Res. Drug induced toxicities with biologic therapeutics (i.e., protein-based products) are generally a result of exaggerated pharmacology as these products are specific for a particular target and are seldom associated with off target toxicity. As a result, attrition of biotherapeutics due to safety issues has traditionally been uncommon, especially in comparison to that of small molecule therapeutics (i.e., traditional pharmaceuticals). However, innovation in therapeutic design has necessitated some changes in de-risking strategies and to reduce concerns of potential liabilities (e.g. immune reactions, infections, cytokine storm, immunogenicity). Industry is now challenged to provide diligence to discharge liabilities and adverse events that may impede progression of novel biologics through the course of drug development. Using past experiences as guidance, evaluation and application of new approaches may help to understand mechanistic processes involved in unintended effects in in vitro models and non-clinical species, and their relevance to humans. Implementation of these new approaches in the early stages of development for a biotherapeutic can minimize delays or terminations, and can also identify human hazards that cannot be efficiently screened in pre-clinical species. Overall, a comprehensive predictive safety strategy for novel biotherapeutics should include considerations for both target/unintended target- and modality-driven putative liabilities.
SYMPOSIUM 40: TRANSLATIONAL MEDICINE Abstracts not available at time of printing.
SYMPOSIUM 41: INNATE LYMPHOID CELLS 128 FUNCTION AND PLASTICITY OF HUMAN ILC SUBSETS Hergen Spits AMC University of Amsterdam, Amsterdam, The Netherlands Innate lymphoid cells are emerging as important regulators and effectors in innate immunity and tissue homeostasis. Three groups of ILC, ILC1, ILC2 and ILC3 have now been identified on the basis of cytokine production profiles, dependence on signature transcription factors and developmental pathways. ILC1 are defined by their dependence on Tbet and capacity to produce Interferon gamma but are less well defined than ILC2 that depend on GATA3 and produce IL-4, IL-5 and IL-13 and ILC3 that are RORgt dependent and produce IL-17 and/or IL-22. I will give an overview of our present knowledge of human ILC subsets and discuss data indicating that ILC highly plastic and that ILC subsets can shuttle between different functional states dependent on environmental cues. I will also discuss the functional interactions of ILC with other hematopoietic cells in particular between class II MHC expressing ILC3 and T cells
129 FUNCTIONS OF IL-33 AND GROUP 2 INNATE LYMPHOID CELLS IN CONTROLLING ALLERGIC AIRWAY INFLAMMATION AND ASTHMA Hirohito Kita Mayo Clinic, Rochester, MN, USA
S101 Innate lymphoid cells (ILCs) have emerged recently as an important component of the immune system and the cell type that regulates mucosal immune responses and tissue homeostasis. Group 2 ILCs (ILC2s) are a subset of ILCs, are resident in various tissues, and are characterized by their capacity to produce type 2 cytokines and tissue growth factors. These ILC2s play an important role in allergic immune response by linking the signals in the atmospheric environment to the immune system. Fungi are one of the major allergens associated with human asthma, and animal and in vitro models using the fungal allergens have provided significant information to understand the mechanisms of allergic disease. In mouse models of fungusinduced allergic airway inflammation, IL-33, IL-25, and TSLP are released by airway epithelial cells. Lung ILC2s that respond to these cytokines quickly produce a large quantity of type 2 cytokines, resulting in airway eosinophilia, mucus production, and airway hyperreactivity even in the absence of adaptive immune cells. Evidence also suggests that ILC2s interact with conventional immune cells, such as CD4+ T cells, and facilitate development of adaptive immune response and persistent airway inflammation. ILC2s are also present in peripheral blood and respiratory mucosa in humans. Further investigations into biology of ILC2s and their roles in the pathophysiology of allergic diseases will provide major conceptual advances in the field and may provide useful information toward development of new therapeutic strategy for patients.
130 LIPID MEDIATOR REGULATION OF ILC2 TH2 CYTOKINE RESPONSE David Broide University of California San Diego, San Diego, USA Th2 cytokines (IL-4, IL-5, IL-9, IL-13) play an important role in the pathogenesis of allergic inflammation and asthma. Although allergen activated antigen specific CD4+ T cells are a significant source of Th2 cytokines in asthma, recent studies suggest that innate lymphoid cell type 2 (ILC2) may also be a significant source of Th2 cytokines in allergy and asthma. In contrast to allergen activation of CD4+ T cells to express Th2 cytokines, ILC2 do not respond to allergen as they do not express antigen receptors and are activated by cytokines including IL-33, TSLP, and IL-25 to express IL-5 and IL-13 but not IL-4. In addition to these cytokines, lipid mediators such as prostaglandin D2 (PGD2) and leukotrienes are able to regulate ILC2 responses including chemotaxis and expression of Th2 cytokines. PGD2 binds to CRTH2 expressed on human ILC2 and induces their chemotaxis. As PGD2 is released from allergen/IgE activated mast cells, the initial PGD2 release within minutes from mast cells following exposure to allergen may play an important role in chemotaxis of ILC2 at sites of allergic inflammation. PGD2 also potentiates the production of IL-13 by peripheral blood ILC2, whereas lipoxin A4 reduces levels of IL-13 produced by ILC2. Levels of cysteinyl leukotrienes (LTC4, LTD4, and LTE4) are increased in asthma and chronic rhinosinusitis. Mouse lung ILC2 express the CysLT1receptor, which when activated mediates ILC2 calcium influx and Th2 cytokine production. Interestingly, in contrast to IL-33 which does not induce ILC2 to express IL-4, LTD4 induces ILC2 to express high levels of IL-4 as well as other Th2 cytokines such as IL-5 and IL-13. As IL-4 is an important switch factor for IgE synthesis, ILC2 activation by LTD4 may contribute to IgE production at sites of allergic inflammation. In addition, leukotriene induced ILC2 production of IL-4 may play an important role in differentiation of CD4+ cells to Th2 cells to amplify Th2 responses. In an innate mouse model of asthma induced by the mold Alternaria (epidemiologically associated with severe human asthma),
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LTD4 enhances ILC2 proliferation and eosinophilia. Thus, overall lipid mediators such as PGD2 and leukotrienes are highly expressed at sites of allergic inflammation including the lower airway in asthma. In addition to the effect of lipid mediators on structural cells such as smooth muscle to induce bronchial constriction in asthma, lipid mediators such as PGD2 and leukotrienes may also serve an important role in chemotaxis and activation of ILC2 to express Th2 cytokines including IL-4 (in response to LTD4) which may amplify IgE and Th2 responses.
131 PATHWAYS THAT GOVERN ILC2S FUNCTION AND HOMEOSTASIS Omid Akbari University of Southern California, Los Angeles, CA, USA Type 2 Innate lymphoid cells (ILC2s) are a newly identified subset of immune cells that play important roles in the pathogenesis of allergic asthma by producing large amounts of IL-5 and IL-13. ILC2 lack lineage markers but express CD45, IL-2R, IL-33R and IL-7R. We will discuss few recently identified pathways that modulate ILC2 function and homestasis. All murine ILC2s and a significant portion of human ILC2s express Inducible T-cell COStimulator (ICOS) that is essential for T cell activation and function, however, the role of ICOS in ILC2s remains unknown. We investigated the role of ICOS in the function and survival of murine and human ILC2s using ICOS-/- and RAG2-/- and humanized mice in different experimental setups. We found that ICOS-/- mice show lower IL-33-induced airway hyperreactivity (AHR) and eosinophilia than wild type (WT) mice. Lack of ICOS decreased survival and impaired the production of IL-5 and IL-13 by ILC2s. We also for the first time introduced ILC2 humanized mice and showed that blocking ICOS:ICOS-Ligand interactions impair the production of IL-5 and IL-13, reduced IL-33 induced AHR and eosinophilia. Finally our data suggest that both human and murine ILC2s express ICOS-Ligand and cis or trans interactions of ICOS/ICOS-L, provide STAT5 dependant survival for the cells. These results indicate that ICOS is required for the ILC2mediated AHR and thus, therapeutic manipulation of the ICOS pathway is an attractive target for future strategies of treatment and prevention of ILC2 dependent asthma.
POSTERS Angiogenesis B001 SERUM CIRCULATING ANGIOGENESIS INHIBITOR ANGIOSTATIN LEVELS IN PATIENTS WITH FAMILIAL MEDITERRANEAN FEVER Goksal Keskin1, Ali Inal2, Lale Ozısık3, Mehmet Yildiz3 1
Medical School of Ankara, Department of Immunology, Ankara, Turkey; 2Baskent University, Department of Immunology, Ankara, Turkey; 3DYBEAH, Department of Internal Medicine, Turkey Background: Familial Mediterranean Fever (FMF) is an autosomal recessive genetic disorder characterized by recurrent episodes of
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febrile serositis, peritonitis, arthritis and pleuritis. Many studies have been performed as an attempt to understand the basis of the inflammatory attacts in FMF. During the acute attacks, elevations in acute phase reactant levels and increased several proinflammatory cytokines and anti-inflammatory peptides have been described. Angiostatin is a physiologic angiostatic factor derived from the proteolytic cleavage of plasminogen. Angiostatin is also known as a potent antiangiogenic mediator that can be found in increased levels in the patients during various states of inflammation. It has been reported that angiostatin directly inhibite neutrophil migration and neutrophil mediated angiogenesis and also might inhibit inflammation. The purpose of this study was to determine serum levels of angiostatin in patients with FMF Material and method: In this study, 58 patients with FMF (38 female, 20 male, mean age 21.3 ± 4.1 years, mean disease duration 5.6 ± 2.8 years) and 22 healthy controls (14 female, 8 male, mean age 20.8 ± 2.9 years) were included. Serum angiostatin levels were measured by ELISA. Results: The mean serum angiostatin levels were 188.4 ± 33.7 ng/ml in patients with FMF and55.6 ± 19.5 ng/ml in the healthy controls. The mean levels of serum angiostatin were 274.9 ± 36.1 ng/ml in active stage and 104.4 ± 30.5 ng/ml in inactive stage. According to these results; serum angiostatin levels were significantly higher in patients with FMF compared with healthy controls (p \ 0.001). In addition, serum angiostatin levels were significantly higher in active stage compared to in inactive stages (p \ 0.001). In the inactive patients with FMF, serum angiostatin concentrations were found to be higher compared to healthy controls s (p \ 0.01). In active patients, the mean ESR was 56.4 ± 3.7 mm/h, the mean serum CRP level was 38.4 ± 3.9 mg/L and the mean serum fibrinogen level was 504.2 ± 96.1 mg/dl. In active FMF patients, the mean serum angiostatin level was correlated with the mean serum fibrinogen level (r = 0.592 p = 0.012), serum CRP level (r = 0.534, p = 0.022) and ESR (r = 0.522, p = 0.028). Conclusion: The high levels of serum angiostatin, in active and inactive patients with FMF suggest that angiostatin may play a significant role of in the pathogenesis of FMF.
B002 SYNCHROTRON MICROBEAM IRRADIATION INDUCES INFLAMMATION AND SELECTIVE VASCULAR DAMAGES IN ADULT ZEBRAFISH Daniel Bro¨nnimann1, Audrey Bouchet1, Werner Graber1, Jean A. Laissue1, Valentin Djonov1, Raphael Serduc2, Elke Bra¨uer2 1
University of Bern, Institute of Anatomy, Bern, Switzerland; European Synchrotron Radiation Facility, Grenoble, France
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Background: Microbeam radiation therapy (MRT) is a new promising tumor treatment strategy used at preclinical stage. MRT is based on the spatial fractionation into arrays of parallel microbeams, which are typically separated by few hundred micrometers. We have used the zebrafish caudal fin model to study the effects of synchrotron based microbeam irradiation on the mature and immature vasculature in vivo. Method: We used transgenic fli1:eGFP zebrafish to visualize endothelial cells in vivo. The ventral part of the caudal fin was partially amputated to trigger regeneration and outgrowth of immature blood vessels. At 6 days post amputation, the caudal fin was irradiated with three parallel microbeams of 50 lm widths and 400 lm spacing. Results: Three hours after the irradiation, the immature vasculature was characterized by severe blood flow disturbances and fragmented endothelial cells in vivo. By the use of correlative microscopy
Inflamm. Res. (in vivo and electron microscopy), we revealed the presence of vacuolated endothelial cells and a tremendous inflammatory response inside the beam path. At 6 h post irradiation, the blood vessels were characterized by the presence of activated macrophages phagocytizing damaged endothelial cells. At this time point the capillary plexus adopted a striated metronomic pattern, with alternating destroyed and intact zones, corresponding to the beam paths. At later time points, activated macrophages were as well found in the loose connective tissue surrounding the vasculature. In contrast, the mature vasculature remained intact in terms of blood perfusion, endothelial cell coverage and inflammatory response. Conclusions: Vascular toxicity and physiological effects of microbeam irradiation depend on the stage of capillary maturation and appear in the first hours after irradiation. Newly formed, immature capillaries are highly sensitive to microbeam irradiation, whereas mature vessels are barely affected. The selective vascular damage could serve in the future to create new and promising anti-angiogenic treatment strategies.
B003 SUPPRESSIVE EFFECTS OF SOLUBLE VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR-2 ON LYMPHOGENIC METASTASIS Shotaro Maehana1, Rimika Imai2, Rei Murakami2, Masaki Nakamura2, Fumihiro Ogawa3, Fumiaki Kojima4, Masataka Majima3, Hidero Kitasato2 1
Department of Environmental Microbiology, Kitasato University Graduate School of Medical Sciences, Minato, Japan; 2Department of Microbiology, Kitasato University School of Allied Health Sciences, Minato, Japan; 3Department of Pharmacology, Kitasato University School of Medicine, Minato, Japan; 4Department of Pharmacology, Kitasato University School of Allied Health Sciences, Minato, Japan Objective: Vascular endothelial growth factor (VEGF) family is known to key factors of the vascular and lymph vessel formation. Among VEGF families, VEGF-C is one of most important factor for lymphangiogenesis via binding with those receptors VEGF-R2 and -R3. At the same time, soluble VEGFR-2 (sVEGFR-2) is known to have a potential activity for corneal alymphaticity in association with binding with VEGF-C and selectively inhibit lymphangiogenesis but not angiogenesis. In this study, we transduced sVEGFR-2 into lung cancer cells and evaluated the effects on cancer progression including suppressor of genes regulating for lymph vessel formation and metastasis in vivo. Methods: VEGF-C and sVEGF-R2 genes were individually transduced into Lewis lung carcinoma cells (LLC) using defective retrovirus vector, then designed as LLC/VEGF-C and LLC/sVEGFR2. The secreted proteins in supernatant of cultured these cells were detected by western blotting using specific antibodies (rabbit polyclonal anti-mouse VEGFR-2 and mouse polyclonal anti-mouse VEGF-C), respectively. To examine the lymphangiogenesis in primary lesion of tumor in vivo, LLC/sVEGFR-2 and LLC/VEGF-C cells were subcutaneously injected to C57/BL6 mice, respectively. After 14 days of the injection, immunohistochemical analysis was carried out by using antibody against lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), a lymphatic vessel marker. And then, the mRNA expression of VEGFR-2, VEGFR-3 and matrix metalloproteinase 9 (MMP-9) were also determined by real-time PCR. In addition, LLC/ sVEGFR-2 and LLC/VEGF-C were directly inoculated into left lung of C57/BL6 mice, respectively, in order to determine the number of the micro-metastatic colonies in lung lymph node. Results: sVEGFR-2 and VEGF-C proteins were detected in cultured supernatant of LLC/sVEGFR-2 and LLC/VEGF-C cells, respectively,
S103 in vitro. The number of LYVE-1 positive lymphatic vessels were reduced in LLC/sVEGFR-2 inoculated on comparison with control group in vivo. In addition, the expression of VEGFR-2, VEGFR-3 and MMP-9 genes were also suppressed in primary lesion of LLC/ sVEGFR-2 group, compared to that in control group. Furthermore, the number of micro-metastatic colonies in lymph node were reduced in LLC/sVEGFR-2 group inoculated mice in comparison with those control mice, when these cells directly inoculated in lung. Conclusion: The present study indicated that intervention of sVEGFR-2 suppressed lymphangiogenesis in primary lesion and lymphogenic metastasis of lung tumor through depriving VEGF-C, followed by down regulation VEGFR-2, VEGFR-3 and MMP-9. sVEGFR-2 might be one of the prominent target for the treatment of cancer by regulating lymphangiogenesis and lymphogenic metastasis.
B004 MLN4924 INHIBITION OF CULLIN-RING LIGASES ACTIVATES A HYPOXIC RESPONSE AND SECRETION OF ANGIOGENESIS-PROMOTING CYTOKINES IN HUMAN MCF-7 BREAST CANCER CELLS David Finkel, James Rivard, Amy James, Erin Eliria, Neal Lee, Kathy Brumbaugh, Greta Wegner R&D Systems Inc, Minneapolis, MN, USA Hypoxia-inducible factors (HIFs) are the key transcription factors that mediate the cellular response to low oxygen tension. During normoxia, HIF activity is tightly controlled by its oxygen-dependent prolyl hydroxylation which allows polyubiquitination by von HippelLandau tumor suppressor (VHL), leading to HIF degradation by the ubiquitin–proteasome pathway. During hypoxia, low oxygen tension prevents HIF prolyl hydroxylation resulting in HIF accumulation and subsequent transcription of HIF target genes. VHL is a known CullinRING E3 ubiquitin ligase (CRL) that is dependent on Neddylation of its Cullin-2 subunit by NEDD8 Activating Enzyme (NAE) for enzymatic activity. NEDD8, NEural precursor cell expressed Developmentally Down-regulated-8, is an ubiquitin-like molecule that is attached to its substrates in a manner similar to ubiquitin. The small molecule inhibitor MLN4924 inhibits NAE Neddylation of Cullins by covalently binding the nucleotide-binding site of NAE. HIF-1a is a well-known CRL substrate that increases in abundance upon MLN4924 treatment. This report presents evidence that MLN4924 activates a hypoxic (1 % O2, 48 h) response in MCF-7 human breast cancer cells with secretion of cytokines known to promote angiogenesis and inflammation. Cell lysates tested using the Human Oncology Antibody Array and Western blot showed that both hypoxia and MLN4924 induced accumulation of HIF-1a protein, with higher protein levels in MLN4924 treated cells. This correlated with the expected attenuation of HIF-1a ubiquitination during hypoxia and MLN4924 treatment, as detected by the Human Ubiquitin Antibody Array. Western blots showed that MLN4924 specifically inhibited the Neddylation of Cullin-2 and Cullin-1. Western blots also revealed that MLN4924 indirectly inhibited mTORC1 complex-mediated Thr389 phosphorylation of p70 S6K, known to induce autophagy. Analysis of cell culture supernates from normoxia, hypoxia, and MLN4924treated cells using the Human XL Cytokine Antibody Array revealed protein expression of several known hypoxia-activated targets. The relative levels of Angiogenin, MIF, PDGF-AA, and VEGF were found to be highest in MLN4924 treated cells. Subsequent ELISA analysis confirmed these array data. The observations reported here are consistent with previous reports showing the mechanism of action
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Apoptosis B005 L-AMINOACID OXIDASE, THE MAJOR COMPONENT OF B LEUCURUS VENOM, INDUCES DIRECT NEPHROTOXICITY THROUGH ROS, MITOCHONDRIAL DYSFUNCTION AND CASPASES PATHWAY Isabel Cristina Oliveira de Morais1, Gustavo Jose´ da Silva Pereira2, Mar Orza´ez3, Marcos Hikari Toyama4, Soraya Soubhi Smaili2, Enrique Pe´rez Paya´3, Alice Maria Costa Martins5
Inflamm. Res. the endoplasmic reticulum, reactive oxygen species (ROS) generation, mitochondria dysfunction with enhanced expression of Bax protein levels, caspase-3 and caspase-7 activation, suggesting that LAAO-Bl causes nephrotoxicity by acting in multiple cell death pathways. Taken together our results suggest that LAAO-Bl is responsible for the nephrotoxicity observed in the envenomation by snakebites.
B006 BOTHROPOIDES PAULOENSIS VENOM CYTOTOXICITY IN RENAL TUBULAR EPITHELIA CELLS: CELL DEATH SIGNALING PATHWAYS Aline Diogo Marinho, Isabel Cristina Oliveira Morais, Roberta Jeane bezerra Jorge, Ramon roseo Paula Pessoa bezerra Menezes, Joao alison moraes Silveira, Danya Bandeira Lima, Alice Maria Costa Martins, Helena Serra Azul Monteiro
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Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of Ceara´, Fortaleza, Ceara´, Brazil; 2Department of Pharmacology, Federal University of Sa˜o Paulo (UNIFESP), Brazil; 3Department of Medicinal Chemistry, Centro de Investigacio´n Prı´ncipe Felipe, Valencia, Spain; 4Sa˜o Vicente Unit, Paulista Coastal Campus, Sa˜o Paulo State University (UNESP), Sa˜o Paulo, Brazil; 5 Department of Clinical and Toxicological Analysis, Federal University of Ceara´, Fortaleza, Ceara´, Brazil The pit viper Bothrops leucurus (White-tailed-jararaca) is a poisonous snake habituating area in the northeast of Brazil. The biological effects due envenomation have similar profile than those observed with other Bothrops, such as coagulant activity, hemorrhagic, fibrinolytic, and acute renal failure (ARF). ARF is a common complication caused by Bothrops snakebite with relevant morbidity and mortality. Pathogenesis of ARF in snakebite envenomation may be related to hypovolemia and hypoperfusion secondary to cardiovascular disturbances, deposit of fibrin in the glomerular capillaries leading thrombotic microangiopathy and high venom concentration at the renal tissue, direct venom action on the tubular cells and oxidative stress. Recently, we observed that Bothrops leucurus venom induces nephrotoxicity in the isolated perfused kidney of rats associated with cytotoxicity against renal tubular epithelia cells. In this study, it was evaluated the direct nefrotoxicity of a main component of B. leucurus venom called L-aminoacid oxidase (LAAO-Bl) by using tubular epithelial cell lines MDCK and HK-2. In these cells treated with LAAO-Bl, 1.56–100 lg/mL for 12 h, there was a decrease in their viability in a concentration-dependent manner. We next evaluated if necrosis was implicated in the cellular viability decrease observed by analyzing lactate dehydrogenase (LDH) release. In MDCK cells LDH release was not observed after 12 h of LAAO-Bl exposure while LAAO-Bl induced an apparent membrane rupture in HK-2 cells at the highest concentrations studied. Annexin V/PI staining was applied to detect apoptotic/necrotic cells after LAAO-Bl treatment. In MDCK cells, LAAO-Bl significantly increased the percentage of early apoptotic (Annexin-V+, PI-), necrotic (Annexin-V-, PI+) and secondary necrotic cells (Annexin-V+ , PI+) when compared with control untreated cells. In HK-2 cells, in accordance with data obtained in the LDH-release assay, the Annexin-V-PI loading cell analysis demonstrated an increase in necrotic (PI+ cells) and secondary necrotic cells (Annexin-V+ , PI+) in a concentration-dependent manner. MDCK and HK-2 apoptosis induction was accompanied with Ca2+ release from
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Federal University of Ceara´, Fortaleza, Brazil Snakebite envenomation constitutes a serious medical condition common in tropical countries. In Brazil, snakes of the genus Bothrops are the main cause of venomous snake accidents. Acute renal failure is a common complication caused by Bothrops snakebite with relevant morbidity and mortality. The aim of the present study was to investigate the effects of the Bothropoides pauloensis venom (BpV) in cultured renal tubular cells of the type Epithelial Madin–Darby Canine Kidney (MDCK). The determination of the cytotoxic potential was conducted by MTT reduction assay. Lactate dehydrogenase (LDH) activity was determined in culture supernatants from experimental groups for investigation of cell lysis induced by BpV. The mechanism of cell death induced by BpV was assessed by flow cytometry, by staining with propidium iodide (PI) and annexin V-FITC. To verify the BpV effects on mitochondrial transmembrane potential (DWm) cells were stained with the mitochondrial specific probe, tetramethylrhodamineethyl ester (TMRE) and analyzed by flow cytometry. Cytosolic ROS was measured using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) and analyzed by flow cytometry. The expressions of Caspase-3 and -7 were analyzed by Western blotting. Treatment with BpV caused decrease in cell viability until 6.25 lg/mL concentration with an IC50 of 7.5 lg/mL. The results indicated an apparent membrane rupture in MDCK cells at the highest concentrations studied [7.5 lg/mL (25.71 + 4.66); 15 lg/mL (39.10 + 13.01); 25 lg/mL (49.36 + 14.67); 50 lg/mL (65.62 + 6.28), control negative (0.0001 + 0.001); control positive (100 + 0.001)], showing a increasing of LDH release. Annexin-PI loading cell analysis demonstrated an increase mainly in AX+/PIcells at the concentrations of 7.5 lg/mL, as well as AX+/PI+ cells in both concentrations (15 and 7.5 lg/mL), suggesting the participation of apoptosis and late apoptosis on BpV-induced cell death. BpV 7.5 lg/mL (IC50) produced ROS overproduction and mitochondrial membrane potential decrease. The BpV (15 and 25 lg/mL) treatment induced activation of caspase 3 and caspase 7 [7.5 lg/mL (1.08 + 0.06); 15 lg/mL (3.04 + 0.12); 25 lg/mL (4.61 + 0.53); 50 lg/mL (5.17 + 0.36); control negative (0.87 + 0.24)]. In summary, we have reported that BpV induced MDCK death, which involves an increase in ROS production and mitochondrial dysfunction, with participation of apoptosis and late apoptosis mechanisms.
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B007 HYPERGLYCEMIA FAVORS TH17 DIFFERENTIATION AFTER PHAGOCYTOSIS OF MRSA-INFECTED APOPTOTIC CELLS Naiara N. Dejani1,2,3, Stephanie Brandt3, Nicole L. Glosson-Byers3, Soujuan Wang3, Alexandra I. Medeiros1,2, C. Henrique Serezani3 1
University of Sa˜o Paulo, USP, Ribeira˜o Preto, SP, Brazil; Department of Biological Sciences, School of Pharmaceutical Sciences, UNESP, Universidade Estadual Paulista, Araraquara, SP, Brazil; 3Indiana University School of Medicine, Indianapolis, IN, USA 2
Clearance of infected apoptotic cells is an essential component of the host defense. Efferocytosis containing microbial products triggers the production of cytokines crucial to T cells differentiation. Staphylococcus aureus infection leads to neutrophil abscess formation with increased necrosis and apoptosis. In addition, S. aureus is frequently isolated in the skin lesions of diabetic patients with chronic uncontrolled hyperglycemia. It has been shown that IL-17 production in the skin is crucial for neutrophil migration and bacterial clearance. Therefore, we hypothesized that during active S. aureus infection in diabetics, the clearance of infected apoptotic cells drives dendritic cells-mediated Th17 generation, favoring overwhelming inflammatory response and increased lesion development. Bone marrowderived dendritic cells (BMDCs) from C57BL/6 mice were differentiated in the presence of GMCSF with high glucose (25 mM) or low glucose (5 mM). After differentiation, BMDCs were co-culture with MRSA-infected apoptotic neutrophil cell line (HL-60) for 18 h and the supernatants were collected to detect cytokines by ELISA and to evaluate the differentiation of naı¨ve CD4+ T cells. Our results show that high glucose does not influence BMDC efferocytosis (*40 % in both low and high glucose). The abundance of Th17-driven cytokines TGF-ß, IL-1ß and IL-6 were slightly enhanced in high glucose. When naı¨ve CD4+ T cells were differentiated in the presence of supernatants from co-culture of DC with MRSA-infected apoptotic cells, the percentage of IL-17A-producing lymphocytes was 2-folder higher in high glucose. Indeed, we isolated naı¨ve CD4+ T cells from streptozotocin-induced diabetic mice and observed increased Th17 differentiation using supernatants from co-culture or Th17-polarizing conditions. These data suggest that hyperglycemia favors Th17 responses and may increase inflammation during recognition of MRSA-infected apoptotic cells by DCs. Besides, overwhelming IL17A production may contribute to neutrophil recruitment, abscess formation and pathology in skin lesions in diabetics. Financial support: FAPEP 14/17374-3, 12/23580-0, 11/17611-7; NIH HL-103777; R01HL124159-01; T32AI060519; T32DK007519.
B008 MPO-DERIVED CHLORINATED LIPIDS INDUCE ENDOPLASMIC RETICULUM STRESS IN BRAIN MICROVASCULAR ENDOTHELIAL CELLS 1
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Nora Kogelnik , Christoph Nusshold , Christine Rossmann , Gerald Rechberger2, Helga Reicher1, Pierre-Oliver Courad3, Ernst Malle1, Wolfgang Sattler1 Medical University of Graz, Graz, Austria; 2University of Graz, Graz, Austria; 3University Paris Descartes, Paris, France 1
Most regions of the brain operate within a well-controlled environment separated from the milieu of the peripheral circulation by the
neurovascular unit where brain microvascular endothelial cells (BMVEC) form the morphological basis of the blood–brain barrier (BBB). Under inflammatory conditions peripheral leukocytes can exacerbate neurovascular dysfunction by the release of cytotoxic mediators that induce BBB breakdown. One of these mediators is hypochlorous acid (HOCl), which is formed via the myeloperoxidase (MPO)/H2O2/chloride system of activated leukocytes. In vitro treatment of BMVEC with the MPO/H2O2/chloride system or activated neutrophils results in oxidative modification of the endogenous cellular ether-phospholipid (plasmalogen) pool. This reaction generates chlorinated fatty aldehydes (including 2-chlorohexadecanal; 2-ClHDA) and the corresponding remnant lysophospholipid. During the present study we aimed to explore the hypothesis that 2-ClHDA and its metabolite 2-chlorohexadecanoic acid (2-ClHA) induce sustained and unresolved activation of the endoplasmic reticulum (ER) stress response ultimately culminating in barrier dysfunction and BMVEC apoptosis. Using the human BMVEC hCMEC/D3 line as in vitro BBB model we investigated whether 2-ClHDA can initiate the unfolded protein response (UPR)-mediated signal transduction pathway during ER stress. Using Western blot analysis we analysed whether 2-ClHDA and its redox metabolite 2-chlorohexadecanoic acid (2-ClHA) can initiate the UPR via PERK mediated signalling. Treatment of hCMEC/D3 cells with 2-ClHDA and 2-ClHA induced phosphorylation of eukaryotic elongation factor 2a (eIF2-a) and elevated protein expression of activating transcription factor 4 (ATF4) and representing crucial events in the PKR-ER like kinase (PERK) -mediated UPR pathway activation. Furthermore 2-ClHDA and 2-ClHA stimulation induced the expression of pro-apoptotic transcription factor C/EBP homologous protein (CHOP) and mediated caspase-3 cleavage, demonstrating 2-ClHDA and 2-ClHA induced apoptosis in hCMEC/D3. To further examine ER function in 2-ClHDA/2-ClHA stimulated cells we investigated lipid composition in these cells as many of the enzymes involved in lipid homeostasis reside in the ER. These analyses revealed decreased cellular concentrations of C16 and C18 triglyceride and cholesterylester species. In addition, 2-ClHDA treatment resulted potently increased the concentration of several ceramide species, findings accompanied by a concomitant decrease in the corresponding sphingomyelin classes. In conclusion the current data provide evidence that a-chloro fatty aldehydes and a-chloro fatty acids that are generated under inflammatory conditions in vivo are able to induce BBB dysfunction via induction of ER stress by the PERK-mediated signalling cascade.
Autoimmune Diseases: RA, Psoriasis, SLE, IBD, MS B010 THE TYROSINE KINASE INHIBITOR PREVENTS CYTOKINE INDUCTION IN THE PROGRESSION OF ADJUVANT INDUCED ARTHRITIS Sheikh Fayaz Ahmad, Mushtaq Ahmad Ansari, Khairy M A Zoheir, Saleh A. Bakheet, Sabry M. Attia King Saud University, Riyadh, Saudi Arabia Rheumatoid arthritis (RA) is one of the major autoimmune diseases of global prevalence. Irrespective of much research in RA disease, no drugs with capable safety profiles are yet available. Protein tyrosine kinases help to regulate the expression of many genes that play important roles in arthritis. We investigated the possible antiarthritic effects of the tyrosine kinase inhibitor in a mouse model of
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S106 adjuvant induced arthritis (AIA). We report here that tyrosine kinase inhibitor exerts potent anti-arthritic effects in animal model of AIA in vivo. In this study, we examined the effects of tyrosine kinase inhibitor on the key mediators of arthritic inflammation. Tyrosine kinase inhibitor treatment significantly attenuated the severity of AIA, reduced the arthritis scores, a substantial reduction in the levels of cell surface receptors, chemokines, and as well as the proinflammatory mediators. However, tyrosine kinase inhibitor significantly up-regulated the number of anti-inflammatory mediators levels. Our results suggest that treatment with tyrosine kinase inhibitor attenuated AIA in mice might offer a promising alternative/adjunct treatment for RA.
B011 ANTI-INFLAMMATORY EFFECTS OF TRIPEPTIDE WOL074-019 IN MURINE MODELS OF PSORIASIS AND COLITIS Michael Soeberdt1, Carlo Sternemann2, Christoph Abels1, Thomas A. Luger2, Karin Loser2 1
Dr. August Wolff GmbH & Co. KG Arzneimittel, Bielefeld, Germany; Department of Dermatology, University of Mu¨nster, Mu¨nster, Germany
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KdPT is a tripeptide with broad anti-inflammatory activity. It was shown to be effective in different murine models of intestinal inflammation and psoriasis. Oral KdPT proved to be safe and well tolerated in a single and two multiple ascending dose clinical studies. Moreover, in a phase II trial it was shown to significantly accelerate disease remission in patients with mild to moderate active ulcerative colitis. However, KdPT’s phys.-chem. properties limit its oral bioavailability and are not suitable for development of a topical formulation. Thus, we have designed and synthesized numerous analogues of KdPT with optimized phys.-chem. properties. Effects determined on protein and gene level in vitro (murine and human T cells, HaCaT) showed anti-inflammatory activity of WOL074-019 (19) comparable to KdPT and thus 19 was selected for in vivo studies. The anti-inflammatory and immunomodulatory potential of 19 in vivo was investigated in the mouse model of imiquimod-induced psoriasis-like skin inflammation (topical application for 8 days). At d 5 and 6 after the start of treatment mice were injected with either PBS, betamethasone dipropionate (BMDP), KdPT or 19 (5 lg, i.v.). Interestingly, similar to KdPT, treatment with 19 ameliorated ongoing skin inflammation as shown by the reduced thickness of epidermal rete ridges and the decreased levels of pathogenic Th1 as well as Th17 cells in regional lymph nodes and lesional skin quantified by flow cytometry, real-time PCR and immunofluorescence staining. This effect was mediated by the reduction of pro-inflammatory cytokines like IL-1b, IL-6 or TNF-a and the expansion of immunosuppressive Treg in 19-treated mice versus controls. In addition, 19 was tested in the dextrane sodium sulphate (DSS)-induced colitis model in mice. Mice that were injected with 19 (5 lg, i.p.) at d 4–7 were protected from weight loss and moreover, did not show any signs of diarrhea or rectal bleeding. At day 8 (end of experiment) 19-treated mice looked like BMDP-treated controls. Quantitative real-time PCR as well as immunofluorescence staining of colonic tissue revealed decreased levels of IL-6, IL-1b, TNF-a and IFN-c. Moreover, the numbers of neutrophils and macrophages were significantly reduced in mesenteric lymph nodes and the colon from 19-treated mice versus PBS-injected controls.
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Inflamm. Res. In summary, these data show that 19, similar to the original tripeptide KdPT, is able to effectively ameliorate ongoing inflammation in skin and gut. Because of its improved phys.-chem. properties 19 may be formulated as topical drug. Moreover, its oral bioavailability is likely to be improved compared to KdPT.
B012 CYTOKINE PRODUCTION AND CALCIUM INFLUX OF T LYMPHOCYTES UPON KV1.3 AND IKCA1 CHANNEL INHIBITION IN RHEUMATOID ARTHRITIS AND ANKYLOSING SPONDYLITIS Gergely Toldi1, Luis Munoz2, Martin Herrmann2, Georg Schett2, Attila Balog3 1 Semmelweis University, Budapest, Hungary; 2University of Erlangen-Nu¨rnberg, Erlangen, Germany; 3University of Szeged, Szeged, Hungary
Voltage-sensitive Kv1.3 and calcium-dependent IKCa1 lymphocyte potassium channels have been implicated as important targets of selective immunomodulation in autoimmune disorders. The transient increase of the cytoplasmic free calcium level, which is a prerequisite of the downstream events of lymphocyte activation, is maintained by the function of potassium channels. They conserve the electrochemical potential gradient via the efflux of potassium from cytoplasm. The relationship between the influx of calcium through the cell membrane and the efflux of potassium makes proliferation and activation of lymphocytes sensitive to pharmacological inhibition of Kv1.3 and IKCa1 channels, and provides a potential opportunity for targeted intervention. We compared the alterations in cytokine production (IL-1b, IL1RA, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IL-17A/F, IFN-g, TNF-a) upon selective inhibition of Kv1.3 or IKCa1 channels (by MGTX and TRAM, respectively) in 8 healthy donors (HD), 15 rheumatoid arthritis (RA) and 10 ankylosing spondylitis (AS) patients. We also determined calcium influx kinetics and its sensitivity to Kv1.3 and IKCa1 channel inhibition following PHA activation in Th1, Th2 and CD8 cells. The application of TRAM resulted in a lower production of TNF-a and IL1-RA in all three study groups. Inhibition by TRAM had contrary effects on the production of IL-1b and IL-5: while their production was increased by PBMCs of RA patients, this effect was not observed in HD and AS PBMCs. While treatment with MGTX resulted in a similar decrease of calcium influx in the CD4 and Th2 subsets across all study groups, TRAM treatment had opposite effects on RA and HD samples: it decreased calcium influx in the Th2 and CD8 subsets in RA, while only Th1 cells were affected in HDs. PBMCs isolated from RA and AS patients react with a different pattern of alterations in cytokine production and calcium influx kinetics compared to HDs. The application of lymphocyte potassium channel inhibitors has controversial effects on cytokine production and calcium influx patterns in patients with RA and AS, questioning its therapeutic relevance. TRAM decreases the secretion of TNF-a and increases the secretion of the Th2 cytokine IL-5 in patients with RA. This would contribute to the shift of the immune balance towards less tissue damage and cellular paucity in RA. Nevertheless, TRAM increases the secretion of IL-1b and at the same time decreases that of IL1-RA which is unfavorable, since it supports an IL-1b mediated pro-inflammatory response. Additionally, TRAM
Inflamm. Res. also inhibits the short-term activation of Th2 lymphocytes in RA, leaving that of Th1 cells unaffected, probably further contributing to a shift of the inflammatory balance to a pro-inflammatory profile. The differences observed in cytokine response upon inhibition of Kv1.3 and IKCa1 channels support the differential pathomechanisms of RA and AS.
B013 SUPPRESSING INFLAMMATORY BOWEL DISEASE BY MODULATING PROINFLAMMATORY GENE EXPRESSION AND IMMUNE CELL POPULATIONS Anh T. Do, Praveer Gupta, Abishek Iyer, David P. Fairlie University of Queensland, St Lucia, QLD, Australia Inflammatory bowel diseases (IBDs) are a group of complex inflammatory conditions affecting colon and rectum. Different subsets of T-lymphocytes (Th1, Th2, Th17 and Treg) have been implicated in inflammatory bowel diseases (IBDs), with various inflammatory cytokines and chemokines (IL-1, IL-6 and TNFa) involved in the disease pathogenesis. Anti-TNF therapy (e.g. Infliximab, Enteracep) is currently used as last resort treatment for refractory IBD patients. However, these drugs are expensive, not effective in everyone and have inherent serious side-effects. To treat IBD more effectively, we must first understand the nature of immune responses occuring from the acute stages through progression to chronic disease when inflammation can trigger tumor formation. Using RT-PCR arrays and flow cytometry techniques we have serially characterized inflammatory changes in the progression of early to late stage of IBD and tumorigenesis in a dextran sulfate sodium (DSS)-induced IBD model in mice. As disease progressed, cytokine expression pattern with more involvement of lymphocyte cytokines, and progressively reduced monocyte cytokines involvement. At the most chronic stage, 20 % of animals developed dysplastic lesions, associated with increases in Il13, Il10 and Il4 mRNA, along with Cxcl13, Cxcl16, Ccl20 and GCsf. These are chemoattractants for T- and B-lymphocytes, tumorassociated neutrophils and macrophages, suggesting involvement of different immune cells in dysplastic stages of disease. Furthermore, a lysine deacetylase inhibitor, N-Hydroxy-7-[5-(4-tertbutoxycarbonylaminophenyl)-3-isoxazolecarboxamido]heptanamide (BML-281) altered these immune responses with suppression of multiple inflammatory cytokines/chemokines and reduced infiltrated B-lymphocytes by 40 % in both colon and lymph node, leading to delay and reduction of the dissease symptoms.
B014 THE CLINICAL FEATURES OF TAKAYASU’S ARTERITIS IN OUR HOSPITAL (53 CASES) Takumi Nagamoto1,2, Kenta Misaki1,2, Rintaro Saito1,2, Yuri Nakamura1,2, Yoshie Gon1,2, Toshihiko Yokota1,2 1 Kurashiki Central Hospital, Kurashiki, Japan; 2Department of Endocrinology and Rheumatology
Objectives:To reveal the clinical findings, and the long-term prognosis of Takayasu’s arteritis (TA).
S107 Methods:We retrospectively analyzed the data from the medical records of 53 patients with TA (male 11, female 42) who received treatment at our hospital From January, 2000 to September, 2014. Results:The average age at onset of disease was 28.6 ± 1.4 (mean ± SE) years old, period from onset to diagnosis 14.8 ± 7.43 months, and observation period was 181.8 ± 17.1 months, respectively. Disease classifications were type I (13), type II a (5), type II b (10), type III (n = 1), type IV (n = 1), and type V (n = 21). In adults, left subclavian artery involvement was common (86.4 %), however, was less common in child (37.5 %). Celiac, mesentric and renal artery lesions were remarkably present in children compared with those of in adults (75.0 and 18.2 %, respectively). Stenosis was found in 43 cases, and in 37 patients stenotic lesions were found at time of diagnosis. Aneurysm was found in 8 cases, of which 1 burst. Cerebral infarction was observed in 5 patients, 3 of which were on the first visit. Aortic regurgitation (AR) was found in 13 cases on the first visit, and 2 were found after treatment. Revascularization or valve replacement was performed 30 times in 13 patients. Combination therapy with immunosuppressant and glucocorticoid was administered in 12 patients with 3 biologic agents. Relapse was revealed 23 times in 11 cases. Duration from first induction therapy to relapse was 26.6 ± 23.1 months. New vascular involvement or progression of stenosis under the negative CRP or ESR was occurred in 7 cases. The survival was 97.5 % at 5 years, 96.9 % at 10 years, and 81.3 % at 20 years. Five patients died at 19.1 years of age after a diagnosis (3 infections, 1 cerebral infarction, and 1 myocardial infarction). Conclusion: Our findings suggested that the long-term outcome was good, and TA patients with cerebral infarction and AR were infrequent after immunosuppressive treatment. It is necessary to commence treatment before severe stenosis, ischemia and AR occur.
B015 C-REACTIVE PROTEIN COULD PROMOTE OSTEOCLASTOGENESIS Sang-Heon Lee1, Chong-Hyeon Yoon2 1
Konkuk University School of Medicine, Seoul, South Korea; 2The Catholic University of Korea, Colleg of Medicine, Seoul, South Korea Background: C-reactive protein (CRP) is one of the biomarkers for the diagnosis and assessment of disease activity in rheumatoid arthritis (RA). CRP is not only the by-product of inflammatory response, but also plays pro-inflammatory and pro-thrombotic roles. Objectives: This study aims to determine the role of CRP on bone destruction in RA. Methods: CRP levels in RA synovial fluid (SF) and serum were measured using the immunoturbidimetric method. The expression of CRP in RA synovium was assessed using immunohistochemical staining. CD14 + monocytes from peripheral blood were cultured with CRP, and RANKL expression and osteoclast differentiation were evaluated by using real-time PCR, counting TRAP-positive multinucleated cells and assessing bone resorbing function. CRP-induced osteoclast differentiation was also examined after inhibition of Fcgamma receptors. Results: There was a significant correlation between CRP levels serum and SF in RA patients. The SF CRP level was correlated with interleukin (IL)-6 levels, but not with RANKL levels. Immunohistochemical staining revealed that CRP was more abundantly expressed in the lining and sublining areas of the RA synovium, compared with the osteoarthritis synovium. CRP promoted RANKL
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production in monocytes, which in turn, induced osteoclast differentiation from monocytes and increased bone resorption in the absence of RANKL. Conclusions: CRP could play an important role in the bony destructive process in RA through the induction of RANKL expression and promote direct differentiation of osteoclast precursors into mature osteoclasts. In the treatment of RA, lowering CRP levels is a significant parameter not only for improving disease activity but also for preventing bone destruction.
Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.
B016 A NOVEL APPROACH FOR TREATMENT OF RHEUMATOID ARTHRITIS: COMBINED TNF-A AND SCLEROSTIN INHIBITION BLOCKS INFLAMMATION AND RESTORES BONE IN A MOUSE COLLAGEN INDUCED ARTHRITIS MODEL
Susanne Schiffmann4,2, Julia Barthelmes2, Max Eberle2, Yael Pewzner-Jung1, Anthony Futerman1, Philipp Ebel3, Klaus Willecke3, Gerd Geisslinger2, Sabine Gro¨sch2
Wendy O. Waegell, Alexander V. Ivanov, Regina N. Mario, Shaughn Bryant, Trudi M. Veldman, Lisa M. Olson AbbVie Bioresearch Center, Worcester, MA, USA One of the hallmarks of RA is progressive bone erosion that is clinically managed by TNF blockade. In the majority of RA patients, anti-TNF therapy halts bone loss but little healing of bone erosions is observed. Sclerostin acts as an inhibitor of Wnt signaling, inhibiting osteoblast differentiation and function. Two genetic diseases, Van Buchem’s disease and sclerosteosis, result from loss of function mutations in the SOST gene encoding sclerostin, and result in overproduction of bone. Recent reports suggest that sclerostin is up-regulated in the synovium of RA patients as well as in the HuTNF transgenic mouse model of RA. Blocking the inhibitory action of sclerostin and restoring Wnt signaling will therefore lead to new bone formation. This has been demonstrated clinically in a phase I trial in which a single dose of anti-sclerostin mAb increased BMD in the lumbar spine and hip. We propose a hypothesis that combined blockade of TNFa and sclerostin may provide greater efficacy and DMARD activity by combining the anti-inflammatory/ bone sparing of TNF inhibition with bone anabolic activity induced by sclerostin inhibition, leading to bone healing, cartilage protection and improved joint function. To test this hypothesis we have evaluated the efficacy of single agent versus dual agent inhibition of TNF and sclerostin in collagen induced arthritis, a mouse model of RA. Anti-TNF treatment initiated at first clinical signs of arthritis, a time-point with no bone loss, was effective in reducing inflammation either alone or in combination with anti-sclerostin mAb, whereas sclerostin inhibition alone did not impact inflammation. In contrast, when treatment was started 5 days after onset of clinical signs, a time-point with moderate bone loss, neither TNFa nor sclerostin inhibition had an effect on inflammation. However, sclerostin blockade prevented additional bone loss after treatment initiation based on micro-computed tomography evaluation. In an additional study, we observed that sclerostin blockade in combination with IL-a and IL-b blockade, which is able to block inflammation when treatment is initiated at the peak of inflammation, led to bone restoration in the arthritic joint. Hence, blocking inflammation and bone resorption by inhibiting TNF or IL-1 and promoting bone formation by inhibiting sclerostin could lead to healing of bone erosions and offer a promising new approach as a novel treatment for RA.
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B017 THE OPPOSING EFFECTS OF CERS2 AND CERS6 IN THE DEVELOPMENT OF EAE
1
Weizmann Institute of Science, Rehovot, Israel; 2University of Frankfurt, Frankfurt, Germnay; 3University of Bonn, Bonn, Germany; 4 Fraunhofer Institute IME-TMP, Frankfurt am Main, Germnay Ceramide synthases (CerS) synthesize ceramides of defined acyl chain lengths, which are thought to mediate cellular processes in a chain length-dependent manner. In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), we observed a significant elevation of CerS2 and CerS6 and its products, C24-/16-ceramides, in CD11b+ cells (monocytes and neutrophils) isolated from blood. This result correlates with the clinical finding that CerS2 and CerS6 mRNA expression and C24-/C16-ceramide levels were significantly increased in white blood cells of MS patients. The increased CerS2 mRNA/C24-ceramide expression in neutrophils/monocytes seems to mediate pro-inflammatory effects, while the increased CerS6 mRNA/C16-ceramide expression in neutrophils/monocytes seems to mediate anti-inflammatory effects. The genetic deletion of CerS2 or Cer6 significantly ameliorates or worsens the clinical symptoms, respectively, due to a reduced or increased infiltration of immune cells, in particular neutrophils, into the central nervous system. CXCR2 chemokine receptors, expressed on neutrophils, promote the migration of neutrophils into the central nervous system, which is a prerequisite for the recruitment of further immune cells and the inflammatory process that leads to the development of MS. Interestingly, neutrophils isolated from CerS2 or CerS6 null EAE mice, as opposed to WT EAE mice, were characterized by significantly lower or higher CXCR2 receptor mRNA expression resulting in their reduced or increased, respectively, migratory capacity towards CXCL2. Most importantly, G-CSF-induced CXCR2 expression was significantly reduced or increased in CerS2 null and CerS6 null neutrophils, respectively, and their migratory capacity was significantly impaired or improved, respectively. In conclusion, our data strongly indicate that CerS2 promotes inflammatory processes, while CerS6 inhibits them presumably via a feedback mechanism. Thus, CerS2 and CerS6 have opposing effects during the development of EAE and MS.
B018 ADA2 DEFICIENCY ATTENUATES M2 MACROPHAGES DIFFERENTIATION Dan Yang1, Guibin Chen1, Qing Zhou2, Yuting Huang1, Zhen Yu1, Avram Walts1, Alejandra Negro1, Cynthia St. Hilaire1, Ivona Aksentijevich2, Manfred Boehm1 NHLBI, Bethesda, MD, USA; 2NHGRI, Bethesda, MD, USA
1
Inflamm. Res. Background: CECR1/ADA2 (ADA2) belongs to the adenosine deaminase family. Loss-of-function mutations in the CECR1 gene (Deficiency of ADA2; DADA2) have been linked to early-onset stroke, vasculopathy and inflammation. Mutant ADA2 protein and ADA2 knockdown in U937 cells have been shown to inhibit monocyte to macrophage differentiation. The mechanism of how ADA2 affects monocyte/macrophage differentiation is largely unknown. Methods: We utilized human induced pluripotent stem cell (hiPSC)-derived monocytes, together with human primary monocytes and human myeloid U937 cell line, to study monocyte/macrophage differentiation. Flow cytometry, cytokine profiling, phagocytosis assay, and immunofluorescence staining were used to characterize cell markers and functions. Results: In DADA2 patients’ monocytes, expression of M-CSF-induced M2 macrophage markers CD163 and CD206 were decreased. GM-CSF-induced patients’ M1 macrophages had higher expression of pro-inflammatory cytokines upon LPS stimulation compared to control cells. This suggests an M1/M2 differentiation imbalance in patients’ cells. Consistent with data from the primary cells, hiPSC-derived patients’ macrophages expressed lower levels of the M2 markers CD206, CD163, CD204, and CD209 compared to control hiPSC-derived cells. Besides, the expression of monocyte maturation markers HLA-DR, CD11c and CD64 also decreased in patients’ cells. This indicates that the ADA2 deficiency leads to a disrupted M-CSF signaling. Furthermore, we observed that the addition of adenosine leads to a down regulation in the expression of M2 markers, while recombinant human ADA2 partially rescued this phenotype. We found that the A3 adenosine receptor (A3AR) and A2bAR agonists partially mimic the effect of adenosine. Inversely, the addition of A3AR antagonist rescued expression of the M2 marker in patient’s hiPSC-derived monocytes. This suggests that the adenosine-mediated pathways at least partially contribute to the impaired differentiation of M2 macrophages in patients with DADA2. Similarly, ADA2 knockdown in U937 cells decreased M2-like macrophage markers. Meanwhile, reduced activation of PKC-delta and p38 suggesting a role for PKC/p38 signaling. Conclusion: deficiency of ADA2 attenuates M2 macrophage differentiation. Data from ADA2-knockdown in U937 cells suggest the mechanism by which ADA2 regulates M2 macrophage differentiation via PKC/p38 signaling. Data from primary monocytes suggests a role for adenosine signaling in this process. Together this data demonstrate a role for adenosine and PKC/p38 signaling pathways in the ADA2mediated macrophage differentiation. The altered ratio of tissue M2/ M1 macrophages likely contributes to inflammation and the vasculopathic/vasculitis phenotype in patients with DADA2.
B019 FLAVONOLS MODULATE THE PRODUCTION OF REACTIVE OXYGEN SPECIES AND THE CHEMOTACTIC ACTIVITY OF PERIPHERAL BLOOD NEUTROPHILS FROM RHEUMATOID ARTHRITIS PATIENTS Larissa F. Marchi1, Adriana P. Paschoalato2, Reneˆ R. Oliveira3, Ana Elisa C. Azzolini4, Eduardo A. Donadi5, Yara L. Valim6 1
University of Sa˜o Paulo, School of Pharmaceutical Sciences of Ribeira˜o Preto, Sa˜o Paulo, Brazil; 2University of Sa˜o Paulo, Ribeira˜o Preto Medical School, Sa˜o Paulo, Brazil; 3University of Sa˜o Paulo, Ribeira˜o Preto Medical School, Sa˜o Paulo, Brazil; 4University of Sa˜o
S109 Paulo, School of Pharmaceutical Sciences of Ribeira˜o Preto, Sa˜o Paulo, Brazil; 5University of Sa˜o Paulo, Ribeira˜o Preto Medical School, Sa˜o Paulo, Brazil; 6University of Sa˜o Paulo, School of Pharmaceutical Sciences of Ribeira˜o Preto, Sa˜o Paulo, Brazil Introduction and objective: Rheumatoid arthritis (RA) is a chronic disease characterized by synovial tissue inflammation, where neutrophils are the most abundant cells and are important mediators of tissue destruction. Tissue damage in AR involves excessive production of reactive oxygen species (ROS) triggered by immune complexes (IC) and neutrophils interactions via receptors FccR and complement receptors (CR). Modulation potential of ROS generation may be relevant to the maintenance of body homeostasis. Compounds as flavonols have been considered helpful to inhibit and/or modulate the neutrophils functions. In this study, we evaluated the oxidative burst from peripheral blood neutrophils (PBN) from AR patients, stimulated via FccR and FccR/CR receptors, the chemotactic activity this cells and the ability of flavonols (quercetin and galangin) to inhibit these two functions. Methods: Human peripheral blood was collected from the healthy volunteers and RA patients treated with methotrexate (MTX). The measurement total ROS production of neutrophils was taken with luminol (CL-lum) chemiluminescence assays. We evaluated the neutrophils chemotactic activity in vitro using acrylic migration chambers and zymosan (Zy), a polysaccharide responsible for cell activation, was employed as complement activator for chemotactic fragments generation from NHS (normal human serum) and rheumatoid arthritis human serum (RAHS). Results: Comparison of the oxidative burst mediated by FccR (IC alone) and FccR/CR (IC opsonized with serum) in neutrophils from RA patients and healthy volunteers resulted in increased responses only in neutrophils from patients with RA (p \ 0.001). Galangin and quercetin (mean IC50 = 2.5 lM; 1.75 lM respectively) suppressed nearly 65 % CLlum of RA patients’ neutrophils. The chemotactic activity of neutrophils also was investigated. When neutrophils were incubated with NHS without the complement activator, the cells did not display increased migration, whereas enhanced migration was observed when cells were incubated with RAHS. These results indicate that RAHS contains chemoattractant factors even before Zy activation occurs. The galangin diminished chemotactic activity when cells were incubated with RASH or Zy (p \ 0.01), indicated the possible modulation in complement activation. Conclusion: Taken together the results showed that the flavonols galangin and quercetin partially inhibited the oxidative metabolism neutrophils and only the galangin inhibited chemotactic activity as consequence of complement activation. The use these compounds to modulate neutrophil functions can be a promising safe therapeutic strategy to control inflammation in RA patients. Support: FAPESP, CNPq.
B020 AMELIORATION OF CHRONIC COLITIS IN MICE BY GENETIC DELETION OF THE IL-13 DECOY RECEPTOR Trisha Pasricha, Thirumalai Ramalingam, Thomas Wynn National Institute of Allergy and Infectious Disease, NIH, Bethesda, MD, USA Introduction: The mainstays of therapy in inflammatory bowel disease, anti-TNF-alpha agents, are ineffective in a significant proportion of patients, underlying a great need to explore broader therapeutic
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options within the inflammatory signaling pathways that govern immune dysregulation. We hypothesized that IL-13Ra2 knock-out (KO) mice would demonstrate restored homeostatic activity of IL-13 in a chronic inflammatory disease model and lead to improved physiological outcomes. Objective: To assess the effects of genetic deletion of the IL-13 decoy receptor, IL-13Ra2, in a chronic model of inflammatory bowel disease. Methods: Chronic colitis was induced in wild type (WT) C57B/6 mice and IL-13Ra2 KO C57B/6 mice via three 7-day cycles of 3 % dextran sodium sulfate (DSS) over the course of 5 weeks. Control mice of both strains were administered normal water throughout the course of the experiment. At day 35, colon tissue was harvested to analyze the severity of chronic colitis. Parameters assessed included weight loss, qPCR analysis of inflammatory markers, colon collagen content measured by hydroxyproline assay. Two independent blinded reviewers performed histological scoring. Colon sections were graded on a 0–12 scale of increasing severity, assessing mucosal architecture, lymphocytic infiltration, epithelial defects, and goblet cell loss. Results: All animals survived the study. On day 35 of the experiment, IL-13Ra2 KO mice on DSS demonstrated greater weight than WT mice on DSS (p = 0.04). In qPCR analysis of distal colonic tissue, IL-13Ra2 KO mice on DSS compared to WT mice demonstrated a significantly reduced induction of TNF-alpha (p \ 0.0001), IFNgamma (p = 0.02), IL-1B (p = 0.0009), and TGF-beta (p = 0.0008). For all qPCR studies, there were no significant differences between WT mice on normal water, IL13Ra2 KO mice on normal water, and IL-13Ra2 mice on DSS. Additionally, IL-13Ra2 KO mice on DSS had a significantly less hydroxyproline content within equal length segments of colon tissue compared to WT mice on DSS (p = 0.004). Lastly, histological scores of IL-13Ra2 KO mice on DSS trended lower than WT mice on DSS. Conclusions: We have shown that genetic deletion of the IL-13Ra2 receptor in mice can serve a protective role in a chronic inflammatory bowel disease model across several physiological parameters. This data suggests that the IL-13 decoy receptor may be an important therapeutic target for patients by restoring the normal homeostatic function of IL-13. Larger additional studies to confirm this regulatory pathway are currently underway.
collagen-induced arthritis (CIA) is widely accepted as a valid RA animal model for mimicking human RA. Thus, we aimed to investigate the role of HQ exposure on CIA in Wistar rats and the involved mechanisms. Methods: Animals were immunized subcutaneously at the tail base by using 200 lg of bovine type-II collagen emulsified 1:1 with complete Freund’s adjuvant (CFA). A booster injection of 100 lg of bovine type-II collagen emulsified in CFA was administered 7 days later. The rats were divided into three groups according to exposition with saline, HQ vehicle (saline ethanol solution 1:20) or HQ 25 ppm. All animals were exposed for 35 consecutive days, 1 h/day, using a nebulization chamber. CIA was induced on day 7th and booster on day 14th. Animals were submitted to clinical evaluation (score scale of 0–4: 0 = no arthritis; 1–2 = weak arthritis, with inflamed digits; 3 = medium arthritis, with more than 2 digits and an inflamed footpad; 4 = strong arthritis, with all inflamed digits and paws), hematological parameters and histological analysis of synovial membranes. Data were obtained in nine animals per group. Results: Data obtained showed that HQ exposure caused a score 3–4 of CIA; HQ vehicle 2–3 and saline 1–2. HQ exposure caused weight body loss (D day 35-day 1: saline = +14.92 %; vehicle = -3.53 %; HQ = -9,66 %), no altered the numbers of circulating cells, and marked increased leukocyte numbers into the synovial fluid and neutrophils influx into the synovial membrane. Moreover, HQ exposure caused pannus formation and hyperplasia of the synovial cells. Conclusion:: Our data clearly show that HQ exposure aggravates the development of RA, suggesting that HQ may be a determinant component of cigarette on RA induction. Supported by Capes.
B021 HYDROQUINONE EXPOSURE AGGRAVATES COLLAGEN INDUCED ARTHRITIS (CIA) DEVELOPMENT IN RATS
Merck Research Laboratories, Merck & Co., Inc, Rahway, NJ, USA
Cintia S. Heluany1, Leoanrd k. Kupa1, Mariana N. Viana2, Cristina M. Fernandes2, Sandra P. Farsky1 1
Pharmaceutical Sciences Faculty, University of Sa˜o Paulo, Sa˜o Paulo, Brazil; 2Pharmacology Laboratory, Butantan Institute, Sa˜o Paulo, Brazil Background: Rheumatoid arthritis (RA) is a chronic autoimmune disease and smoking is assumed to be the main etiological factor in the pathogenesis of RA. Studies have shown that smoking is a trigger factor in the development of RA and also it also worsens pre-existing disease. However, the importance of cigarette components on the overall effects of RA remains unknown. Hydroquinone (HQ) is a phenolic compound of natural or anthropogenic source, also found in high concentrations in cigarette (is the major oxidative component found in cigarette), as well as it is a benzene metabolite. Type II
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B022 MULTI-MODALITY PATHWAY CHARACTERIZATION AND DISEASE EVALUATION IN LUPUS MODEL Jie Zhang-Hoover, Alan Byford, Mark Zielstorff, Robert Faltus, Joseph Eckman, Kimberly Bettano, Gain Robinson, Raquel Sevilla, Adam Hartigan, Weisheng Zhang, Milenko Cicmil
MRL/lpr model mimics some aspects of genetics and phenotypes of human lupus disease in various tissues and has been used extensively for pharmacological evaluation of new therapies. We sought to validate utility of the model for potential pathways by measuring immune inflammation related gene expression levels in kidneys and characterizing immune cell phenotypes in peripheral blood of mice at various stages of disease development. In addition to routine in life biochemistry and terminal histology analyses, we monitored disease phenotypes longitudinally in vivo using multi-modality imaging: (1) tissue reactive oxygen species (ROS) level by luminol-bioluminescence imaging (luminol-BLI); (2) glomerular filtration rate (GFR) tracked by contrast agent Omnipaque washout using micro-computed tomography (micro-CT); (3) cerebral cortical thickness by brain magnetic resonance imaging (MRI). MRL/lpr mice showed a disease associated change of CD3+ B220+ cells (potential pathogenic immune cells) in peripheral blood and up-regulation of PD1/PD-L2, BTLA/HVEM, GITR/GITR ligand, and TIGIT gene expression in kidneys. Flow cytometry analysis showed that CD3+ B220+ cells increased from around 2 % at 8 weeks of age to over 30 % of peripheral blood leukocytes at 19 weeks of age
Inflamm. Res. in MRL/lpr mice. ROS production in the skin and hind limbs visualized by luminol-BLI was detectable at 12 weeks of age and preceded gross skin lesion observation at 14 weeks of age. MicroCT detected a GFR decrease as early as 14 weeks of age, compared to the blood urea nitrogen level increase at 16 weeks of age. Furthermore, brain MRI detected cerebral cortical thinning as early as 10 weeks of age. Cyclophosphamide treatment reduced disease progression significantly across multiple readouts. For the first time we report a comprehensive approach by profiling potential disease modulating gene signatures and immune cell phenotypes and using clinically relevant PD markers to enhance the pharmacological translatability of compound evaluation in a lupus pre-clinical model.
B023 INCREASED SERUM CHEMERIN LEVELS MIGHT PREDICT FLARES OF SYSTEMIC LUPUS ERYTHEMATOSUS Omer N. Pamuk, Mehmet S. Uyanik, Gulsum E. Pamuk Trakya University Medical Faculty, Edirne, Turkey Background and Objectives: Recent studies reported that the receptor for chemerin, Chem R23 was expressed intensively on plasmocytoid dendritic cells (DC); that chemerin was a chemoattractant factor for DC; and chemerin/Chem23R axis was responsible for especially the renal recruitment of DC in systemic lupus erythematosus (SLE). Retinol-binding protein-4 (RBP-4) is also secreted by adipocytes; and it is related to atherogenesis and insulin resistance. It was observed that anti-TNF therapy reduced RBP-4 levels significantly in ankylosing spondylitis (AS). In RA, the increased expression of glypican-4 was found to be related to angiogenesis in inflamed synovitis. Until now, these cytokines have not been evaluated in systemic lupus erythematosus (SLE). We determined serum chemerin, RBP-4, and glypican-4 levels in SLE; and investigated clinical features associated with them. Methods: The study includes age-and-sex matched 46 patients with SLE (45 F, 1 M, mean age: 40.5 ± 12) and 26 apparently healthy subjects (25 F, 1 M, mean age: 40.4 ± 10 years). The demographic and clinical features of SLE patients were recorded from medical charts. Serum chemerin, RBP-4 and glypican-4 levels were determined with the ELISA method. SLE disease activity index (SLEDAI) score was calculated at the time of inclusion into the study. SLE flare was assessed by the SELENA-SLEDAI flare index. Results: Serum chemerin and glypican-4 levels in SLE were significantly higher in SLE patients than in healthy controls (p values \ 0.001). RBP-4 level was similar in SLE patients and in controls (p [ 0.05) (Table 1). In the SLE group which was active according to SLEDAI score ([4), serum chemerin level was significantly higher when compared to the inactive group (191.4 ± 159 vs. 120.5 ± 72.5, p = 0.048). Serum glypican-4 level was significantly lower in SLE patients with thrombocytopenia when compared to nonthrombocytopenic SLE patients (3.34 ± 2.84 vs. 3.86 ± 0.63, p = 0.041). ROC curve analysis revealed that the area under the curve values for glypican-4 and chemerin levels in SLE were, respectively, 0.903 (95 % CI 0.823–0.984) and 0.774 (95 % CI 0.656–0.893) (p values \ 0.001). Glypican-4 level of 3.1 had 88 % sensitivity and 83 % specificity for SLE. For chemerin, a level of 62.6 had 74 % sensitivity and 72 % specificity.
S111 During a median follow-up of 64 months (range 3–120 months), there were 12 flares according to the SLE flare index. SLE group with serum chemerin levels above the cut-off value had significantly shorter relapse-free survival than the other group (p = 0.05). Table 1. The comparison of biochemical parameters of SLE and controls. SLE Glypican-4 (ng/ml)
3.76 ± 0.65
Chemerin (lg/L)
138.2 ± 115.2
RBP-4 (ng/ml)
54.39 ± 54.8
Controls
p
2.73 ± 0.52
\0.001
58 ± 53.3
\0.001
48.16 ± 81.3
0.72
Conclusions: Serum chemerin and glypican-4 levels were increased in SLE patients; and they might be related to disease pathogenesis. Increased chemerin levels in SLE might be predictive for future flares.
B024 THE EFFECTS OF THE SPLEEN TYROSINE KINASE INHIBITOR, FOSTAMATINIB, ON AN IMMUNE THROMBOCYTOPENIA MOUSE MODEL Gulsum E. Pamuk, Mehmet S. Uyanik, Turan Karaca, Selim Demirtas, Ahmet M. Demir, Omer N. Pamuk Trakya University Medical Faculty, Edirne, Turkey Background: Immune thrombocytopenia (ITP) is the most common autoimmune bleeding disorder. The major mechanism involves the phagocytosis of antibody-coated platelets by Fc-gamma receptor (FccR)-bearing macrophages. Many of the FccR-mediated activities are dependent on the spleen tyrosine kinase (Syk) pathway. Objectives: The aims of the present study were to test the hypotheses of whether (1) The Syk pathway is involved in both the pathogenesis of ITP and in the treatment response to ITP, (2) Fostamatinib (Fos, R788), a novel Syk inhibitor drug, has any effects on the complement system, lymphocytes, and macrophages in ITP, and whether (3) Fos decreases the migration of T lymphocytes to the bone marrow and the spleen in ITP. Methods: Six to eight week-old BALB/c mice were divided into eight groups: (1) Sham-operated; (2) isolated ITP; (3) Fos (3 g/kg, p.o.); (4) Fos + Liposomal clodronate (LC) (0.2 ml/U/mouse, i.p.); (5) Fos + cobra venom factor (CVF) (25 U/mouse, i.p.); (6) Intravenous immunoglobulin (IVIG) (2 g/kg, i.m.); (7) IVIG + LC; and (8) IVIG + CVF. ITP model was formed by the i.v. injection of 4 lg of anti-CD41/Integrin alpha-2b antibody. Spleen tissue sections were cut and stained with Syk, phospho (p)-Syk, PECAM, and CX3CR1. Results: Administration of IVIG (p \ 0.001) and Fos (p \ 0.001) prevented the fall in platelet counts in an ITP mouse model. Macrophage depletion by LC, and complement depletion by CVF did not provide any extra increments in the platelet counts. The intensity of Syk (p = 0.0001) and p-Syk (p = 0.0001) stainings were significantly higher in the isolated-ITP group than in the sham-operated group. Groups treated with Fos demonstrated less Syk and p-Syk staining scores than ITP group (p \ 0.001 for Fos and Fos + LC;
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S112 p = 0.028 for Fos + CVF). Syk staining was significantly less intense in the IVIG group than in the ITP group (p = 0.0001). P-Syk levels in all IVIG groups were significantly higher than in other groups (p \ 0.001). The intensity of PECAM staining was significantly higher in the ITP group than in the sham group (p = 0.0001). In ITP and all treatment groups, PECAM staining score was higher than in healthy controls (p \ 0.001). PECAM scores of all Fos groups (Fos, Fos + LC, and Fos + CVF) were similar to that of the ITP group. CX3CR1 staining in spleens of the ITP group was more intense than the sham group (p = 0.0001). CX3CR1 staining in Fos and Fos + LC groups was less intense than in ITP group (p \ 0.001). Fos + CVF and ITP groups had similar CX3CR1 expression. CX3CR1 staining in the Fos group was less intense than in the IVIG group (p \ 0.001). Conclusions: Our study showed that the Syk pathway played a role in the pathogenesis of ITP. The Syk inhibitor, fostamatinib, improved the thrombocytopenia in ITP by acting on the Syk pathway. In addition, fostamatinib decreased the migration of T lymphocytes to the spleen, but not to the bone marrow.
B025 THE SYK INHIBITOR FOSTAMATINIB LIMITS TISSUE DAMAGE AND FIBROSIS IN A BLEOMYCIN-INDUCED SCLERODERMA MOUSE MODEL: EFFICACY IN SYK INHIBITION IN ANIMAL SCLERODERMA Omer N. Pamuk1, Guray Can1, Sulayman Can1, Turan Karaca1, Gulsum E. Pamuk1, Selim Demirtas1, George C. Tsokos2 1 Trakya University Medical Faculty, Edirne, Turkey; 2Harvard University Medical Faculty, Boston, MA, USA
Background/Objectives: The pathogenesis of fibrosis in scleroderma (SSc) is unknown. TGF-b and platelet-derived growth factor are important in the development of fibrosis and tyrosine kinases are involved in these pathways. Spleen tyrosine kinase (Syk) is a protein tyrosine kinase which activates intracellular signal transduction pathways and has been claimed to be involved in the pathogenesis of systemic autoimmune diseases. We investigated the ability of a small drug Syk inhibitor, fostamatinib (Fos), to protect mice from bleomycin-induced SSc. Methods: Four study groups of Balb/c mice were included in this study: control, bleomycin (administered subcutaneously to BALB/c mice for 21 days), bleomycin and Fos (mice fed with chow containing a Syk inhibitor for 21 days) and Fos alone groups. Skin and lung tissue specimens were obtained and evaluated histologically. Results: Mice treated with bleomycin alone had significantly more skin thickness (416.1 ± 6.1) compared to control (260.1 ± 10.1) and Fos (254.3 ± 7.9) treated mice (p \ 0.001). Mice subjected to bleomycin and fed with Fos-containing chow generated more (312.3 ± 4.4) dermal thickness than control and Fos-treated mice (p values \ 0.001) but, significantly less when compared to mice treated with bleomycin alone (p \ 0.001). Alveolar hemorrhage, edema, damage and leukocyte scores in the lungs of mice treated with bleomycin were significantly higher v compared to control or Fos alone-treated mice (p values \ 0.001). Mice though which were exposed to bleomycin and treated with Fos had significantly less alveolar damage and leucocyte infiltration compared to mice exposed to bleomycin and fed with regular chow. At the end of the 21-day bleomycin administration, there was apparent prominent fibrosis which was reduced significantly in the group of mice which received in parallel Fos. Syk expression in both the lung and the skin was noted to be intense in the bleomycin-treated mice compared to control mice.
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Inflamm. Res. Interestingly, treatment of mice with the the Syk inhibitor Fos decreased significantly Syk expression in both skin and lung. The active form of Syk is phospho-Syk, and its expression is suppressed by Syk inhibitor therapy. Bleomycin induced the expression of phospho-Syk but in the presence of the Syk inhibitor its expression was limited sginifcantly. Mice treated with bleomycin displayed more prominent TGF-b staining of skin and lung tissues. The administration of Syk inhibitor to the bleomycin-treated mice abolished TGF-b staining. The number of mast cells were higher in the group of mice that were treated with bleomycin alone (p \ 0.001) and the administration of Fos reduced significantly the number mast cells. Conclusions: The Syk inhibitor Fos prevented bleomycin-induced fibrosis and inflammation in the skin and the lung. The anti-fibrotic effect of Fos is linked to reduced Syk phosphorylation and TGF-b expression. The Syk pathway appears as a potential molecular target for therapeutic intervention in SSc.
B026 SERUM AND SYNOVIAL FLUID C5A LEVELS IN PATIENTS WITH RHEUMATOID ARTHRITIS AND OSTEARTHRITIS Dilek Keskin1, Go¨ksal Keskin2, Ali Inal3 1 University of Kırıkkale, Kırıkkale, Turkey; 2Ankara Univeristy, Ankara, Turkey; 3University of GATA, Turkey
Rheumatoid arthritis (RA) is a chronic inflammatory disease, with unknown etiology. Compleman 5a (C5a) which is generated during acute flare up of Rheumatoid Arthritis (RA) the has a n important role in the pathogenesis of RA. Purpose: The aim of this study was to determine C5a levels in the plasma and synovial fluid (SF) samples. Patients and methods: Thirty patients with RA and Thirty-five patients with Osteoarthritis (OA) were enrolled to the study. The mean duration of the disease was 6.7 ± 3.42 years in patients with RA and 8.3 ± 4.7 years in and patients with OA. All patients with RA were in active period. Synovial fluid was aspirated from knee joint and plasma samples were also taken at the same time and C5a levels were determined by ELISA. Results: The mean plasma C5a levels were in patients with active RA and OA were 107.3 ± 18.2 lg/L and 88.5 ± 23.1 lg/L respectively. The mean SF C5a levels were 168.4 ± 41.7 lg/L in patients with active RA and 103.8 ± 33.1 lg/L in patients with OA. There was no statistically significant difference in plasma C5a levels in both of the groups. The SF C5a levels were significantly high in patients active RA compared to patients with OA. Conclusion: The high levels C5a in SF suggests that C5a is intensively synthesized in the region of inflammation.
B027 THE ROLE OF NEUTROPHILS AND G-CSF IN THE K/BxN SERUM-TRANSFER ARTHRITIS MODEL Anne Deen Christensen1,2, Claus Haase2, Andrew D. Cook1, John A. Hamilton1 1
Department of Medicine, University of Melbourne, Royal Melbourne Hospital, Royal Parade, Parkville, Victoria, Australia; 2Novo Nordisk A/S, Novo Nordisk Park, Ma˚løv, Denmark
Inflamm. Res. Neutrophils are an important cell type in many autoimmune diseases such as rheumatoid arthritis. Furthermore, mobilization of neutrophils from the bone marrow to the circulation during an inflammatory response is controlled by granulocyte-colony stimulating factor (GCSF). In the K/BxN serum-transfer arthritis (STA) model, mimicking the effector phase of rheumatoid arthritis, neutrophils have been shown to play a key role. In this model neutrophils have been depleted using either a rat anti-mouse LyG/C monoclonal antibody (mAb) (clone RB6.8C5) or a rat anti-mouse Ly6G mAb (clone 1A8). However, the two antibodies have recently been shown to deplete with different specificity as well as efficacy. In contrast, the impact of G-CSF on arthritis in this model has not been investigated. The objective of this study was to compare depletion of neutrophils with the two different neutrophil-depleting antibodies, as well as to investigate the role of G-CSF in the K/BxN STA model. For induction of arthritis, K/BxN serum was injected intraperitoneally into C57BL/6 mice on day 0 and day 2. On day 0–10 after the first serum transfer, arthritis was assessed by a clinical score as well as by measurement of paw swelling in the rear paws. On day 1, 4 and 7 levels of G-CSF, IL-1b, IL-10, CXCL10, CXCL2 and CXCL1 were analysed in inflamed paws. Additionally, levels of G-CSF in serum were measured on day 1, 2, 3, 4, 7 and 10. Neutrophils were depleted by injecting 1 mg/mouse of either rat anti-mouse Ly6G/C mAb or rat anti-mouse Ly6G mAb intraperitoneally on day-1 and -2. On day 1 and 7 depletion was confirmed with flow cytometry. G-CSF was blocked by injection of a rat anti-mouse G-CSF mAb (clone 67604) one day prior to the first serum injection. On day 3 and 7, a blood analysis was performed by flow cytometry to quantify the absolute number of neutrophils in peripheral blood. First, it was found that the neutrophil chemoattractants, CXCL1 and CXCL2, were significantly increased after induction of arthritis compared to naı¨ve controls. We also found increased levels of G-CSF both in serum and in inflamed tissue during progression of arthritis. After injection of the two neutrophil depleting antibodies, flow cytometric analysis showed that anti-Ly6G mAb only partially depletes neutrophils in C57BL/6 mice, while anti-Ly6G/C mAb, in addition to completely depleting neutrophils, also depletes a fraction of monocytes. Treatment with anti-Ly6G mAb reduced arthritis significantly, while anti-LyG/C mAb completely blocked arthritis. Interestingly, blockade of G-CSF led to an almost complete absence of arthritis as well as a reduced level of neutrophils in peripheral blood. This study emphasizes the importance of neutrophils in the K/BxN STA model and demonstrates a crucial role for G-CSF in this model with possible significance for human disease.
B028 ENHANCEMENTS TO THE PREDICTABILITY AND CLINICAL RELEVANCE OF TWO MURINE MODELS OF CHRONIC INFLAMMATION: ADOPTIVE TRANSFER-INDUCED COLITIS AND GVHD Dominic R. Beal, Sean M. Graham, Brett Van Dam, Gregory D. Lyng, Stephen T. Sonis Biomodels LLC, Watertown, MA, USA Mouse models are widely used to assess new therapies for autoimmune pathologies such as colitis and GVHD. We now report enhancements which increase the translational value of 3 such models. Adoptive transfer of naı¨ve THcells into immunodeficient mice is one of the best characterized immunological models of chronic colitis (CC). However, traditional disease severity endpoints of weight loss
S113 (WL) and histopathology can be ambiguous as inflammation may occur independent of WL, and WL and colitis may respond differently to treatment. To overcome these limitations, we used serial FACS analyses of peripheral blood (PB) to assess engraftment, and performed serial endoscopy (SE) to track onset and severity of CC. Naive TH cells from C57Bl/6 donors, sorted as either CD4+, CD45RBHIGH, or CD4+, CD44-, CD62L+ were injected i.p. into RAG2-/- recipients. Onset of colitis, determined by endoscopy (E), corresponded with onset of WL (d21-28). PB CD4+ cell counts assessed prior to appearance of observable disease (d14) were highly correlative with final (d56) WL (R2 = 0.74) and final E score (R2 = 0.79). Positive control therapy (anti-TNFa mAb) had little effect on terminal WL but significantly reduced E score (2.0 ± 0.15 vs. 1.1 ± 0.14 at d49). These results and the method’s clinical relevance confirm that SE is a useful addition to WL as an enhanced readout for in-life disease severity and indicate that d14 CD4+ count is a reliable early predictor of disease severity. In the standard murine GVHD model, endpoints of WL and GVHD score often fail to respond to positive control therapies (FK506 and anti-p40 mAb), highlighting the need for improved translatability. We evaluated SE as an additional method to directly assess onset and progress of intestinal GVHD. GVHD was induced by harvesting bone marrow of male C57Bl/6 mice, depleting it of CD3+ cells and transferring it along with splenocytes into lethally irradiated (8 Gy) Balb/c hosts. Subsequent SE showed disease levels that strongly correlated with WL and GVHD score (R2 = -0.84 and 0.79). We next evaluated the utility of using serial peripheral blood (SPB) FACS analysis of human leukocyte count (HLC) as a correlate of WL and GVHD score in a humanized model in which GVHD was induced by the adoptive transfer of human PBMCs into NSG mice. SPB FACS analyses demonstrated a strong correlation between HLC and WL and GVHD score (R2 = -0.85 and 0.67 on d42) indicating that HLC may be applicable to the evaluation of therapies directly targeting these cell types. Early (d7) HLC predicted final WL and GVHD score with high correlation (R2 = -0.86 and 0.90). We conclude that SE and FACS analyses are valuable additions to enhance the translatability, clinical relevance, and predictability of these adoptive transfer-based inflammatory disease models.
B029 IL-7 RECEPTOR POLYMORPHISMS IN MULTIPLE SCLEROSIS: MECHANISM AND THERAPEUTIC IMPLICATION Wenqing Li1, Julie A. Hixon1, Julia Tritapoe1, Joao T. Barata2, Scott K. Durum1 1
Laboratory of Molecular Immunoregulation, Cancer and Inflammation Program, CCR, NCI; 2Cancer Biology Unit, Instituto de Medicina Molecular, Lisbon University Medical School, Lisbon, Portugal IL-7 is required for survival of most T cell subsets and generally promotes T cell immune responses. IL-7 acts on lymphocytes by binding with high affinity to IL-7 receptor a chain (IL-7R) and recruiting c, and then initiating the signaling cascade. Loss-offunctions of IL-7R in human has, for some time, been known to cause severe combined immunodeficiency. We find that 9 % of childhood T-cell acute lymphoblastic leukemia (T-ALL) has somatic gain-offunction mutations of IL-7R. These mutations, usually involved insertions of three or more amino acids immediately before or after residue 244 in the exon 6, create oncogenes driving T-ALL. More recently, polymorphisms in IL-7R have been shown be a risk factor
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S114 for multiple sclerosis (MS) and a number of diseases that are autoimmune or involve excess immune and inflammatory responses. The polymorphysims that affects risk to most of these immunopathologies is T244/I in exon 6, the same region we show to harbor gain-of-function mutations in T-ALL. To investigate the mechanism of IL-7R pathway association with MS and seek new therapeutic approaches directed to IL-7R and its downstream signaling component, we hypothesize that the functional significance of the 244T/I polymorphism in IL-7R is related to increased signaling. To test our hypothesis, the T244 and I244 alleles of IL7R are transfected into BaF3 cells. The high risk allele T244 induces stronger Stat5 activation and promotes better survival of BaF3 cells. We further examine the strength of signaling in vivo in mice. IL-7R deficient bone marrow hematopoietic progenitors are transduced by retroviral infection with T244 or I244 alleles of human IL-7R and transferred to Rag1 deficient mice. Human IL-7R rescues T cell development in this model. Consistent with the in vitro study, the CD8 cells harboring the high risk allele T244 display stronger Stat5 activation compared with cells with I244. Our preliminary study suggests that high risk allele T244 of IL-7R signals stronger than allele I244, and this may promote activation of autoimmune T calls in MS and other autoimmune diseases.
B030 SERUM 14-3-3 ETA IS AN RA SPECIFIC MECHANISTIC MARKER Anthony Marotta1, Yauheniya Cherkas2, Bidisha Dasgupta2, Sarah Lamberth2, Karen Hayden2, Carrie Brodmerkel2, Mark Curran2 Augurex Life Sciences Corp, Vancouver, BC, Canada; 2Janssen R&D LLC, Raritan, NJ, USA
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Background: 14-3-3g is an emerging soluble rheumatoid arthritis (RA) biomarker that activates intracellular pathways that lead to the upregulation of inflammatory and joint damage factors. It is reported to be highly specific and sensitive for RA as a diagnostic marker, higher levels are associated with greater joint damage progression risk1,2 and 14-3-3g’s modulation with treatment suggests a role in disease monitoring. Objectives: In this study, we examine the specificity of 14-3-3g in an independent, clinically well-characterized cohort of moderate to severe RA and disease control subjects. Methods: Serum 14-3-3g levels were measured in a total of 147 patients using the Augurex 14-3-3g ELISA. The patient set comprised 36 with RA and 111 controls consisting of 20 with asthma (A), 20 with Crohn’s disease (CD), 12 presumed healthy (H), 16 with psoriasis (PsO), 20 with sarcoid arthritis (S), and 23 with spondylarthropathies (SpA). Sample testing was done independently of Augurex. Mann–Whitney testing together with Kruskal–Wallis analysis with the post hoc Dunn’s multiple comparison test was performed to assess group differences. Receiver operator characteristic curve (ROC) analysis was performed to assess the specificity of 14-3-3g for RA. Results: Median (IQR) serum 14-3-3g levels were significantly higher in RA [2.35 ng/ml (0.28-19.41)] than all controls [0 (0–0)], p \ 0.0001. ROC curve analysis further underscored this differential expression yielding a significant area under the curve (AUC) of 0.86, p \ 0.0001. At the diagnostic positivity cut-off of C0.19 ng/ml, the ROC curve delivered a sensitivity of 81 % with a corresponding specificity of 84 %. Kruskal–Wallis testing revealed that serum 14-33g levels were significantly higher in RA in comparison to all other diseases, p \ 0.0001. This differential expression is illustrated in Figure 1.
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Fig. 1 Serum 14-3-3g expression by disease. Conclusions: Serum 14-3-3g is a highly specific RA biomarker. As a novel mechanistic disease factor, 14-3-3g is expected to provide new insights and approaches to RA management and clinical studies.
B031 MULTIPLE MESENTERIC LYMPHATIC ANOMALIES IN MURINE CROHN’S DISEASE Pierre-Yves von der Weid, Sonia Rehal University of Calgary, Calgary, AB, Canada Background: Lymphatic pumping is the main mechanism used for propelling lymph and immune cells from peripheral tissues to lymph nodes and back to the blood stream. Decreased lymphatic pumping causes poor lymph drainage and accumulation of fluid, immune cells and macromolecules in the tissues, ultimately leading to edema that may contribute to the perpetuation of inflammation. Importantly, we have demonstrated that TNBS-induced inflammation of the ileum alters pumping in the mesenteric lymphatic vessels via increased production of nitric oxide (NO) and prostaglandins caused by the upregulation of the enzymes inducible nitric oxide synthase (iNOS), and cyclooxygenases (COX1 and COX2). Our aim here was to further assess how inflammation impairs lymphatic contractile function, and to investigate whether TNF-a, a potent pro-inflammatory cytokine upregulated in intestinal inflammation such as Crohn’s disease, alters lymphatic pumping and what are the mechanisms involved. Methods: Rats were euthanized and the small intestine with its attached mesentery rapidly isolated. Mesenteric lymphatic vessels were dissected out, and incubated in physiological and sterile conditions for 24 h with TNF-a or vehicle (sham vessels) with or without pharmacological inhibitors. Vessels were then either mounted on a pressure myograph to measure contractile activity induced by the luminal pressure, or processed for protein and mRNA analysis by western blot and quantitative real-time PCR, respectively. Data obtained from sham and TNF-a-treated vessels were compared. Results: TNF-a significantly decreased lymphatic contraction frequency in a concentration-dependent manner. Contractile activity was restored following administration of pyrrolidine dithiocarbamate (PDTC), an inhibitor of the master regulator of inflammation NF-jB acting downstream of TNF-a. The involvement of NF-jB activation in lymphatic dysfunction was supported by western blot data, which
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revealed an increase in phospho-IjB in TNF-a-treated vessels. Quantitative real-time PCR showed upregulation of iNOS mRNA in TNF-a-treated vessels that was minimized in the presence of PDTC. Furthermore, pumping was restored by pharmacological inhibition of the NO pathway with the iNOS inhibitor 1400 W or the soluble guanylate cyclase inhibitor ODQ, but not with the cyclooxygenase inhibitor indomethacin. Lymphatic pumping was also reestablished in the presence of glibenclamide, a KATP channel blocker, suggesting activation of these channels and hyperpolarization of the lymphatic muscle as a likely cause of the contractile dysfunction. Conclusions: The NF-jB–iNOS–NO-KATP channel pathway is involved in lymphatic pumping decrease via caused by TNF-a. Lymphatic dysfunction may contribute to the perpetuation of inflammation.
Conclusion: The finding that targeted deletion of IRP1 but not IRP2 completely abolished the inflammation in the TNFDARE/+ mouse suggests that IRP1 may be a target for specific inhibition to treat inflammatory bowel diseases.
B032 A NOVEL IRON-MEDIATED MECHANISM FOR DEVELOPMENT OF INFLAMMATORY BOWEL DISEASE
Ribeirao Preto Medical School, Ribeirao Preto, Brazil
Shirly M. Belizowski1, Abraham Nyska2, Avi Zuckerman3, Fabio Cominelli4, Orly Savion1, Esther Meyron-Holtz1 1 Technion Israel Institute of Technology, Haifa, Israel; 2Tel Aviv University and Consultant in Toxicologic Pathology, Timrat, Israel; 3 Aviv Projects, Ness Ziona, Israel; 4Case Western Reserve University, Cleveland, Ohio, USA
Background: Iron accumulation in inflammatory lesions has been described in many settings but a molecular mechanism for this accumulation was not completely elucidated. We hypothesized that an iron independent activation of RNA-binding activity of the Iron Regulatory Protein (IRP)1, via reactive oxygen and nitrogen species is the driving force for this iron accumulation and for the exacerbation of inflammation. Methods: To test this hypothesis, we examined a mouse-model of inflammatory bowel disease, the TNFa overexpressing mouse (TNFDARE/+) and an epithelial cell model for changes in iron homeostasis during inflammation and analyzed the effect of IRP1 and 2 deletions on the course of the inflammation. Differential analysis of the different cell-types within the inflamed tissue was performed. Results: We found that the terminal ileitis, which is developed in the TNFDARE/+ mouse, was accompanied not only by a local iron accumulation but also by profound iron redistribution within the inflamed tissue. While the local and infiltrating macrophages accumulated iron, the inflamed epithelial cells showed a state of significant iron deficiency and elevated iron flux, which was mimicked in the epithelial cell model of inflamed Caco-2 cells. The significant iron redistribution was accompanied by elevated levels of the transcription factor Hypoxia inducible factor (HIF)2a, by distinctive altered RNA-binding activity of both IRP1 and IRP2 in the different local cell types, and by altered levels of the proteins involved in the cellular iron homeostasis. These new dynamics of iron homeostasis were completely reversed by the deletion of IRP1 in the TNFDARE/+ mouse and the Crohn’s like picture of inflammation of the TNFDARE/+ mouse was not detectable in the TNFDARE/+ x IRP1-/- mice. In contrast, TNFDARE/+ x IRP2-/- mice showed severe transmural ileitis. Discussion: The non-iron mediated activation of IRP1 during inflammation triggered an impaired iron homeostasis within the inflammatory lesion that led to the accumulation of iron in the local immune cells. The iron accumulation in these cells and the iron depletion of the epithelial cells both support the recruitment of a systemic immune response and the propagation of an initially local and close to physiologic inflammation of the gut to a severe inflammation with massive infiltration of systemic immune cells.
B033 C-KIT IS EXPRESSED ON A SUBSET OF TH17 CELLS AND REGULATES THEIR IL-22 PRODUCTION Kalil A. de Lima, Jhimmy Talbot, Paula Barbim, Thiago Cunha, Jose´ Carlos Alves-Filho, Paulo Louzada, Fernando Q. Cunha
The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor. It is well know its involvement in TH17 cells polarization, but the AHR downstream signaling remains unclear. Previous data in RORct-dependent group 3 innate lymphoid cells suggest the receptor tyrosine kinase c-kit as a possible downstream target of AHR. Recently, c-kit expression was described in a proinflammatory human TH17 cells, however the c-kit/AHR interaction remains unclear in these cells. Thus, the aim of this study was to investigate the role of c-kit expression in TH17 cells and correlate it with the AHR pathway. We found that naive CD4+CD62L+CD44lo T cells differentiated in vitro into TH17 cells originated around 7–8 % of c-kit+ cells. Investigating the kinetics of c-kit expression under TH17-polarizing conditions we observed that c-kit expression was substantially upregulated 12 h after activation in the presence of TGF-b1 and IL-6, and its expression remained throughout the TH17 differentiation. The real time analysis to kit gene expression corroborate with protein analysis. To characterize the relevance of c-kit to TH17 differentiation during the course of an immune response, we studied the in vivo population expansion of TH17 cells after immunization. We immunized wild-type mice with MOG (35–55) and assessed the c-kit+ cells frequencies in the lymph node and central nervous system-infiltrating CD4+ T cells. Interestingly we also detected an expansion of c-kit-expressing TH17 cells. Additionally, we investigated the effect of c-kit signaling on TH17 differentiation using c-kit ligand and inhibitor, SCF and ISCK03 respectively. We did not observe any difference considering IL-17 production or expression. However, cells treated with c-kit inhibitor showed significant improvement in their IL-22 expression, as shown by their higher expression of Il22 and secretion of IL-22 into culture supernatants. Finally, to determine if AHR pathway has a role in c-kit expression during TH17 cells differentiation, we expose naive T cells to the AHR ligand FICZ and we observed that AHR activation enhanced c-kit expression as well as the frequency of c-kit+ cells. These results suggest that c-kit signaling can be important to negatively regulate IL-22 production on TH17 whereas AHR activation can modulate c-kit expression in these cells.
B034 WONDERBUMIN: A FULLY SYNTHETIC ALTERNATIVE TO IgT FOR AUTOIMMUNE DISEASE Anne S. De Groot1,2, Filipa Antunes3, Eduardo Guillen1, Sandra Lelias1, Ryan Harvey1, Christine Boyle1, William Martin1, Paul Barrett3, Darrell Sleep3
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EpiVax, Inc., Providence, USA; 2University of Rhode Island, Rhode Island, USA; 3Novozymes Biopharma Ltd, Nottingham, USA
Purpose: Immunoglobulin G Therapy (IgT) is the standard of care for several autoimmune diseases but it is not well tolerated by patients due to systemic affects associated with the injection of large amounts of Ig. The use of IgT is expanding because it activates and expands Tregs, which are critical to the control of autoimmune disease (AID). We identified Treg epitopes, now called Tregitopes, contained in conserved framework regions of IgG Fab and Fc. Tregitopes exhibit high affinity binding to multiple human HLA Class II DR and are conserved across mammalian species. Tregitopes activate CD4+/ CD25hi/FoxP3+ Tregs and expand antigen-specific iTregs. We report here on the development of a Tregitope-albumin fusion as a potential alternative to IgT. Methods: EpiVax applied in silico immunoinformatics tools to develop a polypeptide concatamer of Tregitopes for incorporation into Novozymes’ VeltisTM platform—a drug delivery platform that combines therapeutically active molecules with the long half-life of human serum albumin. This Tregitope-Albumin fusion product was evaluated for efficacy in a mouse model of OVA immunogenicity where OVA elicits a robust T cell response. T cell proliferation (CFSE) and phenotyping assays were performed following treatment of OVA-immunized mice with Tregitopes. Results: The mechanism of Tregitope-mediated tolerance induction appears to be as follows: (1) Tregitopes are delivered to antigen presenting cells, possibly through their FcRN, which then present Tregitopes on MHC Class II, (2) Treg recognizes them through their T cell receptors and are activated, proliferate and produce IL-10, (3) Treg provide tolerogenic feedback signals to APC, modulating the APC phenotype, and (4) Treg and tolerogenic APC work together to suppress antigen-specific T cell responses. Conclusion: Tregitopes have been shown to be equivalent to IVIG in a number of murine models, and to induce bystander tolerance in OVA immunization studies in mice. In addition to these pre-clinical studies, one clinical study has been performed using a single Tregitope, demonstrating 30 % efficacy (Sthoeger et al. Journal of Autoimm. 2009). Tregitopes formulated as an albumin fusion may represent an important advance toward a fully synthetic alternative to IgT. Supported by NIH SBIR Phase II R44 DK081261
Inflamm. Res. that murine memory CD4+ T lymphocytes migrate specifically in response to 7 a25-OHC via EBI2. Indeed, IL-17 producing EBI2-/memory CD4+ T cells depicted delayed migration to the central nervous system compared to their wild type counterparts. However, the expression and the role of EBI2 in human T lymphocytes, which are crucial cells in driving the adaptive immune response, have not been studied. Methods: the expression of EBI2 on healthy human peripheral blood mononuclear cells was measured by flow cytometry using a specific anti-human EBI2 antibody. The function of EBI2 in cell migration was assessed using a transwell assay. Results: We observed maximal EBI2 expression on memory CD4+ T cells; memory subsets of B and CD8+ T cells also depicted a modestly increased EBI2 expression compared to naı¨ve populations. Transwell migration assay experiments showed maximal migration of memory CD4+ T cells in response to 7a25-OHC. Even if globally less responsive to 7a25-OHC than the latter, memory subsets of B and CD8+ T cells were also found to migrate more strenuously than their naı¨ve counterparts. This chemotaxis was specific to EBI2 as selective EBI2 inhibition unequivocally abrogated migration. Conclusion:: These data are in line with our murine results and suggest an important role for EBI2 in human T cells migration. Selective targeting of immune cells trafficking has become an important tool in the clinical setting to dampen autoimmunity, for example in the context of neuroinflammation such as multiple sclerosis. Uncovering the role of EBI2 in T cells trafficking, in particular its ability to direct human CD4+ T cells chemotaxis, may open new avenues for understanding the pathogenesis of autoimmunity and may promote novel therapeutic approaches.
B036 HEALTHCARE RESOURCE USE AND COSTS OF ADRENOCORTICOTROPIC HORMONE IN RELAPSES OF MULTIPLE SCLEROSIS Laurie S. Gold1, Patricia Schepman2, John Niewoehner2, Michael Philbin2, Ryan Hansen1 1
University of Washington, Seattle, WA, USA; 2Mallinckrodt Pharmaceuticals, Ireland, Republic of Ireland
B035 OXYSTEROLS AND HUMAN MEMORY LYMPHOCYTES: WHEN INTERACTION MEANS ATTRACTION Aure´lie Clottu1, Fanny Chalmin1, Caroline Pot1,2 1
University of Geneva, Geneva, Switzerland; 2Geneva University Hospitals, Geneva, Switzerland Objectives: to study the expression and function of the oxysterols receptor Epstein-Barr virus induced gene 2 (EBI2) in human lymphocytes. Background: Oxysterols, hydroxylated cholesterol metabolites, have recently been ascribed a role in promoting inflammation and in modulating the immune response. In this line we are interested in studying the role of oxysterols, in particular 7a25-hydroxycholesterol (7a25-OHC), which is the strongest ligand of the G-coupled receptor EBI2. EBI2 is functionally expressed on human macrophages, astrocytes and B cells, and participates to the latter positioning in the lymph nodes during an immune response. In the animal model experimental autoimmune encephalomyelitis, we recently showed
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Background: Multiple Sclerosis (MS) is an autoimmune, neuro-inflammatory disorder characterized by acute exacerbations (‘relapses’), interspersed between remissions. MS relapses impose a heavy economic burden on society and are commonly treated with intravenous methylprednisolone (IVMP), and for some difficult-tomanage relapses, other hormonal therapies such as, adrenocorticotropic hormone (ACTH, H.P. Acthar Gel), intravenous immunoglobulin (IVIG; off label) and plasmapheresis (PMP). Objective: We explored the economic burden accrued among patients with difficult to treat relapses, comparing ACTH versus IVIG or PMP in this study over a 12- and 24-month follow-up period. Method: A retrospective analysis of commercial health insurance claims of MS relapses was conducted using Truven Health Analytics MarketScan database. Patients with C2 MS relapse episodes between 2007 and 2012 were identified; the first relapse was treated with IVMP and following relapses were treated with ACTH, IVIG, or PMP. The first calendar date of the second treated relapse was the index date. Patients with continuous health plan enrollment for 6-months prior and 12-months post index date were included, with a subset who were able to be followed for 24 months. We estimated the healthcare resource use and costs (inpatient, outpatient, and pharmacy) separately for patients whose second relapse was treated with
Inflamm. Res. each treatment option and then compared ACTH to patients who received either IVIG or PMP. Multivariate linear regression models were constructed to adjust for measured baseline patient characteristics and prior resource use. Results: A total of 445 MS patients had 12 months of continuous enrollment after their second MS relapse. 219 (49 %) patients were treated with ACTH and 226 (51 %) patients with IVIG/PMP for their second relapse. Of those, 231 had 24 months of continuous follow-up data, 99 (43 %) Acthar and 132 (57 %) IVIG/PMP. Patients who were treated with ACTH had a significantly lower number hospitalizations (-0.4, 95 % CI -0.6 to -0.2) and outpatient visits (-17, 95 % CI -22 to -11) in the first 12 months, along with accompanying lower costs for those resources, with similar total costs at 1 year. ACTH patients also had significantly fewer relapses (-0.9, 95 % CI -1.2 to -0.6). In multivariate linear regression, the significant differences in reduced inpatient costs, relapses, outpatient encounters, and outpatient costs remained. Additionally, total costs were still similar between the two groups in adjusted analyses. The findings of reduced outpatient services and costs and comparable total costs were consistent among the subgroups with 24 months of continuous follow-up data. Conclusion: Difficult to manage MS relapses pose a challenge to clinicians and patients. We found that treating these relapses with ACTH is associated with decreased resource use and similar costs compared to IVIG or PMP, thereby supporting the value of ACTH in MS relapse.
B037 USE OF A BALANCED DUAL CYCLOOXYGENASE-1/2 AND 5-LYPOXYGENASE INHIBITOR IN EXPERIMENTAL COLITIS Alessandra Bitto1, Giovanni Pallio1, Gabriele Pizzino1, Domenica Altavilla2, Francesco Squadrito1 1 Department of Clinical and Experimental Medicine, Section of Pharmacology, Medical School, University of Messina, Messina, Italy; 2Department of Paediatric, Gynaecological, Microbiological and Biomedical Sciences, University of Messina, Messina, Italy
Cyclooxygenase (COX) and 5-lipoxygenase (5-LOX), and their products PGE-2 and LTB-4, play an important role in inflammatory bowel diseases (IBDs). Therefore we investigated the effects of Flavocoxid, a dual COX/LOX inhibitor, in experimental colitis induced with either dinitrobenzenesulfonic acid (DNBS), or dextrane sulphate sodium. Flavocoxid effect was compared with the 5-LOX inhibitor, Zileuton. In the DNBS model, colitis was induced in 21 rats by a single intra-colonic instillation (25 mg in 0.8 mL 50 % ethanol); after 24 h animals were randomized to receive, by gavage twice a day, either Flavocoxid (10 mg/kg), or Zileuton (50 mg/kg), or vehicle. Sham (n = 7) or DNBS-vehicle (n = 7) animals received 0.8 ml of saline or 50 % ethanol, respectively. Rats were sacrificed 4 days after induction and samples were collected for analysis. In the DSS model, colitis was induced in 21 rats by the administration of 8 % dextran sulfate sodium dissolved in drinking water; after 24 h animals were randomized to the same above reported treatments. Sham animals (n = 7) received standard drinking water. Rats were sacrificed 5 days after induction and samples were collected for analysis.
S117 Flavocoxid and Zileuton improved weight loss, reduced colonic myeloperoxydase activity, macroscopic and microscopic damage, LTB-4, and TNF-a serum levels. Flavocoxid was the only treatment effective in reducing also malondialdheyde, PGE-2 levels, and apoptosis. In addition, histological features were strongly improved with Flavocoxid, as compared to Zileuton. Our research demonstrate the protective effect of Flavocoxid in IBDs, suggesting that this dual balanced inhibitor of COX and 5-LOX may represent a future treatment for inflammatory bowel diseases.
Biologic Therapies for Targeting Inflammatory and Immune Mechanisms B038 MYRIANTHUS ARBOREUS EXHIBITED ANTIINFLAMMATORY PROPERTIES IN EXPERIMENTAL MODELS Oluwafemi G. Oluwole1, Akinyinka Alabi2, Olatunde Ganiyu2 1 Department of Pharmacology and Therapeutics, Faculty of Basic Medical Science, College Of Medicine, University Of Ibadan, Ibadan, Nigeria; 2Department of Pharmacology and Therapeutic, College of Medicine, Olabisi Onabanjo University, Ago Iwoye, Ogun State, Nigeria
This study investigated the anti-inflammatory effects of ethanolic extracts of Myrianthus arboreus leaves in carrageenin-induced paw edema acute inflammation, formaldehyde- induced sub-acute inflammation, and lipopolysacharide (LPS) induced granuloma air pouch chronic inflammation in rats. Its membrane stabilizing activity was also evaluated. Rats (n = 5) were treated orally with M. arboreus (125, 250 and 500 mg/kg), Indomethacin (10 mg/kg) or distilled water (3 ml/ kg). Thirty minutes after treatment, acute inflammation was induced with sub-plantar injection of 0.1 ml of 1 % carrageenin into the right hind paw. The paw edema sizes were measured with the aid of plethymometer Ugo basile over a period of 3 h. Using similar technique, 0.1 ml of 2.5 % formaldehyde was induced for 3 days in sub-acute inflammation. Thereafter, anti-inflammatory effects of Marboreus was assessed based on neutrophil migration, volume of fluid exudates, also by free radicals scavenging activities based on the levels of MDA, GSH, Catalase, Nitric oxides and Prostaglandin released in the inflammatory fluids derived from 5 day LPS induced air pouch granuloma chronic inflammation. Membrane stabilizing activity was evaluated spectrophotometerically using RBC lysis induced phenlhydazine, heat, and then hypotonic medium, Histology of pouch tissues and stomach mucosa sections were also examined. M. arboreus (250, 500 mg/kg) produced a significant inhibition of acute and sub-acute inflammation when compared with distilled water control; it also significantly stabilized membrane by preventing RBC lysis, as well having antioxidant properties and modulates markers of inflammation significantly. The results of this investigations suggested that M. arboreus certainly contain active compounds therapeutically useful as anti-inflammatory agents. Keywords: M. arboreus, anti-inflammatory, antioxidant, membrane stabilizing.
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B039 A MODIFIED GLYCOSAMINOGLYCAN, GM-0111 INHIBITS INFLAMMATORY MOLECULES RELATED TO PERIODONTITIS Won Yong Lee1, Justin R. Savage1, Abigail Pulsipher1,2, Narayanam Rao1, Thomas P. Kennedy3,1, Maria E. Ryan4, Glenn D. Prestwich2,1 1
GlycoMira Therapeutics, Inc., San Diego, USA; 2University of Utah, Salt Lake City, Utah; 3Tulane University, New Orleans, Louisiana; 4 Stony Brook University, New York, USA While approximately 30 % US adults are suffer from moderate to severe form of periodontitis, the available treatment options are far too limited. This chronic disease occurs due to a disruption of homeostasis between the microorganisms residing in the oral cavity and the host immune system. GM-0111 is a modified glycosaminoglycan and it has demonstrated anti-inflammatory effects on various inflammatory diesease models molecular targets and possibly offer alternative therapeutic choices for treating periodontitis. We investigated whether GM-0111, a modified GAG, could block molecular events important for the development of periodontitis and the resulting bone loss. First, we tested GM-0111 on RANKL-induced osteoclast formation and bone resorption in cultured mouse pre-osteoclasts. Next, we tested whether GM-0111 could block proinflammatory TLR2 and TLR4 signaling pathways in mouse macrophage RAW 264.7 cells and heterologously expressed HEK293 cells. Lastly, we investigated the antibacterial effects of GM-0111 on Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. GM-0111 blocked RANKL-induced osteoclast formation even at 300 ng/mL and it also reduced the resulting bone resorption. The antiosteoclastic effects of GM-0111 were independent of RANKL to RANK signaling. We also found that GM-0111 selectively targets TLR2 (IC50 of 1–10 ng/mL) than TLR4 (IC50 [ 100 lg/mL). By blocking TLR-mediated inflammatory pathways and inhibiting key process leading bone loss, we suggest that GM-0111 can provide a therapeutic potential to treat periodontitis.
B040 GM-0111, A MODIFIED GLYCOSAMINOGLYCAN, PREVENTS RADIATION-INDUCED MUCOSITIS Abigail Pulsipher1,2, Justin R. Savage1, Thomas P. Kennedy1,3, Glenn D. Prestwich1,2, Won Yong Lee1 1
GlycoMira Therapeutics, Inc., Salt Lake City, UT, USA; Department of Pharmacy, University of Utah, Salt Lake City, UT, USA; 3School of Medicine, Tulane University, New Orleans, LA, USA
2
Mucositis is an inflammatory disease that frequently develops during radiation and chemotherapy, posing serious challenges to patients undergoing cancer treatments. GM-0111 is a modified glycosaminoglycan that blocks various innate immune molecules such as TLRs and selectins. GM-0111 also broadly suppresses bacterial growth present in the oral cavity. We investigated whether these anti-inflammatory and anti-bacterial effects of GM-0111 could be exploited to prevent radiation-induced oral mucositis by testing in vitro and in vivo radiation-induced mucositis models (1). In vitro studies: human oral epithelial cells (HOEPs) and mouse macrophage cells were treated with GM-0111 and irradiated with X-rays (10–15 Gy), and cell viability was determined by staining cells with Annexin V/7AAD. (2) In vivo studies: BDF1 mice were subcutaneously treated
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Inflamm. Res. with saline or 30 mg/kg of GM-0111 once daily (Days 2–7). On Day 0, mice were irradiated with a single dose of x-rays at 20 Gy. On Day 8, we harvested tissues and determined the degree of inflammation by gross and histological examinations, as well as detection of myeloperoxidase (MPO). GM-0111 at 100 lg/mL significantly reduced x-ray-induced cell death in both HOEPs and macrophage cells. Mice treated with GM-0111 showed markedly reduced signs of inflammation in the tongue, with decreased infiltration of polymorphonuclear leukocytes in the mucosa, thicker epithelial layer, and normal mucous glandular morphology than the saline-treated group. MPO analysis confirmed our histological observations, with significantly (p \ 0.01) lower concentrations of MPO in GM-0111 treatment groups compared to vehicle-treated groups (saline/20 Gy 206 ± 77 vs. GM-0111/20 Gy 104 ± 27 mU/mg protein; normal 62 ± 23 mU/mg protein). We suggest that the cytoprotective effects of GM-0111 potentially reduce clinical signs of oral mucositis that frequently occur during cancer therapy.
B041 THE EFFECT OF DUPILUMAB ON BIOMARKERS IN THE PERIPHERAL BLOOD AND NASAL SECRETIONS IN THE TREATMENT OF CHRONIC SINUSITIS WITH NASAL POLYPOSIS Brian N. Swanson1, Leda Mannent2, Jennifer D. Hamilton3, Donghui Zhang1, Nian Tian1, Ying Wang1, Gabriele Holtappels4, Lars-Olaf Cardell5, Jeffrey E. Ming1, Neil Graham3, Gianluca Pirozzi1, Claus Bachert4 Sanofi, Bridgewater, NJ, USA; 2Sanofi, Chilly Mazarin, France; Regeneron Pharmaceuticals, Inc., Tarrytown, NY, USA; 4Ghent University Hospital, Ghent, Belgium; 5Karolinska Institute, Stockholm, Sweden 1
3
Background: Dupilumab, a fully human monoclonal antibody against interleukin-4 receptor alpha (IL-4Ra), potently inhibits both IL-4 and IL-13 signaling, and has shown efficacy in asthma and atopic dermatitis clinical trials. In patients with chronic sinusitis with nasal polyposis (CSwNP), dupilumab significantly reduced polyp scores, sinus CT scans (Lund-Mackay scores), and suppressed symptoms (ClinicalTrials.gov: NCT01920893). We now report the effect of dupilumab treatment on biomarkers related to type 2 helper (Th2) cell cytokine activity and eosinophil (Eos) inflammation in patients with CSwNP. Methods: Adults with CSwNP were treated with dupilumab (600 mg loading dose, then 300 mg subcutaneously weekly for 15 weeks) or placebo (PBO) for 16 weeks; each was added to daily treatment with intranasal mometasone furoate. Nasal secretions (NS) were collected using bilateral absorbent packings and then eluted into 3 mL of saline. Biomarkers in peripheral blood and NS were assayed using commercially available methodologies. Results: Addition of dupilumab treatment (N = 30), versus PBO (N = 30), significantly suppressed (least squares mean % change from baseline): serum thymus and activation regulated chemokine (TARC) from Week (W) 2 (-26.1 vs. +6.2, respectively; p \ 0.0001) to W12 (-23.1 vs. +6.4; p = 0.0001); plasma eotaxin-3 from W2 (-41.9 vs. -5.8; p \ 0.0001) to W16 (-35.5 vs. +10.0; p \ 0.0001) and serum total immunoglobulin (Ig)E from W8 (-29.1 vs. -2.5; p \ 0.0001) to W16 (-48.4 vs. +7.9; p \ 0.0001). In NS (saline eluates), Th2 biomarkers were lower after dupilumab versus PBO (mean % change from baseline): eotaxin-3 from W8 (-32.4 vs. +74.4, respectively; p = 0.017) to W16 (-38.5 vs. +183.0; p = 0.040); eosinophil cationic protein (ECP) at W12 (-2.5 vs.
Inflamm. Res. +102.6; p = 0.055) and W16 (+7.2 vs. +120.2; p = 0.072); and total IgE from W8 (-15.7 vs. +60.1; p = 0.043) to W16 (-15.9 vs. +43.8; p = 0.025). The mean serum ECP, unlike the mean NS ECP, did not show a decrease during dupilumab treatment. After dupilumab treatment, there were transient increases in blood Eos counts which resolved during treatment (mean % change from baseline: W4, +48.9 dupilumab vs. +0.2 PBO; W16, -5.8 vs, +3.8). Injection site reactions (ISRs), headache, and nasopharyngitis were the most frequently reported adverse events, with ISRs more frequent with dupilumab. Conclusions: Dupilumab suppressed IL-4Ra-dependent biomarkers in the peripheral blood and NS. Reductions in TARC and eotaxin-3 suggested that dupilumab may reduce homing of Th2 cells and Eos. Declines in IgE indicate that dupilumab modulates Ig switching away from an allergic phenotype. Relative decreases in eotaxin-3, ECP, and IgE in NS versus PBO demonstrated a local effect of dupilumab on nasal inflammation.
B042 TSPO GENE EXPRESSION ON 3T3-L1 DIFFERENTIATION PROCESS IS MODULATED BY DIAZEPAM TREATMENT AND LPS Eric D. Barioni, Rodrigo A. Loiola, Edson M. Oliveira, Ana Campa, Sandra HP Farsky University of Sa˜o Paulo, Sa˜o Paulo, Brazil Backgound: Obesity is a chronic disease associated to low intensity chronic inflammation, characterized by influx of macrophages, insulin resistance, hyperglycemia, dyslipidemia, hypertension, and others associated diseases. The 18 kDa translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor, has been implicated in many important physiological functions, including cholesterol transport and steroidogenesis, cellular respiration and immunomodulation. TSPO is expressed in immune cells, and considered a marker of inflammation. Moreover, TSPO is expressed on adipocytes, nevertheless its role in adipose tissue differentiation is not established. Hence, we here proposed to investigate the TSPO expression on adipocytes in different conditions. Methods: TSPO gene expression on the differentiation process of 3T3-L1 lineage was quantified by Real Time-PCR. The growth curve of 3T3-L1 pre-adipocytes treated with TSPO benzodiazepine agonist (diazepam, 1, 10, 100 and 1000 nM) and dimethylformamide (DMF, diazepam vehicle, 0.01 %) was evaluated by optical microscopy. The viability of 3T3-L1 pre-adipocytes treated with diazepam and vehicle was quantified by flow cytometer. TSPO gene and protein expression on the differentiation process of 3T3-L1 lineage treated with diazepam, vehicle and E. coli lipopolysaccharide (LPS, 10 ng/mL) was quantified by Real Time-PCR and western blot, respectively, and 3T3-L1 triacylglycerol storage was quantified by Oil Red staining and colorimetric assay. Results: Our results showed the positive regulation of TSPO complementary tapes on 3T3-L1 differentiation process. Furthermore, diazepam treatment induced TSPO gene expression (diazepam 10 nM) and did not evoke significantly changes in the growth curve and viability of 3T3-L1 pre-adipocytes. Finally, 3T3-L1 triacylglycerol storage was not modified by diazepam or LPS treatment. Conclusion: Our data show that TSPO is expressed on 3T3-L1 cells during the differentiation process, and its modulated by TSPO agonist and LPS. Further studies are being carried out to investigate the role of TSPO activation by different agonist on the adipogenesis process. Supported by FAPESP (2013/11027-7).
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B043 NOVEL ANTI-INFLAMMATORY EFFECT OF CONIFERYL ALDEHYDE BY SELECTIVE INHIBITION OF STAT1-INOS SIGNALING Muhammad Akram, Kim Kyeong-A, Ok-Nam Bae Hanyang University, Seoul, South Korea Coniferyl aldehyde (CA), a phenolic compound, can be found in several medicinal plants. Although the anti-oxidant and anti-radical activities of CA have been previously reported, its therapeutic potential remains to be elucidated. In this study, we tried to determine the anti-inflammatory activity of CA using in vivo and in vitro systems and identify the underlying molecular mechanisms. CA significantly reduced nitrite production, iNOS enzyme activity and iNOS expressions in LPS-stimulated murine macrophages, RAW 264.7 cells. While NF-kB and MAPKs (ERK1/2, JNK 1/2 and p38) pathways, the representative cellular pathways for iNOS induction, were not affected by CA, LPS-induced STAT1 phosphorylation and p-STAT1 nuclear translocation were significantly inhibited by CA. In in vivo mouse models, topical application of CA produced significant protective effects against TPA-induced ear edema. Systemic administration of CA also significantly reduced carrageenan-induced paw edema in rats, confirming its potent anti-inflammatory activity. CA significantly reduced carrageenan-induced iNOS induction and STAT1 phosphorylation in a dose dependent manner in paw edema samples. Taken together, we demonstrated a novel anti-inflammatory activity of CA and its selective inhibition of STAT1-iNOS signaling using in vivo and in vitro models, suggesting its potential therapeutic application in various chronic diseases associated with excessive production of nitric oxide. Keywords: Coniferyl aldehyde, anti-inflammatory activity, STAT1, iNOS Funding Source: None Disclosure Statement: None
B044 AN INVESTIGATION OF ANTI-INFLAMMATORY ACTIVITIES PRODUCED BY SCHISANDRIN A AND SCHISANDRIN B IN VITRO AND IN VIVO Pou Kuan Leong, Kam Ming Ko Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong, China The dried fruits of Schisandra chinensis and Schisandra sphenanthera are commonly used Chinese herbs for the treatment of inflammatory diseases. Schisandrin A (Sch A) and schisandrin B (Sch B) are the active ingredients isolated from the fruits of S. sphenanthera and S. chinensis, respectively. In the present study, we compared the mechanism underlying the anti-inflammatory activities produced by Sch A and Sch B, using cultured lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. The exposure to LPS (1 lg/mL) activated the c-Jun N-terminal kinase 1/2 (JNK1/2), p38 kinase and the nuclear factor-jB (NF-jB), induced the releases of tumor necrosis factor alpha (TNFa) and interleukin-6 (IL-6), with the resultant induction of inducible nitric oxide synthase (iNOS) as well as cyclooxygenase 2 (COX2) and increased production of nitric oxide (NO) in RAW264.7 cells, of which are indicative of an inflammatory response. Pre-incubation with Sch A and Sch B (50 lM, alone) for 6 h produced an anti-inflammatory action in
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S120 LPS-stimulated RAW264.7 cells, as evidenced by significant inhibitions on the release of TNFa and IL-6, the expression of iNOS and COX2 as well as the production of NO. Sch A and Sch B also suppressed the LPS-activated pro-inflammatory signaling cascade, including JNK1/2, p38 kinase as well as NF-jB in LPS-stimulated RAW264.7 cells, with the extent of inhibition afforded by Sch A being more prominent. The effect of Sch A and Sch B on the antioxidant response was also investigated. The result showed that only Sch B but not Sch A activated the nuclear factor (erythroidderived 2)-like 2, the master regulator of antioxidant system, in RAW264.7 cells. At 16 h after the Sch B exposure, the expression of thioredoxin 1 (TRX) was increased by 30 %. The ability of Sch B to induce TRX expression was associated with significant suppressions on the releases of TNFa and IL-6 as well as the inductions of iNOS, COX2 and NO in LPS-stimulated RAW264.7 cells. To confirm the results obtained from the cell-based study, the effect of single bolus dose (1 mmol/kg, p.o.) or long-term low dose (0.25 nmol/kg p.o. for five consecutive days) of Sch A/Sch B on kcarrageenan-induced paw edema in female ICR mice was examined. The long-term low dose treatment with Sch A and Sch B reduced the k-carrageenan-induced paw edema to a similar extent in mice. However, the treatment with a single bolus dose of Sch A (but not Sch B) significantly reduced the extent of paw edema in k-carrageenan-injected mice, suggesting the anti-inflammatory response induced by Sch A being more instantaneous. In conclusion, both of Sch A and Sch B were found to produce a direct anti-inflammatory action by the suppression of pro-inflammatory signaling cascade. In addition, Sch B may also reduce the extent of inflammation in an indirect manner, presumably via the induction of an antioxidant response.
B045 OVERVIEW OF NEW STRATEGIES FOR PATENTING BIOLOGIC AGENTS TARGETING INFLAMMATORY DISEASES
Inflamm. Res. The search results yielded 733 applications, with nearly 90 % of them filed by biotech and pharmaceutical companies. Even if the applications were widely dispersed, clearly the leader in the field is Amgen with about 3 % of the applications. Besides inflammatory diseases, the claims included the treatment of cancer (11.2 %), infections (3.4 %), cardiovascular disorders (2.9 %) and allergies (1.9 %), in addition to diagnostics (2.8 %). Biologicals can be categorized by the way they work in the body, thus in a large amount (29 %) of the applications the main matter found was antibodies, followed by therapeutic and fusion proteins (14 %). The methods of use together with dosage forms and compositions were claimed in 30 % of the applications. Around 25 % of the applications include signal transduction modulators as the core mechanism of action influencing the efficacy of biologic agents. Given that these drugs mainly inhibit specific components of the immune system that play pivotal roles in diseases, mechanisms for neutralizing cytokines were found in almost 30 % of the documents, including antibodies, soluble receptors, receptor blockade, and activation of anti-inflammatory pathways by bioengineered versions of several cytokines. Over the past decade, biologics have accounted for just about onethird of new drug approvals and many studies are under way investigating targets in diseases with inflammatory etiologies. The findings reported here can have important implications for both policy makers and executives involved in making strategic decisions on the pharmaceutical companies, in order to find better therapeutic options for patients, with enhanced clinical efficiency and reduced side effects.
B046 ADP TREATMENT IMPROVES WOUND HEALING IN DIABETIC MICE Claudia F. Benjamim, Paula B. Alvarenga, Janaı´na L. Georgii, Ariane R. Brogliato, Jose´ Roberto Meyer, Robson Coutinho, Josiane S. Neves Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
Iolanda M. Fierro2,1, Rodrigo A. Oliveria1, Fernando Tibau1, Natalia L. Von Ranke2, Adelaide Maria S. Antunes2,3 1
Universidade do Estado do Rio de Janeiro; Rio de Janeiro; Brasil; Instituto Nacional da Propriedade Industrial; Rio de Janeiro; Brasil; 3Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil 2
In 2014 five of the 10 top-selling drugs were biologics, a number expected to grow in the next coming years. Biologic agents (also termed biologicals or biologics) are therapeutics synthesized by living organisms and targeting distinct immune cell receptors or cytokines. Biologicals have changed the way to treat several inflammatory and immune-mediated disorders, including rheumatoid arthritis, inflammatory bowel disease and psoriasis. Patent data can be used as a source of information to measure science and technology activities. This work analyzes patent applications of biologics for the treatment of inflammatory diseases, in order to identify technology trends and to better understand the strategies adopted by the main actors in the development of new formulations of these compounds. The technological forecasting focused on patent applications between 2010 and 2015, considering that most of these medicines have gained approval only within the last few years. The search strategy to recover these documents was based on predefined keywords using the database Thomson Reuters Integrity SM that indexes patent applications in the World Intellectual Property Office in addition to other six patent offices, covering the main world markets in the pharmaceutical field.
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Objectives and Backgrounds: Chronics wounds are a health problem worldwide, which affect 6.5 millions patients in USA. Such problem in association with high global prevalence of diabetes, reflects the increase in diabetic ulcers. Considering the absence of an effective treatment for chronic wound, we investigated the possible beneficial effects of purinergic agonists in tissue repair. In the present work we explore the role of ADP in healing process of skin chronic wounds in diabetic mice. Methods and results: In this study, Diabetes Mellitus was induced by a single intravenous Alloxan injection (65 mg/kg) 8 h after fast in Swiss male mice (20–24 g). Mice were then separated in four groups and treated once a day per 14 days as following: (a) control group: saline was topically applied on wound beds; (b) ADP group, 30 lM of ADP was topically applied on wound beds; (c) clopidogrel + saline: in which clopidogrel was given by gavage and saline was topically applied on wound beds; and (d) clopidogrel + ADP, in which clopidogrel was given by gavage and ADP was topically applied on wound bed. Wound contraction was measured at days 3, 7, 10, and 14 days after skin lesions. Wounds were collected at day 7, formalin fixed and paraffin-embedded. Skin sections were stained with HE (for granulation tissue), Sirius Red (for collagen), modified Sirius Red (for eosinophil), Alcian Blue (for mast cell), or immunostained for a-actin, laminin, macrophages, VEGF and TGF-a. Wounds were also collected at day 7 for MPO activity and ELISA. Our data showed that ADP was able to accelerate the wound healing of diabetic mice, which resembles the healing of non-diabetic mice. Clopidogrel treatment, a P2Y12 receptor antagonist, prevented the lesion closure in diabetic mice
Inflamm. Res. treated with ADP. Interestingly, clopidogrel worsened the lesion closure even in non-diabetic mice. Others nucleotides as adenosine, ATP, AMP50 and AMP30 at 30 lM did not accelerate the lesion healing in diabetic mice, as observed for ADP. It was observed by histological analysis that ADP treatment improved the granulation tissue formation, collagen synthesis and increased the recruitment of eosinophils and neutrophils, and the population of mast cells on the seventh day after the beginning of the lesion. ADP stimulated the release of the cytokine IFN-c on the third day and the IL-10 and IL-13 on the seventh day. In addition, at day 7 ADP was effective in increasing the differentiation of myofibroblasts and the expression of laminin, VEGF and TGF-a. Still in this time point, ADP seemed to increase the arginase + cells and to reduce iNOS + cells, which implies in the increase of M2 macrophages in the wound after ADP. Conclusion: Our results suggest that ADP accelerates the wound healing in diabetic mice and the mechanism seems to be the recruitment of inflammatory cells to the lesion and by positive modulation of the wound repair. Financial support CAPES, CNPq and FAPERJ.
B047 NEUTRALIZING THE PRO-METASTATIC IMMUNE RESPONSES OF MDSC AND T CELLS THROUGH B CELL TARGETING
S121 that control the chain of suppressive and pro-metastatic events, including the activation of MDSCs. This work was supported entirely by the Intramural Research Program of the NIH, National Institute on Aging 1. Olkhanud, P. B. et al. Tumor-evoked regulatory B cells promote breast cancer metastasis by converting resting CD4 T cells to T-regulatory cells. Cancer research. 71, 3505–3515. doi:10.1158/ 0008-5472.CAN-10-4316 (2011). 2. Bodogai, M. et al. Anti-CD20 antibody promotes cancer escape via enrichment of tumor-evoked regulatory B cells expressing low levels of CD20 and CD137L. Cancer research. doi:10.1158/ 0008-5472.CAN-12-4184 (2013). 3. Olkhanud, P. B. et al. Breast cancer lung metastasis requires expression of chemokine receptor CCR4 and regulatory T cells. Cancer Res.69, 5996–6004 (2009).
B048 ROLE OF COLLAGEN VI TO IMPROVE TISSUE INTEGRATION OF DENTAL IMPLANTS Ramesh Tati1, Veronika Dill1, Melissa N. Langer1, Suado M. Abdillahi1, Sara Nordin1, Maria Baumgarten1, Matthias Mo¨rgelin1, Eric Gerner2, Christina Gretzer2 1
Lund University, Lund, Sweden; 2Dentsply Implants, Sweden
Monica Bodogai1, Kanako Moritoh1, Catalina Lee Chang1, Christine M. Hollander3, Li Yang3, Robert Wersto2, Arya Biragyn1 1 NIH, National Institute on Aging, Laboratory of Molecular Biology and Immunology, Bethesda, MD, USA; 2NIH, National Institute on Aging, Flow Cytometry Unit, Bethesda, MD, USA; 3NIH, National Cancer Institute, Laboratory of Cancer Biology and Genetics, Bethesda, MD, USA
Although cancer metastasizes via expanding myeloid-derived suppressive cells (MDSCs) that suppress antitumor effector immune responses, our recent results questioned this simplistic view. The loss of cancer-induced B cells alone is sufficient to abrogate metastasis without affecting the expansion of MDSCs. We report that metastasis of a highly aggressive 4T1 breast cancer requires the help from regulatory B cells (tBregs) converted from resting B2 cells in response to cancer-produced metabolites of 5-lipoxygenase. The tBregs produce high levels of TGFb and convert CD4+ T cells into CCR4+ FoxP3+ Tregs that inactivate NK cells and CD8+ T cells protecting the lungs from metastasizing tumor cells. Here, using modelling studies with ex vivo generated MDSC and mice with different tumors, we report that tBregs play a previously unknown, but essential role in metastasis— they activate the regulatory function of cancer-primed MDSCs. To do this, cancer-induced B cells/tBregs directly activate the regulatory function of both Mo-MDSCs and PMN-MDSCs, requiring at least in part TgfbR signaling. The educated MDSCs increase production of ROS and NO and more efficiently suppress anti-tumor CD4+ and CD8+ T cells and thereby promote metastasis. As such, the loss of B cells or the TgfbR deficiency in MDSCs is sufficient to disable the education of MDSCs and concurrently block metastasis. Thus, cancer induced B cells need to be removed to improve antitumor immune responses. However, the B-cell neutralizing strategy should be approached cautiously, as depletion of B cells with anti-CD20 Ab/ rituximab caused enrichment of CD20Low 4-1BBLLow tBregs and thereby further enhanced lung metastasis by exacerbating tBreg-mediated immunosuppression. In contrast, we show that BLC-arp-coupled CpG-ODN can efficiently abrogate lung metastasis by inactivating tBregs and activating immunostimulatory B cells. Overall, these results further underscore the importance of B cells/tBregs as important factors
Background and objective: Followed by the surgical application of dental implants, oral pathogens are able to infiltrate the damaged tissue leading to infection in the oral cavity. Collagen type VI, a subepithelial extracellular matrix component has been shown to have adhesive properties as well as antimicrobial activity against pathogens. As a part of recent developments in the bioactive surfaces antimicrobial treated implants, made of titanium, may be beneficial in this case. The aims of the present study were to investigate the antimicrobial activity of collagen VI against invading pathogens and also its role in enhancement of tissue integration of dental implants. Methods: Human keratinocytes were cultured on collagen coated titanium discs followed by bacterial inoculation. Scanning electron microscopy was performed to check the keratinocyte morphology on titanium discs and also to detect bacterial killing in order to conform the antimicrobial activity of collagen VI. Results: Results showed that the keratinocytes had shown better attachment on modified titanium surface. In case of infection, keratinocyte survival was increased on collagen VI coated surfaces thereby confirming the antimicrobial activity of collagen VI against oral pathogens. Conclusions: Together, these data suggest that extracellular matrix components are able to protect the tissues from invading pathogens thereby improving the tissue integration onto the implant leading to improved performance.
B049 APPLICATION OF TREGITOPES FOR TREATMENT OF TYPE 1 DIABETES (T1D): OPTIMIZATION OF THE TREGITOPE DELIVERY VEHICLE Ann S. De Groot1,2, Leslie Cousens1, Christine Boyle1, Guilhem Richard1, William Martin1 1
EpiVax, Inc, Providence, USA; 2University of Rhode Island, Rhode Island, USA
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S122 HLA class II-restricted regulatory T cell (Trig) epitopes in IgG (‘‘Tregitopes’’) have been reported to suppress immune responses to co-administered antigens by stimulating expansion of natural Tregs (nTregs). In our previously published work we show that co-administration of Tregitopes and T1D antigens delayed development of hyperglycemia and reduced T1D incidence in NOD mice. T1D suppression was observed even following disease onset.1 Current studies aim to identify a suitable delivery vehicle and dosing regimen for Tregitope therapy. Using both an OVA-mouse model and NOD T1D mouse model, different delivery vehicles were used to co-deliver Tregitopes and antigen. The effects of these in vivo regimes on antigen-specific immune responses was measured in vitro. From these studies, a human serum albumin (HSA) Tregitope fusion emerged as our lead candidate. To examine the therapeutic potential of this delivery vehicle, HSA-Tregitope fusion was administered to NOD mice with blood glucose level between 200-350 mg/dL on two consecutive days. Mice that received the HSA-Tregitope fusion exhibit prolonged adjusted survival compared to other treatment groups. These results show promise for implementation of these defined regulatory T cell epitopes for therapy of T1D, and the HSA-Tregitope fusion as a potential treatment platform for the application of Tregitope therapy to other autoimmune diseases. 1. Cousens LP, Su Y, McClaine E, et al. Application of IgG-Derived Natural Treg Epitopes (IgG Tregitopes) to Antigen-Specific Tolerance Induction in a Murine Model of Type 1 Diabetes. Journal of Diabetes Research. 2013;2013:621693. doi:10.1155/2013/621693.
Cartilage and Bone Remodeling B050 THE ROLE OF RESOLVIN D1 IN THE REGULATION OF INFLAMMATORY AND CATABOLIC FACTORS IN OSTEOARTHRITIS Houda Benabdoune1,2, Shi Qin2, Julio Fernandes2, Pierre Ranger3, Hassan Fahmi1,4, Mohammed Benderdour1,2 1
Pharmacology department, Universite´ de Montre´al, Montreal, QC, ˆ pital du Sacre´-Cœur de Canada; 2Orthopedic research laboratory-Ho ˆ pital du Sacre´-Cœur de Montre´al, Montreal, QC, Canada; 3Ho Montre´al, Montreal, QC, Canada; 4Research Center-Centre hospitalier de l’universite´ de Montre´al, Montreal, QC, Canada Introduction: Osteoarthritis (OA) is a degenerative joint disease, characterized by cartilage and bone damage and accompanied by increased production of inflammatory and catabolic mediators such as prostaglandin-E2 (PGE2), nitric oxide (NO), metalloproteinase-13 (MMP-13), tartrate-resistant acid phosphatase (TRAP) and cathepsinK. Despite its important consequences, therapeutic modalities for OA are limited to symptomatic treatments. Resolvin-D1 (RvD1) is a derivative of the omega-3 fatty acids and a potent anti-inflammatory agent synthesized during the resolution phase of inflammation. The overall objective of this study is to investigate the anti-inflammatory, anti-catabolic and anti-resorptive role of RvD1 in OA. Methods: Post-surgery discarded human OA articular cartilage was obtained from OA patients who underwent total knee arthroplasty. First passage human OA chondrocytes, were treated with IL-1B with or without RvD1 (1-10 uM) for 24 h. The expression of cyclooxygenase-2 (Cox-2) and inducible NO synthase (iNOS) was determined by western blot and real-time PCR. Levels of PGE2, MMP-13 and NO were measured by EIA, ELISA and Greiss reaction, respectively. To investigate the signaling pathways, p38 MAPK, SAPK/JNK1/2, NF-kB/p65 activation was determined by
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Inflamm. Res. western blot. To induce osteoclasts (OC) recruitment, RAW264.7 cells were incubated with 50 ng/ml LPS with or without RvD1 (1-10 uM) for 24 h. OC phenotype markers, namely, TRAP and cathepsin-K were assessed by western blot, enzymatic staining and immunocytochemistry. Results: Our results show that RvD1 inhibits COX-2 and iNOS expression as well as PGE2, MMP-13 and NO generation. These effects are likely to be attributed to the blocking of the activation of p38 MAPK, SAPK/JNK1/2 and NF-kB/p65. Besides, RvD1 strongly reduces OC recruitment as indicated by the inhibition of TRAP and cathepsin K expression. Conclusion: Our in vitro results clearly show that RvD1 presents very interesting anti-inflammatory, anti-catabolic and anti-resorptive properties in OA. Additionally, our recent data that are not presented showed a remarkable anti-apoptotic and antioxidant potential of RvD1 in OA chondrocytes. Taken together, these findings suggest that RvD1 may offer a novel and original perspective to make a real contribution to OA therapy. Acknowledgement Centre of excellence for arthroplasty research (RACE).
B051 EFFECT OF ATORVASTATIN ON INFLAMMATORY BONE LOSS IN RATS WITH GLUCOCORTICOID-INDUCED OSTEOPOROSIS: PARTICIPATION OF WNT/ß-CATENIN PATHWAY Karuza MA Pereira1, Karianne M. Mendonc¸a1, Luzia HT Sousa1, Ana L. Franc¸a1, Eveline Linhares1, Pedro H. Isaias1, Ana GF Linhares1, Danielle RD Val2, Raul S. Freitas1, Mirna M. Bezerra1, Hellı´ada V. Chaves1, Gerly Anne DC Brito1, Conceic¸a˜o DS Martins1, Joanna T. Magalha˜es1, Paula Goes1 Federal University of Ceara´, Fortaleza, Brazil; 2Federal University of Pernambuco, Recife, Brazil 1
Periodontal disease (PD) is a chronic infectious-inflammatory disease that causes loss of connective tissue and alveolar bone loss. Glucocortioid-induced osteoporosis (GIOP) is the main cause of secondary osteoporosis. Atorvastatin (ATV) is a hypolipemiant drug that has also present anti-inflammatory activity and bone anabolic effects. It has been recently reported the WNT/b-catenin signaling might have a role on osteoblastic differentiation. Therefore the aim of this work was to evaluate the effect of Atorvastatin on inflammatory bone loss in rats with glucocorticoid-induced osteoporosis and the participation of WNT/b-catenin pathway on this condition. Experiments were approved by the Institutional Animal Care and Use Committee of the Federal University of Ceara´, Fortaleza, Brazil (49/2012). The animals were divided in four groups: PD receiving 0.9 % saline solution (2 mg kg-orally); GIOP: (7 mg/kg of dexamethasone once/wk for 5 weeks – IM); GIOP + PD; ATV: GIOP + PD (27 mg/kg of ATVorally). Parameters evaluated: (1) Alveolar bone loss (ABL), through macroscopic and radiographic analysis; (2) Radiographic density of femur; (3) Leukogram; (4) Bone-Specific Alkaline Phosphatase (BALP); (5) Transaminases (TGO/TGP); (6) Histological analysis; (7) Immunohistochemistry for WNT 10b, b-catenin, DKK1; (8) Gingival cytokine level IL-1b and TNF-a; (9) Myeloperoxidase levels in gingival tissue. The animals from GIOP + PD group showed greater ABL (7.53 ± 0.75 mm2) compared to PD (5.24 ± 0.39 mm2) and ATV prevented ABL (4.68 ± 0.39 mm2) compared to GIOP + PD (p \ 0.05). The radiographic density analysis of maxillae corroborated the macroscopic analysis. ATV prevented the reduction of radiographic density of femur (173.91 ± 4.87 grey tones) when
Inflamm. Res. compared to GIOP + PD (158.48 ± 5.38). ATV prevented the leukocytosis/neutrophilia (L = 8.5 ± 0.75 cell/mm3/N = 3.1 ± 0.53) seen in GIOP + PD group (L = 15.89 ± 1.78/N = 6.3 ± 0.46). ATV treatment did not alter transaminases serum levels. ATV prevented BALP reduction (25.56 ± 1.73 U/L) when compared baseline (29.52 ± 0.93 U/L). GIOP + DP showed intense inflammatory infiltrate and bone loss (3 [3–3]) and ATV prevented these microscopic findings (1 [1–2]) (p \ 0.05). It was seen greater immunolabeling for WNT and beta-catenin and reduced immunolabeling for DKK on ATV group when compared to GIOP + PD group. ATV prevented the increase on TNF (2.53 ± 0.09 pg/ml) and IL-1 (4.50 ± 0.17) levels on gingival tissue, when compared to GIOP + PD (TNF = 4.61 ± 0.53; IL-1 = 6.33 ± 0.31) (p \ 0.05). ATV prevented the increase on MPO levels in gingiva. In this way we can conclude that ATV has an anti-inflammatory effect and prevents alveolar bone loss in GIOP + PD conditions through WNT/b-catenin signaling pathway. Funding Sources FUNCAP, CNPq, CAPES.
Cell Adhesion and Leukocyte Migration B052 ACUTE EXPOSURE TO APOLIPOPROTEIN A1 INHIBITS MACROPHAGE CHEMOTAXIS IN VITRO AND MONOCYTE RECRUITMENT IN VIVO Asif J. Iqbal1, Tessa J. Barrett3, Lewis Taylor1, Eileen McNeill1, Carlota Reico1, Gemma E. White1, Maximillian H. Brodermann1, Dianne Cooper2, Keith M. Channon1, David R Greaves1 1 University of Oxford, Oxford, UK; 2Queen Mary’s University of London, London, UK; 3NYU School of Medicine, New York City, NY, USA
Apolipoprotein A1 (apoA1) is the major protein component of high density lipoprotein (HDL) and has been reported to have anti-inflammatory properties in addition to its role in reverse cholesterol transport. To better understand the anti-inflammatory activity of apoA1 we explored the effects on monocyte and macrophage chemotaxis in vitro and monocyte trafficking in vivo. Acute apoA1 treatment (20–60 min) induced a substantial (50–90 %) decrease in murine macrophage chemotaxis in real-time assays to a range of pathophysiologically relevant chemoattractants (CCL2, CCL5, chemerin and complement C5a). In addition, human monocyte chemotaxis to CCL2 was abrogated by short-term treatment with apoA1, and human PBMC exposed to apoA1 for 45 min showed reduced rolling, adhesion and transmigration in experiments with activated HUVEC monolayers under physiological flow conditions. Remarkably, acute pre-treatment of mouse GFP+ monocytes with apoA1 ex vivo reduced recruitment by 65 % (p \ 0.01) following adoptive transfer into an on-going peritonitis model. Macrophages exposed to acute pre-treatment with the non-specific cholesterol depleting agent methyl-ß-cyclodextrin or macrophages from ATP-binding cassette transporter A1 (ABCA1) null mice display a similar reduction in macrophage migration, indicating that the mechanism for A10 s effects is independent of its role in reverse cholesterol transport. We also demonstrate that the rapid effects of apoA1 on monocyte/macrophage recruitment occur via reducing the activity of PI3kinase, a signalling pathway important for actin-cytoskeleton reorganisation. Collectively our data support a model in which rapid depletion/reorganisation of membrane cholesterol independent of ABCA1
S123 transporters renders monocytes/macrophages no longer sensitive to chemoattractants. Our data suggests that acute infusion of apoA1 or apoA1 mimetics could significantly reduce inflammatory myeloid cell recruitment via effects on responsiveness to multiple chemoattractants.
B053 TRPV4 MEDIATES MATRIX STIFFNESSDEPENDENT ENDOTHELIAL ACTIVATION Harry A. Scott, Xiao Yang, Soroush Ardekani, Andrea Cabrera, Kaustabh Ghosh University of California Riverside, Riverside, CA, USA Endothelial activation during inflammation, resulting from impaired activity of eNOS, is a key determinant of various conditions such as emphysema, diabetes and atherosclerosis. Past efforts to understand impaired NO-dependent endothelial activation have focused on genetic factors, soluble cues, and shear stress. However, these inflammatory conditions are also marked by abnormal subendothelial matrix. To determine whether matrix stiffness alone regulates endothelial activation, ECs were plated on fibronectin-coated polyacrylamide (PA) gels to normal (1000 Pa), softer (200 Pa), and stiffer sub-endothelial matrices (4000 Pa). Here we found, abnormal matrix stiffness induces increased monocyte-EC adhesion which correlates strongly with decreased phospho-eNOS and NO production. Importantly, this adhesion, at least in part, is mediated through stiffness-dependent biphasic ICAM-1 clustering with increased clustering on soft and stiff matrices. Notably, we show for the first time, stiffness-dependent variations in TRPV4 activity. This differential activity influences NO production, as determined by TRPV4 inhibition. Further, we show that stiffness-dependent endothelial activation can be reversed by TRPV4 modulation. These findings indicate TRPV4 as a key mediator in matrix stiffness-dependent endothelial activation and implicate it as a therapeutic target for antiinflammatory strategies.
B054 DOCK8 EXPRESSION IN DENDRITIC CELLS DIFFERENTIALLY REGULATES CYTOTOXIC T LYMPHOCYTE RESPONSES DURING INNOCUOUS AND PATHOGENIC CHALLENGES Manuela Sales Lima Nascimento1,2, Dong Liu1, Pei Chen1, Lan Xu1, Jayendra Kumar Krishnaswamy1, Samuele Calabro1, Gowthaman Uthaman1, Antonia Gallman1, Joa˜o Santana da Silva2, Stephanie C. Eisenbarth1 1
Yale University School of Medicine, New Haven, Connecticut; Ribeira˜o Preto Medical School, University of Sa˜o Paulo, Sa˜o Paulo, Brazil
2
DOCK8 is a guanine nucleotide exchange factor (GEF) that activates Rho GTPase CDC42, playing an important role in controlling cell cytoskeletal function and migration capacity. DOCK8 deficiency leads to an immunodeficiency syndrome characterized by severe atopy, early malignancy and recurrent viral infections in humans. We have previously shown that the absence of Dock8 results in defective DC migration, which also abrogates CD4+ T cell activation. The role of DOCK8 on DC migration and the subsequent impact on CD8+ T
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S124 cells responses, however, remain unclear. To study this, we took advantage of protein antigen immunization and a bacterial infection murine model. By adoptively transferring OVA-specific TCR transgenic OT-1 T cells into both WT and Dock8-/- mice, we demonstrated that CD8+ T cell priming is not impaired in contrast to a highly attenuated CD4+ OT2 T cell response in Dock8-/- mice after OVA immunization. Moreover, ovalbumin coated splenocytes (OCS) immunized Dock8-/- mice generated comparable amount of cytotoxic T lymphocytes (CTLs), which produce normal levels of IFN-c and granzyme-B, and are equally capable killing target cells in vivo. Surprisingly, our preliminary results suggest that Dock8-/mice infected with Listeria monocytogenes (L. monocytogenes) showed a defect in CD8+ T cell proliferation. Altogether, these data suggest that although DOCK8 is dispensable for CD8+ T cell priming during a noninvasive antigen challenge, it might be required for CTL priming to bacterial-derived antigens. Therefore, our data highlight the critical role of DOCK8 for an effective adaptive immune response.
Inflamm. Res. this pivotal enzyme in mediating some of Met’s anti-inflammatory effects. Conclusion: Protection of the glycocalyx by Met via activation of AMPK may contribute to its anti-inflammatory activities and help explaining, in part, its cardiovascular benefits. Financial support Canadian Institutes of Health Research (CIHR), Canadian Foundation for Innovation (CFI), Fonds de la Recherche du Que´bec-Sante´ (FRQS) and the Wilbrod-Bherer Foundation.
B056 CHEMERIN: INFLAMMATORY BIOMARKER OR ANTI-INFLAMMATORY MEDIATOR? Daniel Regan-Komito, Asif J. Iqbal, David R. Greaves University of Oxford, Oxford, UK
B055 METFORMIN MODULATES NEUTROPHIL MIGRATION: IMPLICATION OF ENDOTHELIAL CELL AMPK, ADHESION PROTEIN EXPRESSION AND GLYCOCALYX INTEGRITY Genevie`ve Bertheau Mailhot, Giambelluca S. Miriam, Cynthia Laflamme, Nathalie Bourcier, Aline Dumas, Benoıˆt Mailhot, Luc Vallie`res, Steve Lacroix, Marc Pouliot Centre de recherche du CHU de Que´bec-Universite´ Laval, Quebec City, Que´bec, Canada Introduction: Metformin (Met), a drug of the biguanide class, is a first-line treatment for diabetes mellitus type 2. Beside its bloodglucose controlling effect, Met ameliorates a number of associated complications such as cardiovascular diseases, suggesting anti-inflammatory properties. In the present study, we investigated the impact of metformin on acute inflammatory responses. Methods: Air pouch, and dextran sulfate sodium (DSS) murine models were used to measure neutrophils migration, viability and activation. Mice received DSS (3.5 %) in drinking water, with or without Met 50 mg kg-1. Myeloperoxidase assays and immunohistochemistry were performed on colon tissues. In adhesion experiments, human microvascular cells (HMEC-1) were stimulated with LPS (0.01–5 lg mL-1), TNF (1–30 gg mL-1) or IL-1b (3–100-gg mL-1) with or without Met (0.001–3 mM). Human neutrophils were obtained from healthy donors and incubated with Calcein-AM. ELISAs quantified the surface expression of adhesion proteins and glycocalyx components. Results: In the air pouch model, Met reduced neutrophils migration by up to 50 % to the site of injection. We also observed a reduction in the DSS model, with significant decreases of myeloperoxidase activity and neutrophil numbers in colon tissues, in presence of Met. In vitro, Met reduced adhesion of neutrophils to HMEC-1 in a concentration-dependent manner, by up to 50 %, without significantly affecting the expression of adhesion proteins. Instead, Met appeared to prevent degradation of the endothelial cell glycocalyx induced by inflammatory agonists, as measured by a reduction in the release of glycocalyx components. Incubation of HMEC-1 with an activator of 50 adenosine monophosphate-activated protein kinase (AMPK), 5-amino-4-imidazole carboxamide riboside (AICAR), reproduced the effects of Met in that system, supporting a role for
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Background: Human pro chemerin (E21-S163) is a 142 amino acid protein that circulates in plasma in an inactive form. During inflammation, the carboxyl terminus is cleaved by serine proteases to generate shorter bioactive forms of the protein which act as chemoattractants for macrophages, immature DCs, pDCs, and NK cells. Chemerin has been reported to play a role in the recruitment DCs, pDCs and NK cells to secondary lymphoid organs as well as sites of inflammation. A number of studies have reported elevated chemerin levels in patients with inflammatory and metabolic diseases while others have reported anti-inflammatory effects of chemerin. We have analysed endogenous chemerin levels during murine inflammatory disease models as well as the effects of modulating endogenous chemerin levels therapeutically. We hypothesised that during inflammation active murine chemerin T17-S156(corresponding to Chemerin E21-S157 in humans) is further cleaved by inflammatory proteases to generate shorter bioactive peptides with anti-inflammatory properties. Materials and methods: C57Bl6/J mice were injected intraperitoneally with 100 lg of zymosan. Mice were treated with chemerin, dexamethasone or PBS at different time points. The peritoneal cavity was lavaged with ice cold PBS supplemented with 2 mM EDTA and peritoneal exudate cells were quantified using flow cytometry. Chemokines, cytokines and chemerin levels were measured using ELISA/Luminex. Plasma was also analysed for chemerin levels. Results: Injection of 100 lg zymosan elicited a sterile inflammatory response in which neutrophils peaked at 4 h and monocytes/macrophages peaked at 8 h. T and B cells peaked in the peritoneum at later stages (9 and 13 days). Endogenous chemerin levels increase both locally and systemically during the onset of inflammation while dexamethasone significantly decreased chemerin levels. Pre-treatment with recombinant murine chemerin had no effect on monocyte recruitment. However, when administered as an intervention into an inflammatory environment chemerin decreased monocyte recruitment by *27 % (P B 0.001) as well as CCL4 levels by *30 % (P B 0.05). Using mass spectrometry we have demonstrated that murine chemerin can indeed be further processed to shorter bioactive peptides by the inflammatory neutrophil derived proteases elastase and cathepsin-G. Conclusions: When chemerin was administered as a pre-treatment, there were no significant effects on monocyte recruitment. However, when administered into an inflammatory environment, we observed significant decreases in monocyte numbers and chemokine levels. We have also demonstrated the ability of neutrophil derived proteases to cleave active chemerin into shorter bioactive chemerin peptides. Taken together our experimental data are consistent with further processing of chemerin (T17-S156) by proteases present at sites of
Inflamm. Res. inflammation generating shorter anti-inflammatory peptides, which may prove to have therapeutic benefit.
B057 SPIN90 DEFICIENCY INCREASES CXCL13MEDIATED B CELL MIGRATION Sang-Heon Park Gwangju Institute of Science and Technology (GIST), Gwangju, Korea Cell migration is important wound healing and immune Responses. T cells and B cells migrate to regions of the lymph nodes where they can interact with each other and facilitate T cell-dependent antibody production. SPIN90 is a ubiquitously expressed. SPIN90 binds to actin related proteins, such as WASP, which mediates the effect of CDC42 on actin cytoskeleton reorganization, and SPIN90-C-termius interacts with Arp2/3 complex, and G- and F-actin, and participates in the regulation of actin dynamics. CXCL13 is one of the lymphoid chemokines. CXCR5 is the receptor for CXCL13, required for lymphoid follicle formation, follicular helper T cell (Tfh) and T celldependent B cell activation. In addition, the chemokine-receptormediated signal attracts B cells and T cells into lymph nodes or spleen, towards the B cell or T cell zone of lymphoid follicles. CXCR5 deficiency in T cells leads to failure of plasma cell later stage and germinal centre reponses. CXCL13-mediated B cell migration into lymphoid follicles is important during B cell-mediated immune reponses. In this study, we investigated the role of SPIN90 in CXCL13mediated B cell migration using Spin90 gene-deficient mice. Our chemokinesis analysis and transwell cell migration assay showed that SPIN90 is involved in CXCL13-mediated B cell migration without affecting B cell development. Moreover, the level of CXCR5, which is CXCL13 receptor, was increased in SPIN90-deficient B cells compared with wild-type B cells. Overall, our data suggest that SPIN90 plays an important role in B cell immune responses through the regulation of CXCL13-mediated B cell migration.
B058 DIFFERENT MODES OF LFA-1 INHIBITION TRANSLATE INTO DIFFERENT DOWNSTREAM EFFECTS IN VITRO Riccardo V. Mancuso1, Stephan Kra¨henbu¨hl1, Gabriele Weitz-Schmidt2 1
University Hospital Basel, Division of Clinical Pharmacology and Toxicology, Hebelstrasse 20, Basel, Switzerland; 2AlloCyte Pharmaceuticals AG, Hochbergerstrasse 60C, Basel, Switzerland Objectives: The integrin lymphocyte function-associated antigen-1 (LFA-1) mediates the recruitment of leukocytes to sites of inflammation and is a promising therapeutic target. Three classes of LFA-1 inhibitors with different modes of action have been identified: (1) Monoclonal antibodies (mAbs), (2) ligand mimetics, and (3) allosteric inhibitors. The objective of this study was to compare these different types of LFA-1 inhibitors for downstream effects in vitro. Methods: The effects of the LFA-1 inhibitors on (1) cell adhesion, (2) the LFA-1 conformational status and (3) the surface expression of LFA-1 and other immune receptors were analysed. Cell adhesion was
S125 quantified by measuring the binding of fluorescently labelled leukocytes to immobilized intercellular adhesion molecule-1 (ICAM-1), the major ligand of LFA-1. The activation status of LFA-1 was monitored by quantifying the binding of the conformation-sensitive mAb MEM148. The impact of the inhibitors on the expression of LFA-1 and other immune receptors was investigated by flow cytometry and ImageStreamxtechnology. Results and conclusion: All inhibitors had in common that they blocked LFA-1-mediated leukocyte adhesion to ICAM-1, as expected. However, the ligand mimetic induced the LFA-1 activation epitope MEM148 in contrast to the allosteric inhibitor and the antiLFA-1 antibody. Moreover, the antibody and the ligand mimetic triggered internalization of LFA-1 while the allosteric inhibitor did not affect the surface expression of LFA-1. Changes in LFA-1 surface expression were found to be associated with changes in T-cell receptor expression. In conclusion, the differential effects of these LFA-1 inhibitors representing different modes of action may need to be taken into account during their further pre-clinical and clinical development.
B059 THE INFLUENCE OF MELANOCORTINS ON PLATELET LEUKOCYTE INTERACTIONS Felicity NE Gavins, Paul M. Holloway, Helen K. Smith, D.Neil Granger LSUHSC-S, Shreveport, LA, USA Aberrant inflammatory responses play a significant role in the initiation, maintenance and progression of a wide variety of both chronic and acute diseases. Further to their crucial role in haemostasis, it has become evident that platelets display a variety of inflammatory functions, acting both as target and effector cells, allowing cross talk between inflammatory and thrombotic processes. The melanocortin receptor system has demonstrated impressive anti-inflammatory properties and has been proposed as an endogenous system for the resolution of inflammation. Activation of melanocortin receptors can modulate a variety of inflammatory molecules also implicated in platelet functioning, however despite the intimate association between inflammation and thrombosis, the influence of melanocortin signaling on platelet functioning has not yet been investigated. As such we have utilized both in vivo and in vitro models of platelet-leukocyte cross talk to evaluate the potential influence of melanocortins on platelet inflammatory and thrombotic functioning. Melanocortin receptor dependant and independent effects were investigated using the super potent melanocortin receptor agonist, NDP-a-MSH and the melanocortin peptide terminal sequence KPV, which has previously been suggested to act independently of melanocortin receptor binding, instead acting as an IL-1b receptor antagonist. In a static leukocyte-platelet adhesion assay, treating murine neutrophils with either the melanocortin receptor agonist NDP-aMSH, or the terminal melanocrtin sequence KPV, was found to significantly reduce murine neutrophil adherence to IL-1B stimulated platelet monolayers. Furthermore platelets treated with KPV prior to IL-1B stimulation showed significantly reduced affinity to untreated neutrophils, further supporting KPV as acting as an IL-1b antagonist. However, neither treatment influenced thrombin stimulated or vehicle treated platelets. To further investigate leukocyte platelet interactions in vivo, intravital microscopy was performed in the inflamed cerebral microvasculature. C57BL/6 mice were treated with lipopolysaccharide (LPS) 4 h prior to visualizing the cerebral vessels and leukocytes
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S126 were visualized by i.v infusion of rhodamine 6G. Donor platelets were labeled with carboxylfluorescein succinimidyl ester (CSFE) and infused 5 min prior to imaging. LPS treatment was found to significantly increase both leukocyte and platelet adherence to cerebral microvessels. Treatment with KPV was found to significantly reduce LPS induced platelet adherence whilst also reducing both leukocyte rolling and adherence on the inflamed vasculature. This data suggests that melanocortin peptides may suppress platelet leukocyte interactions and that the terminal melanocortin sequence KPV may directly influence platelet function. The mechanisms by which melanocortins influence these events and the consequence on platelet thrombotic function are the subject of our on going investigations.
Chemokines and Chemokine Receptors B060 CCL3 DELETION PROTECTS MICE AGAINST ORAL SQUAMOUS CARCINOGENESIS
Inflamm. Res. molecules shaped by pathogens to escape the immune system, we previously identified a small compound, the chalcone 4. By analogy to the effect of neutralizing antibodies, this molecule is called neutraligand, as it binds to the chemokine CXCL12 but not to the cognate receptor CXCR4. Besides the regulation of homeostatic processes, CXCL12 is implicated in skin inflammation, in particular the Th2mediated atopic dermatitis (AD) disease. Given that this chemokine is involved in AD on one hand, and the number of pharmacological tools to investigate its function or to correct for defects is limited, it seems very interesting to use specific chemokine inhibitors like the chalcone 4 neutralizing CXCL12. So, we evaluated the in vivo activity of this compound in a MC903-induced AD model, and we showed that the topical application of chalcone 4 (350 lmol/kg) significantly reduced ear redness and thickness, as well as the infiltration of inflammatory cells into the skin. In addition, the expression of the Th2-related cytokines (L-4 and IL-5) in the skin, as well as plasma IgE levels were decreased in chalcone 4-treated mice. Altogether, our results showed that Th2 skin inflammation could be pharmacologically controlled with anti-inflammatory CXCL12 neutraligands. Chemical probes with such neutralizing activity could therefore be useful tools to understand and/or treat chronic inflammation.
Tarcilia A. Silva1, Janine M. Silva1, Aline C. Batista2, Mauro M. Teixeira1, Milene A. Rachid1, Remo C. Russo1
UFMG, Belo Horizonte, Brazil; 2UFG, Goiaˆnia, Brazil
1
The chemokine CCL3 displays a diversity of roles that may contribute to worse prognosis in oral carcinogenesis. The aim of this study was to evaluate the role of CCL3 in 4-nitroquinoline-1-oxide (4NQO)induced oral carcinogenesis. C57BL/6 (WT) and CCL3 deficient mice (CCL3-/-) male mice were treated with 4NQO during 20 or 28 weeks. The tongues were collected for macroscopic, histopathological and immunohistochemical analysis. Cytokine levels in tongue lesions were evaluated by ELISA. Our results showed a significantly higher incidence of tumors in tongue of WT- in comparison with CCL3-/–treated mice. Consistently, microscopic analysis demonstrated a pronounced cytological atypia in the WT-treated group. The immunoexpression of PCNA and Ki67 revealed a significant increase of the proliferative index in WT compared with CCL3-/- treated group. The concentration of CCL3, TNF-a, CCL5 and CCL11 was significantly enhanced after 4NQO treatment, but CCL3-/- treated mice showed lower TNF-a level when compared with WT. In conclusion, these data suggest a protective effect of CCL3 deletion in oral tongue carcinogenesis which may be associated with local changes in TNF-a production.
B061 COMBATING SKIN INFLAMMATION WITH CXCL12 CHEMOKINE NEUTRALIGANDS Dayana Abboud1,2 1
Biotechnologie et Signalisation Cellulaire, UMR 7242 CNRS/ Universite´ de Strasbourg, and Labex Medalis, ESBS, Illkirch, France; 2 Laboratoire d’Innovation The´rapeutique, UMR 7200 CNRS/ Universite´ de Strasbourg, and Labex Medalis, Faculte´ de Pharmacie, Illkirch, France Excessive signalling by chemokines has been associated with chronic inflammation or cancer, thus attracting substantial attention as promising therapeutic targets. Inspired by chemokine-clearing
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B062 ROLE OF INFLAMMATORY CHEMOKINE, CCL3, IN THE DOMINANT PROLIFERATION OF LEUKEMIA INITIATING CELLS IN CML BM Tomohisa Baba, Naofumi Mukaida Cancer Research Institute, Kanazawa University, Kanazawa, Japan Accumulating evidence suggests that a macrophage-derived inflammatory chemokine, CCL3, has multiple functions. Besides its proinflammatory activities, CCL3 can in vitro inhibit the proliferation of hematopoietic stem/progenitor cells (HSPCs). Based on this unique function, CCL3 is alternatively called as ‘‘stem cell inhibitor (SCI)’’. Moreover, CCL3 can in vivo rapidly induce the mobilization of HSPCs from bone marrow (BM) to peripheral blood (PB). Thus, CCL3 can potentially influence the homeostasis of HSPCs. In sharp contrast, CCL3 has few effects on leukemia initiating cells (LICs) in chronic myeloid leukemia (CML), which share several characteristic capabilities including self-renewal and cellular quiescence, with normal HSCs. It was reported that BCR-ABL-mediated tyrosine kinase activity can directly desensitize LICs to the SCI activity of CCL3. Thus, CCL3 can be a potent mediator to induce the dominant proliferation of LICs in the CML BM. However, it still remains to be investigated on the precise functions of endogenously-produced CCL3 in the CML pathophysiology and normal hematopoiesis. In the present study, we at first determined the dynamics of the BM HSPCs and PB cell population in the CCL3-deficient mice or BM chimeric mice arising from the transplantation with CCL3-deficient BM cells, in order to examine the role of endogenously-produced CCL3 in physiological hematopoiesis and hematological reconstitution. Of interest is that PB cell populations were excessively reconstituted in the BM chimeric mice transplanted with CCL3-deficient BM cells, despite of similar PB cell numbers in the physiological conditions between CCL3-deficient and wild-type (WT) mice. The HSPCs in BM transiently proliferated after BM transplantation with WT BM cells and this proliferation was further augmented when CCL3-defcient BM cells was used for BM transplantation. Hence, we assume that endogenous CCL3 has SCI activity only when the proliferation status of HSPCs is enhanced under the
Inflamm. Res. stress conditions such as hematopoietic reconstitution after sub-lethal irradiation. In order to delineate the role of CCL3 in the dominant proliferation of leukemia cells among normal hematopoietic cells, we established the non-irradiated mouse CML model, in which BCR-ABL gene-transduced LICs were directly transplanted into the BM cavity of non-irradiated mice. In this model, an initial CML-like leukocytosis was accompanied with a transient increase in serum CCL3 concentration. Moreover, the ablation of the CCL3 gene in LICs dramatically inhibited the development of CML. Furthermore, the genetic loss of the gene of either CCL3-specific receptor, CCR1 or CCR5, induced normal HSPCs to directly impede the maintenance of LICs in BM. Thus, these observations strongly suggest the crucial role of CCL3-mediated SCI activity in the competitive interaction between normal HSPCs and LICs in the CML BM.
B063 CCL4-CCR5 INTERACTIONS HAVE PIVOTAL ROLES IN SURVIVAL OF A MURINE BREAST CANCER CELL LINE IN OSSEOUS ENVIRONMENT Soichiro Sasaki Divison Molecular Bioregulation, Cancer Research Institute, Kanazawa University, Ishikawa, Japan Triple negative breast cancer (TNBC) is a subtype of breast cancer, which lacks the expression of estrogen receptor, progesterone receptor, and HER2. TNBC has poor prognostic outcome compared with other types of breast cancer, and frequently metastasizes to bone, thereby deteriorating the patients’ quality of life. However, the lack of curative therapy for bone metastasis necessitates understanding on the precise mechanisms controlling bone metastasis of breast cancer. Intravenous administration or intracadiac injection of breast cancer cells is widely used as bone metastasis model. However, with these maneuvers, a large number of tumor cells enter into bone even in the early phase of metastasis, whereas only a small number of tumor cells are presumed to be present in bone in the early phase. Thus, it is necessary to establish a breast cancer cell line, which can metastasize to bone upon its orthotopic injection into mammary fat pad, in order to elucidate the molecular and cellular mechanisms of bone metastasis, particularly at its early phase. From a murine TNBC breast cancer cell line, 4T1, we established a subclone which can metastasize to bone with a high probability upon its orthotopic injection. The resultant clone, 4T1.3, proliferated in vitro and in vivo at the primarily injected site, mammary fat pad, with similar rates as the parental clone did. However 4T1.3 cells exhibited resistance to anoikis and an increase in CD44+ CD24-/low cell population, which can represent cancer stem cells (CSCs). Moreover, compared with the parental clone, 4T1.3 cells exhibited a higher survival rate when injected directly into tibial osseous tissue, but no enhanced short-time migration ability, compared with the parental clone. Microarray analysis on in vitro cultured cells revealed that mRNA expression of a chemokines, CCL4, was enhanced in 4T1.3 clone, compared with the parental clone. CCL4 shRNA transfection reduced its tumor formation compared with control shRNA transfection, when injected into osseous tissues, whereas the same treatment failed to modulate CSC phenotypes of 4T1.3 cells. Tumor growth was reduced in mice deficient in CCR5, a specific receptor for CCL4, or wild-type (WT) mice transplanted with CCR5-deficient mouse-derived bone marrow cells, compared with WT mice or WT-derived bone marrow
S127 cell-transplanted mice, when 4T1.3 cells was injected into tibial osseous tissue. Given the lack of CCR5 expression by 4T1.3 clone, tumor cellderived CCL4 can act on CCR5-expressing bone marrow-derived normal cells to promote survival of 4T1 cells in bone marrow, and to accelerate bone metastasis.
B064 PROTECTIVE ROLE OF THE ATYPICAL CHEMOKINE RECEPTOR D6 DURING EXPERIMENTAL SEPSIS Fernanda VS Castanheira1, Fabiane Sonego1, Alexandre Kanashiro1, Vanessa F. Borges1, Paulo H. Melo2, Remo C. Russo3, Flavio A. Amaral4, Mauro M. Teixeira4, Gerard J. Graham5, Massimo Locati6, Thiago M. Cunha1, Jose´ C. Alves Filho1, Fernando Q. Cunha1 1
Department of Pharmacologym, School of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo-Brazil, Sa˜o Paulo, Brazil; 2 Department of Immunology, School of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo-Brazil, Sa˜o Paulo, Brazil; 3Department of Physiology and Biophysics, University of Minas Gerais-Brazil, Gerais, Brazil; 4Department of Biochemistry and Immunology, University of Minas Gerais-Brazil, Gerais, Brazil; 5Institute of Infection, Immunity and Inflammation, University of GlasgowScotland, Glasgow, Scotland; 6Department of Medical Biotechnologies and Translational Medicine, Humanitas Clinical and Research Center, Milan, Italy Introduction: Sepsis is a systemic inflammatory response resulted from the inability of the immune system to control infections. During an infection, neutrophils are the first cell line to reach the primary focus, and chemokines have a fundamental role in this process. However, in sepsis, these chemokines also contribute to the neutrophil infiltration to remote organs and to multiple organ failure. Under normal physiological conditions neutrophils do not express the CC chemokine receptor subfamily and, as consequence, do not respond to the CC chemokines. On the other hand, our group has shown that during sepsis neutrophils become responsive to these chemokines and express CC chemokine receptors, as CCR2 and CCR5. Recently, a new chemokine receptor named D6 has been described. It is an atypical receptor due to its involvement in the removal and degradation of CC inflammatory chemokines. However, to date, there are no studies showing the involvement of D6 in sepsis physiopathology. In this way, the aim of this study is to show the role of D6 during experimental sepsis. Methods:All experiments were performed according to our institution’s ethical guidelines (169/2011). C57BL/6 and D6 deficient mice (D6-/-) were used to induce sepsis using cecal ligation and puncture (CLP) model. Neutrophil migration to the peritoneal cavity, bacteremia, markers of organ damage, neutrophil infiltration and chemokine levels on remote organs were determined 24 h after sepsis induction. The survival rate of animals was assessed twice a day, until the 100day after sepsis induction. The means of the parameters evaluated in WT and D6 mice submitted to CLP were compared by ANOVA, followed by Bonferroni test, or by t test and the survival rate by the Mantel-Cox log rank test. Results and discussion: it was observed that the D6-/- mice under sub-letal CLPsurgery exhibited a significant reduction in the survival rate, as compared to WT animals. However, neutrophil migration to the peritoneal cavity, bacterial load in the peritoneal exudate, blood and vital organs were similar between WT and D6-/- mice, 24 h after CLP. On the other hand, we showed increased levels of CC
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S128 chemokines in the lung, heart and kidney of the D6-/- mice and, as consequence, increased neutrophil infiltration in these organs, assessed by MPO, and increased levels of organs damage markers, when compared to WT animals. In conclusion, our data indicate that D6 has a protective role during sepsis, mediating the reduction of chemokine levels in remote organs and, consequently, the reduction of organ damage. Financial support CNPq, FAPESP, CAPES, FAEPA.
Co-Stimulatory and Co-Inhibitory Pathways B065 DICAM ATTENUATES INFLAMMATORY MACROPHAGE DIFFERENTIATION VIA SUPPRESSION OF INTEGRIN AVß3–DEPENDENT TYPE I INTERFERON SYSTEM Seungwoo Han1,2, Youn-Kwan Jung1, Min-Su Han1, Eun-Ju Lee1, Hye-Ri Park1, Jiae Jang1, Gun-Woo Kim1,2 1 Laboratory for Arthritis and Bone Biology, Fatima Research Institute, Daegu, Republic of Korea; 2Department of Internal Medicine, Daegu Fatima hospital, Daegu, Republic of Korea
Objective: DICAM, a dual Ig domain containing adhesion molecule, is involved in cell–cell adhesion through a direct interaction with aVb3 integrin. In our previous study showing the inhibitory role in osteoclastogenesis, we found a clue that DICAM also has a suppressive role in macrophage differentiation. However, it remains still obscure the role of DICAM in macrophage differentiation and M1/M2 polarization. Method: To induce differentiation into resting M0 macrophage, THP1 cells were cultured with 100 nM PMA for 24 h, and then rested for 6 days. For M1/M2 polarization, resting M0 THP-1 macrophages were treated with IFN-c or IL-4 for 24 h. To investigate the role of DICAM during THP-1 macrophage differentiation, THP-1 cells were infected with 50 moi of control LacZ adenovirus or with DICAM adenovirus. Results: The expression of DICAM was increased during PMA-induced THP-1 differentiation, and DICAM was slightly decreased by IFN-c for M1 polarization and increased by IL-4 for M2 polarization. The overexpression of DICAM in THP-1 cells suppressed PMA-mediated macrophage differentiation in the number of activated branched macrophage and macrophage marker expression, CD14 and CD68. However, DICAM does not affect the viability and proliferation of PMA-stimulated THP-1 macrophage. Functionally, DICAM attenuated the TNF-a secretion of differentiated THP-1 cells and their phagocytic activity as well. To investigate the molecular mechanisms for DICAM-mediated suppression of macrophage differentiation, we conducted microarray analyses, which revealed that DICAM overexpression significantly suppressed type 1 interferon system. Among interferon regulatory factors (IRFs) family, IRF7 was most significantly reduced by DICAM. DICAM also attenuated Akt phosphorylation and increased a nuclear translocation of FoxO3a that is known to be a critical negative regulator of IRF7. Consistently, DICAM also decreased total integrin b3 level and integrin-linked kinase (ILK) phosphorylation, the major adaptor molecule of integrin b3. Based on the fact that type 1 interferon is important in M1 macrophage polarization, we investigated the role of DICAM in M1/M2 polarization of macrophage. Overexpression of DICAM induced downregulation of M1-associated genes such as IL-12b p40, IL12 p19, TNFa, IL-6, and IL-1b but did not affect M2 genes, Arg1 and Fizz1. In addition, DICAM increased IL-10, but decreased TNFa and INF-b.
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Inflamm. Res. Conclusion: DICAM potently reduces differentiation and function of THP-1 macrophage and skews a THP-1 polarization into M2-like macrophage via suppression of integrin aVb3-dependent AktFoxO3a-IRF7 pathway.
B066 ABROGATION OF OX40 AND CD30 SIGNALING REDUCES HOUSE DUST MITE DRIVEN ALLERGIC LUNG INFLAMMATION Donald T. Gracias1, Hideo Yagita2, Michael Croft1 1
La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA; Juntendo University, Tokyo, Japan
2
Allergic asthma is an inflammatory disease that is induced by immune responses to airborne allergens in the lungs. Several studies have identified T cells as being important players in asthma, with Type 2 helper T cells (Th2) being a critical subset. The selective removal of pre-existing pathogenic memory Th2 cells could potentially be a key process resulting in enhanced tolerance. We have attempted to identify signaling molecules, in the TNF superfamily that regulate persistence or activity of memory Th2 cells in a house dust mite (HDM) model of allergic inflammation. C57BL/6J mice given a single intranasal instillation of HDM showed increased expression of several TNF superfamily and cytokine genes in the lungs. Among these, up-regulation of OX40L was significantly higher than other TNF family members measured. We then tested whether OX40-OX40L interactions were required for memory Th2 responses and lung inflammation driven by HDM. While OX40-deficient mice displayed strongly reduced overall lung inflammation, eosinophilia, and Th2 development in recall responses to HDM challenge, therapeutic blocking of OX40L at the time of memory T cell reactivation did not result in similar inhibition of allergic responses, implying OX40L may have been important for initiation of the naı¨ve response but was not needed for the memory response. Similar negative results were gained by targeting another TNF superfamily protein CD30L that was also upregulated by HDM exposure. However, we observed significant reduction in airway inflammation and the Th2 response when OX40L was simultaneously neutralized with CD30L. This suggests that memory responses to complex allergens may be controlled by several costimulatory interactions, and only abrogation of signaling from multiple TNF superfamily members may promote airway tolerance.
Cytokines in Inflammatory Disease B067 TH17 CYTOKINES REGULATE OSTEOCLASTOGENESIS IN RHEUMATOID ARTHRITIS Sang-Heon Lee1, Kyoung Woon Kim2, Hae-Rim Kim1 1 Konkuk University School of Medicine, Seoul, Korea; 2The Catholic University of Korea, Seoul, South Korea
Background: Th17 cells, which are defined by a selective interleukin (IL)-17 secretion, are considered as a distinct lineage of CD4+ helper T cells which are regulated by the other Th1 and Th2 cytokines. IL17A is a dominant proinflammatory cytokine produced by the Th17 cells. IL-21, IL-22 and IL-26 are also produced by the Th17 cells. In
Inflamm. Res. RA, IL-17A induces the production of proinflammatory mediators, such as IL-1 and tumor necrosis factor (TNF)-a from synovial fibroblasts, macrophages, and chondrocytes. Objectives: This study aimed to determine the regulatory effect of Th17 cytokines on the osteoclastogenesis in rheumatoid arthritis (RA). Methods: The expression of IL-17 and RANKL was determined in synovial tissue, fibroblast-like synoviocytes (FLS) and synovial fluids of RA patients using immunohistochemical staining, ELISA and realtime PCR. The Th17 cytokines inducedRANKL expression was studied in RA FLS using real-time PCR, luciferase activity, and western blot. Human peripheral blood monocytes were cultured with M-CSF and Th17 cytokines, and then osteoclastogenesis was determinedby counting the number TRAP-positive multinucleated cells. The osteoclastogenesis was also determined after humanmonocytes were co-cultured with IL-17-prestimulated FLS. Results: There was a significant correlation between RANKL and IL17 levels in RA synovial fluid. After RA FLS werestimulated with IL17, IL-21 and IL-22, the expression of RANKL mRNA increased and the IL-17-induced RANKL expression was decreased by the inhibition of Act1, TRAF6, NF-jB and the AP-1. Th17 cytokines and IL17-prestimulated FLS induced osteoclastogenesis from monocytes in the absence of osteoblasts or RANKL. Conclusions: Th17 cytokines have a dual effect on osteooclastogenesis in RA; direct induction of osteoclastogenesis from monocytes and upregulation of RANKL production in RA FLS. Th17 cytokines/ RANKL axis could be a potential therapeutic target for bone destruction in RA.
B068 IL-38: A NEW FACTOR IN RHEUMATOID ARTHRITIS Shinjiro Kaieda, Shin-ichi Takenaka, Tomoaki Hoshino Kurume University School of Medicine, Kurume, Japan Background: IL-38 (IL-1F10) was originally described as an IL-1 family cytokine, and named IL-1HY2. The IL-38 gene is located in the IL-1 family cluster on chromosome 2, next to the genes encoding the IL-1 receptor antagonist (IL-1Ra) and the IL-36 receptor antagonist (IL-36Ra). IL-38 shares 37 % homology with IL-1Ra, 43 % homology with Il-36 Ra, and has a three-dimensional structure similar to IL-1Ra. Il-38 was recently shown to inhibit Candida albicans-indeuced IL-17 and Il-22 production by human memory T cells. However, the role of IL-38 in inflammatory diseases such as RA remain unclear. Methods: We established several clones of mouse anti-human IL-38 mAb. One anti-human IL-38 mAb can be used for sandwich ELISAs and immunohistochemistry. To determine the expression pattern of the IL-38 gene, a panel of cDNA derived from normal lung, pancreas, spleen, muscle and synoviocytes was analyzed by qRT-PCR. The serum levels of IL-38 in 137 RA and 26 OA patients, and in 56 healthy donors, were determined by ELISA. Synovial tissue samples were also obtained from7 RA and 3 OA patients. Arthritis was also initiated in mice lacking IL-38, as well as their wild-type littermates, via intraperitoneal administration of K/BxN serum. Total RNA was isolated from mouse ankle joints and IL-1b and IL-6 mRNA were quantified by qRT-PCR. Results: The IL-38 gene were strongly expressed in the lung, spleen and synoviocytes. The serum levels of IL-38 were 5.7 ± 0.4, 2.8 ± 0.8, and 2.8 ± 0.7 pg/ml in RA patients, OA patients, and healthy donors, respectively. Twenty-one of the 137 RA patients, 1 of 26 OA patients, and 5 of the 56 controls had IL-38 levels that was above the detection limit our ELISa system (10 pg/ml).
S129 Immunohistochemical approach revealed the prominent expression of IL-38 protein in the synovial lining of RA synovium. In contrast, IL38 was barely expressed in the synovial lining of OA synovium. Moreover, we found that IL-38-deficient mice exhibited siginificant exacerevation of their arthritis scores, compared with their wild-type littermates. Correspomdingly, histological measures of arthritis were also exacerbated, and accompanied by increasedIL-1b and IL-6 gene expression in the ankles. Conclusion: Our presentstudy is the first to identify the presence of soluble OL-38 protein in the serum andits expression in the synovium of RA patients. IL-38 may play a role as an inhibitor in the pathogenesis of RA.
B069 OPPOSITE ROLE OF SOLUBLE FORMS OF IL-6 RECEPTOR, SIL-6R AND SGP130, ON THE REGULATION OF INFLAMMATORY STATUS IN RHEUMATOID ARTHRITIS Kazuyuki Yoshizaki, J Song Soken-Nakazawa, Kazuko Uno Osaka University, Suita, Japan The contribution of Interleukin 6 (IL-6) is well known in inflammation, especially in chronic inflammatory status. IL-6 signal is mediated into cells through two receptors, an IL-6 binding receptor (IL-6R) and an IL-6 signal transducer, gp130, which binds the complex of IL-6/IL-6R. Soluble forms of IL-6R (sIL-6R) and gp130 (sgp130) are presented in serum. sIL-6R is produced by the proteinase cleavage of membrane IL-6R and the other sgp130 is secreted by splicing gp130 gene. Our previous in vitro study showed that sIL-6R with IL-6 induced and augmented the production of CRP, SAA, and hepcidin from hepatocyte, and that sgp130 inhibited the IL-6 signal and suppressed the production of acute phase proteins. Norwell et al. previously reported that sIL-6R enhanced IL-6 activity in the RA synovial, but that the development of arthritis could be blocked by Fc coupled sgp130 in mouse model. However, less is known about the function of soluble receptors in inflammatory condition in human. Then, in order to know the significance of the soluble forms of receptors in chronic inflammatory disease, we analyzed these soluble receptors in the patients with rheumatoid arthritis (RA) who were treated with an anti-IL-6 receptor antibody, Tocilizumab. Biomarkers including 4 soluble receptors with 19 cytokines and 8 chemokines were measured in serum before starting Tocilizumab therapy by Bio-Plex200 and Milliplex MAP. Simple and multiple linear regression analysis were used to determine if any relationship existed between pretreatment biomarkers and patient’s week 16 DAS28-CRP score. These values and clinical variables underwent a stepwise multiple linear regression analysis. The resulting parameters with P \ 0.05 were considered significant. To find biomarkers that may contribute to 16 week DAS28 score, we performed single linear regression analysis of 16 week DAS28 score. We found that sgp130, sTNFR-1 and sIL-6R significantly coincided with the 16 week DAS28 score. Then multi linear regression analysis of biomarker levels was performed to determine the best equation of DAS28 improvement. We found that eight biomarkers including sgp130 values were significantly expressed in biological naı¨ve Tocilizumab treated patients (R2 = 0.646, P \ 0.0001). In these markers, sgp130 level was a key biomarker in naı¨ve and non-naı¨ve patients. In fact, the distribution for remission and non-remission patients according to their pretreatment serum sgp130 level showed that among naı¨ve patients 59.2 % of those who experienced remission and 19.0 % of nonremission patients showed sgp130 level over 0.2 lg/ml. On the other
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hand, among non-naı¨ve patients 66.6 % of remission and 19.3 % of non-remission patients had sgp130 levels exceeding 0.2 lg/ml. These results suggest that sgp130 is an important predictor of RA patient’s clinical outcome of Tocilizumab therapy, indicating that sgp130 may be a natural IL-6 inhibitor inflammatory status.
These results suggest that IL-6 signaling pathway can differ in each RA patients with similar disease activity and further suggesting the applicability of pStat3 as a biomarker to adjust TCZ interval in RA patients. Measurement of pSTAT3 in RA patients treated with TCZ could be a promising strategy to optimize treatment.
B070 ALTERATION OF IL-6 SIGNALING IS USEFUL IN RHEUMATOID ARTHRITIS TREATMENT: ADJUSTMENT OF DOSING INTERVAL OF TOCILIZUMAB, A IL-6 RECEPTOR-INIHIBITOR
B071 CIGARETTE SMOKE ENHANCES ADAPTIVE IMMUNE RESPONSE IN MURINE MODEL OF ALLERGIC AIRWAY INFLAMMATION
Shuntaro Saito, Keisuke Izumi, Yuko Kaneko, Katsuya Suzuki, Kunihiro Yamaoka, Tsutomu Takeuchi Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan Introduction: Interleukin-6 (IL-6) is a key cytokine in the pathogenesis of rheumatoid arthritis (RA). Tocilizumab (TCZ) binds to membrane and soluble forms of human IL-6 receptor, efficiently inhibiting IL-6-STAT3 signaling with standard every 4 weeks intravenous administration. However, subset of patient show poor response or become tolerant to TCZ, reminiscent of the strength of IL-6 signaling could differs in each individual. Therefore, we measured phosphorylated STAT3 (pSTAT3) in RA patients treated with TCZ, with those whose administration interval was adjusted. Methods: Three hundred fifty-five patients were treated with TCZ (8 mg/kg) at our institution between 2008 and 2014. Patients treated with dosing interval of 3 weeks (shortened group, n = 25), 4 weeks (normal group, n = 266) and C5 weeks (prolonged group, n = 64). Whole blood from 10 patients in each group and methotrexate treated patients (control group, n = 10) with low disease activity (CDAI \ 10, CDAI = Clinical Disease Activity Index) was stimulated with different concentration of recombinant human (rh) IL-6 (0, 0.1, 1, 10, 100 ng/ml). Proportion of pSTAT3 positive CD4+ T cells was measured by phosflow cytometric analysis. Results: A.
Clinical efficacy of adjusting administration interval of TCZ Twenty-five patients (7.0 %) were in shortened group due to insufficient response to 4-week dosing for mean of 33.8 weeks. Disease activity improved after two administrations with CDAI changing from 20.8 to 7.8 (p \ 0.05). Sixty-four patients (18.0 %) were in prolonged group, after achieving remission for mean of 70 weeks. Among them, 51 patients (79.7 %) continued prolonged interval, while 13 (20.3 %) were placed back to 4-week interval due to exacerbation of disease activity after a mean of 41.9 weeks. B. Assessment of IL-6 induced pSTAT3 in CD4+ T cell Proportion of pSTAT3 positive CD4+ T cells increased in a dose dependent manner in each treatment group. Although, all patients were in low disease activity, pSTAT3 was significantly increased in control group from the lowest concentration (0.1 ng/mL) of rhIL6 compared to TCZ treated patients. Among the TCZ treated patients, pSTAT3 was significantly suppressed in shortened group with low detectable pSTAT3 even when stimulated with the highest concentration (100 ng/mL) of rhIL-6. On the other hand, pSTAT3 was increased from low concentration (1 ng/mL) of rhIL-6 in prolonged group and normal group resulted in inbetween of prolonged and shortened group. Conclusion: Our study demonstrated that the strength of IL-6-STAT3 inhibition was altered depending on the adjustment of TCZ administration interval, resulting in low disease activity in RA patients.
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Thayse R. Bruggemann, Paula Fernandes, Jessica M. Oliveira, Beatriz M. Saraiva-Romanholo, Mı´lton A. Martins, Fernanda M. Arantes-Costa Laboratory of Experimental Therapeutics, LIM20, School of Medicine of Sa˜o Paulo, Sa˜o Paulo, Brazil Introduction: Several studies have shown that cigarette smoke interacts with the asthma phenotype and has the potential to interfere in the physical, chemical and immunological barrier. Moreover, asthmatics subjects who have smoke habits have more severe symptoms and higher number of hospitalizations. Methods: Male balb/c mice (n = 8/group) were sensitized with two intraperitoneal injections of ovalbumin (OVA, 20 lg) and alum (3 mg) on days 0 and 14 and were challenged with an aerosol of OVA (1 %, 30 min) on days 21, 23, 25 and 27 for OVA and OVA + CS groups. The CS and OVA + CS groups were exposed to cigarette smoke once a day (7 cigarettes/session) for twelve consecutive days (from 16 to 27). CS and Control (SAL) groups received saline and alum intraperitoneal injection and were challenged with saline 0.9 %. 24 h after the last challenge the levels of IL-5, IL-10, IL-13 and IFN-c were measured in bronchoalveolar lavage fluid (BALF) by cytometric bead array (CBA). ELISA measured TSLP levels in lung homogenate and edema area and number of eosinophils were measured by histological lung stained slides with Luna. Results: OVA-sensitization caused rich pulmonary inflammatory infiltrate of eosinophils, an increase in inflammatory cytokines such as IL-5 and IL-13 and an increase in peribronchoalveolar edema that were all even enhanced when was co-exposition to cigarette smoke. We showed a remodeling process been initiated by increased edema on peribronchoalveolar space on OVA + CS group. TSLP levels showed increased in animals exposed to cigarette smoke but decreased when OVA-sensitized. The regulatory cytokine IL-10 increased only on CS group. Levels of the Th1 cytokine IFN-c, that acts in a allergic inflammation with a regulatory manner, showed increased in OVA and OVA + CS groups. Conclusion: In summary, our study showed that smoking acts as an aggressive agent that worsens the precondition of asthma. Furthermore, we could see that neither IL10 nor IFN-c managed the inflammation based on increased levels of this cytokines and the inflammatory mediators in same groups.
B072 THE ANTI-INFLAMMATORY ROLE OF ADIPONECTIN IN PATIENTS WITH CHRONIC CHAGAS DISEASE Ka´rita CF Lidani, Thaisa L. Sandri, Marcia H. Beltrame, Renato M. Nisihara, Iara JT Messias-Reason Laboratory of Molecular Immunopathology, Clinical Hospital, Federal University of Parana´ (HC-UFPR), Curitiba, PR, Brazil
Inflamm. Res. Chagas disease (CD) is a tropical neglected disease caused by Trypanosoma cruzi, which affects more than eight million people in Latin America and represents an emerging problem in North America and Europe due to international migrations. Although most individuals remain asymptomatic in the indeterminate form all lifelong, around 2–5 % of them progress each year, to one of the symptomatic forms of the disease, developing cardiomyopathy, digestive megasyndromes, or both. In chronic CD, adipose tissue and adipocytes have been related as a long-term storage site of T. cruzi, where the adipokines have a critical role in inflammatory process. Thus, this study investigated the levels of adiponectin in patients with indeterminate or cardiac clinical forms of chronic CD compared to controls, besides the contribution of the adipokine in chronic chagasic cardiomyopathy (CCC) progression. Adiponectin plasma levels were determined in chronic CD women (n = 64) from Southern Brazil using an ELISA commercial kit. The cardiac patients were graded according to the cardiac insufficiency classification of the American Heart Association, adapted for Chagas disease (2005) in A, B1, B2, C, and D groups. Demographical, laboratorial, and clinical findings of the specific CD forms were obtained from medical records. Moreover, 80 Southern Brazilian women with negative T. cruzi serology and without clinical complaints were used as control subjects. All parameters were analyzed with adjustment for age and Body Mass Index (BMI) using logistic regression analysis. Formal written consent was obtained from each individual and this study was approved by local ethics committee. Our results demonstrated higher levels of adiponectin in controls than chronic CD patients (p \ 0.0001, means 15316.5 vs. 2217.48 ng/lL, respectively) and higher in controls when compared to indeterminate form (p \ 0.0001, means 15316.5 vs. 2208 ng/lL, respectively). Besides, higher levels of adiponectin were observed in controls than in patients with cardiac form (p = 0.0001, means 15316.5 vs. 2113.24 ng/lL, respectively). Although there was no difference between indeterminate and cardiac clinical forms (p = 0.32, means 2356.5 vs. 2113.24 ng/lL, respectively) the adiponectin levels were negatively correlated with cardiometabolic parameters, such as triglycerides (q = -0.41 and p = 0.034) and cholesterol (q = -0.45 and p = 0.018). Patients with arterial hypertension presented higher adiponectin levels than those non hypertensive (p = 0.03, medians 3060 vs. 2062 ng/lL, respectively). In addition, comparing adiponectin levels in patients with cardiac insufficiency (C + D forms) and without it (A + B), no significance difference was observed (p = 0.94, means 2202 vs. 2155 ng/lL, respectively). As the chronic Chagas disease is associated with low levels of adiponectin, its anti-inflammatory role inT. cruzi infection might contribute to CCC or risk of metabolic syndrome in all clinical forms.
S131 important role in immunity and inflammation, and its expression has been reported in most barrier tissues of the body, such as lung, gut and skin as well as in the placenta. Natural killer (NK) cells express AhR and AhR activation stimulates antitumor activity as well as resistance to infections. However, the exact mechanism by which NK cells contribute to obesity with severe sepsis remains unknown. The purpose of this study was to investigate the role of NK cells in the inflammatory response and also evaluate the potential protective effect of AhR activation by6-formylindolo[3,2-b]carbazole (FICZ) during cecal ligation and puncture (CLP) induced sepsis on high-fat diet-fed mice. Methods: Male C57BL/6 mice at 6 weeks of age were randomized to a high-fat diet (HFD) or standard chowdiet (SD) for 10 weeks. After 10 weeks of feeding, polymicrobial sepsis was induced by CLP followed by a peritoneal injection of FICZ or saline. Twenty-four hours after CLP, AhR expression of lung, spleen tissues and splenic NK cells were analysed. The effect of FICZ administration on early immune response and survival rate was investigated as well. Results: Lung and spleen tissue from HFD mice with sepsis expressed significantly less AhR than SD controls (P \ 0.05). We also found that purified splenic NK cells from HFD mice expressed far lower level AhR, displaying a significant decrease of their capacity to produce IFN-c compared with that of SD mice after CLP (P \ 0.01). IFN-c synthesis of HFD NK cells was partly restored by addition of IL-15 and FICZ in vitro. Accordingly, HFD mice had a higher mortality than SD mice. Treatment of HFD mice with FICZ augmented NK cell function with increased IFN-c production and improved sepsis survival. Either AhR antagonist or NK cells depletion reversed the beneficial effects of AhR activation on immune defense to CLP induced sepsis. Conclusions: These data demonstrate that HFD induced obesity have detrimental effects on experimental polymicrobial sepsis due to impairedIFN-cproducing NK cells. AhRactivation by FICZ could increases survival of obesity with sepsis by improving NK cells function with increased IFN-c production during early inflammation response.
B074 CHRONIC LEVELS OF IFN GAMMA DECREASED CHEMOTAXIS OF BONE MARROW DERIVED MACROPHAGES AT SITES OF INFLAMMATION Julio C. Valencia
B073 ARYL HYDROCARBON RECEPTOR ACTIVATION BY 6-FORMYLINDOLO[3,2-B]CARBAZOLE (FICZ) INCREASES SURVIVAL IN HIGH-FAT DIET-FED MICE WITH SEPSIS: CRITICAL ROLE FOR NK CELLS Min Zhu1, Peng Xiong1, Wei Zhang2, Hai-Wei Sang1, Xiang-Ning Fu1 1 Department of Thoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; 2Wuhan Institute of Burn, Wuhan No. 3 Hospital, Wuhan, China
Background: Sepsis is a worldwide life-threatening disease, especially in criticallyillobesepatients. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor which plays an
LEI, NCI-Frederick, Frederick, MD, USA Interferonc (IFNc, also called Type II interferon or immune interferon, is a critical factor in several autoimmune disorders. IFNc is a modulator of inflammation capable of influencing the clinical outcome of autoimmune disorders with both pro- and anti-inflammatory functions. IFNc signaling activates both innate and adaptive immune cells such as macrophages and T-cells. Recent evidence indicates that immune activated macrophages are first recruited and later reprogrammed by the microenvironment at sites of inflammation. Given that chronic inflammatory responses are associated with a macrophage-mediated inflammatory response, we examined the role of chronic IFNc exposure on the activation and trafficking of macrophages. Bone marrow (BM) macrophages were isolated from a mouse model of chronic IFNc expression generated by deletion of the IFNc 30 UTR AU-Rich element (ARE-DEL). Isolated BM macrophages from ARE-DEL+/+ wild type (WT) or ARE-DEL-/- (KO) C57B6/L6 mice were immortalized using an avian retrovirus (J2) carrying the Myc and Raf oncogenes. FACS analysis confirmed that immortalized
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S132 WT and KO macrophages expressed the markers CD11b/MAC1 and F4/80 consistent with their BM origin, while the cells were negative for CD11c and GR-1, markers for dendritic cells and neutrophils, respectively. Combined FACS and immunoblotting analysis revealed that KO macrophages had higher basal STAT1 protein levels and produced higher levels of phosphorylated STAT1 (Y701) after short (10 min) or long (24 h) stimulation with mouse recombinant IFNc (rIFNc), compared to WT macrophages. These results suggest that chronic exposure to IFNc primed KO macrophages for enhanced responsiveness to further stimulation. To examine the consequences of IFNc-mediated priming on macrophage motility, transwell migration assays were carried out over a period of 18 h in the presence or absence of mouse rIFNc. Interestingly, although in a primed state, KO macrophages had a significantly lower motility than WT macrophages (p \ 0.05, ANOVA). Furthermore, pre-treatment with 50, 100 and 200 U/mL of mouse rIFNc for 72 h decreased significantly the motility of WT macrophages (p \ 0.05, ANOVA) compared to untreated controls. Collectively, these data indicate that macrophage chemotaxis is severely decreased after long term exposure to IFNc at the site of inflammation. As a result, this mechanism has potential therapeutics implications to limit further macrophage recruitment at sites of chronic inflammation.
B075 HAPLOTYPE ANALYSIS OF INTERLEUKIN-6 GENE PROMOTER POLYMORPHISMS IN PATIENTS WITH CHRONIC PERIODONTITIS AND DIABETES MELLITUS Simona Valova1, Petra Borilova Linhartova1,2, Katerina Kankova1, Hana Poskerova2, Jan Vokurka2, Antonin Fassmann2, Lydie Izakovicova Holla1,2 1
Department of Pathophysiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic; 2Clinic of Stomatology, Institutions Shared with St. Anne’s Faculty Hospital, Faculty of Medicine, Masaryk University, Brno, Czech Republic Objectives: Strong evidence confirms a bidirectional relationship between chronic periodontitis (CP) and diabetes mellitus (DM). Pathogenesis of both diseases has been associated with high levels of interleukin-6 (IL-6), pleiotropic cytokine involved in immune regulationand control ofinflammatory processes. The IL-6 expression is influenced by polymorphisms in the IL6 gene promoter region. The aim of our study was to investigate the association of single nucleotide polymorphisms at positions -174G/C (rs1800795), -572G/C (rs1800796) and -597G/A (rs1800797), and their combinations (haplotypes) in patients with CP, type 1 DM (T1DM) and type 2 DM (T2DM) in comparison with healthy controls. Methods: 900 Czech subjects were enrolled in this case–control study: 381 diabetic patients without periodontitis (84 T1DM and 297 T2DM patients), 115 subjects suffered both from diabetes and periodontitis (37 T1DM + CP and 78 T2DM + CP), 223 patients had only CP and 181 subjects were healthy controls. The individual polymorphisms in IL6 gene promoter (-174G/C, -572G/C and -597G/A) were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) assay. Results: The allele and genotype frequencies of the studied polymorphisms between groups of patients and healthy controls did not differ statistically significantly. However, we proved significant differences in haplotype frequencies between groups of patients with CP, T1DM, T2DM and healthy controls. The GGA haplotype acts as a
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Inflamm. Res. protective haplotype in patients with CP (OR = 0.159, 95 % CI = 0.035–0.728, p = 0.0054) and in patients with T2DM (OR = 0.179, 95 % CI = 0.049–0.654, p = 0.0039). On the contrary, the GGG haplotype is associated with an increased risk for T1DM (OR = 1.870, 95 % CI = 1.035–3.379, p = 0.0399). Conclusions: Our results indicate that different IL6 promoter haplotypes can affect the risk for CP and DM development. However, the individual IL6 gene promoter polymorphisms do not act independently and may affect each other. This study was supported by thegrantsGACR GB14-37368G, IGA NT11405-6 and by the project MUNI/A/1359/2014.
B076 PLATYMISCIUM FLORIBUNDUM VOG, A MEDICINAL PLANT, DECREASES THE PRODUCTION OF PRO-INFLAMMATORY TNF-a AND IL-1ß IN A RAT MODEL OF LIGATUREINDUCED PERIODONTITIS Karuza MA Pereira1, Luzia H. Teixeira1, Danielle RD Val2, Hellı´ada V. Chaves1, Gerly Anne DC Brito1, Conceic¸a˜o DS Martins1, Paula Goes1, Antonia TA Pimenta1, Mary Anne S. Lima1, Alrieta H. Teixeira1, Jordaˆnia MDO Freire1, Alice RD Freitas1, ˆ nia TDPP Parente1, Mirna M. Bezerra1 Janine M. Bastos1, Anto 1 Federal University of Ceara´, Fortaleza, Brazil; 2Federal University of Pernambuco, Recife, Brazil
Periodontitis is known to be one of most prevalent worldwide chronic inflammatory diseases. During periodontitis course, it has been shown that proinflammatory cytokines such as TNF-a and IL-1b modulate bone loss. Platymiscium floribundum Vog. is a tree that occurs in Northeast Brazil, where it is known as ‘‘sacambu’’ and ‘‘jacaranda-dolitoral’’, that has been used in traditional medicine as anti-inflammatory. Some authors have reported that this species are a source of isoflavonoids that possess various biological effects such as antifungal, antimitotic, and anticancer properties. Since a few of studies have been developed in the search of others chemotherapeutic values from this plant, the aim of this study was to evaluate the effects of Platymiscium floribundum Vog. on alveolar bone loss in a rat model of ligature-induced periodontitis, investigating the involvement of TNF-a and IL-1b. Methods: This study was conducted with the approval of the Ethics Committee of the UFC, Fortaleza, Brazil (CEPA no. 08/13). Periodontitis was induced by placing a nylon thread (3.0) in the upper molars of female Wistar rats (180-200 g). Rats (6 per group) were treated (per os) daily with Platymiscium floribundum Vog (0.1, 1 or 10 mg/kg) or vehicle (saline) for 11 days. The rats were killed on day 11 after ligature placement, and the maxillae were removed and processed for macroscopic analyses of alveolar bone loss (mm2) using the ImageJ software. Gingival samples were collected to evaluate TNF-a and IL-1b levels (pg/ml) by ELISA. Rats were weighed daily. Results: Platymiscium floribundum Vog (1 or 10 mg/kg) reduced (P \ 0.0001) alveolar bone loss (2.33 ± 0.59 and 1.99 ± 0.34 mm2, respectively), compared to the vehicle group (4.17 ± 0.19). Further, Platymiscium floribundum Vog decreased (P \ 0.05) both TNF-a and IL-1b levels in gingival tissues (3.88 ± 0.13 and 4.44 ± 0.29 pg/ml, respectively), compared to the vehicle group (6.83 ± 0.36 and 9.43 ± 0.42 pg/ml, respectively). Conclusion: These findings reveal that Platymiscium floribundum Vog decreases bone loss in ligature-induced periodontitis in rats by reducing, at least in part, TNFa and IL-1b levels. Funding Sources RENORBIO, CNPq, CAPES, and INCT-IBISAB.
Inflamm. Res.
B077 GLYCOGEN SYNTHASE KINASE-3 SIGNALLING AND REGULATION OF TNF MRNA TRANSLATION IN HUMAN NEUTROPHILS Miriam S. Giambelluca1, Genevie`ve Bertheau Mailhot1, Emmanuelle Rollet-Labelle1, Cynthia Laflamme1, Marc J. Servant2, Marc Pouliot1 1
Centre de Recherche du CHU de Que´bec-Univesite´ Laval, Quebec City, QC, Canada; 2Universite´ de Montre´al, Faculty of Pharmacy, Montreal, QC, Canada Introduction: Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase involved in the regulation of multiple cellular processes. Its two known isoforms, a and b, are differentially expressed in tissues throughout the body and exert overlapping, but sometimes distinct functions. GSK-3 is typically active in resting cells, inhibition by phosphorylation of Ser21 (GSK-3a) or Ser9 (GSK-3b) being the most common regulatory mechanism. GSK-3 activity has been linked recently with immune system functions, yet little is known about the role of this enzyme in neutrophils, the most abundant type of leukocytes. In this study, we examined GSK-3 expression, signaling and impact of GSK-3 inhibition by lithium or specific GSK-3 inhibitors, on the production of TNF by neutrophils. Methods: Freshly-isolated human neutrophils and the murine airpouch model of inflammation were used for this study. GSK-3 isoform identification and activation were monitored by western blots, whereas the effects of GSK-3 inhibition on mRNA, cytokine production, transcriptional and translational pathways were studied by RT-PCR, ELISAs, Western Blots and quantitative mass spectrometry. Results: GSK-3a was found to be the predominant isoform. It was constitutively expressed and cell stimulation with different agonists did not alter its expression. The main pathways contributing to the control of GSK-3 activity were: Src kinase, PKC, PI3K/AKT and ERK/RSK in lipopolysaccharides (LPS)-stimulated neutrophils, PI3 K/AKT, ERK/RSK and PKC in GM-CSF-stimulated neutrophils. In the murine air pouch model of inflammation, injection of lithium chloride potentiated TNF production induced by LPS. In human neutrophils, GSK-3 inhibitors mimicked the impact of lithium chloride, also potentiating TNF release. When cellular mechanisms were investigated, no alteration in the NF-Jbor CREB pathways activation, or in the levels of TNF mRNA, was observed but an association with the p38/MNK1/eIF4E translation pathway and RNAbinding proteins activity was revealed. GSK-3 inhibition potentiated de novo TNF synthesis. Conclusions: GSK-3a emerges as a central kinase in human neutrophil physiology, being regulated by a number of different stimuli and playing a central role in regulating TNF mRNA translation. Financial support Canadian Institutes of Health Research (CIHR), Canadian Foundation for Innovation (CFI), Fonds de la Recherche en Sante´ du Que´bec (FRSQ) and the Wilbrod-Bherer Foundation.
B078 EFFECTS OF A STEMODIA MARITIMA LINN. EXTRACT ON BONE RESORPTION AND TNF-A AND IL-1ß LEVELS IN RATS WITH LIGATUREINDUCED PERIODONTITIS Karuza MA Pereira1, Luzia H. Teixeira1, Danielle RD Val2, Raul S. Freitas1, Hellı´ada V. Chaves1, Paula Goes1, Alrieta H. Teixeira1, Jordaˆnia MDO Freire1,
S133 Francisca RLD Silva1, Francisco EA Rodrigues1, Jair Mafezoli1, Vicente de Paulo T. Pinto1, Gerardo C. Filho1, Virgı´nia CC Gira˜o1, Ba´rbara M. Bastos1, Angela MC ARRIAGA1, Aline CD Medeiros1 1 Federal University of Ceara´, Fortaleza, Brazil; 2Federal University of Pernambuco, Recife, Brazil
Periodontitis is an inflammatory condition that result in the destruction of the supporting structures of the dentition. Though the etiology of periodontitis is bacterial, some evidences strongly suggest that the pathogenesis of the disease is mediated by the host immuneinflammatory response. Stemodia maritima Linn. (Scrophulariaceae) is a very common shrub that grows wild in northeastern Brazil near the sea, where it is known as ‘‘melosa’’. It is used to treat stomachache and swelling by the local population. This study aimed to evaluate the effects of a hydro-alcoholic extract of Stemodia maritima Linn. (Sm) on alveolar bone loss in a rat model of ligatureinduced periodontitis, investigating the involvement of TNF-a and IL-1b. Methods: The experimental protocol was conducted with the approval of the Ethics Committee of the UFC, Fortaleza, Brazil (CEPA no. 08/13). Periodontitis was induced by placing a nylon thread (3.0) in the upper molars of female Wistar rats (180–200 g). Rats (n = 6 per group) were treated (per os) daily with Sm (0.1, 1 or 10 mg/kg) or vehicle (non-treated group) for 11 days. The rats were killed on day 11 after ligature placement, and the maxillae were removed and processed for macroscopic analyses of alveolar bone loss (mm2) using the ImageJ software. Gingival samples were collected to evaluate TNF-a and IL-1b levels (pg/ml) by ELISA. Rats were weighed daily. Results: Sm (10 mg/kg) reduced (P \ 0.05) alveolar bone loss (1.93 ± 0.54 pg/ml), when compared to non-treated group (4.39 ± 0.31 pg/ml). Further, Sm (10 mg/kg) decreased (P \ 0.05) both TNF-a and IL-1b levels in gingival tissues (3.53 ± 0.15 and 5.90 ± 0.36 pg/ml, respectively), compared to the non-treated group (6.83 ± 0.36 and 9.43 ± 0.42 pg/ml, respectively). Conclusion: These findings suggest that Stemodia maritima Linn. might have a protective potential on gingival tissue inflammation and alveolar bone loss during the process of periodontitis by inhibiting pro-inflammatory cytokines, resulting in improved periodontal tissue in experimental periodontitis, which is an attractive model to evaluate inflammatory bone loss. Funding Sources RENORBIO, CNPq, CAPES, and INCT-IBISAB.
B079 HIGH DOSE OF LEPTIN INDUCES FIBROTIC CONVERSION OF ENDOTHELIAL CELLS INTO FIBROBLAST Alvaro Becerra1, Macarena Rojas1, Alejandro Vallejos1, Felipe Simon1,2 1
Departamento de Ciencias Biolo´gicas, Facultad de Ciencias Biolo´gicas and Facultad de Medicina, Universidad Andres Bello, Santiago, Chile; 2Millennium Institute on Immunology and Immunotherapy, Santiago, Chile Introduction: During sepsis syndrome progression, a number of inflammatory mediators are released into the blood vessels, exposuring a wide range of inflammatory mediators to endothelial cells (ECs). Several evidences have been shown that inflammatory mediators induce a fibrotic conversion to become ECs into activated fibroblasts through a process known as endothelial-to-mesenchymal transition (EndMT). It has been demonstrated that during the course of sepsis the adipokine leptin is increased in serum. Despite that it has been described that leptin exert a modulatory function on immune
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S134 system, the role played by leptin during sepsis is poorly understood. Thus, our aim was to investigate whether leptin, similarly to inflammatory mediators, induces conversion of ECs into activated fibroblasts. Methods and Results: Using primary cultures of rat mesenteric endothelial cells (RMEC), we demonstrated that RMEC exposed to high doses of leptin (50–100 ng/ml) for 72 h exhibits a conversion of ECs in activated fibroblasts. Leptin-induced fibrotic conversion was determined by changes in morphology and protein expression pattern. Results showed a leptin-induced fibrotic-like ECs morphology compared to cells in absence of leptin. It was found a decreasing of the endothelial markers VE-Cadherin and CD31/PECAM and a increasing in the expression of the fibroblast-specific proteins, a smooth muscle actin (a-SMA) and fibroblast-specific protein-1 (FSP-1). Furthermore, extracellular matrix (ECM) proteins, fibronectin (FN) and collagen type III (Col III), were also increased upon exposition to high doses of leptin. In addition, changes on intracellular distribution of the endothelial, fibrotic and MEC protein in ECs exposed to high doses of leptin were studied. ECs exposed to high doses of leptin increased the secretion of TGFß-1, suggesting an autocrine/paracrine effect to induce conversion of ECs. Using an inhibitor of the ALK5 activation, we observed that ALK5 is also involved in the sepsisinduced EC fibrosis. Conclusion: Our data demonstrated that ECs exposed to high doses of leptin acquire fibroblasts-like features mediating the conversion of ECs in activated fibroblasts through a TGF-ß1/ALK5-dependent pathway. Results provided here contribute to understand the pathophysiology of sepsis revealing a novel photogenic mechanism involved in sepsis and other inflammatory diseases.
B080 MEASURING RECEPTOR DIMERIZATION TO CREATE FUNCTIONAL CELL-BASED ASSAYS FOR [ 85 % OF THE INTERLEUKIN RECEPTOR FAMILY Scott Gridley, Hyna Dotimas, Sangeetha Gunthuri, Hanako DainoLaizure, Albert Doan, Phil Achacoso, Jane Lamerdin DiscoveRx Corporation, San Francisco, CA, USA Ligand-induced receptor dimerization is the first functional step in receptor activation, representing the most proximal, functional readout for receptor activation. It is well understood that the family of Interleukin receptors will dimerize with the other members of its family leading to a complicated signaling cascade that is critically involved in a variety of auto-immune, inflammatory and oncogenic diseases. Surprisingly, existing cellular assays have been unable to faithfully monitor these interactions in a proximal manner to the receptor. Here we present a novel application of the Enzyme Fragment Complementation system to monitor receptor-receptor interactions at the surface of intact live cells, applicable to diverse receptor types such as Interleukin receptors, BMP receptors, receptor tyrosine kinases and cytokine receptors, with a specific focus on the interleukin family of receptors. The high specificity, simplicity of the assay protocol, large signal to noise ratio, serum tolerance and reproducibility of these assays has enabled their use in cell-based screening, functional characterization, QC lot release assays and neutralizing antibody studies. Examples discussed here include assays for the IL-1, IL-2, IL-6, IL-10 receptor families, with assays developed for over 85 % of the Interleukins and their receptors.
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B081 CYTOKINES AND CHEMOKINES ANALYSIS IN PREGNANT WOMEN WITH ERYTHROCYTE ALLOIMMUNIZATION Juliana AC Schettini1,2, Thoma´s V. Gomes1, Alexandra Karla S. Barreto1, Claudeir D. Silva Ju´nior1, Marina C. da Matta1, Isabela C. Coutinho2, Maria do Carmo VC Oliveira3, Leuridan C. Torres1 1
Translational Research Laboratory, Instituto de Medicina Integral Prof. Fernando Figueira (IMIP), Recife, Brazil; 2Department of Obstetrics and Gynecology of the Instituto de Medicina Integral Prof. Fernando Figueira (IMIP), Recife, Brazil; 3Fundac¸a˜o de Hematologia e Hemoterapia de Pernambuco (HEMOPE), Recife, Brazil Cytokines and chemokines analysis in pregnant women with erythrocyte alloimmunization. Introduction: It is not clear if there is any difference in chemokine and cytokine profile according to the presence or absence of maternal RhD alloimmunization. Objective: Evaluate the levels of inflammatory cytokines and chemokynes in RhD negative pregnant women with erythrocyte alloimmunization. Methods: The study was carried out the Outpatient Clinic for Pregnancies (OCP) and Translational Research Laboratory of the Instituto de Medicina Integral Professor Fernando Figueira (IMIP) between August 2013 to December 2014. Cytokines (IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, TNF-a and IFN-c) and chemokines (RANTES, MIG, MCP1, IP10) were measured in culture supernantants. Maternal plasma RHD genotyping was performed for RhD negative sensitized mothers. About 103 women were divided in groups of pregnants: 16 RhD (-) alloimmunized [indirect antiglobulin test = IAT (+)] and 43 RhD (-) non-sensitized; and groups of controls: 24 RhD (+) IAT (-) pregnant and 20 non-pregnant women. Analyses of groups was used the Mann–Whitney test two-tailed. The correlation between variables was determined by calculating the Spearman correlation coefficient r. A p value \ 0.05 was considered significant. Results: IL12p70 concentration was higher in RhD(-) IAT(-) and RhD(+) IAT(-) compared with those in women control (p** = 0.005 and p* = 0.02, respectively). MCP1 level was increased in the RhD (-) IAT (+) (median = 20648 pg/mL) when compared to RhD(-) IAT(-) (median = 16136 0 g/mL, p \ 0.0001) and women control (median = 476 pg/mL, p \ 0.0001). In the RhD(-) IAT(+) group IL-8 was positively correlated with IL-10 (r = 0.51, p = 0.04) and IL-6 (r = 0.32; p = 0.01). IL1-b was positively correlated with IL-6 (r = 0.57, p = 0.02) and IL-10 (r = 0.57, p = 0.02). Anti-D alloantibody was present in 10 mothers and anti-D and C in 6 mothers. RHD genotyping showed that 12 fetus were RHD positive and 4 RHD negative. There were two neonatal death and 1 stillbirth secondary to fetal hydrops. Conclusions: There is a increase in proinflammatory cytokines in sensitized pregnant women.
B082 COMBINATORIAL TARGETING OF TSLP, IL-25, AND IL-33 IN TYPE 2 CYTOKINE-DRIVEN INFLAMMATION AND FIBROSIS Kevin M. Vannella1, Thirumalai R. Ramalingam1, Allen W. Cheever2, Lee A. Borthwick3, Luke Barron1, Kevin M. Hart1, Robert W.
Inflamm. Res. Thompson1, Kristen N. Kindrachuk1, Sandra White1, Alison L. Budelsky4, Michael R. Comeau4, Dirk E. Smith4, Thomas A. Wynn1 1
National Institute of Allergy and Infectious Disease, Bethesda, USA; Biomedical Research Institute; 3Newcastle University, Newcastle upon Tyne, England; 4Amgen Inc., Thousand Oaks, USA
2
Thymic stromal lymphopoietin (TSLP), IL-25, and IL-33 are important initiators of type 2-associated mucosal inflammation and immunity. However, their role in the maintenance of progressive type 2 inflammation and fibrosis is much less clear. Here, using chronic models of helminth infection and allergic lung inflammation, we show that collective disruption of TSLP, IL-25, and IL-33 signaling suppresses chronic and progressive type 2 cytokine driven inflammation and fibrosis. In a schistosome lung granuloma model or during chronic S. mansoni infection in the liver, individual ablation of TSLP, IL-25, or IL-33/ST2 had no impact on the development of IL-4/IL-13-dependent inflammation or fibrosis. However, significant reductions in granuloma-associated eosinophils, hepatic fibrosis, and IL-13-producing group 2 innate lymphoid cells (ILC2s) were observed when signaling of all three mediators was simultaneously disrupted. Combined blockade via mAb treatment also reduced IL-5 and IL-13 expression during primary and secondary granuloma formation in the lung. In a model of chronic house dust mite-induced allergic lung inflammation, combined mAb treatment did not decrease established inflammation or fibrosis. TSLP/IL-33 double-knockout mice treated with anti-IL-25 mAb during priming, however, displayed decreased inflammation, mucus production, and lung remodeling in the chronic phase. Together, these studies reveal partially redundant roles for TSLP, IL-25, and IL-33 in the maintenance of type 2 pathology and suggest that in some settings, early combined targeting of these mediators is necessary to ameliorate progressive type 2-driven disease.
B083 DECIPHERING THE DELETERIOUS EFFECT OF IFN-C IN A NEW RODENT MODEL FOR EXPERIMENTAL SEVERE MALARIA Norinne Lacerda-Queiroz, Nicolas D. Riteau, Richard Eastman, Alan Sher, Dragana Jankovic, Xin-Zhuan Su NIAID/NIH, Rockville, MD, USA Immune response plays an important role in controlling malaria infection; however, excessive inflammatory response can lead to severe disease. Here we have investigated the mechanism of pathogenesis in mice after infection with a virulent rodent malaria parasite, Plasmodium yoelii nigeriensisstrain N67C. Previous studies showed that C57BL/6-infected mice display high parasitemia and a strong pro-inflammatory response, culminating in extensive splenic damage and host lethality at around 7 days post-infection. The present work aims to characterize the cellular and molecular events associated with host immune-mediated pathology following intravenous infection with 106 N67C infected red blood cells. We confirmed that infected mice develop a progressive inflammation in the spleen, characterized by an intense cellular death and an up-regulation of pro-inflammatory cytokines (notably IFNc, IL-6, CCL2 and CXCL1). Interestingly, we identified splenic CD4+ and CD8+ T cells as major sources of IFN-c as early as day 4 post-infection. Additionally, we demonstrated that IFN-c is a major trigger of tissue pathology since its absence or blockade prolonged host survival and prevented excessive host inflammatory response. Moreover, by employing chimeric mice, we showed that the absence of IFN-c receptor in the non-hematopoietic compartment protect mice from early death. Since IFN-c signaling in
S135 stromal radio-resistant cells can impact myeloid progenitors, we are currently testing whether IFN-c can shape the development and/or the mobilization of aberrant inflammatory monocytes during N67C infection, which may lead to immune-mediated splenic pathology and rapid mortality, in the absence of parasite clearance. The elucidation of this IFN-c signaling mechanism may enable further development of interventions to decrease human morbidity and mortality associated with severe malaria.
B084 IMMUNE RESPONSE TO LATENCY ASSOCIATED MYCOBACTERIUM TUBERCULOSIS ANTIGEN AMONG LATENT AND ACTIVE TB PATIENTS Manju Namdeo1, Dipendra Kumar Mitra1, Deepshi Thakral1, Steven G. Reed2, Rhea Coler2 1 All India Institute of Medical Sciences, New Delhi, India; 2Infectious Disease Research Institute, Seattle, WA, USA
Purpose: Polyfunctional T cells are believed to be critical for the immune containment of Mycobacterium tuberculosis (Mtb) infection. To address the role of latency associated Mtb antigen (Rv1813) in breach of latency, we intended to delineate the host polyfunctional CD4+ T cell response in latent TB infection (LTBI) versus active TB patients. Methods: We evaluated polyfunctional CD4+ T cell response in LTBI and active TB patients. PBMCs isolated from Mtb infected subjects were stimulated in vitro with Mtb antigen Rv1813 and CD4+ T cell response was assessed for the production of IFN-c, IL-2, TNF-a at single cell level. Using flow cytometry, we identified single (IFN-c+ , IL-2+ , TNF-a+), dual (IFN-c+ TNF-a+ , IFN-c+ IL-2+ , and TNFa+ IL-2+) and triple (IFN-c+ TNF-a+ IL-2+) producer T cells specific to Rv1813 among LTBI versus active TB patients. Results: We observed a decrease in polyfunctional CD4+ T cell response (Dual and Triple cytokine producers) in active TB patients as compared to LTBI. Among the dual cytokine producers, IFNc+ TNF-a+ phenotype was significantly reduced in active TB patients. This could imply that polyfunctional CD4+ T cells response in TB patients is impaired, presumably due to immune evasion strategies by Mtb. We have previously shown that Programmed death1 receptor (PD-1) plays a critical role in inhibition of effector T cell response. Therefore, we envisage that blocking PD-1 could rescue the polyfunctional T cell response. This strategy may be used for rescuing host polyfunctional T cell response against Mycobacterium tuberculosis, thereby preventing the breach of latency. Conclusion: Our results suggest that polyfunctional CD4+ T cell response among infected subjects against Rv1813 may serve as immunological correlates of protection, preventing disruption of latency. Rescuing polyfunctional T cell response may have implications for vaccination strategies.
B085 PORPHYROMONAS GINGIVALIS’ NUCLEOSIDE DIPHOSPHATE KINASE CONTRIBUTES TO IL-1ß PRODUCTION AND SECRETION IN MACROPHAGES Ca´ssio L C Almeida-da-Silva1, Gabrielle C. Rocha1, Erivan S. Ramos-Ju´nior1, Ana Carolina F. Morandini1, Ju´lio Scharfstein1, O¨zlem Yilmaz2, David M. Ojcius3, Robson Coutinho-Silva1
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1
Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; University of Florida, Gainesville, FL , USA; 3University of the Pacific, Stockton, CA, USA
2
Porphyromonas gingivalis (Pg) are Gram-negative bacteria associated with periodontitis. One of Pg’s virulence factors is a homolog of nucleoside disphosphate kinase (NDK) enzyme, which is secreted by Pg. NDK cleaves extracellular ATP (eATP), suppressing ATP-induced P2 9 7-dependent apoptosis and ATP-induced reactive oxygen species (ROS) production via P2X7-NADPH-oxidase, and contributes to Pgpersistence in gingival epithelial cells. Our group showed that ATP via P2X7 controls infections with different kind of intracellular parasites such as Leishmania amazonensis and Toxoplasma gondii via IL-1b production. We also showed that IL-1b secretion induced by P2X7 signaling in macrophages is impaired by Pg’s fimbriae. Here, we investigated the role of Pg’s NDK in infection in vitro. To analyze whether IL-1b secretion is modulated by Pg’s NDK, we infected bone marrow derived macrophages (BMDM) with wild-type Pg or NDKdeficient (Pg D ndk) (MOI of 100) for 6, 18 or 24 h, and treated the cells or not with 5 mM ATP during the last 30 min. BMDM infected with Pg D ndk showed less IL-1b secretion by ELISA than wild-type Pg at all times tested. IL-1b secretion was dependent on P2X7 signaling since there was no secretion in BMDM from P2X7-/- mice infected with Pg. To verify immature pro-IL-1b production, we analyzed IL1-b in BMDM infected with Pg by Western blotting. By densitometry, we quantified 46 % less pro-IL-1b in cellular extracts and 46 % less IL-1b in supernatants of BMDM infected with Pg D ndk than wild-type Pg. Next, we tested the invasion capacity of Pg strains using the antibiotic protection assay. Pg strains infected at similar levels after 2 h of infection. Further, to verify whether Pg’s NDK hydrolyzes eATP, we quantified this nucleotide in BMDM supernatants after Pg infection. We detected more ATP in BMDM supernatants infected with Pg D ndk than wild-type Pg. Recently, adenosine (Ado) was shown to potentiate IL-1b secretion in macrophages treated with LPS/ATP. Therefore, we evaluated IL-1b release in BMDM supernatants after infection with Pg and treatment with ATP, Ado or its pan-antagonist receptor (CGS15943). Ado potentiated IL-1b secretion induced by Pg/ATP, and this enhancement was abolished by CGS15943. Together, these results indicate that Pg’s NDK contributes to IL-1b production and secretion in BMDM, and that the mechanism might be due to Ado production and signaling via Ado receptor after ATP cleavage by Pg’s NDK and other BMDM ectonucleotidases. Financial support CAPES, CNPq, FAPERJ, National Institutes of Health.
B086 ARF6 INHIBITION STABILIZES THE VASCULATURE WITHOUT COMPROMISING THE IMMUNE RESPONSE OF CYTOKINES Dean Li1,2, Weiquan Zhu1, Kirill Ostanin2, Alan Mueller2 1
2
University of Utah, Salt Lake City, UT, USA; Navigen Pharma, Salt Lake City, UT, USA The innate immune system is the first line of defense that facilitates recognition and clearance of invading microbes and ensures tissue repair. During infection, macrophages release cytokines to activate the nuclear factor-jB (NF-jB)-dependent signaling pathways, leading to recruitment of inflammatory cells. Cytokines also activate pathways that disrupt the endothelial barrier, facilitating clearance of infection by leukocytes. However, dysregulated cytokine release in conditions like chronic inflammation and sepsis causes excessive
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tissue degradation, actually worsening the disease. Current therapies target cytokines upstream of NF-jB, rendering the patient immunocompromised and susceptible to reactivation of infection. Therefore, a therapeutic strategy to combat pro-inflammatory manifestations of cytokines, without affecting the immune response, could be effective in preventing vascular destabilization. By dissecting pathways that trigger degradation of barrier function, we have identified that the small GTPase ADP-ribosylation factor 6 (ARF6) is a convergence point of several destabilization cues: the cytokine interleukin-1b (IL-1b), the endotoxin LPS, and proliferation signals such as vascular endothelial growth factor (VEGF). We report that IL-1b and ARF6 control the permeability of cultured human dermal microvascular endothelial cells, and that ARF6 is a critical regulator of VE-cadherin localization on the cellsurface. More importantly, inhibition of the NF-jB canonical pathway by siRNAs does not affect ARF6 activation or vascular permeability, implying that cytokine-induced ARF6 activation and downstream signaling are independent of NF-jB-mediated immune responses. Collectively, these results suggest that cytokine signaling bifurcates into NF-jB-mediated immune activation and ARF6-mediated VE-cadherin endocytosis and membrane hyperpermeability. We have verified this hypothesis by blocking ARF6 activation in animal models of inflammation. ARF6 inhibition after the onset of collageninduced arthritis in mice reduced vascular permeability in the joints, but had no effect on cytokine levels at 24 h after treatment. We also confirmed the efficacy of ARF6 inhibition in animal models of other conditions that result from compromised vasculature. Blocking ARF6 was sufficient to reduce vascular leak and enhance survival during endotoxic shock in mice, without inhibiting the host cytokine response. In addition to reducing cytokine- and endotoxin-mediated endothelial permeability, we report that ARF6 also blunts the internalization of VEGFR, dampens signaling to multiple downstream cascades including MARCKS and ERK, and mutes pathologic endothelial hyperpermeability in mouse models of diabetic retinopathy. Collectively, these data identify ARF6 activation to be a promising target to modulate the inflammatory process by stabilizing vasculature in conditions such as arthritis, acute respiratory distress syndrome, and vascular eye diseases.
B087 DIFFERENT PHENOTYPES OF LUNG PARENCHYMAL CD4+ T CELLS ACTIVATION DURING AGGRESSIVE FORMS OF TUBERCULOSIS Eduardo P. Amaral1, Simone C.M. Ribeiro2, Caio Cesar Bomfim1, Rafael M. Salgado1, Veronica R. Lanes2, Mario Hirata3, Jose M.M. Alvarez1, Elena Lassounskaia2, M. Regina D’Imperio-Lima1 1
Departament of Immunology, Biomedical Science Institute, University of Sa˜o Paulo, Sa˜o Paulo, Brazil; 2Laboratory of Biology of Recognition, State University of North Fluminense, Campos dos Goytacazes, Brazil; 3Departament of Clinical Chemistry and Toxicology, Faculty of Pharmaceutical Sciences, University of Sa˜o Paulo, Sa˜o Paulo, Brazil Severe tuberculosis (TB) is characterized by lung necrosis, pneumonia, atelectasis and bacillus dissemination. However, the mechanism by which hypervirulent mycobacteria induce severe disease and how the host immune response is activated during that process is not so clear. In this study, we sought to determine the major features of
Inflamm. Res. bacillus-host interaction responsible for immune response activation induced by hypervirulent strains (Beijing 1471, MP287/03 and B2 strains). C57BL/6 mice were infected with a low-dose of bacteria (*100 bacilli) and evaluated bacteria burden, cytokine production and CD4+ T cells activation in the lung parenchyma and pulmonary vasculature by using intravenous CD45 staining. We found that disease severity correlated directly with the intensity of bacillus multiplication in vivo and in vitro. C57BL/6 mice suffering from severe TB showed extensive necrotic areas in the lungs, pneumonia and bacillus dissemination. Either extremely low or high productions of pro-inflammatory mediators. In contrast, during mild disease (induced by H37Rv strain), the production of intermediate levels of proinflammatory cytokines was associated with low bacillus burden. Moreover, mice infected with hypervirulent mycobacteria (Beijing 1471 and MP287/03) showed similar frequencies of CD4+ CD44+ T cells. All CD4+ T subsets that presented expression of CD69 were found inside the lung parenchyma. Mice infected with Beijing 1471 showed increased frequency of lung parenchymal CD4+CD44+CD69+ T cell subset, whereas reduced frequencies of that subset were found in mice infected with MP287/03. These phenotypes of lung parenchyma CD4+ T cells reflected in the production of IFN-c and number of Mtb-ESAT-6 tetramer+ CD4+T cells in the tissue. This study shows that several pathological manifestations of severe TB can be reproduced in mice infected with hypervirulent mycobacteria. Furthermore, the lung parenchyma millieu can impact the CD4+ T cell activation, contributing to the outcome of aggressive TB.
Epigenetics and Regulation of the Immune Response B088 LYSINE METHYLTRANSFERASE SETD7 REGULATES OXYGEN HOMEOSTASIS THROUGH OXIDATIVE STRESS DETOXIFICATION AND MITOCHONDRIA FUNCTIONS Shuying He, Dafydd Owen, Scott Jelinsky, Lih-Ling Lin Pfizer, New York City, NY, USA Introduction: Lysine methylation is critical for signaling regulation. Lysine methyltransferase SETD7 has been found to methylate lysine residues in both histone and non-histone proteins. Reactive oxygen species (ROS) have profound disease implication in neurodegeneration, cardiovascular defects, metabolic dysfunction, respiratory diseases and cancer. Our hypothesis is that SETD7 is required for cellular oxidative stress defense. Understanding the functions of SETD7 in ROS homeostasis will provide new insight into the mechanisms of lysine methylation and oxidative stress response, and identify novel therapeutic targets against ROS-associated diseases. Methods: In order to study the functions of SETD7, siRNA knockdown was performed in human lung bronchial epithelial cells (Beas2B) and primary lung fibroblasts. Cells were stimulated by cigarette smoke extract, hydrogen peroxide or TGFb1 to induce oxidative stress. Gene expression was quantified by western blot and qPCR. Cellular ROS and mitochondrial ROS levels were measured after stimulation. Similar experiments were performed in the presence of SETD7 inhibitor to evaluate compound potency. Results: Expression of NRF2, PGC1a, MnSOD and Catalase was elevated in both siSETD7- and compound- treated cells. Inflammatory cytokine production was inhibited in the knockdown cells. Both
S137 cellular and mitochondrial ROS were attenuated by either RNA knockdown or SETD7 inhibitor under different stress conditions. Conclusions: In this study we identify novel roles of SETD7 in oxidative stress response. Our data suggest that SETD7 is important in fine-tuning cellular oxygen signaling and protecting cells against excessive production of ROS.
B089 EFFECT OF HYDROGEN SULFIDE (H2S) ON HISTONE METHYLATION OF LPS-STIMULATED MACROPHAGES Ricardo C. Petroni, Denise F. Barbeiro, Thais M. de Lima, Suely K. Ariga, Francisco G. Soriano University of Sa˜o Paulo, Medical School, Sa˜o Paulo, Brazil Hydrogen sulfide (H2S) is an endogenously produced gaseous messenger that has gained increasing recognition as an important player in modulating inflammatory mediator production. Recent studies have shown the role of H2S in chromatin modulation by histone acetylation. However, its role in histone methylation (associated with gene repression and hallmark of condensed chromatin at silent loci) and its effects on inflammatory cytokine production is currently unknown. In this study, we investigated the role of H2S on histone methylation in an invitro model of lipopolysaccharide (LPS)-induced inflammation and evaluated its effects on inflammatory cytokine production. Macrophage-differentiated THP-1 cells were pre-treated with propargylglycine (PAG) (inhibitor of enzyme CSE) at 1 mM for 90 min or sodium hydrosulfide (NaHS) (an H2S donor) at 1 mM for 30 min. To stimulate cytokine production, the cells were challenged with bacterial LPS (1 lg/ml) for 24 h. Histone H3 and H4 methylation was analyzed by western blotting and cytokine production was measured by ELISA. H2S inhibition increased the production of interleukin-6—IL6(692.2 ± 1.6) and tumor necrosis factor-a—TNFa(9790 ± 1318) compared to NaHS group (4.1 ± 1) and (163.5 ± 61), respectively. This effect was associated with adecreased H3 methylation at lysines 9 (26,5035.9 ± 17880) and 27 (73,1460 ± 73004.7) compared to NaSH treated cells (40,1700 ± 5032) and (1,735,050 ± 5783.2) respectively and H4 Lys 20 methylation (75,161 ± 2473.7) in H2S inhibited cells compared to NaSH cells (312,985.5 ± 3923.7). Thus, the findings of the present study suggest that H2S inhibition suppresses histone methylation, which, in turn, induce chromatin openness, leading to an increase in the gene transcription of pro-inflammatory cytokines.
B090 DUAL TRANSCRIPTOME SEQUENCING REVEALS RESISTANCE OF TLR4 LIGAND-ACTIVATED BONE MARROW-DERIVED MACROPHAGES TO INFLAMMATION MEDIATED BY THE BET INHIBITOR JQ1 Amitabh Das1, Jin Choul Chai2, Chul-su Yang2, Young Seek Lee2, Kyoung Hwa Jung3, Young Gyu Chai1,2 1 Department of Bionanotechnology, Hanyang University, Seoul, 133-791, Republic of Korea; 2Department of Molecular and Life Sciences, Hanyang University, Ansan, 426-791, Republic of Korea;
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Institute of Natural Science and Technology, Hanyang University, Ansan, 426-791, Republic of Korea The persistence of macrophage activation is associated with the production and secretion of various pro-inflammatory genes, cytokines and chemokines, which may initiate or amplify inflammatory disorders. A novel synthetic BET inhibitor, JQ1, was proven to exert immunosuppressive activities in macrophages. However, a genome-wide search for JQ1 molecular targets is largely unexplored in macrophages. The present study aimed at evaluating the anti-inflammatory function and underlying genes that are targeted by JQ1 in lipopolysaccharide (LPS)stimulated primary bone marrow-derived macrophages (BMDMs) using two transcriptomic techniques:global transcriptomic RNA sequencing and quantitative real-time PCR. Among annotated genes, transcriptional sequencing of BMDMs treated with JQ1 revealed selectively reduced expression of cytokines/chemokines, interferonstimulated genes, and prominent (transcription factors) TFs. Additionally, we found that JQ1 reduced the expression of previously unidentified genes that are important in immune regulation. Importantly, these inflammatory genes were not affected by JQ1 treatment alone. Moreover, the biological pathways and functional genomics obtained in this study may facilitate the suppression of different key inflammatory genes through JQ1 treatment in BMDMs. These unprecedented results suggest that the BET inhibitor JQ1 is a candidate for the prevention or therapeutic treatment of inflammatory disorders.
Fibrosis and Tissue Remodeling B091 IDENTIFICATION OF BOTH IMMUNOMODULATORY AND ANTI-FIBROTIC ACTIONS OF NINTEDANIB AND PIRFENIDONE USING BIOMAPÒ HUMAN PRIMARY CELLBASED DISEASE MODELS Sharlene Velichko, Sylvie Privat, Dat Nguyen, Hannah Cho, Alison O’Mahony BioSeek, a division of DiscoveRx, South San Francisco, CA, USA Idiopathic pulmonary fibrosis (IPF) is a chronic progressive disease that is characterized by pleural scarring of the lung, a result of an aberrant wound healing response to acute lung injury. Given the unfavorable response of IPF patients to both corticosteroid treatment and other anti-inflammatory agents in clinical trials, the impact of inflammation on IPF, particularly at end-stage disease, remains unclear. However, recent reports on the presence of inflammatory cell types in IPF lung tissue as well as in the periphery support the contribution of inflammation in disease pathogenesis. Agents targeting the anti-fibrotic effects, such as TGFbR signaling inhibitors, have also failed in IPF trials and are actually pro-inflammatory. Collectively, these data suggest that inhibition of both fibrotic and inflammatory processes may have a greater impact on IPF pathology than targeting either aspect alone. Recently, nintedanib (Ofev), a multi-angiokinase inhibitor targeting downstream mediators of VEGF, PDGF and bFGF signaling, and pirfenidone (Esbriet), an anti-fibrotic small molecule with an unknown mechanism of action, were approved by the FDA and the EMA for the treatment of IPF. We used the BioMAP technology platform to predict how these agents work both mechanistically and biologically to improve therapeutic responses in IPF patients. The BioMAP technology platform enables target agnostic phenotypic screening of agents in human primary cell-based disease
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Inflamm. Res. models called BioMAP systems. Each system consists of complex cocultures of human primary cell types, stimulated in a manner designed to recapitulate the complex signaling and multi-component biology contributing to inflammation, fibrosis, and wound healing. Screening of nintedanib and pirfenidone across a wide panel of BioMAP systems revealed, as expected, potent anti-fibrotic activities in systems modeling fibroblast biology. Interestingly, both compounds also displayed potent immunomodulatory activities on both adaptive and innate immune cell types, including T cells, B cells and monocytes/macrophages. Activities include: suppression of proinflammatory chemokine and cytokine production by immune cells as well as anti-proliferative activity, which could result in significant immune cell skewing. These data indicate the efficacy of these drugs is due to combinatorial effects impacting both anti-fibrotic and antiinflammatory aspects involved in IPF. The effectiveness of these agents supports either further development of drugs with this desired polypharmacology, or novel combination approaches designed to target both the fibrotic and inflammatory components driving IPF pathogenesis.
B092 DISEASE RELEVANT IN VITRO AND IN VIVO MODELS FOR LUNG FIBROSIS Jeroen DeGroot1, Krista Ouwehand1, Blandine Mille-Baker1, Alan Young2, Mary McElroy3, Joe Cornicelli4, David Bonnel5, Jonathan Stauber5, David F. Fischer1 1
Charles River Laboratories Leiden, the Netherlands; 2Charles River Laboratories Oxford, UK; 3Charles River Laboratories Edinburgh, UK; 4Charles River Laboratories, Wilmington, MA, USA; 5 ImaBiotech Lille, France Objectives: Fibrosis is characterized by excessive deposition of extracellular matrix due to exaggerated repair in response to damage. While the initiating event(s) and the underlying pathophysiological processes may vary between organs and diseases, common features include the involvement of inflammation, appearance of myofibroblasts, and changes in tissue architecture and function. In lung fibrosis, persistent and non-resolving injury to the alveolar epithelium is thought to drive the disease. Because of the complex interactions between various cell types require state-of-the art in vitro and in vivo models are needed that capture disease relevant processes to enable the discovery and development of new drugs for fibrotic diseases. Methods: Primary human bronchial epithelial cells and fibroblasts, isolated from IPF donors and control donors were cultured in 96-well format. Trans-differentiation was induced with TGF-b and assessed by high content imaging (INCell 2200) following immunostaining for fibronectin (EMT in the epithelial cells) and aSMA (FMT in the fibroblasts). The effect of SB525334 (ALK5 inhibitor; assay positive control), perfenidone, nintedanib, imatinib, MB06322, thalidomide, N-acetyl cysteine, tofacitinib, and GSK2126458 was evaluated. In vivo, lung fibrosis was induced in rats by seven daily bleomycin doses (at 1 mg/kg) that were delivered by oropharyngeal aspiration. Readout parameters (day 7 and 22) included body weight, clinical signs, respiratory parameters, histopathology and molecular MALDI imaging. Results: Both EMT and FMT assays were compatible with high throughput screening of small molecules and RNAi vectors. Signal/ background ratio, vehicle tolerance, assay window and intra and interassay variability all passed pre-set QC criteria. Nintenadib and GSK2126458 inhibited EMT and FMT. Imatinib and MB06322 inhibited FMT but not EMT. The other compounds did not modulate trans-differentiation (up to 10 lM). In vivo, lung function (mean tidal
Inflamm. Res. volumes) decreased and respiratory rate increased statistically significantly following repeated bleomycin administration. Inflammatory and fibrotic changes at necropsy were consistent with progressive fibrosis. A combination of MALDI imaging and microscopy allowed monitoring the distribution of bleomycin in the lung, as well as the dynamics of (novel) fibrosis markers. Conclusions: The primary human cell-based assays allow evaluation of compounds targeting various molecular mechanisms. In vivo, the combination of mass spectrometry imaging and histological staining allowed detailed assessment of disease processes by identifying disease markers and studying their co-distribution at sites of active disease.
B093 LINEAGE TRACING OF RESIDENT FIBROBLASTS BY THE INTRATRACHEAL CELL TRANSFER ELUCIDATES THE CELLULAR ORIGIN OF ACTIVATED FIBROBLASTS IN PULMONARY FIBROSIS Tatsuya Tsukui1,3, Satoshi Ueha1,3, Shigeyuki Shichino1,3, Yutaka Inagaki2,3, Kouji Matsushima1,3 1 Department of Molecular Preventive Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; 2Center for Matrix Biology and Medicine, Graduate School of Medicine, Tokai University, Kanagawa, Japan; 3Japan Science and Technology Agency-CREST Program, Tokyo, Japan
Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease with few therapeutic options, characterized by architectural destruction of alveoli and formation of fibroblastic foci. Activated fibroblasts, such as myofibroblasts that express a-smooth muscle actin (a-SMA), accumulate in fibroblastic foci and produce excessive amounts of extracellular matrix components. To develop specific treatments for IPF, the pathologic nature and origin of activated fibroblasts need to be clarified. One problem in recent lineage tracing studies is that the rigorous lineage tracing of resident fibroblasts, which are classically suggested as progenitors of activated fibroblasts, is still lacking. Here we report lineage tracing of resident fibroblasts by using the intratracheal adoptive cell transfer method in bleomycininduced lung injury. Histological analysis of GFP reporter mice of type 1 collagen showed that activated fibroblasts migrated into epithelium-denuded alveolar airspaces in the early phase of bleomycin-induced pulmonary fibrosis. Purified resident fibroblasts delivered into injured alveoli through an intratracheal route displayed similar activated signatures and comprised fibroblastic foci. Neither pericytes nor epithelial cells had the same potential. Transferred resident fibroblasts proliferated and highly up-regulated pro-fibrotic genes including a-SMA. These data may suggest resident fibroblasts as the major origin of activated fibroblasts in pulmonary fibrosis.
B094 PERICYTE MYD88 CONTROLS INFLAMMATORY AND FIBROTIC RESPONSES TO TISSUE INJURY Irina Leaf1,3, Ivan Gomez1,2, Shunsaku Nakagawa2, Bryce Johnson1,2, Jin Joo Cha2, Julia Lichtnekert2, Kristen Mittelsteadt2, Alan Aderem3, Bill Altemeier2, Jeremy Duffield1,2 Biogen, Cambridge, MA, USA; 2University of Washington, Seattle, WA, USA; 3Seattle BioMed, Seattle, WA, USA 1
S139 Sterile inflammation is increasingly recognized as a central problem in many acute and chronic diseases but the mechanisms controlling responses to sterile inflammation are poorly understood. Injured or dying cells release products, or Danger Associated Molecular Patterns (DAMPs), recognized by immune receptors contributing to disease progression. Here we show that DAMPs isolated from injured kidney activate TLR2/4 and MyD88 dependent transcription of immune genes in non-immune kidney resident cells, pericytes. When treated with extracellular DAMPs pericytes form an active NLRP3 inflammasome, process pro-IL1 and pro-IL18 to their active secreted forms and undergo pyroptosis, thereby contributing to inflammation and leukocyte trafficking. Recent studies have indicated that kidney pericytes represent a major source of interstitial myofibroblasts in renal fibrogenesis. We show that treatment with DAMPs in vitro activate pericyte to myofibroblast transition indicated by upregulation of transcription of fibrotic genes Col1a1 and Acta2 and production of SMA. MyD88-/pericytes showed significant reduction in fibrogenesis markers. In wound healing assays DAMPs caused pericyte migration, which was significantly reduced in the absence of MyD88. Cell-specific ablation of MyD88 in perivascular and stromal cells in models of ischemic acute kidney injury in mice significantly attenuates innate immune activation, injury and fibrogenic responses. In addition, we show that human pericytes respond to DAMPs in a similar manner by activating immune genes, secreting pro-inflammatory cytokines and inducing pyroptotic cell death. In conclusion, pericytes respond to sterile inflammation via two divergent mechanisms both of which are controlled by MyD88: activation of immune signaling which enables detection and amplification of the inflammatory signal; and activation of fibrogenesis contributing to pathology. Therefore, this regulation could be an important new therapeutic target for tissue injury.
B095 POLYCYSTIC LIVER DISEASE IS SUPPRESSED BY IL-4/IL-13 AND TGF-ß SIGNALING David A. Cantu1, Kristen Kindrachuk1, Lee Borthwick1, Doug Morris2, Danielle Donahue2, David Dorwood3, Brenda Klaunberg2, Thirumalai Ramalingam1, Sandy White1, Robert Thompson1, Thomas Wynn1 1
Immunopathogenesis Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH), Bethesda, MD, USA; 2Magnetic Resonance Imaging Research Facility/Mouse Imaging Facility, National Institutes of Health (NIH), Bethesda, MD, USA; 3Microscopy Unit, Research Technologies Branch, NIAID/Rocky Mountain Laboratories Hamilton, MT, USA
Introduction: Polycystic liver disease (PLD) is a relatively rare but devastating condition that causes cysts—fluid filled sacs—to grow throughout the liver. The immunological mechanisms controlling liver cyst formation, however, remain unclear. Methods: Briefly, IL-17A knockout (KO), IL-4/IL-13 double KO (dKO) mice, and IL-4/IL-13/IL-17A triple KO (tKO) were infected with 35 cercariae of the Puerto Rican (NMRI) strain of Schistosoma mansoni (S. Mansoni) harvested from biomphalaria glabrata snails (Biomedical Research Institute Rockville, MD). Mouse livers were harvested 7-12 weeks post-infection for hydroxyproline content, flow cytometry, gene expression, and histological analysis. Results: We show that during infection with the helminth parasite S. Mansoni, severe PLD-like disease develops when infected mice are deficient in both IL-4 and IL-13, suggesting that Stat6-mediated
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S140 signaling is critically required to prevent the development of PLD. We hypothesized that the parasite eggs might be contributing to cyst formation by promoting damage to microvessels in the liver, which are not efficiently repaired when mice are deficient in both IL-4 and IL-13. Because IL-4 and IL-13 are linked with wound repair and are potent inducers of transforming growth factor-beta (TGF-b) expression, we examined whether cyst formation in IL-4/IL-13-deficient mice was linked with uncontrolled type 1-associated inflammation and/or maladaptive repair. Although IL-17A was up-regulated in IL4/IL-13 double knockout (dKO) mice, supporting the emergence of a Th17-driven inflammatory response, IL-17A KO and IL-4/IL-13/IL17A triple KO (tKO) were equally susceptible to the development of PLD, although the tKO displayed slightly improved survival. Histological analysis of liver tissues from IL-4/IL-13 dKO mice revealed an elevated infiltration of F4/80+ macrophages that co-localized with TGF-b-inducible protein along the cyst border, suggesting TGF-b signaling was elevated in the dKO mice. To elucidate the role of TGF-b, infected IL-4/IL-13 dKO mice were treated with a pan-neutralizing TGF-b monoclonal antibody (mAb). Strikingly, the number and size of cysts was dramatically increased when type-2 cytokines and TGF-b signaling were simultaneously blocked, suggesting that IL-4, IL-13, and TGF-b collaborate to prevent PLD during chronic S. mansoni infection. Conclusion: These studies suggest that therapeutic strategies that increased IL-4/IL-13 and/or TGF-b signaling may provide a novel approach to treat patients with progressive PLD. References [1] Lee et al. IL-13 Induced Tissue Fibrosis by Selectively Stimulating and Activating Transforming Growth Factor b1. J. Exp. Med. 2001; 194(6): 809–821 [2] Chiaramonte et al. An IL-13 inhibitor blocks the development of hepatic fibrosis during a T-helper type 2-dominated inflammatory response. J. Clin. Invest. 1999; 104: 777–785.
B096 EVALUATING IL-13RA2 AS A THERAPEUTIC TARGET FOR PULMONARY FIBROSIS Nikhil Jiwrajka1, Thirumalai Ramalingam1, Josh Sciurba1, Sandy Oland1, Rafael Prado1, Kevin Hart1, Trisha Pasricha1, Kevin Vannella1, Bernadette Gochuico2, Marion T. Kasaian3, Thomas A. Wynn1 1
Immunopathogenesis Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA; 2Undiagnosed Diseases Program, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA; 3Department of Inflammation and Immunology, Pfizer Research, Cambridge, MA, USA Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal disease that remains incurable despite recent therapeutic advances. Gene expression profiling studies in IPF patients have repeatedly revealed increased expression of IL-13Ra2, a high-affinity receptor that binds IL-13 and sequesters it from the surrounding milieu. We sought to examine the role of IL-13Ra2 in pulmonary fibrosis by interrogating the murine bleomycin-induced lung injury model. Previous work has indicated that fibrosis in this model is driven by the inflammatory cytokine IL-17A. Recently, several groups have shown that Th17 cells express a functional IL-13 receptor, IL-13Ra1, that serves to downregulate the production of IL-17A and upregulate the production
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Inflamm. Res. of IL-10, suggesting that Th2 cytokines may help regulate Th17mediated inflammation. To investigate the interplay between IL13Ra2 and Th17/Th2 immunity, wild-type (WT) C57BL/6, IL-13Ra2 KO, and IL-10 KO mice were subjected to intratracheal bleomycin injections and were subsequently evaluated for inflammation and fibrosis over a 30-day period. Temporal gene expression analysis in WT mice showed a very early increase in IL-13Ra2 expression that was rapidly accompanied by increased expression of IL-17A, TNF-a, and IP-10, in the absence of a prominent early Th2 signature. IL13Ra2 isolated from the bronchoalveolar lavage fluid of bleomycintreated animals remained unsaturated throughout the 30-day period, suggesting that IL-13Ra2 could be contributing to the development of pulmonary fibrosis by mitigating the potential dampening effects of IL-13 on Th1/Th17-mediated inflammation during the early inflammatory stage following injury. Though our studies with IL-13Ra2 KO mice did not show statistically significant changes in fibrosis, gene expression analysis in these mice suggested increased IL-13 effector functions and IL-10 expression compared to WT mice, supporting the notion that IL-13Ra2 suppresses both IL-13 and IL-10 bioactivity. Furthermore, our findings contradict earlier observations showing that IL-13Ra2, through its induction of TGFb, is required in bleomycininduced lung fibrosis, as we observed no impairment in the expression of TGF-bi, a surrogate marker for TGFb activity, in the lungs of IL13Ra2 KO mice. Future studies in a more chronic model of pulmonary inflammation and fibrosis may better address the role of IL-13 and IL-13Ra2 in established and progressive pulmonary fibrosis.
B097 CROSSTALK BETWEEN MACROPHAGES AND HUMAN HEPATIC FIBROBLASTS SHOW THAT INFLAMMATORY RESPONSE ATTENUATE THE ACTIVATION OF FIBROBLASTS IN THE FIBROSIS RESPONSE Sacha Robert1, Thomas Gicquel1, Aude Bodin1, Alain Fautrel2, Vincent Lagente1, Elisabeth Boichot1 1
UMR991 INSERM, Universite´ de Rennes 1, Rennes, France; 2H2P2, Histopathological Platform, Universite´ de Rennes 1, Rennes, France Liver fibrosis is a physiological response due to acute injury and resulting in an inflammatory process, imbalance between matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs), and the production of scar tissue (collagen). When the injuries persist, excessive collagen deposition could lead to serious pathologies. At cellular level, collagen production is mediated by activated fibroblasts, also known as hepatic stellate cells. This activation occurs after crosstalk between fibroblasts and liver macrophages which express pro-inflammatory cytokines such as interleukin-1 beta (IL-1ß) cleaved into active form by caspase-1 associated with NLRP3-inflammasome pathway. Driving a coculture model of macrophages and hepatic fibroblasts, we put on light that the inflammatory response and especially IL-1ß seems to attenuate the activation of fibroblasts in the fibrosis response. THP-1 cell line or human peripheral blood monocyte cells were differentiated into macrophages with respectively phorbol-myristateacetate at 10 ng/mL for 3 days and GM-CSF at 50 ng/mL for 7 days, then treated with lipopolysaccharide at 0,1 lg/mL and followed after 18 h by monosodium urate crystals at 300 lg/mL for 6 h. Two coculture models were chosen with two types of liver fibroblasts. We used the LX-2 cell line, which are human immortalized liver myofi-
Inflamm. Res. broblasts, or human hepatic stellate cells from liver biopsies. Firstly, macrophages were placed in coculture at the top of inserts with liver fibroblasts in the bottom of the multi-well dish. Secondly, the supernatant of macrophages was removed and put on fibroblasts. After 24 h, mRNA expression was evaluated by RT-qPCR and after 48 h, proteins secretion was measured in the supernatant using ELISA method and MMPs activities by zymography. Cocultures of liver fibroblasts with macrophages result in increased mRNA levels of MMP-1, MMP-3 and MMP-9 and also their collagenase activity whereas there is a decrease in a-SMA, type I and IV collagens, endothelin-1 and PDGF-BB. IL-1ß from the macrophages also exacerbates the pro-inflammatory environment by stimulating IL-1ß, IL-6, IL-8 and chemokines such as GRO-a and MCP1 expression and/or release. These effects were partially reduced by pre-treatment of macrophages with an inhibitor of inflammasome activation (by targeting caspase-1), the Z-YVAD-FMK, or pre-treatment of fibroblasts with an antagonist of IL-1ß receptor, Anakinra. IL-8 and TNF-a seems also to participate in the process as they show similar effects. Taken together, these results demonstrate that the inflammatory response, mostly involving IL-1ß, trigger an anti-fibrogenic environment on liver fibroblasts and that regulation of the differentiation state of macrophages is important for the production of collagen in the control of chronic liver fibrosis.
B098 PERICYTE AND MYOFIBROBLAST ACTIVATION REGULATED BY THE TWEAK/FN14 PATHWAY Ivan G. Gomez1,2, Naoki Nakagawa2, Timothy S. Zheng1, Jeremy S. Duffield1,2, Linda C. Burkly2 1 Biogen, Cambridge, MA, USA; 2Renal Division, University of Washington, Seattle, WA, USA
Background: Pericytes, mesenchymal cells that partially wrap capillary endothelium and are critical for supporting their integrity, have been shown by genetic fate-mapping to be a major progenitor pool for myofibroblasts in a number of fibrotic contexts. Pericytes provide directional cues that route the innate immune cells extravasating into injured tissue. Previously it was shown that TWEAK, a TNF family cytokine produced largely by leukocytes, promotes macrophage infiltration and fibrosis in the UUO model of renal injury. We hypothesized that TWEAK mediates its effect through its injury-inducible receptor, Fn14. Since Fn14 is expressed by mesenchymal lineage cells and regulates their fate, we hypothesized that TWEAK/ Fn14 might be a key element for pericyte activation in response to tissue injury. Results: We show that Fn14 deficiency ameliorates myofibroblast appearance and renal fibrosis in the UUO model, phenocopying TWEAK deficient mice. TWEAK activates primary murine cultured renal pericytes through Fn14, inducing low level proliferation, migration and production of proinflammatory mediators, as well as pericyte to myofibroblast transition measured by induction of a-smooth muscle actin expression and stress fiber formation. Furthermore, myofibroblasts are activated by TWEAK/Fn14 signaling, exhibiting higher-level proliferation, migration and cytokine production as well as cytoskeletal changes. Transcriptional profiling of TWEAK-stimulated myofibroblasts supports its multifaceted role and identified TWEAK-response genes. Conclusions: These findings suggest that targeting the TWEAK/Fn14 pathway is an approach to modulate pericyte and myofibroblast activation and a novel way to target both inflammatory and fibrotic aspects of fibrotic disease.
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GPCRs in Inflammation B099 ACETATE PROTECTS THE ISCHEMIC INTESTINE FROM REPERFUSION INJURY Zoe V. Schofield1, Reena Halai1, Matthew A. Cooper1, Trent M. Woodruff2 1
Institute for Molecular Bioscience, St Lucia, QLD, Australia; School of Biomedical Sciences, The University of Queensland, St Lucia, QLD, Australia
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It has long been advocated that acetate has therapeutic benefits however, the mechanism of action for this has not been determined. SCFA’s can transiently modulate cell function or alter cell function through the free fatty acid 2 receptor (FFA2) [1]. In the past decade FFA2 has been shown to be involved in several diseases including cancer, obesity, diabetes mellitus, asthma and inflammatory bowel disease [2, 3]. FFA2 is abundantly expressed on neutrophils [3] which are the primary responders in ischemia reperfusion injury (IRI) and represent important components in the prolonged inflammatory response and severity associated with these conditions [4]. IRI is a common, damaging and untreatable condition that is common during surgery, trauma and stroke [4] with a 50 % mortality rate. In this study, we investigated the role of acetate in a mouse model of mesenteric IRI to address the potential anti-inflammatory effects of acetate in IRI. Untreated mice showed significant damage of the villi in the small intestine, whereas protection was afforded to the villi by acetate. Interestingly despite this protective effect, neutrophil infiltration was significantly increased in the intestine after dosing with acetate. Our data suggests that acetate may modulate neutrophil function and equally protect cells against ischemic damage. References 1. Vinolo M, Hosana AR, Rodrigues G, Hatanaka E, Sato FT, Sampaio SC, Curi R. Suppressive effect of short-chain fatty acids on production of proinflammatory mediators by neutrophils. J Nutr Biochem 2011. 2. Ulven T. Short-chain free fatty acid receptors FFA2/GPR43 and FFA3/ GPR41 as new potential therapeutic targets. Front endocrinol. 2012. 3. Maslowski KM, Vieira AT, Ng A, Kranich J, Sierro F, Yu D, Schilter HC, et al. Regulation of inflammatory responses by gut microbiota and chemoattractant receptor GPR43. Nature. 2009. 4. Schofield ZV, Woodruff TM, Halai R, Wu MC-L, Cooper MA. Neutrophils-a key component of ischemia–reperfusion injury. Shock. 2013.
B0100 PRIMARY MICROGLIA ADOPT A PROINFLAMMATORY PHENOTYPE AFTER EXPOSURE TO INCREASED LPA LEVELS Joanna Plastira1, Eva Bernhart1, Astrid Hammer2, Wolfgang Sattler1 1
Institute of Molecular Biology and Biochemistry, Medical University Graz, Graz, Austria; 2Institute of Cell Biology, Histology and Embryology, Medical University Graz, Graz, Austria Microglia, the immunocompetent cells of the CNS, are rapidly activated in response to brain injury and disease. Microglial migration towards and homing at damaged tissue plays a key role in CNS
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S142 regeneration. Classically activated microglia (M1) are able to synthesize pro-inflammatory factors that inflict neuronal damage, while the production of trophic and anti-inflammatory factors by M2 microglia can support neuronal survival and regeneration. Under chronic inflammation as observed in many neurodegenerative diseases, microglia are characterized by over activation and secretion of proinflammatory and neurotoxic factors that can induce neuronal damage. Lysophosphatidic acid (LPA) is produced via the autotaxin pathway or by phospholipase A-mediated pathways. LPA has diverse biological functions mediated by downstream signaling through different receptors. These receptors play prominent role in the central nervous system, and signaling is amplified at sites of inflammation where LPA concentrations are increased. During earlier work we could show that LPA regulates microglia protein expression and affects their migrational response in a PKD dependent pathway. Western blotting for MAPKs and PKDs revealed that lysophosphatidic acid (LPA) can potently activate these pathways and confocal microscopy analysis depicted LPA induced changes in microglia cytoskeleton. Using flow cytometry, we obtained evidence that microglia (both BV-2 and primary) polarized towards an M1 proinflammatory phenotype. LPA treatment induced high CD40 (M1 marker) expression while the levels of CD206 (M2 marker) dramatically decreased. Western blot analysis demonstrated that LPA increased protein expression of iNOS and COX-2 (both M1). On the contrary, the basal expression of arginase I and RELMa (FIZZ) was gradually suppressed (both M2). Immunofluorescence for iNOS, COX2, Arginase I and RELMa confirmed these results. These findings were accompanied by increased IL-6 secretion and nitric oxide (NO) production further supporting LPA-mediated polarization towards M1. A better understanding of the role of bioactive lipid mediators in microglia physiology and pathophysiology and the characterization of signaling cascades that drive microglia polarization could open new possibilities to modulate microglia function.
B101 PLATELET ACTIVATING FACTOR RECEPTOR CONTROLS ADIPOSE TISSUE MACROPHAGES PHENOTYPE AND GLUCOSE METABOLISM Luciano R. Filgueiras1, Francisco J. Rios1,2, Marianna M. Koga1, Paula G. Quaresma3, Edson K. Ishizuka1, Marlise BA Montes1, Patricia O. Prada3,4, Mario S. Saad3, Sonia Jancar1 1 Department of Immunology, Institute of Biomedical Sciences, University of Sa˜o Paulo, Sa˜o Paulo, Brazil; 2Institute of Cardiovascular and Medical Sciences, British Heart Foundation Glasgow Cardiovascular Research Centre, University of Glasgow, Glasgow, United Kingdom; 3Department of Internal Medicine, State University of Campinas (UNICAMP), Campinas, SP, Brazil; 4School of Applied Sciences, State University of Campinas (UNICAMP), Limeira, SP, Brazil
Luciano R Filgueiras and Francisco J Rios contributed equally Introduction: Metabolic dysfunction is associated with inflammatory response in adipose tissue mediated by activated macrophages. We previously showed that the interaction between oxidized lipids and platelet activating factor receptor (PAFR) shifted macrophages towards anti-inflammatory phenotype. We thus questioned whether in vivo deficiency of PAFR changes the activation profile in adipose tissue macrophages and influences metabolic parameters in these animals.
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Inflamm. Res. Methods: PAFR deficient (PAFRKO) and wild type animals (WT) were fed standard and high fat diet (HFD) for 16 weeks or aged in standard diet for 40 weeks. Glucose and insulin tolerance tests were performed by blood monitoring. Macrophages were isolated from epididymal withe adipose tissue (WAT) and evaluated by FACS. Gene and protein expression was investigated by PCR and westernblot, respectively. Results: Macrophages infiltrated in WAT showed a pro-inflammatory M1-phenotype, characterized by increased population of F4/ 80+CD11c+ cells (twofold) and increased gene expression of Ccr7 (twofold), Nos2 (3-fold), Il6 (threefold), and Il12 (twofold); whereas IL10 gene was less expressed (p \ 0.05 vs. WT animals). This was observed in animals fed standard or high fat diet and in aged mice. WAT from PAFRKO and WT showed similar levels of adiponectin and chemerin, anti- and pro-inflammatory adipokines, respectively. Compared to WT, PAFRKO presented significant (p \ 0.01), increased weight (25 %) and adipocyte size (30 %), which are characteristics of obesity; higher fasting serum glucose levels, followed by decreased glucose tolerance and insulin resistance; impaired insulininduced AKT phosphorylation in the liver and hepatic damage. When fed with HFD, PAFR deficient mice presented increased weight gain, insulin resistance that affected the liver and skeletal muscle and developed liver steatosis. Conclusion: Our results indicate that PAFR expressed by adipose tissue macrophages have an important role in maintaining their antiinflammatory profile and this process is essential to preserve adipose tissue homeostasis and balanced glucose metabolism.
B102 GUT MICROBIAL METABOLITES PROTECT AGAINST AUTOIMMUNE DIABETES BY PRESERVING GUT HOMEOSTASIS AND PROMOTING PERIPHERAL TREG BIOLOGY Eliana Marino1, James L. Richards1, Keiran H. McLeod1, Dragana Stanley2, Yu Anne Yap1, Hoey Yein Goh1, Jose M. Polo1, Jan Kranich3, Ana Carolina Oliveira4, Alan G. Baxter5, Trevor J. Lockett6, Julie M. Clarke7, David L. Topping7, Leonard C. Harrison8, Charles R. Mackay1,9 1 School of Biomedical Sciences, Faculty of Medicine, Nursing and Health Sciences, Monash University, Wellington Road, Clayton, Victoria, Australia; 2Central Queensland University, School of Medical and Applied Sciences, Rockhampton, Queensland, Australia; 3 Institute for Immunology, Ludwig Maximilians University, Munich, Munich, Germany; 4Instituto de Biofı´sica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, Brazil; 5Comparative Genomics Centre, Molecular Sciences Bldg 21, James Cook University, Townsville, Queensland, Australia; 6CSIRO Preventative Health National Research Flagship, North Ryde, NSW, Australia; 7CSIRO Preventative Health National Research Flagship, Adelaide, SA, Australia; 8Walter & Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia; 9Charles Perkins Centre, The University of Sydney, Camperdown NSW, Australia
Diet and its effects on the composition of the gut microbiota may underlie the increased incidence of certain inflammatory diseases in western countries. Type 1 diabetes (T1D) in humans is an autoimmune disease with a genetic basis, but incidence around the world is influenced by as yet unknown environmental factors. Here we used the non-obese diabetic (NOD) mouse model, to demonstrate that pathogenesis T1D is critically dependent on bacterial metabolites, particularly the short chain fatty acids (SCFA) acetate and butyrate.
Inflamm. Res. We found high concentrations of bacterial metabolites acetate and butyrate in blood and faeces of male but not female mice, and in NOD.MyD88-/- mice, which correlated with protection from disease. We employed specialised high acetate- and butyrate-yielding diets, which significantly reduced the high diabetes incidence in female NOD mice, through changes in intestinal microbial composition and improved gut epithelial integrity. This protection was associated with both acetate and butyrate promoting the expansion of colonic as well as peripheral Treg cells, and unresponsiveness of islet-reactive (IGRP-reactive TCR transgenic) CD8+ T cells in NOD mice. Acetate and butyrate reduced inflammatory cytokines IL-21 and TNFa, and increased IL-22 and TGFb. Protection relied on the metabolite sensor GPR43, a receptor for both acetate and butyrate. Hence diet and metabolites can dramatically influence disease pathogenesis, and autoantigen reactivity, in the NOD model of autoimmune diabetes. These results fit with the notion that altered dietary habits in humans from western countries may also lead to an altered gut microbial ecology and reduced SCFAs, important for immune regulation and tolerance. We propose these factors favour the development of T1D in genetically susceptible individuals. Thus diet and bacterial metabolites may represent an effective, non-pharmacological means to modulate gut and immune homeostasis, to treat autoimmune diabetes.
B103 EXPRESSION AND ACTIVATION OF THE LONGCHAIN FATTY ACID RECEPTOR GPR40 IN HUMAN NEUTROPHILS Patricia RS Souza, Hefin R. Jones, Lucy V. Norling, Mauro Perretti William Harvey Research Institute/QMUL, London, UK Omega-3 fatty acids are essential polyunsaturated fatty acids with a double bond between the third and fourth carbon atoms from the methyl end of the carbon chain. Nutritionally important omega-3 fatty acids include eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are correlated with lower incidence of chronic diseases. Beside recent ground-breaking work on resolvins and protectins as bioactive derivatives of omega-3 fatty acids, there are still molecular mechanisms to be unveiled. DHA and EPA can activate the long-chain free fatty acid receptors GPR40 and GPR120, two GPCRs with a poorly investigated biology. In this work, we focused on GPR40 by using selective agonist GW9508. Using real-time PCR analysis, we detected GPR40 transcript in human neutrophils, a result confirmed at the protein level by flow cytometry and image stream analysis. Expression of GPR40 was up-regulated after 10 min cell stimulation with platelet-activating factor (PAF, 10 nM) or leukotriene B4 (LTB4, 10 nM). To establish the affinity of GW9508 for GPR40, coupled to Gaq/11 protein, intracellular calcium fluxes were assessed. Tested on human neutrophils, GW9508 elevated intracellular calcium when applied within the 0.1–10 lM range. The up-regulation of GPR40 expression by pro-inflammatory stimuli suggested to us potential counter-regulatory roles for this receptor during inflammation. We therefore investigated whether the GPR40 agonist GW9508 could counter-regulate neutrophil adhesion molecules expression induced by TNF-a (10 ng/ml, 15 min). However, GW9508 did not modulate the expression of CD11b and L-selectin. According, GW9508 was inactive when tested neutrophilendothelial interaction under flow. However, 1 and 10 lM GW9508 increased neutrophil chemotaxis in response to the cytokine IL-8 (30 ng/ml). In addition, GPR40 activation by GW9508 modulated phagocytosis of E. coli by human neutrophils by approximately 50 % when tested at 0.1 and 1 lM.
S143 In summary, the chemotaxis and phagocytosis data suggest that activation of GPR40 by GW9508 may have subtle and selective effects on neutrophil biology, and possibly, the resolution of inflammation. Supported by Science Without Borders/CNPq/Brazil and William Harvey Research Institute Founding.
B104 IMPORTANCE OF THE S1P1 PHOSPHORYLATION DOMAINS AND RECEPTOR INTERNALIZATION IN REGULATING INFLAMMATORY RESPONSES Manisha Menon, Leigh Maher, Kamal M. Khanna University of Connecticut Health Center, Farmington, CT, USA Identification of novel mediators of inflammation is important for development of new therapeutics. The sphingosine 1 phosphate (S1P)—sphingosine 1 phosphate receptor-1(S1P1) signaling axis has been implicated in regulating inflammatory responses. Previous reports have shown that the phosphorylation-deficient mutant S1P1S5A exacerbates experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis (MS). Here, we demonstrate a potential anti-inflammatory role for the phosphorylation domain of S1P1. S1P1-S5A mice exhibit inflammatory phenotypes characterized by enlarged spleen and intestinal tissue damage. H&E stained images of the small bowel and the colon show significant damage to the villi architecture. By using intravital two-photon dynamic imaging of live mouse gut we demonstrate that S1P1-S5A mice show compromised barrier function that likely contributes to intestinal tissue damage and heightened inflammation. Interestingly, the tissue damage appears to be exasperated in S1P1S5A mice with age. Furthermore, we used bone marrow chimeras to isolate the receptor internalization deficiency specifically in the stromal versus hematopoietic compartment to determine the mechanism responsible for the aforementioned observations. These results indicate that proper regulation of S1P1 receptor internalization even under steady state conditions is essential to maintain immune and intestinal homeostasis.
Immunotherapeutics B105 DECIPHERING THE HUMAN ANTICARBOHYDRATE REPERTOIRE OF IGG, IGA AND IGM Christoph Schneider1, Marc Wehrli1, David F. Smith2, Richard D. Cummings2, Alex Straumann3, Adrian Zu¨rcher4, Stephan von Gunten1 1
Institute of Pharmacology, University of Bern, Bern, Switzerland; Protein-Carbohydrate Interaction Core H, Consortium for Functional Glycomics in the Department of Biochemistry, Emory University School of Medicine, Atlanta, GA, USA; 3Department of Gastroenterology, Kantonsspital, Olten, Switzerland; 4Research and Development, CSL Behring AG, Bern, Switzerland
2
Despite the paradigm that carbohydrates are T cell-independent antigens, isotype-switched glycan-specific IgG antibodies and polysaccharide-specific T cells are found in humans. We employed a
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S144 systems level approach combined with glycan array technology to decipher the repertoire of carbohydrate-specific antibodies. A strikingly universal architecture of this repertoire with modular organization among different donor populations revealed an association between immunogenicity or tolerance and particular structural features of glycans. Antibodies were identified with specificity not only for microbial antigens, but for a broad spectrum of host glycans that serve as attachment sites for viral and bacterial pathogens and/or exotoxins. The comparison of IgG with IgA and IgM further reveals novel insights into the isotype-specific glycan-recognition in human sera, breast milk, saliva and in the gastrointestinal tract. Our study highlights the power of systems biology approaches to analyze immune responses and reveals potential glycan antigen determinants that are relevant to vaccine design, diagnostic assays, and antibodybased therapies.
B106 SITE-TARGETING NITROGLYCERIN NANOTHERAPEUTIC FOR LOCAL IMMUNOSUPPRESSION WITHOUT INDUCTION OF TOLERANCE Soroush Ardekani1, Shane Eum1, Sharad Gupta3, Harry A. Scott1, Xiao Yang1, Alexander R. Brunelle2, Sean M. Wilson2, Umar Mohideen1, Kaustabh Ghosh1 1 University of California, Riverside CA, USA; 2Loma Linda University School of Medicine, Loma Linda CA, USA; 3Indian Institute of Technology, Indore, India
Nitroglycerin (NTG) markedly enhances nitric oxide (NO) bioavailability. However, its ability to mimic the anti-inflammatory properties of NO remains unknown. Here, we examined whether NTG can suppress endothelial cell (EC) activation during inflammation and developed NTG nanoformulation to simultaneously amplify its antiinflammatory effects and ameliorate adverse effects associated with high-dose NTG administration. Our findings reveal that NTG significantly inhibits human monocyte adhesion to NO-deficient human microvascular ECs in vitro (EC50 = 0.64 lM) through an increase in endothelial NO and decrease in endothelial ICAM-1 clustering, as determined by NO analyzer, microfluorimetry, and immunofluorescence staining. Nanoliposomal NTG (NTG-NL) was formulated by encapsulating NTG within unilamelar lipid nanoparticles that were * 150 nm in diameter and readily uptaken by ECs, as determined by dynamic light scattering and quantitative fluorescence microscopy, respectively. More importantly, NTG-NL produced an approximately two orders of magnitude greater anti-inflammatory effect than free NTG while preventing excessive mitochondrial superoxide production and loss of arterial vasorelaxation associated with high NTG doses. Finally, to facilitate targeting of NTG-NL to inflamed ICAM-1-expressing vessels, we have tethered a non-immunogenic fragment of ICAM-1 antibody to the surface of NTG-NL. Our preliminary in vitro studies show that NTG-NL modified with anti-ICAM-1 fragment exhibits sixfold greater binding to inflamed (ICAM-1-expressing) ECs than to normal ECs. Thus, by identifying the superior therapeutic effects of NTG nanoformulation and conferring potent site-targeting capability to it, this study provides the rationale for detailed investigation of NTG nanotherapeutic as a potentially superior anti-inflammatory therapy.
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Inflamm. Res.
B107 INTERFERON BETA CELL THERAPY SHOWED ANTI-TUMOR EFFECTS ASSOCIATED WITH INHIBITION OF TUMOR CELL PROLIFERATION AND ACTIVATION OF IMMUNE CELLS Masaki Nakamura1, Lisa Kagawa1, Norihiro Nakada2, Masashi Satoh3, Shotaro Maehana6, Fumiaki Kojima5, Hideki Amano4, Yoshiki Murakumo2, Kazuya Iwabuchi3, Masataka Majima4, Hidero Kitasato1 1 Department of Microbiology, Kitasato University School of Allied Health Sciences, Sagamihara, Japan; 2Department of Pathology, Kitasato University School of Medicine, Sagamihara, Japan; 3 Department of Immunology, Kitasato University School of Medicine, Sagamihara, Japan; 4Department of Pharmacology, Kitasato University School of Medicine, Sagamihara, Japan; 5Department of Pharmacology, Kitasato University School of Allied Health Sciences, Sagamihara, Japan; 6Department of Environmental Microbiology, Kitasato University Graduate School of Medical Sciences, Sagamihara, Japan
Objectives: Interferon beta (IFNb) is known to have anti-tumor effects via suppression of tumor cell proliferation, activation of immune cells, and inhibition of tumor angiogenesis. IFNb has been clinically used for the treatment of several types of cancers including melanoma and brain tumor. However, the clinical benefit by the systemic administration of IFNb itself is limited because of its short half-life and sever systematic side effects such as hepatic dysfunction and thrombocytopenia. In this study, we showed a novel IFNb cell therapy, in which therapeutic cells highly producing IFNb were injected around the tumor as cancer therapy. We demonstrated the efficacy and safety of the IFNb cell therapy using a murine melanoma model, and histologically investigated the mechanism of anti-tumor effects in this therapy. Methods: To establish IFNb therapeutic cells, murine fibroblasts were transfected with murine IFN-b gene by using a retrovirus vector. To evaluate directly suppression of tumor cell proliferation, the supernatant of therapeutic cell was added into B16 murine melanoma cells, then the proliferation of B16 cells was evaluated with MTT assay in vitro. As a in vivo study of murine tumor model, C57BL/6 mice were subcutaneously inoculated with B16 murine melanoma cells. Then IFNb therapeutic cells were injected locally around the B16 tumor as cell therapy. After a week, the tumor tissue was excised and analyzed histologically. Subsets of lymphocyte infiltrating surround the B16 tumor were also analyzed by flow cytometry. In addition, the concentration of IFN-b in blood and tumor tissues were determined to estimate the efficacy and side effects of the IFNb cell therapy. Systematic side effects including hepatic dysfunction and thrombocytopenia were also examined. Results: Interferon beta cell therapy significantly suppressed the proliferation of the B16 cells in vitro, and the progression of the B16 tumors in a murine tumor model in vivo. By histological analysis, proliferation of tumor cells was suppressed and cell degeneration was also observed after the IFNb cell therapy. Interestingly, significant lymphocyte infiltration was observed around the tumor in respones to the IFNb cell therapy. Flow cytometry analysis further revealed that NK cells was the most dominant population among the infiltrating lymphocytes. The level of IFN-b in the tumor tissue was high enough to suppress tumor growth, while blood level of IFNb was not elevated. Systemic side effects including hepatic dysfunction and thrombocytopenia were not detected after the IFNb cell therapy, suggesting safety and efficacy of this therapy.
Inflamm. Res. Conclusion: The present study indicates that novel IFNb cell therapy suppresses the tumor growth both in vitro and in vivo. In addition, the activation of NK cells by IFNb cell therapy partly plays an important role for anti-tumor effects. IFNb cell therapy might be one of the prominent strategy for the treatment of cancer.
B108 EVALUATION OF MOLECULAR MARKERS AND THERAPEUTIC TARGETS IN TNBS-MODEL OF INTESTINAL INFLAMMATION Aline Witaicenis Fantinati, Alexandre S. Chagas, Alexandre Tanimoto, Luiz C. Di Stasi Sao Paulo State University, UNESP, Araraquara, Brazil Introduction: Ulcerative colitis and Crohn’s disease are major forms of Inflammatory Bowel Diseases (IBD) in humans; both diseases are characterized by excessive immune response against normal constituents of the gastrointestinal tract. Recent studies show that different genes are involved in the initiation, amplification and perpetuation of intestinal inflammation. Based on these, the present study aims to identify new molecular markers and/or future therapeutically target in animal models of intestinal inflammation. Methods: Anaesthetised rats (n = 8) were daily treated (p.o.) with coumarin derivatives with proven intestinal anti-inflammatory activity in rats, such as 4-methylesculetin (4-M, 5 mg/Kg), esculetin (5 mg/ Kg), esculin (25 mg/Kg), or with reference drugs: sulphasalazine (50 mg/Kg), azathioprine (2 mg/Kg) or prednisolone (2 mg/Kg) for 4 days and 2 h prior to TNBS administration into the colon (preventive protocol) or 2 h after TNBS instillation and daily until the 7th day (curative protocol). After the animals were killed, colon segments (100 mg) were collected and regulation of mRNA expression of HSP70, HPRT, NF-kB p50 and p65, JAK1, 2, 3; TYR2, STAT1, 2, 3, 4, 6; MAPK1, 3, 6, 9; and TIMP1 were analyzed by qRT-PCR. Results: In preventive protocol, TNBS-control group showed increased expression of HSP70 and TIMP1 and decreased NF-kB p65, JAK1, JAK3, STAT3 and MAPK9 expressions in comparison to noncolitic animals; all treatments reduced HSP70 mRNA levels; esculetin increased JAK3 and MAPK1 expressions; esculin, azathioprine and prednisolone increased the JAK3 and MAPK6; and sulphasalazine reduced STAT6 and increased MAPK1 and TIMP1 mRNA levels. In curative protocol, only expression of MAPK6 and MAPK9 were decreased and TIMP1 was increased in TNBS-control group. Treatments with esculetin, 4-M and prednisolone decreased TIMP1 gene expression and 4-M increased MAPK9 expression. Conclusion: Gene expression alterations occur mostly in the early phase of inflammatory process. The genes HSP70, JAK3, MAPK6 and TIMP1 are altered after TNBS administration; therefore these genes might be new molecular markers and/or future therapeutical targets in IBD. The treatment with the coumarins derivatives regulated somehow the altered gene expressions in intestinal inflammation. Financial Support FAPESP (11/50824-4; 11/50512-2).
B109 LEUKOTRIENE B4 IS A HOMEOSTATIC COMPONENT OF METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS SKIN INFECTION Stephanie Brandt, Soujuan Wang, Sebastian Carrasco, Stacy Blank, Nathan Delafield, C. Henrique Serezani
S145 Indiana University School of Medicine, Indianapolis, IN, USA Methicillin-resistant Staphylococcus aureus (MRSA) is the major cause of skin and soft tissue infections. Due to antibiotic resistance, MRSA infections are challenging to treat which demonstrates a need for novel therapeutic strategies. Leukotriene B4 (LTB4) is a bioactive lipid mediator produced by the 5-lipoxygenase (5-LO) metabolism of arachidonic acid. LTB4 signals through its high affinity receptor LTB4 receptor 1 (BLT1) and induces leukocyte recruitment and enhances macrophage antimicrobial effector functions. We hypothesize that LTB4 is a homeostatic regulator of skin host defense by increasing different arms of the innate immune response. MRSA skin infection leads to macrophage-dependent enhanced 5-LO and BLT1 expression and LTB4 abundance. 5-LO-/- and BLT1-/- mice infected subcutaneously with MRSA had larger abscesses and higher bacterial burden than wild type mice at day nine post infection, suggesting an impairment in immune response. BLT1-/- mice had less neutrophil recruitment and more apoptotic cells compared to wild type mice at day two post infection. Mice treated with a pharmacological BLT1 inhibitor had more apoptotic cells in the skin, suggesting that LTB4 has a protective role in both neutrophil recruitment and survival during MRSA skin infection. Endogenously produced LTB4 enhanced MRSA ingestion and killing, which correlated with enhanced generation of reactive oxygen species and antimicrobial molecules in response to MRSA. Additionally, BLT1-/- mice produced similar TNFa levels but less IL1b than wild type mice in response to in vivo MRSA infection. IL1b is an important inflammatory cytokine involved in controlling MRSA infections. IL1b requires maturation through caspase-1 activity. Inhibiting BLT1 reduced MRSA-induced caspase-1 activity, suggesting that LTB4 promotes IL1b production and processing. Topical treatment with an ointment containing LTB4 greatly improved bacterial clearance and accelerated healing in both 5-LO-/- mice and wild type mice. These findings show that LTB4 is vital for skin host defense mechanisms and suggests that topical LTB4 treatment is a likely candidate for an immunotherapeutic agent to control MRSA infections. Financial support NIH Immunology and Infectious Diseases T32 AI060519, R00HL103777, and R03 AI110990-01A1
B110 TARGETING NORMAL AND NEOPLASTIC HUMAN B CELLS IN VITRO AND IN VIVO USING ANTIBODY-MEDIATED DELIVERY OF SIRNA Gary P. Sims, Fred Karnell III, Lena Shirinian, Vladimir Voynov, Daniel C. Rowe, Marlon C. Rebelatto, Elizabeth Ward, Bo Chen, Nazzareno Dimasi, Changshou Gao, Ronald Herbst Research and Development, MedImmune, Gaithersburg, MD, USA The use of synthetic siRNAs to specifically silence gene expression in key pathological pathways provides appealing therapeutic opportunities. However, without an efficient cell-specific delivery system, the clinical use of siRNA therapeutics has been restricted. Herein, we combined the precise cell recognition and internalization properties of anti-CD22 antibody with the nucleic acid-binding capacity of protamine, to specifically deliver siRNA to B cells. Blimp-1 siRNA delivered to primary B cells, directed the knockdown of the target mRNA, and inhibited plasma cell differentiation and immunoglobulin secretion. Increasing the siRNA payload by conjugating additional protamine peptides to the antibody increased the potency 20-fold. Delivery of siRNAs targeting the oncogenes Bcl-2 and c-myc inhibited growth of acute lymphoblastic leukemia cells in vitro, and
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S146 systemic administration resulted in target knockdown and the remarkable regression of established subcutaneous tumors in vivo. This study demonstrates the therapeutic potential of cell-specific antibody-mediated delivery of siRNA for the targeted, functional inhibition of normal and neoplastic B cells.
B111 A NONHUMAN PRIMATE MODEL OF ALLERGIC RHINITIS AS A MECHANISTIC MODEL FOR FOOD ALLERGY Joanne Schiding, Nick Colletti, Mike Shaw, Neil Fitch
Inflamm. Res. The objective of this animal study and laboratory investigation were to investigate whether the decrease in the rate of mortality caused by lipopolysaccharide (LPS) tolerance may be associated with an increased population of CD4+ T regulatory lymphocytes and Th17. Male black C57/6 mice received subcutaneous (s.c.) injections of LPS (1 mg/kg) for 5 days, followed by cecal ligation and puncture (CLP). Cytokines and marked lymphocytes were measured after tolerance and CLP challenge. Both of T reg subpopulation (induced and natural) and Th17 lymphocytes, showed increase in cells in the spleen and plasma after tolerance. Mortality reduced in tolerant animals. This study demonstrated that reduced mortality after tolerance may be associated with increasing population of Treg and Th17 cells due to immunoregulation of the hyperinflammatory response and neutrophil recruitment.
Sanofi US Inc, BioInnovation, Cambridge, MA, USA Introduction: Food allergy affects approximately 8 % of US children and is thus a major public health concern. The most common food allergens induce an IgE-mediated response which can sometimes be severe enough to cause fatalities. While children will outgrow their allergies to some foods, other food allergies persist over a lifetime. Current strategies to treat food allergy involve specific immunotherapy to desensitize the patient to the food allergen. We are looking at a novel strategy involving the use of immunomodulators to divert the immune response away from the IgE-mediated Th2 phenotype. Hypothesis: A nonhuman primate natural allergic rhinitis model, (which is IgE mediated and driven by a Th2 cytokine phenotype), can act as both a surrogate as well as a mechanistic model for food allergy in assessing therapeutic benefits of an immunomodulator. Methods: A clinical acoustic rhinometer was adapted for use with the nonhuman primate. The rhinometer transmits a sound wave into the nasal cavity and measures the reflected signal. Algorithms are used to calculate nasal volume and the minimum cross-sectional area of the nasal cavity. Intranasal delivery of allergen induces nasal congestion which is evidenced by a reduction in both volume and area. Animals were treated with the pro-Th1 TLR-4 agonist Glucopyranosyl lipid A (GLA) in an attempt to ameliorate the allergic response to antigen. Results: Treatment with 10ug GLA co-administered with 10ug antigen intramuscularly once weekly for 4 weeks blocked the response to intranasal antigen challenge when measured 24 h after the final treatment. Efficacy was maintained 2–4 weeks after the final treatment. If the antigen was administered at a distal site relative to the GLA, there was no significant effect. FACS analysis indicated that GLA treatment reduced (1) basophil responsiveness (demonstrated by reduced histamine release on stimulation) and (2) antigen-specific CD4+ effector T-cells. Cytokine analysis of serum suggested that C-reactive protein (CRP) may be a valuable biomarker for GLA dose monitoring. Conclusion: This nonhuman primate allergic rhinitis model was immunomodulated by a Th1-inducing TLR4 agonist. This suggests that this mechanism may translate into efficacy in animal models of food allergy and in patients with food allergy.
B112 THE LPS TOLERANCE AFFECTS ON CD4, TREG AND TH17 LYMPHOCYTES Mariana M. Andrade, Andre Botega, Denise F. Barbeiro, Hermes Barbeiro, Francisco G. Soriano Medicine Faculty of Sa˜o Paulo Univerity, Sa˜o Paulo, Brazil
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B113 THE EFFECT OF TRANSCUTANEOUS CERVICAL ELECTRICAL VAGAL NERVE STIMULATION ON AUTONOMIC INDICES IN HEALTHY HUMANS: A POTENTIAL ANTI-INFLAMMATORY THERAPY? JP Errico3, Christina Brock1, Bruce Simon3, Qasim Aziz2, Asbjorn M. Drewes1, Adam D Farmer2,1 1 University of Aalborg, Aalborg, Denmark; 2Barts and the London School of Medicine, London, UK; 3ElectroCore Medical LLC, Basking Ridge, NJ, USA
Background: The autonomic nervous system is a bidirectional hierarchically controlled brain body nexus that integrates the external environment with the internal milieu. The parasympathetic nervous system (PNS), whose main neural substrate is the vagus nerve, influences inflammation through the cholinergic anti-inflammatory pathway. A non-invasive transcutaneous cervical vagus nerve stimulator (t-VNS), gammaCore (Basking Ridge, NJ), has been recently developed for the treatment of epilepsy and migraine. If this new technology has the potential to be used as a modulator of the immune system, it is important to first establish that it can modulate cardiac vagal tone in a measurable manner. Aim: To investigate the effect of t-VNS on cardiac vagal tone (CVT) in healthy subjects. Methods: 20 healthy subjects (13 female, median age 34 years, range 23–56) had heart rate (HR), blood pressure (BP) and CVT, a validated real time non-invasive parameter of brain stem mediated efferent PNS tone (1), measured at baseline, during 4 min of bilateral t-VNS and post t-VNS at 90 min and 24 h. Venous blood was also sampled at these time points to assess the effect of t-VNS on inflammatory cytokine expression. Results: All subjects tolerate t-VNS well except one subject (1 female, 30 years old) who felt light headed during the stimulation. A repeated measures analysis of variance (ANOVA) was conducted to ascertain the effect of t-VNS on HR, systolic BP and diastolic BP. T-VNS had no effect on HR (F (3,76) = 2.5, p = 0.07) or diastolic BP (F (2.6, 49.1) = 2.2, p = 0.1). T-VNS had a small effect in reducing systolic BP (F (2.7, 52.4) = 4.5, p = 0.01). A non-parametric Friedman test was conducted to assess the effect of t-VNS on CVT, yielding a Chi square of 28.7, p \ 0.0001, see figure 1. Conclusions: t-VNS raises CVT, suggesting that it is sufficient to potentially stimulate the cholinergic anti-inflammatory pathway. Should the cytokine expression data from this study demonstrate an anti-inflammatory effect of t-VNS, further work will be warranted to translate theses findings in clinical disorders such inflammatory bowel disease.
Inflamm. Res. Figure 1 The effect of t-VNS on cardiac vagal tone. The maximal effect of t-VNS on CVT was seen 90 min post stimulation, with an effect still present at 24 h, **** p \ 0.0001, *p \ 0.05.
Reference 1. Borokova et al. Nature 2000. 2. Farmer et al. Ann Gastro 2014.
Inflammasomes in Health and Disease B114 INVOLVEMENT OF P2X4 AND P2X7 PURINERGIC RECEPTORS IN IL-1ß PRODUCTION AND NLRP3INFLAMMASOME PATHWAY ACTIVATION IN HUMAN MACROPHAGES Thomas Gicquel1,2, Sacha Robert1, Pascal Loyer1, Tatiana Victoni1, Aude Bodin1, Catherine Ribault1, Gleonnec Florence1, Isabelle Couillin3, Elisabeth Boichot1, Vincent Lagente1 1
UMR991 INSERM, Universite´ de Rennes 1, Rennes, France; Laboratoire de toxicologie biologique et me´dico-le´gale, CHU Rennes, France; 3UMR-IEM CNRS6218, Universite´ d’Orle´ans, France
2
The Nod-like receptor family protein 3 (NLRP3)-inflammasome pathway is known to be activated by danger signals such as monosodium urate (MSU) or adenosine triphosphate (ATP). Here, we investigated the role of P2X4R and P2X7R, two P2 purinergic receptors in the activation of NLRP3-inflammasome pathway in primary human monocyte-derived macrophages (MDMs). After initial stimulation with a low concentration of LPS (0.1 lg/mL), a 6-h treatment with ATP agonists, 20 ,30 -O-(4-benzoylbenzoyl) adenosine 50 -triphosphate (BzATP), Adenosine 50 -[c-thio]triphosphate tetralithium salt (ATPcS) or MSU crystals induced the MDMs to release IL-1b in a dose-dependent manner. Moreover, the caspase-1 inhibitor Z-YVAD-FMK reduced production of IL-1b in a dose-dependent manner after LPS + MSU treatment. We used RT-qPCR to show that treatment with MSU (500 lg/mL) or BzATP (250 lM) induced significantly the expression of NLRP3 after LPS priming or not. We also found that MSU treatment, but not BzATP, induced P2X7R mRNA expression. Using flow cytometry and immunoblotting analysis, we
S147 showed that a 24-h treatment with MSU crystals induced the expression of P2X7R. Furthermore, addition of the P2X7 purinergic receptor antagonist A-740003 significantly impeded IL-1b production after treatment with LPS + MSU or LPS + BzATP. Contrarily, addition of the P2X4R antagonist 5-(3-bromophenyl)-1,3-dihydro2H-benzofuro-3,2-e]-1,4-diazepin-2-one (5-BDBD) did not affected IL-1b release after treatment with LPS + MSU whereas 5-BDBD significantly reduced production of IL-1b after treatment with LPS + BzATP. Remarkably, RNA silencing of P2X7R (but not P2X4R) inhibited significantly the release of IL-1b and other M1 macrophage cytokines (such as IL-1a, IL-6 and TNF-a) from MDMs stimulated with LPS + MSU. Our findings confirm the quantitative difference between NLRP3-inflammasome activation by MSU crystals and activation by exogenous ATP, and suggest that blockade of the NLRP3-inflammasome pathway or the P2 purinergic P2X7R receptors is a novel potential therapeutic approach to control the inflammatory process in several associated pathologies.
B115 INFLAMMASOME ACTIVATION BY LANTHANIDE UPCONVERSION NANOPARTICLES AND ITS ABROGATION BY A SURFACE COATING PEPTIDE Longping Wen, Yunjiao Zhang, Han Yao School of Life Science, University of Science and Technology of China, Hefei, Anhui, China Lanthanide upconversion nanoparticles (UCNs) hold great promise for in vivo theranostic applications. It is known that a variety of nanocrystals, including rare earth oxide nanoparticles, elicits potent inflammatory response through activation of NLRP3 inflammasomes, but whether UCNs have the similar effect remains to be shown. Here we have assessed the ability of UCNs to activate inflammasomes and investigated in detail the underlying mechanism. UCNs triggered inflammasome activation, characterized by the activation of caspase 1 and release of IL-1 and IL-18, in a dose- and time-dependent fashion in macrophages, including human THP-1 cells, mouse bone marrow derived macrophage (BMDM) cells, and mouse peritoneal microphage cells. Cellular internalization, reactive oxygen species (ROS) and lysosomal damage were all critical, while potassium efflux only played a minimal role, for UCN-induced inflammasome activation. Consistent with published reports, NLRP3 inflammasome were mainly responsible for UCN-elicited inflammation, as the IL-1 release was largely reduced in nlrp3-/- mouse BMDM cells treated with UCN. However, significant IL-1 release, corresponding to approximately 30 % level to that seen in the wild-type cells, was still observed in the UCN-treated nlrp3-/- BMDM cells, indicating the involvement of non-NLRP3 inflammasomes during UCN-elcited inflammatory response. RE-1, a lanthanide-specific surface coating peptide we discovered previously, significantly abrogated the inflammasome-activating activity of UCN. RE-1 coating did not affect the ability of the nanocrystals to enter cells through phagocytosis, but led to significant reduction in UCN-induced ROS generation and lysosomal damage. In conclusion, we demonstrated that UCNs were potent inflammasome activators in macrophages, and the ability of RE-1 peptide to abrogate this effect may be of great value for in vivo applications of UCNs and other rare earth-based nanomaterials.
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B116 WHOLE EXOME ANALYSIS OF INDIVIDUALS AND FAMILIES WITH CHRONIC RECURRENT MULTIFOCAL OSTEOMYELITIS (CRMO) Allison J. Cox1,2, Benjamin W. Darbro1, Xinyu Bing1, Alexander G. Bassuk1, Polly J. Ferguson1 1
Department of Pediatrics, University of Iowa, Iowa City, IA USA; Interdisciplinary Graduate Program in Genetics, University of Iowa, Iowa City, IA, USA
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Chronic recurrent multifocal osteomyelitis (CRMO) is a rare, autoinflammatory bone disease presenting in infancy and childhood. While CRMO is characterized by painful bone lesions, it is often comorbid with psoriasis or Crohn disease and many patients have close relatives with either of the more common disorders. Some syndromic cases of CRMO are caused by truncation mutations in the interleukin-1 receptor agonist (IL-1RA) gene and these children respond very well to treatment with recombinant IL-1RA. However, most cases of CRMO are non-syndromic with an unknown genetic cause, although many patients respond to TNF-a blocking agents, implicating the pathway in the disease. Using whole exome sequencing paired with genetic analyses based on inheritance patterns in affected families, we have determined that the genetic basis of CRMO is heterogeneous and complex. However, for several families, we have determined the most likely causative mutations, in both novel and previously characterized genes affecting a shared pathway. Our research demonstrates how multiple approaches to candidate gene selection may be merged to help determine the underlying cause of a rare and complex genetic disease.
Inflamm. Res. expression in human coronary artery specimens obtained from 10 explanted hearts. In advanced atherosclerotic plaques (AHA IV-VI), a significant upregulation of 12 target genes was found compared to early lesions (AHA I-III), among them several core components of the inflammasome pathway. Immunohistochemical stainings of the advanced plaques revealed the presence around necrotic cores of macrophage foam cells positive for NLRP3 inflammasome components. The PCR array target p38delta mitogen-activated protein kinase (MAPK) was upregulated in advanced coronary plaques and expressed in macrophage foam cells of the plaques. In cultured human monocyte-derived macrophages, activators of the NLRP3 inflammasome, cholesterol crystals and ATP, triggered strong p38delta MAPK activation. The activation of p38delta was required for inflammasome-mediated IL-1beta secretion and depended on intracellular stress signals previously linked to NLRP3 inflammasome activation. Conclusions: Our data revealed upregulation of several core components of the inflammasome pathway in advanced coronary plaques compared to early lesions from the same patients, implying a role for the inflammasome pathway in disease progression. Among the upregulated genes, p38delta MAPK was identified as a novel mediator of NLRP3 inflammasome activation, and thus represents a potential target for modulation of atherosclerotic inflammation.
B118 NLRC4 IS INVOLVED IN ACTIVATION OF THE NLRP3 INFLAMMASOME IN RESPONSE TO LEISHMANIA AMAZONENSIS INFECTION Alexandre L.N Silva1, Djalma S. Lima-Junior1, Larissa D. Cunha1,2, Dario S. Zamboni1
B117 P38DELTA MAPK – A NOVEL EFFECTOR IN NLRP3 INFLAMMASOME ACTIVATION UPREGULATED IN HUMAN CORONARY ATHEROSCLEROTIC LESIONS Kristiina Rajamaki1, Mikko I. Mayranpaa2, Jarno Tuimala4, Katariina Nurmi1, Kari K. Eklund3, Katariina Oorni1, Petri T. Kovanen1 1
Wihuri Research Institute, Helsinki, Finland; 2Department of Pathology, Haartman Institute, University of Helsinki and HUSLAB Division of Pathology, Meilahti Laboratories of Pathology, Helsinki University Central Hospital, Helsinki, Finland; 3Division of Rheumatology, Department of Medicine, Helsinki University Central Hospital, Helsinki, Finland; 4RS Training, Helsinki, Finland Background: Inflammation is recognized as a major driving force in atherogenesis, yet the mechanisms involved remain elusive. Recently, cholesterol crystals commonly found in atherosclerotic plaques were shown to trigger a strong proinflammatory response via the NLR family pyrin domain containing 3 (NLRP3) inflammasome in macrophages, the key immune cells in atherosclerotic plaques. The NLRP3 inflammasome regulates the proteolytic maturation and secretion of potent proinflammatory and proatherogenic cytokines, interleukin (IL)-1beta and IL-18. Aims: The aim of the current study was to comprehensively characterize, for the first time, the expression of inflammasome pathway components and regulators in human atherosclerotic lesions. Methods and Results: A quantitative PCR array targeting 88 inflammasome pathway-related molecules was employed to analyze mRNA
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1
University of Sa˜o Paulo, Medical School Ribeira˜o Preto, Sa˜o Paulo, Brazil ; 2St Jude Children’s Research Hospital, Memphis, TN, USA
NLRs are a group of intracellular receptors that recognize endogenous and exogenous danger signals. Some members of this family participate of the inflammasome formation. The inflammasome is able to activate inflammatory caspases, such as caspase-1 that is responsible for processing and secretion of IL-1b and IL-18, as well to induce an inflammatory form of cell death called pyroptosis. Leishmaniasis is a group of diseases caused by protozoa of Leishmania genus. These parasites exhibit a remarkable capacity to survive and proliferate within the phagolysosome of host macrophages. Macrophages are critical for effective and protective immune responses to different diseases, including Leishmaniasis. Macrophages express different pattern recognition receptors (PRR) responsible for recognition of molecular patterns associated with microbes (PAMPs). While many publications emphasize the importance of TLRs in the susceptibility and control of Leishmania infection, the role of NLRs is still unclear. We have recently demonstrated that the NLRP3 inflammasome is activated in response to L. amazonensis infection in a process that is required for restriction of parasite infection. However, the molecules involved in activation of NLRP3 in response to L. amazonensis infection are unknown. In this study, we found that NLRC4 is involved in activation of the NLRP3 inflammasome in response to Leishmania infection. By using mice deficient in NLRC4, we found that NLRC4 is activated in response to L. amazonensis infection and plays an important role in controlling infection in macrophages and in vivo. We also demonstrate that the NLRC4 receptor is phosphorylated after infection with L. amazonensis and found that the NLRC4 inflammasome is assembled in response to L. amazonensis infection in macrophages when macrophages are transduced with retrovirus encoding NLRC4-GFP. Collectively, these studies identify NLRC4 as
Inflamm. Res. an important molecule participating on NLRP3 activation in response to L. amazonensis infection.
B119 L. AMAZONENSIS ELIMINATION BY P2X7 RECEPTOR AND LEUKOTRIENE B4 REQUIRES NLRP3 INFLAMMASOME ACTIVATION AND IL1R SIGNALING Mariana M. Chaves1, De´bora A. Sinflo´rio1, Maria Bellio1, Dario Zamboni2, Cla´udio Canetti1, Robson Coutinho-Silva1
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Inflammation and Aging B120 MEASUREMENT OF NITRIC OXIDE METABOLITES AS POTENTIAL MARKERS OF INFLAMMATION IN DEMENTIA Annie Knight1, Miranda J. Smallwood1, Emma Taylor1, David Llewellyn1, Patrick G. Kehoe2, Paul G. Winyard1 1
University of Exeter Medical School, Exeter, EX2 4TE, UK; University of Bristol, School of Clinical Sciences, Learning and Research Building, Southmead Hospital, Bristol, UK
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Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; University of Sa˜o Paulo, Sa˜o Paulo, Brazil
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Objectives: P2X7 receptors activation via extracellular ATP (eATP) has been reported as an important molecule signal to intracellular parasites elimination. After activation, the P2X7 receptor can lead to apoptosis, and cytokines release such as IL-1b, among others mechanisms. Recently, it was demonstrated that IL-1b release, after NLRP3 inflammasome activation, participates of the resistance to Leishmania amazonensis. Furthermore, our group showed that L. amazonensis elimination through P2X7 receptor activation depends of leukotriene B4 (LTB4) production. Thus, we investigated whether L. amazonesis elimination by P2X7 receptor and LTB4 involves NLRP3 inflammasome activation and IL-1 receptor (IL-1R) signaling. Methods and Results: Peritoneal macrophages from C57Bl/6 (C57Bl/ 6), P2X7 receptor (P2X7-/-), NLRP3 (NLRP3-/-), ASC (ASC-/-) and IL-1R (IL-1R-/-) deficient mice, 2–7 months, were infected or not with L. amazonensis at 10:1 MOI ratio. These were tested for parasitic load presence or absence of 500 lM ATP, 100 nM LTB4, or 100 pg/mL IL-1b for 30 min at 37 C. After 24 h, infected macrophages were fixed and stained with Panotic kit and analysed by optical microscope. C57Bl/6 and P2X7-/- mice were infected in the footpad with 106 L. amazonensis promastigotes for 7 days. After, P2X7-/mice were treated in infect footpad twice a week with 5 ng of LTB4 for 3 weeks and thickness paw and parasitic load were determined. Paw’s thickness was accompanied with thickness gauge and the parasitic load was established with limiting dilution assay (LDA). Nitric oxide (NO) levels were measured by Griess assay. The graphs were generated and analyzed using the GraphPad Prism 5.0. Our results showed that infected macrophages from NLRP3-/- mice treated with ATP and LTB4 did not decrease parasitic load (difference between means 4 %, n = 4; 5 %, n = 3, respectively). However, when NLRP3-/- infeceted macrophages were treated with IL-1b, a decrease in parasitic load was noted (20 %; n = 3). Furthermore, ASC-/- infected macrophages treated both ATP and LTB4 also did not diminish parasitic load (13 %, n = 3; 7 %, n = 3, respectively). The same happened with IL-1R-/-infected macrophages (3 %, n = 3; 3 %, n = 3, respectively). Similarly, infected macrophages from P2X7-/- mice treated with IL-1b decreased parasitic load (difference between means 39 %, n = 3). Furthermore, P2X7-/- mice infected with L. amazonensis in the footpad treated with exogenous LTB4 showed more resistance to infection, because their footpad had lower parasite load (difference between means 1.1x109 ± 4.7 9 108 parasites; n = 8) and lower lesion (37.94 ± 11.45 mm, n = 8) when compared to untreated P2X7-/-mice. This resistance does not seem to be via NO. Conclusion: Thereby, these data suggest the involvement of LTB4 in resistance of WT mice when compared with P2X7-/- mice, and that L. amazonensis elimination by P2X7 receptor mediated by LTB4 depends of NLRP3 activation and IL-1R signaling. Financial Support CNPq, CAPES, FAPERJ
Inflammation is present in the Alzheimer’s disease (AD) brain, with activation of complement, cytokine and chemokine pathways. Activated microglia and astrocytes release reactive oxygen species, such ¯ as nitric oxide (NO) and superoxide (O2). These species react to ¯ form peroxynitrite (ONOO) leading to nitration of tyrosine residues (i.e. 3-nitrotyrosine; Tyr-NO2) within brain proteins. Tyrosine residues are also nitrated by the myeloperoxidase (MPO)–H2O2-nitrite (NO2-) system. Immunohistochemical studies have suggested increased Tyr-NO2 in AD brain tissue. However, there is limited quantitative data on Tyr-NO2 levels in AD brain, compared with normal brain tissue. If Tyr-NO2 is elevated in the AD brain, this would suggest that serum Tyr-NO2 should be tested as a biomarker in future studies. In order to assess if Tyr-NO2 is a potential marker of inflammation in AD, Tyr-NO2 was measured along with the stable oxidation endproducts of •NO, NO2- and NO3- (nitrate). Frontal lobe homogenates from people with AD, vascular dementia (VaD) and non-demented elderly controls (n = 15 for each group) were analysed. NO2- and NO3- were measured by ozone based chemiluminescence and TyrNO2 was measured using a novel electrochemiluminescence based ELISA. Brain NO2- levels were significantly different between the three groups (P = 0.03, Kruskal–Wallis test) with AD and VaD significantly higher than non-demented controls, P \ 0.05 (median (IQR): non-demented 0.08 (0.07–0.09); AD 0.1 (0.08–0.18) and VaD 0.1 (0.08–0.12) lmol/g protein). There were no statistically significant differences between groups for NO3- levels: controls 2.1 (1.8–2.3); AD 2.6 (1.8–4.9); VaD 2.4 (1.9––3.3) lmol/g protein. Levels of Tyr-NO2 in brain tissue showed no statistically significant differences between the three groups: normal volunteers 0.296 (0.224–0.548); AD 0.292 (0.185–0.572) and VaD 0.357 (0.182–0.402) pmol BSA-Tyr-NO2 equiv/mg protein. There were no statistically significant correlations between NO2-, NO¯3-or Tyr-NO2 and subject age or post-mortem delay. The increased concentration of NO2- in AD and VaD brain tissue suggests increased production of NO. Yet, our data set challenges the current consensus that Tyr-NO2 is increased in AD. A potential mechanism of Tyr-NO2 generation is via the MPO-H2O2-NO2- system. Our results demonstrate that NO2- is available in the AD/VaD brain as a substrate for MPO. However, the known loss of superoxide dismutase activity in the AD brain, may limit the rate of H2O2 generation. This may, in turn, limit MPO catalysed nitration, thereby explaining the lack of increased Tyr-NO2 in AD brain tissue in the current study. Acknowledgement We would like to thank the South West Dementia Brain Bank (SWDBB) for providing the brain tissue homogenates for this study. The SWDBB is supported by BRACE (Bristol Research into Alzheimer’s and Care of the Elderly), Brains for Dementia Research and the Medical Research Council.
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B121 DYSREGULATION OF RESOLUTION OF ACUTE INFLAMMATION IN AGED HUMANS Roel P. H. De Maeyer, Madhur Motwani, Justine S. Newson, Derek W. Gilroy Division of Medicine, Centre for Clinical Pharmacology and Therapeutics, University College London, London, UK Age-related pathophysiologies are becoming an increasing global health concern as the population reaches newfound heights of longevity. Susceptibility of the elderly to severe and lasting infections coincides with a chronic inflammatory state termed inflamm-aging. Thus far, few data are available on whether resolution, or the ability to switch off innate immune-mediated responses, is dysregulated in aged individuals. Therefore, we investigated the inflammatory response in healthy young and aged volunteers using the cantharidin skin blister model of self-resolving, tissue injury driven inflammation. A novel and extensive multi-colour fluorescence staining protocol was designed to identify all cell populations in the blister using flow cytometry. We observed classic neutrophilia associated with inflammatory onset in both age groups, but found a significantly increased number of HLA-DR+/CD14+/CD16lo monocytes/macrophages (Mo/ Mu) present in the blisters of aged volunteers. By resolution, neutrophils cleared from the blisters of young volunteers in a manner that was proportional to Mo/Mu influx and that was associated with robust Mo/Mu p38a/MAPK14 expression as well as prostaglandin E2 (PGE2) synthesis, established molecular consequences of effective Mu phagocytosis of apoptotic neutrophils. However, in blisters of aged volunteers, correlations between neutrophil clearance and Mo/ Mu numbers was lost being associated with a significant (threefold) reduction in PGE2. qPCR on isolated blister Mo/Mu also revealed that, in the aged, these cells fail to upregulate both cyclooxygenase-2 (COX-2) and p38a/MAPK14. These data implicate a dysregulation of efferocytosis, a process which ultimately leads to a pro-resolution phenotypic switch of the Mo/Mu. We therefore speculate that a dysregulated pro-resolution cascade may contribute, at least in part, to the syndrome of inflamm-aging.
Inflammation and Cancer B122 ROLE OF INFLAMMATION IN PROMOTION AND PROGRESSION OF ENDOMETRIAL HYPERPLASIA
Inflamm. Res. Design of investigation includes four groups of 107 female patients: control group with normal endometrium, simple hyperplasia, complex hyperplasia with and without atypia. In tissue samples of endometrium we performed a quantitative analysis of estrogen and progesterone receptors and CD45+ (common leukocyte antigen) expression by immunohistochemical method. The level of estrogens, progesterone and cytokines IL-1ß, IL-6 and TNF-a investigated in uterine flushing’s by ELISA methods. The elastase-like and trypsinelike activities and level of acid-stable inhibitors and antitrypsine activity were measured by enzyme methods. The obtained results showed that more intensive imbalance with prevalence of estrogens and estrogen receptor expression it was showed in simple EH and we have not found the progression of hormonal changes in complex EH. But we found increase of morphological characteristics of chronic inflammatory process in the endometrium and increase common leukocyte antigen CD45+ expression during the transition from normal endometrium (4.2 ± 1.2 %) to complex hyperplasia (57.8 ± 2.4 %). Study of local cytokine levels showed that they are also increased in 3–5 times in simple EH and 15–20 times in both types of complex EH. The activity of elastolytic enzymes significantly increased in 7.5 times in simple EH and in 8–9 times in both forms of complex hyperplasia. Intensity of change depended from presence of infectious and inflammatory processes in the urogenital system and not dependent from the menstrual cycle. This results showed that in simple EH more important progressive factor can be hormonal imbalance, but in complex and atypical EH increase role of inflammatory process. This inflammatory process which follow EH, interpreted in our investigation such as ‘‘endometrial hyperplasia associated’’ or ‘‘endometrial hyperplasia related’’ inflammation. Formation of this inflammatory changes in endometrial hyperplasia can be considered factor of promotion and progression of pathology, as well as attributed risk factor of malignancy in endometrial hyperplasia.
B123 IL-1 RECEPTOR ANTAGONIST (IL-1RA) AND DEXAMETHASONE (DEX) THERAPY IN PATIENTS WITH SMOLDERING/INDOLENT MYELOMA (SMM/IMM) SHOWS IMPROVED SURVIVAL IN PATIENTS WITH A DECREASED C-REACTIVE PROTEIN (CRP): INTERPLAY BETWEEN IL-1, IL-6 AND IL-17 John A. Lust, Kathleen A. Donovan Division of Hematology, Mayo Clinic, Rochester, MN, USA
Anatoliy V. Kubyshkin, Evgeniia Kovalenko, Leonid Aliev, Vladimir Kubyshkin V.I.Vernadsky Crimea Federal University, Simferopol, Republic of Crimea In many non-inflammatory pathologies after some time begin development associated inflammatory processes. This type of inflammation had secondary origin and depended from primary non-inflammatory metabolic or tissue rearrangements in organs and systems. This mechanism takes part in the development of cancer-related inflammation (Colotta et al. 2009; Hanahan 2011). In some investigations described inflammation which also associated with atherosclerosis, obesity, allergic processes, chronic kidney disease, angiopathies and others. In our investigation role of inflammation in the development of endometrial hyperplasia (EH) it was studied.
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In early stage myeloma, IL-6 is a central myeloma growth factor and we have shown that abnormal production of IL-1 in the myeloma microenvironment stimulates the generation of IL-6 in a paracrine fashion. IL-1 has also been shown to be a crucial factor in the induction of IL-17 producing T-cells in vivo. We have completed a Phase II trial using IL-1Ra and dexamethasone, in patients with smoldering/indolent MM (SMM/IMM), showing that IL-1Ra targets the myeloma proliferative component which parallels a decrease in the C-reactive protein (CRP), a surrogate for IL-6 production. Patients that had [10 % bone marrow plasma cells and/or an IgG or IgA M-spike [3 g/dL were eligible. All patients received 100 mg of Anakinra (IL-1Ra) SQ qd for 6 months. Patients with evidence of reduction in M-protein levels continued receiving IL-1Ra alone. Patients with stable disease at 6 months or those with a rising M-protein before 6 months received low dose dexamethasone (20 mg
Inflamm. Res. qweek) in addition. Data were available on 47 patients based on intent to treat, and patients were classified as smoldering (72 %) vs. indolent (28 %). All 47 patients received IL-1Ra initially and 25/47 subsequently received IL-1Ra/Dex. Median follow-up was 7.7 years. Myeloma cell growth rate (PCLI), C-reactive protein (an in vivo marker of IL-6 levels) and IL-17 were measured in patients on trial. Seven patients had a decrease in the plasma cell labeling index (PCLI) on IL-1Ra alone which paralleled a decrease in the C-reactive protein in all cases. Three patients achieved a minor response to IL-1Ra alone and nine patients achieved a PR/MR after addition of dexamethasone. When patients were grouped into whether they exhibited a reduction in the C-reactive protein from baseline after 6 months of therapy, the median PFS for patients without (22 patients) or with (25 patients) a greater than 40 % reduction in baseline CRP was 10.8 months vs. 8.6 years (p \ 0.0001). Similarly, the median OS for patients without or with a 40 % reduction in baseline CRP was 7.9 years vs. median not reached (p = 0.001). Analyses of biomarkers suggested that patients with elevated IL-17 levels may be less likely to respond to IL-1Ra treatment. Sixty percent of patients without a CRP decrease had IL-17 levels [10 pg/mL versus 25 % of patients with a CRP decrease. The median TTP in the IL-17 \10 pg/mL group was 2047 vs. 1367 days in the IL-17[10 pg/ml group. In conclusion, the above results suggest that agents such as IL-1Ra that specifically inhibit IL-1 induced paracrine IL-6 production are effective at targeting the proliferative myeloma component and warrant further investigation in combination with standard myeloma therapies. Elevated IL-17 levels may suggest that the inflammatory process is too far advanced in some individuals to respond to IL-1 blockade, and biomarkers such as CRP and IL-17 may be useful to predict those patients that are most likely to benefit from anti-IL-1 therapies.
B124 ANALYSIS OF ALLELIC AND GENOTYPIC FREQUENCY OF GHRL-4427G [ A POLYMORPHISM IN A SAMPLE OF THE KUWAITI POPULATION AND LYMPHOMA PATIENTS Maryam H. Alrashid, Suzanne A. Al-Bustan, Jeethu Anu Geo Kuwait University, Kuwait City, Kuwait Analysis of allelic and genotypic frequency of GHRL-4427G [ A polymorphism in a sample of the Kuwaiti population and Lymphoma patients Maryam H. Alrashid, Suzanne A. Al-Bustan, Jeethu Anu Geo Introduction: The candidate gene approach is a well-established method to study disease-associated polymorphisms in a population and to investigate the relationship between certain genes underlying complex diseases. Ghrelin is an anti-inflammatory peptide hormone that is produced and released mainly during caloric restriction and weight loss. It has been shown that ghrelin exerts protective effects in various inflammatory disease models. Ghrelin SNP polymorphisms have been reported to be associated with a change in cancer risk, specifically the GHRL-4427G [ A (rs1629816) with non-Hodgkins Lymphoma (NHL). Since association of certain polymorphisms with specific conditions can be population specific, we aimed to determine the genotypic and allelic frequencies for rs1629816 SNP in a sample of Kuwaiti healthy population and lymphoma patients. Methods: Blood samples and relevant biostatistics and background data were collected from healthy Kuwaiti volunteers from public Polyclinics and patients from Sheikha Badriya Al Sabah Medical Oncology Center (18–75 years old, both sexes). DNA was extracted
S151 from the blood samples and the genotypes were analyzed by RealTime PCR and confirmed by DNA sequencing. Allele and genotype frequencies were calculated by the gene counting method and the Hardy–Weinberg equilibrium was estimated using GENEPOP 4.2. Results: Out of the 600 healthy individuals investigated, 228 (38 %) were homozygote for the GG, 109 (18.2 %) for the AA and 263 (43.8 %) were heterozygote. Out of the 145 NHL patients investigated 57 (39.3 %) were homozygote for the GG allele, 18 (12.4 %) were homozygote for the AA allele and 70 (48.3 %) were heterozygote. The minor allele frequency for the A allele was 0.4008 for controls and 0.365 for patients. The Hardy–Weinberg equilibrium was found to be significantly deviated (P = 0.0292) in the healthy controls whereas in the patient samples was found within equilibrium (P = 0.72). Conclusion: Our data shows that the allelic and genotypic frequencies of the investigated SNP are different between the healthy and patient population. This might be an indicator that the polymorphism in this SNP might be associated with a change in the risk to develop lymphoma. Future work investigating other Ghrelin SNPs and additional statistical analysis is needed to establish a relationship between these SNPs and the risk to develop NHL.
B125 CONTROL OF LUNG CANCER WITH ASPIRINTRIGGERED STIMULATION OF RESOLUTION Molly M. Gilligan1,2, Megan L. Sulciner1,2, Sesquile Ramon3, Romain A. Colas3, Sui Huang4, Charles N. Serhan3, Dipak Panigrahy1,2 1 Center for Vascular Biology Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA; 2 Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA; 3Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA; 4Institute for Systems Biology, Seattle, WA, USA
Objective: To establish aspirin-triggered resolvins as a novel treatment to enhance current cancer therapies through the resolution of inflammation and clearance of tumor cell debris inevitably induced by chemotherapy and targeted therapy. Background: Inflammation in the tumor microenvironment is now recognized as a strong promoter of tumor growth. Substantial epidemiological evidence suggests that aspirin, which suppresses inflammation, also reduces the risk of cancer. However, the mechanism by which aspirin inhibits cancer remains unclear and toxicity has limited its clinical use. Aspirin is not only anti-thrombotic and antiinflammatory, but, as more recently discovered, low-dose aspirin also stimulates the production of pro-resolving mediators, such as aspirintriggered resolvins (AT-RvDs). Resolvins are novel pro-resolution lipid mediators derived from omega-3 polyunsaturated fatty acids and are being evaluated as effective anti-inflammatory agents in phase III ocular human clinical trials. We recently discovered that resolvins have potent anti-tumor activity, making resolvins potential candidates to mediate the anti-cancer activity of aspirin. Thus, we hypothesize that aspirin’s tumor-inhibitory effect is mediated in part by aspirintriggered (AT) resolvins via novel anti-inflammatory and pro-resolution mechanisms. Results: Aspirin-triggered resolvins inhibit cancer progression by enhancing endogenous clearance through macrophage phagocytosis of tumor debris at nanomolar concentrations. In addition, AT-resolvins counter-regulated macrophage secretion of cytokines/ chemokine exposed to tumor debris, including PAI-1 and IL1-ra. To abrogate AT-resolvin receptor activity, we utilized a pharmacological antagonist of the AT-resolvin D1 receptor (ALX/FPR-2) termed
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S152 WRW4. WRW4 neutralized both aspirin’s anti-tumor activity and low-dose aspirin’s stimulation of macrophage phagocytosis. Conclusions: Resolvins may provide a novel pathway and molecular mechanism to explain how aspirin reduces cancer risk so broadly. These anti-inflammatory and pro-resolving lipid mediators may complement current therapies for lung cancer with minimal toxicity.
B126 LIPOXIN A4 SELECTIVELY SHIFTS, IN VITRO AND IN VIVO, THE PROFILE OF TUMORASSOCIATED MACROPHAGES, IMPAIRING THE TUMOR GROWTH AND ANGIOGENESIS Christina Barja-Fidalgo, Rafael L. Simo˜es, Natalia M. Brito, Hayandra C. Costa, Veronica Morandi, Iolanda M. Fierro Department of Cell Biology, IBRAG, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil In tumor microenvironments, macrophages acquire anti-inflammatory and pro-tumor characteristics. Tumor-associated macrophages (TAMs) exhibit an M2-like profile, with low cytotoxicity and deficient production of NO and ROS. Lipoxins (LX) are lipid mediators with anti-inflammatory and pro-resolution activities in mononuclear cells. We investigated the effects of 15-epi-LX A4 on modulation of TAM in vivo and in vitro. TAMs, obtained by incubation of human macrophages with conditioned medium of metastatic melanoma cells, exhibited increasing M2 surface markers: CD206+; high Arginase (Arg); and low iNOS expression. Treatment with LX, which shifted cells from M1 (induced by LPS/IFN) to an M2-like profile, have selectively decreased M2 markers in TAM, but not in M2 cells (induced by IL-4). LX also stimulated in vitro NO production, increasing the iNOS/arginase ratio, and activated NADPH-oxidase activity, triggering ROS production. Alterations in TAM profile in vitro reduced their antiapoptotic effects and increased cytotoxicity on melanoma cells. Additionally, in a murine melanoma model, LX induces in vivo a significant reduction in tumor weight and volume. This effect was accompanied by a decrease in CD206 expression on the macrophages associated in vivo to the tumor. Furthermore, LX inhibited the angiogenic process activated by TAM in vitro (tubulogenesis assay), and also diminishing, in vivo, CD105 expression in tumor mass. Together, the results revealed a novel data on the effect of lipoxins in cancer, showing that the pro-resolutive lipid derivatives down-modulate the tumor progression stimulated by TAM, induce the shift of cells from M2- to an M1-like profile, restoring the tumoricidal activity of macrophages. (FAPERJ; Capes; CNPq/Brazil)
Inflamm. Res. Objective: As tumor cells undergo cell death, the resulting cell debris contributes to an underappreciated component of the tumor microenvironment that promotes tumor progression. To stimulate the natural debris-clearing process in breast cancer, we utilized endogenous specialized pro-resolving mediators (SPMs), specifically resolvins and maresins, which are natural tissue products that normally function to terminate inflammation through a process called resolution. Background/methods: Current cancer therapies, including chemotherapy and targeted therapy, reduce tumor burden by killing tumor cells, but as a result, create debris. Breast cancer debris may stimulate tumor growth via associated inflammation and by increasing cancer stem cells. Thus chemotherapy and targeted therapy may be a doubled-edged sword. Therefore, enhancing clearance of tumor debris with SPMs may target novel and endogenous mechanisms that drive tumor growth, resistance, and dormancy escape. Maresin 1 (MaR1), a key mediator of inflammation resolution, is biosynthesized by human macrophages from endogenous docosahexaenoic acid. We hypothesize that maresins represent a novel modality in breast cancer treatment by inhibiting tumor growth through enhancing endogenous clearance of tumor debris by macrophage phagocytosis. Results: Flow cytometry confirmed tumor cell debris generated by chemotherapy and anti-hormone therapy. Co-injection of breast carcinoma debris with a sub-inoculum of viable tumor cells promotes primary breast tumor growth via breast cancer stem cells and inflammation. Specialized proresolving mediators (SPMs) inhibit breast cancer tumors stimulated by cell debris. Systemic administration of anti-hormone therapy can stimulate tumor dormancy escape. SPMs inhibit spontaneous mammary tumor growth (MMTV-PyT) in a genetically engineered model of breast cancer. SPMs promote the clearance of tumor debris by stimulating macrophage phagocytosis of apoptotic tumor cells, and by counter-regulating critical pro-inflammatory cytokines, as well as stimulating natural endogenous antiinflammatory cytokines such as IL-1 RA. Conclusions: The maresin pathway or the enhancement of endogenous resolution processes, may offer an entirely novel approach for clearing tumor cell debris inevitably induced by conventional cancer therapy. Our results suggest that resolution-based therapies may complement current breast cancer treatments, which inevitably produce tumor cell debris.
B128 PERITONEAL LEUKOCYTE BIOLOGY DISRUPTION DURING INFLAMMATORY OVARIAN CANCER James E. Riggs, Kelley DePierri, Naomi Goldman, Gretel Torres, John Somerville
B127 CONTROLLING BREAST CANCER THROUGH THE STIMULATION OF RESOLUTION Kristen A. Lehner1, Megan Sulciner1, Sesquile Ramon2, Sui Huang3, Charles Serhan2, Dipak Panigrahy1 1 Center for Vascular Biology Research, Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA; 2Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA; 3Institute for Systems Biology, Seattle, WA, USA
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Biology Department, Rider University, Lawrenceville, NJ, USA The i.p. transfer of ID8 cells (mouse ovarian surface epithelial cell carcinoma) into intact, syngeneic, C57BL/6 J (wild type, WT) mice leads to hemorrhagic ascites within 5 weeks. CD11b+Gr-1+ (granulocytic) myeloid-derived suppressor cells (MDSCs) arise concomitant with ascites appearance in both the spleen and peritoneal cavity. To determine if pro- (IFNc) or anti- (IL4, IL10) inflammatory cytokines are drivers of disease in this model we transferred ID8 into C57BL/6J mice lacking either these molecules or their receptors. Relative to WT mice, the development of hemorrhagic ascites was accelerated in IL10-/-, IL4-/-, and IFNcR-/- mice. MDSC and ID8 expansion were greatest in IL10-/- mice. ID8 also triggered a marked increase in PerC macrophages in the IL10-/- mice and lymphocytes in the
Inflamm. Res. IFNcR-/- and IL4-/- mice. However, the percent representation of B cells in the total PerC cell pool declined markedly for all mice. B cell subset analysis revealed initial depletion of B-1 (CD11b+IgMhiIgDlo) B cells and persistence of B-2 B cells (CD11b-IgMloIgDhi). Immunization with thymus-independent type 1 (TI-1, FITC-LPS) and type 2 (TI-2, FITC-Dextran) antigens revealed initial loss of the TI-2 response. Beyond the peritoneal locus of disease, both B and T lymphocyte percentages dropped in the spleens of all mice. These observations reveal that inflammation disrupts leukocyte biology during ovarian cancer emergence and might serve to inform strategies to develop biomarkers for this disease. Supported by NIH AREA R15CA173688.
B129 MODULATION OF INFLAMMATORY RESPONSE INDUCED BY INHIBITOR MOLECULES OF THE KINESIN EG5 WITH PRONOUNCED ANTITUMOR ACTIVITY AGAINST BREAST CANCER Luis Felipe F. Silva1, Rafael Correa1, Breno AD Neto2, Jose´ Raimundo Correa3, Kelly G. Magalha˜es1 1 Laboratory of Immunology and Inflammation, University of Brası´lia, Brazil; 2Laboratory of Medicinal and Technological Chemistry, University of Brası´lia, Brazil; 3Laboratory of Electron Microscopy, University of Brası´lia, Brazil
Breast cancer is the second most common type of cancer and responsible for the death of millions of women around the world. In order to find new solutions for the treatment of this disease, our group has previously synthesized and selected 5 new derivatives (4M, 4BT, 4P, 4Bc and 4X) from 3,4-dihydropryrimididone, which is a class of inhibitory molecules of the motor spindle protein Eg5. These molecules have been previously described as potent antitumor compounds against different breast cancer cells lines. To provide a wider understanding on the effects of these drugs, the present study aimed to characterize the modulation of inflammatory response induced by our newly synthesized Eg5 inhibitory compounds (EG5ICs). To address this goal, weinvestigated the effects of EG5ICs on the: (I) biogenesis of cellular lipid droplets, (II) release of nitric oxide (NO), (III) secretion of inflammatory cytokines (IL-6 and TNF-a) and (IV) formation of Reactive Oxygen Species (ROS). RAW 264.7, mice peritoneal macrophages and human monocytes cells were stimulated or not with the lipopolysaccharide (LPS) and treated with EG5ICs (4M, 4BT, 4P, 4Bc and 4X) at different concentrations and time periods. Monastrol, Taxol and LPS were used as assay controls. IL-6, TNF-a and NO were quantified in cells supernatant by ELISA and NO assay, respectively. Lipid droplet biogenesis was assessed by flow cytometry and confocal microscopy. ROS generation was detected by flow cytometry. Our results showed that EG5ICs 4P, 4BC, 4X and Monastrol, but not 4M and 4BT, were capable of inhibit the LPSinduced TNF-a secretion after 24 h of stimulation. All EG5ICs, except 4BT, decreased significantly LPS-induced IL-6 secretion after 24 h of stimulation. Only Monastrol significantly reduced NO levels induced in cells. Lipid droplet biogenesis was decreased only in cells treated with EG5ICs 4X and Monastrol. All EG5ICs increased ROS generation in cells after 3 h of stimulation. Taken together, our results show that most of the EG5ICs tested tend to modulate macrophages and monocytes to an anti-inflammatory profile where the most prominent results were seen by Monastrol, 4BC and 4X. On the other hand, the EG5ICs 4BT has shown a tendency to modulate the macrophages to the pro-inflammatory profile. Therefore, EG5ICs can
S153 have antitumor activity against breast cancer also by modulating tumor microenvironment inflammatory response. Support: CNPq
Inflammation and Metabolic Disorders B130 ADIPOSE TISSUE INFLAMMATION AND REMODELING DURING WEIGHT CYCLING IN MICE Cı´ntia RP Caria, Caroline C. Oliveira, E´rica MF Gotardo, Tanila W. Dos Santos, Alessandra Gambero Sa˜o Francisco University, Sa˜o Paulo, Brazil Expansion of adipose tissue (AT) during obesity induces accumulation of macrophages and other immune cells which produce proinflammatory mediators and contribute to a local and systemic inflammation. AT expansion also triggers events known as AT remodeling that involves adipocytes, immune cells infiltrated and extracellular matrix proteins. Dysfunctional remodeling events are a hallmark of obesity associated with metabolic disease. During weight loss, macrophage infiltration and local inflammation decrease, but several anti-weight loss mechanisms probably acts and weight regain occurs easily in the vast majority of individuals. We studied the inflammation in AT after weight loss and regain focusing adipokine release and remodeling for a better understanding the anti-weight loss mechanisms. Eight-week-old male Swiss mice were fed a high-fat diet (HFD; 60 % of the calories derived from fat) during 8 weeks. Obese mice were subjected to moderate caloric restriction for subsequent 8 weeks (weight loss group, WL). WL was reintroduced to HFD for additional 8 weeks (weight regain group, WR). Group of age-matched obese mice was used as control (Ob16 and Ob24). Glucose homeostasis was evaluated by glucose blood level and insulin tolerance test (ITT). Mice were sacrificed and AT collected. Adipokine and matrix metalloproteinases (MMP) were quantified by multiplex kit. Moderated caloric restriction in mice reduced final body weight (63 ± 2 and 40 ± 1 g for Ob16 and WL, respectively; p \ 0.05) and adiposity (2.5 ± 0.2 and 1.1 ± 0.1 g of epididymal AT for Ob16 and WL, respectively; p \ 0.05). HFD reintroduction resulted in a final body weight of 71 ± 6 and 70 ± 3 for WR and Ob24, respectively) and adiposity gain. Weight loss improved local adipokine production reducing leptin, monocyte chemoattractant protein-1 (MCP-1), plasminogen activator inhibitor-1 (PAI-1) and interleukin-6 (IL-6). Although, MMP-3 expression was also reduced, MMP-2 (4.1 ± 0.5 and 4.0 ± 1.1 ng/mL for Ob16 and WL, respectively) and MMP-12 (27.1 ± 5.6 and 23.6 ± 11.0 ng/mL for Ob16 and WL, respectively) remain elevated in AT after weight loss. Weigh regain resulted in higher levels of IL-6 (641 ± 96 and 351 ± 38 pg/ mL for WR and Ob24, respectively; p \ 0.05) and PAI-1 (3.6 ± 0.9 and 1.7 ± 0.3 ng/mL for WR and Ob24, respectively;p \ 0.05). In WR group, MMPs levels were not different when compared with obese-matched group, but glucose blood levels (234 ± 28 and 195 ± 17 mg/dL of glucose for WR and Ob24, respectively, p = 0.10) and insulin resistance (2.0 ± 0.3 and 3.3 ± 0.4 kITT for WR and Ob24, respectively, p = 0.10) were worsened in WR group. In conclusion, inflammation in AT is resolved during weight loss, but remodeling process is remained. We hypothesized that a new cycle of expansion when AT is in a remodeling process could worsened inflammation establishment and metabolic alteration, resulting that weight cycles are more deleterious that obesity maintenance.
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B131 SERUM ANTIBODIES TO ENTEROBACTERIAL LIPOPOLYSACCHARIDES AND THEIR RELATIONSHIP WITH INFLAMMATORY MARKERS IN DIABETES MELLITUS Anatoliy V. Kubyshkin, Andrey Gordienko, Vladimir Beloglazov, Natalia Khimich, Yuliana Shramko V.I.Vernadsky Crimea Federal University, Simferopol, Republic of Crimea Cardiovascular complications conditioned by the chronic intima’s inflammation of low intensity are considered the main reason of disability and mortality in diabetes mellitus (DM). Enterobacterial lipopolysaccharides are regarded as the potential factor in maintenance of low intensity chronic inflammation in DM. The laboratory study of 51 patient with type 1 diabetes mellitus (DM-1) and 60 ones with type 2 diabetes mellitus (DM-2) was performed. The concentration of serum C-reactive protein (CRP) and levels of serum antibodies to enterobacterial lipopolysaccharides (anti-LPS-Ab) were determined with the enzyme- linked immunosorbent assay (ELISA) in both groups of the patients. Claster analysis determined that in 40.8 % of patient with DM-1 increased concentration of CRP was associated with significant decrease of anti-LPS-IgA, anti-LPS-IgM and anti-LPS-IgG. In 56.7 % of patient with DM-2 accompanied by the high concentration of CRP the rise of anti-LPS-IgA and anti-LPS-IgM was not reliable, whereas the levels of anti-LPS-IgG were reliably increased. At the same time, activization of the inflammatory response with the subsequent rise of serum CRP in the patients with DM-2 was followed by the substantial increase of anti-LPS-IgA and anti-LPS-IgG levels and the tendency of anti-LPS-IgM levels decrease. Consequently, the obtained data indicate the existence of the close interactions between inflammation of low intensity and the immune response to enterobacterial lipopolysaccharides in diabetes mellitus type 1 and 2.
Inflamm. Res. measured in culture supernatants using the AmplexRed assay. Preadipocytes were differentiated into mature adipocytes with rosiglitazone and lipid droplet accumulation, adiponectin production and browning (UCP1 mRNA levels) were assessed. The effect of MB06322 (CS-917; FBP1 inhibitor; hepatocyte assay positive control), BADGE (PPARc antagonist; adipocyte assay positive control), pioglitazone, sitagliptin, salsalate, acetylsalicylic acid, metformin, geldanamycin and rimonabant were evaluated. Results: Both hepatocyte and adipocyte assays were compatible with high throughput screening of small molecules and adenoviral RNAi vectors. Signal/background ratio, vehicle tolerance, assay window and intra- and inter-assay variability all passed pre-set QC criteria. MB06322 and metformin inhibited gluconeogenesis with IC50 values that approximate literature values. Geldanamycin, rimonabant and MB06322 inhibited adiponectin production, with geldanamycin showing signs of cytotoxicity. Rimonabant, MB06322 and pioglitazone modulated lipid droplet formation. Other compounds did not inhibit the readouts at concentrations [ 10-fold higher than the IC50 on their primary target(s). Conclusions: A panel of primary human cell-based assays allows evaluation of compounds targeting various molecular mechanisms, as potential lead molecules to treat metabolic syndrome. In the current study, non-steroidal anti-inflammatory compounds such as salsalate and acetylsalicylic acid that have been reported to potentially modulate aspects of metabolic syndrome (insulin resistance, cardiovascular events) did not modulate the disease phenotype in patient-derived primary human cell-based assays, suggesting a different mode of action in vivo.
B133 INFLAMMATORY MARKERS IN PATIENTS WITH STABLE ANGINA AND DIABETES MELLITUS OF TYPE 2 Ludmila I. Gapon, Tatiana I. Petelina, Natalia A. Musikhina, Irina V. Osipova, Vadim A. Kuznetsov
B132 PRIMARY HUMAN CELL-BASED MODELS FOR METABOLIC SYNDROME Jeroen DeGroot1, Annelieke Strijbosch1, Folkert Verkaar1, Blandine Mille-Baker1, Joe Cornicelli2, David F. Fischer1 Charles River Laboratories. Leiden, the Netherlands; 2Charles River Laboratories. Wilmington, MA, USA 1
Objectives: Metabolic syndrome is a disorder of energy utilization and storage, characterized by obesity, elevated blood pressure, plasma glucose, triglycerides, and decreased HDL cholesterol. Metabolic syndrome increases the risk of developing diabetes and cardiovascular disease. Adipocytes play a pivotal role in the development of metabolic syndrome, since they contribute to the production of circulating proinflammatory cytokines and adipokines (such as TNFa, adiponectin, resistin, and PAI-1). Hepatocytes play a key role in energy metabolism (storage and emergency glucose release). Because of the complex interactions between various cell types, state-of-the art in vitro and in vivo models are needed that capture the disease relevant processes to enable the discovery and development of new disease-modifying drugs. Methods: Human primary hepatocytes (50,000 cell/well) and preadipocytes (4000 cells/well), both from T2D donors were cultured in 96-well plates. Hepatocytes were glucose-starved and subsequently incubated with a gluconeogenic substrate. Glucose levels were
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Tyumen Cardiology Center, Tyumen, Russian Federation Objectives: To study inflammatory markers and lipid profile in group of Stable Angina (SA) patients with and without (type 2 diabetes mellitus) DM. Methods: 86 patients aged 64.3 ± 6.5 years with SA and insignificant coronary stenosis (\75 %) were examined. Group I included 33 patients with SA and DM, group II consisted of 53 patients with SA without DM. All patients received statins, ACE inhibitors, beta blockers, l antiplatelet therapy. In group I all patients received antihyperglycemic therapy. Lipid profile parameters (total cholesterol, triglycerides, LDL cholesterol, VLDL cholesterol, lipoprotein (a), Apo-A, Apo-B), inflammatory markers (hs-CRP, TNF-alpha, homocysteine, interleukine 1 b, 6, 8, sCD40 L, MMP-9, TIMP-1), endothelial dysfunction markers (endothelin-1, nitrites) were measured. Results: There were high levels of hs-CRP, TNF-alpha, lipoprotein (a), MMP-9, triglycerides, and endothelin-1 in both groups. The level of TIMP-1 was significantly reduced in both groups. Patients in group 1 had significantly elevated levels of total cholesterol, LDL cholesterol, homocysteine, Apo-B, Apo-B/Apo A-1 ratio, IL-1 b. In group 1 the following positive correlations were found: between glycohemoglobin and Apo-B, Apo-B/Apo A-1 ratio, homocysteine, IL-1 b, sCD40 L; IL-6 and hs-CRP; homocysteine and LDL cholesterol, MMP-9, duration of CAD; endothelin-1 and sCD40L, TNF-alpha. In group I the following positive correlations were found: between scale of Syntax Score and hs-CRP and homocysteine level.
Inflamm. Res. Conclusion: In patients with SA and DM there was a significant increase in the levels of atherogenic lipid fractions as well as homocysteine, hs-CRP and IL-1 b which may indicate a higher risk of coronary events even in the absence of significant coronary stenosis.
B134 IRON SUPPLEMENTATION EFFECTS ON SYSTEMIC AND ADIPOSE TISSUE INFLAMMATION DURING OBESITY IN MICE Alessandra Gambero, Cintia RP Caria, Erica MF Gotardo Sa˜o Francisco University, Sa˜o Paulo, Brazil The conception of obesity as a chronic low systemic inflammatory condition has been recently linked to the reduction in plasma iron observed in obese subjects. Visceral adipose tissue (AT) releases several pro-inflammatory cytokines, as interleukin (IL)-6 when this local is infiltrated by macrophages. IL-6 induces hepcidin mRNA in macrophages and adipocytes and hepcidin decreases iron release from these cells and reduces iron absorption by intestinal cells resulting in hypoferremia of inflammation in obese patients and experimental models. Iron is important micronutrients but, it also is able to generate dangerous hydroxyl radical in the presence of reactive oxygen species (ROS) and contribute to inflammation exacerbation. In this work, we studied the effects of systemic iron supplementation to obese mice with hypoferremia upon adipose tissue and systemic inflammation. Eight-week-old male Swiss mice were fed a high-fat diet (HFD; 60 % of the calories derived from fat) during 24 weeks. In the last 2 weeks, obese mice received iron citrate (IC; 50 mg/kg/week, i.m.). Agematched lean mice were used as control. Glucose homeostasis was evaluated by glucose blood level and insulin tolerance test (ITT). Mice were sacrificed and AT collected. Serum and AT cytokines were quantified by multiplex kit. Iron supplementation increased iron levels improving hypoferremia (376 ± 34, 172 ± 13 and 217 ± 16 lg/dL for IC, non-treated obese group (NT) and lean control, respectively; p \ 0.05) but it did not promote alterations in insulin tolerance or basal glucose levels altered by obesity. Iron supplementation did not modify the amount of hepcidin, adiponectin, leptin, resistin, tumor necrosis factor (TNF)-a, IL-6 or monocyte chemoattractant protein-1 (MCP-1) in adipose tissue altered by obesity, but we observed a tendency to decrease local production of plasminogen activator inhibitor-1 (PAI-1; 104 ± 21 and 220 ± 76 ng/ml of PAI-1 for IC or NT, respectively). Serum levels of cytokine, including PAI-1 were not altered by iron supplementation. In conclusion, iron supplementation did not worsen adipose tissue or systemic inflammation in a model of hypoferremia associated to obesity in mice. Increased PAI-1 levels were previously described for iron scarce or hypoxic conditions in cancer cells. PAI-1 was increased in AT during obesity and iron supplementation had a tendency to reduced it, suggesting that intracellular iron level or hypoxia in AT could be improved by iron supplementation, but without modify AT inflammation.
B135 EVALUATION OF THE URIC ACID EFFECT IN INFLAMMATORY RESPONSE Larissa AC Carvalho, Eliziane S. Patricio, Joa˜o PP Bonifacio, Flavia C. Meotti Universidade de Sa˜o Paulo, Sa˜o Paulo, Brazil
S155 Introduction: Uric acid is the main antioxidant that accumulates in plasma (200–500 lM). Its mono-anion form, urate (pKa 5.4), chelates transition metals ions, reacts with hydroxyl radical, singlet oxygen and repairs protein radicals. Despite its antioxidant potential, the release of urate from dying cells initiates inflammatory response and neutrophils recruitment. Urate primes immune cells and induces cytokine production through Toll-like receptors. Here, we investigated the effects of urate in superoxide (O2-), hypochlorous acid (HOCl) and cytokines release to understand the redox mechanisms by which urate modulate inflammatory response. Methods and Results: HL-60 cells were differentiated into neutrophils (dHL-60; 5 days, 1.3 % dimethylsulfoxide) and activated with phorbol myristate acetate (PMA; 100 ng/mL). Superoxide production was detected using dihydroethidium probe (DHE). The products of DHE oxidation, 2-hydroethidium (2-OH) and ethidium were separated in a HPLC (column Phenomenex Synergi, 4 lm Polar-RP 80A, 150 9 60 mm) and quantified by fluorescence (kexc 480 nm and kem 580 nm). The consumption of O2 was evaluated by polarography using Clark electrode. Hypochlorous acid was measured by taurine chloroamine-DTNB assay. Tumor necrosis factor alpha (TNF-a) was quantified by Enzyme-Linked Immunosorbent Assay (ELISA). At low concentration (50 lM) urate decreased in 15 % the levels of O2•-. However, at a concentration of 500 lM, urate increased in 51 % the levels of O2-. Urate slightly increased O2 consumption in cells activated with PMA. The incubation with PMA increased five times the levels of HOCl by dHL-60. Urate (0.5–2 mM) significantly decreased HOCl concentration. The PMA also induced a hundred fold increase in TNF-a release (98.8 ± 1.2 pg/mL). Urate (2 mM) caused a slightly but significant decreased in TNF-a release (88.9 ± 3.2 pg/ mL). Conclusion: These results demonstrate that urate could have a dual effect in the redox balance during the inflammatory oxidative burst. If in one hand it increased oxygen consumption and superoxide production, on the other hand it decreased HOCl. In our model, urate slightly decreased TNF-a release. Additional studies are being performed to understand the modulatory mechanisms of urate upon immune cells. Financial support FAPESP (2011/18106-4, 2013/07937-8, 2013/02195-3), CNPq and USP.
B136 PHYSIOLOGICALLY DIGESTED CARRAGEENAN AFFECTS INTESTINAL BARRIER FUNCTION Lulu Fahoum, Uri Lesmes, Esther G. Meyron Technion-Israel Institute of Technologh, Haifa, Isreal Background: Intestinal inflammation is becoming more frequent in the western world. The dramatic increase within all ethnic groups supports the theory that environmental, including nutritional factors contribute to the disease development. One food additive that has been associated with the induction of gut epithelial inflammation is carrageenan (E407) (CGN), a sulfated marine polysaccharide that is widely incorporated in processed foods as a thickener, stabilizer, and texturizer. The use of CGN in processed foods has been limited to high molecular weight CGN in contrast to degraded low molecular weight CGN that is recognized to mediate intestinal inflammation and is designated as a Group 2B carcinogen. We hypothesized that polydisperse high molecular weight CGN permitted in foods may undergo partial physiological digestion (based on a semi-dynamic in vitro digestion system recreating human digestion) and consequently affect the epithelium-integrity and possibly impair the physiologic intestinal homeostasis.
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S156 Objectives: Our study addresses both the functional and the morphological impacts of physiologically digested CGN on an intestinal epithelium model in order to elucidate its effects on intestinal homeostasis and its potential role in the progression of inflammatory bowel diseases. Methods and results: A Caco-2 cell model for intestinal epithelium was incubated with physiologically relevant concentrations of CGN digesta. Barrier-function was analyzed by immunofluorescence and immunoblots of subcellular fractionations of proteins relevant to the tight-junction functionality and by evaluation of apical to basolateral transport of fluorescent dextran molecules in a transwell-system. We detected increased paracellular permeability to macromolecules and disruption of the monolayer structure. Specifically, changes in tight junction protein zonula occludens 1 (Zo1) morphology we detected along with redistribution of the protein in the sub-cellular compartments. The apical actin filaments were also affected by CGN showing phenotypic characteristics typically associated with stress fibers. Conclusion: This study provides new evidence revisiting the safety evaluation of CGN by demonstrating deleterious effects of food-grade CGN after physiologic digestion, on epithelial barrier-function implying a possible deleterious effect of this food additive on consumer health.
Inflamm. Res. IL-1b but not TNF-a expression. Moreover, we found that HMW adiponectin suppressed LPS-induced C/EBPb expression and nuclear translocation. Interestingly, contrary to expectation, inhibition of NF-jB was not necessary for reduced IL-1b expression, because HMW adiponectin did not lead to the significant alteration of NF-jB reporter activity. These results suggest that HMW adiponectin has potent anti-inflammatory activities, as suppressor of IL1b expression, by inhibition of Akt-C/EBPb signaling pathway in macrophages.
Inflammatory Cell Signaling B138 ERYTHROPIETIN PROTECTS ENDOTHELIAL CELL FROM HIGH GLUCOSE INDUCED INJURY Yasunori Iwata, Yasuyuki Shinozaki, Haruka Yasuda, Kengo Furuichi, Norihiko Sakai, Takashi Wada Kanazawa University, Kanazawa, Japan
B137 HMW ADIPONECTIN SUPPRESS LPS-INDUCED IL-1ß EXPRESSION THROUGH INHIBITING AKTC/EBPß INFLAMMATORY SIGNALING IN RAW264.7 MACROPHAGE Toshihiro Tanioka, Yoshinao Tainaga, Michiyo Yamada, Yuuri Kamimura, Mariko Suzuki, Saori Takatori, Yasuko Nakano School of Pharmacy, Showa University, Tokyo, Japan Adiponectin is abundant adipocytokine secreted from adipocytes, which exists as trimer, hexamer, high molecular weight (HMW) adiponectin and proteolytic product, globular adiponectin (gAd) in plasma. Several groups have reported that adiponectin has anti-inflammatory effects on macrophages, however, anti-inflammatory action of adiponectin are still largely controversial, because of the different sources of recombinant adipoenctin were used. Recently, we established a very simple and effective purification method for HMW adiponectin from human plasma. In this study, we compared purified native HMW with various recombinant adiponectin to investigate whether adiponectin induces anti-inflammatory or proinflammatory action on macrophages. Pretreatment with 10 lg/mL HMW for 16 h inhibited LPS-induced IL-1bexpression but not TNF-a expression, whereas treatment with HMW and LPS at the same time showed no inhibitory effects on IL-1b expression. As expected, exposure of macrophages to 10 lg/mL recombinant human adiponectin (rhAd) expressed in CHO cells, which contains mainly hexamer, attenuated LPS-induced IL-1b expression but not TNF-a. Unexpectedly, 10 lg/mL recombinant mouse adiponectin (rmAd) expressed in CHO cells, which contains mainly HMW, markedly inhibited bothIL-1bandTNF-a expression. Recombinant human and mouse globular adiponectin (rhgAd and rmgAd), expressed in E. Coli also significantly suppressed IL-1b expression. In contrast, these molecules showed no effect on TNF-a expression. Further analysis of the underlying molecular mechanisms revealed that HMW adiponectin abrogated the ability of LPS to induce phosphorylation of Akt (Ser 473). Importantly, inhibition of PI3K, as upstream molecule of Akt, by pharmacological inhibitor in combination with adiponectin additively diminished LPS-induced
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Diabetic nephropathy (DN) is a major cause of end stage kidney disease and a strong risk factor for cardiovascular diseases. High glucose induces endothelial injury in vasculature, resulting in tissue injury in diabetic condition. Chronic inflammation has been reported to play an important role for the progression of high glucose induced cell injury. Growing data showed that erythropoietin (EPO) protect the tissues from some kind of injury, such as hypoxia and mechanical stress. However, the contribution of EPO to high glucose induced tissue injury remains to be explored. Therefore, we hypothesized that EPO protects endothelial cells from high glucose (HG) induced injury via the regulation of inflammatory and anti-inflammatory balance. To explore this possibility, we performed genome-wide transcriptome profiling in human umbilical vein endothelial cells (HUVEC), which were stimulated by high glucose (HG) with/without EPO treatment and detected the expression of inflammation associated genes. Hieralchial clustering analysis showed the different pattern of mRNA expression in HG stimulated HUVEC with/without EPO. While inflammatory cytokines/chemokines mRNA expression were increased by the HG stimulation in HUVEC, Th2 related cytokine receptors and intracellular signaling molecules showed the reduced mRNA expression levels. EPO treatment reduced inflammatory cytokines/chemokines mRNA expression and increased Th2 related cytokine mRNA expression levels. Real-time PCR analysis confirmed the increased expression of inflammatory related genes, those were decreased in HG stimulated HUVEC with EPO treatment. Moreover, EPO stimulation increased mRNA expression of EPO receptor and b-common receptor. Taken together, EPO signaling protects high glucose induced cell injury by the regulation of immune balance.
B139 SALT-INDUCIBLE KINASES (SIK) INHIBITION IN HUMAN MYELOID CELLS MODULATES TLR AND IL-1R SIGNALING AND INDUCES AN ANTIINFLAMMATORY PHENOTYPE Maria Stella Lombardi1,2, Corine Gillie´ron1,2, Damien Dietrich1,2, Cem Gabay1,2
Inflamm. Res. 1
Division of Rheumatology, Department of Internal Medicine Specialties, University Hospitals of Geneva, 26 Avenue de BeauSe´jour, 1211 Geneva 14, Switzerland; 2Department of Pathology and Immunology, University of Geneva School of Medicine, 1 Rue MichelServet, 1211 Geneva 4, Switzerland Background: Experiments performed primarily in mouse cells showed that salt-inducible kinases (SIKs) synergize with Toll-like receptor (TLR) signaling to restrict the formation of regulatory macrophages involved in the resolution of inflammation via the production of high levels of anti-inflammatory IL-10 and low levels of proinflammatory IL-12 and tumor necrosis factor (TNF-a) cytokines. Mechanistically, pharmacological inhibition of SIK leads to de-phosphorylation and nuclear translocation of CREB transcriptional co-activator (CRTC3) and class II histone deacetylase HDAC4, respectively. CRTC3 interacts with p-CREB to promote a gene expression program including strong up-regulation of IL-10, whereas HDAC4 deacetylates p65-NF-kB leading to repression of pro-inflammatory cytokines. Objectives: To examine the regulation of expression and function of SIKs in human monocytes and monocyte-derived macrophages (MDM) and dendritic cells (MDC) in culture. Methods: We used two structurally unrelated small molecule SIK inhibitors: HG-9-91-01 and ARN-3236 (Arrien Pharmaceuticals, Salt Lake City, UT). Cells were pretreated 1 h with SIK inhibitors before challenge with TLR4 (LPS) or TLR2 (Pam3CSK4) agonist or IL-1b for 3–24 h. Total mRNA was isolated and gene expression determined by RT-qPCR. Cell lysates were analysed by immunoblotting for the expression of SIK kinases family members (SIK 1-3) or using specific phospho-antibodies for CRTC3 and HDAC4. Cytokines secreted in the supernatants were determined by ELISA. Results: Our results demonstrated that the differentiation from peripheral blood monocytes to MDM or MDC cells induced a marked upregulation of SIK protein expression. We showed that SIK inhibition significantly decreased proinflammatory cytokines (TNF-a, IL-6, IL-1b and IL-12p40) and increased IL-10 secretion by human myeloid cells stimulated with TLR2 and-4 agonists. Differently than in mouse cells, SIK inhibition did not enhance IL-1Ra production in human MDM. Interestingly, SIK inhibition impaired proinflammatory (M1) polarization of MDMs as assessed by the downregulation of different validated markers (including CD80 and CCXL9), and induced a regulatory-like M2b/c (IL-10high/IL-12low) phenotype. More importantly, we showed for the first time that SIK inhibition decreases pro-inflammatory cytokines secretion (TNF-a and IL-6) in human MDMs and MDCs upon IL-1R stimulation. The downstream effects observed with SIK inhibitors on cytokine modulation correlated with direct SIK targets (CRTC3 and HDAC4) dephosphorylation. Conclusion: Altogether our results show that SIK inhibition exerts anti-inflammatory effects in human myeloid cells and expand the therapeutic potential of SIK inhibitors for the treatment of immunemediated inflammatory diseases.
B140 EVALUATION OF GLUTAMINE AS MODULATOR OF THE TRANSCRIPTION FACTOR NF-KB IN MACROPHAGES FROM MICE SUBJECTED TO DIETARY RESTRICTION Dalila C. Oliveira, Ed W, Santos, Jackeline S. Beltran, Primavera Borelli, Ricardo A. Fock University of Sa˜o Paulo, Sa˜o Paulo, Brazil Dietary restriction (DR) modify the innate and adaptive immune response, exerting effects on inflammatory and regulatory cytokine
S157 production also modifying the expression of genes directly involved in immune response. Macrophages play key roles in innate immune response and have high utilization rate of amino acid glutamine (GLN), essential for energy and nitrogen supply. In addition, activation of macrophages in vitro increases the transcription process and secretion of proteins, such as pro-inflammatory cytokines. This type of activation leads to an increased synthesis of mRNA, in which GLN has an important role, acting as a precursor of nitrogenous bases. In this context, GLN metabolism in macrophages is essential for the cytokines synthesis and it is dependent on the extracellular concentration. Considering the modulatory effects of GLN and DR, we proposed addressing some complex aspects of immune regulation of the NF-kB transcription factors, and the influence of GLN supplementation modulating these processes in peritoneal cells from DR mice. Materials and methods: We evaluated the effects of different concentration of GLN (0, 0.6, 2 or 10 mM) in vitro on TNF-a, IL-12 and IL-10 production by peritoneal cells. The expression of NF-jB, IkB and their respective phosphorylated portion were evaluated by Western Blotting (WB), under in vitro stimulation of LPS (1.25 lg), the source of cells were DR BALB/c mice, subjected to a DR for 10 days, reducing their ration consumption in 30 % when compared to the control group. Results: We observed that animal’s weight, Lee index, cholesterol, triglycerides and glucoses serum concentrations were reduced in DR group. Anemia, leucopenia, peritoneal and spleen cellularity reduction in animals submitted to DR was also observed. In peritoneal cells, reduction of IL-12 and TNF-a production were observed when cultivated without GLN or supplemented with 2 or 10 mM of GLN in the DR group. Peritoneal cells from DR group supplemented with 10 mM of GLN showed increased production of IL-10 when compared with cells that did not received GLN, we also observed that cells from control group produced less IL-10 in comparison to cells from DR group when both were cultivated in medium with 10 mM of GLN. WB results showed reduced pIKBa/IKBa ratio expression in cells from both groups cultivated with 10 mM of GLN and compared to the others GLN concentrations studied. In addition, the pNFkB/NFkB expression in cells from both groups cultivated with 2 or 10 mM showed reduced expression when compared to cells cultivated without GLN. Comparison between control and DR groups did not show differences. In conclusion, we can infer that GLN can modulate the IKBa and NF-kB expression in peritoneal cells interfering in cytokines production and increasing IL-10 production in DR mice.
B141 EFFECT OF URIC ACID IN THE BACTERICIDAL ACTIVITY OF INNATE IMMUNE CELLS Joa˜o P. P. Bonifacio, Larissa A. C. Carvalho, Flavia C. Meotti University of Sa˜o Paulo, Sa˜o Paulo, Brazil Introduction: Uric acid (UA) is considered an important antioxidant in human plasma and is also a pro-inflammatory agent. Urate activates inflammatory cytokine production by priming Toll-like receptors. The oxidation of urate in the inflammatory burst generates a pro-oxidant intermediate that could modulate the killing activity of innate immune cells. We have found that urate increases Pseudomonas aeruginosa 14 (PA14) survival by decreasing tumor necrosis factor alpha (TNF-a) and interleukin 1b (IL-1b) release in neutrophils and macrophages. In this study, we investigate the mechanisms by which urate increases bacterial survival.
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S158 Methods and Results: Human THP-1 and HL-60 cells were differentiated into macrophages and neutrophils by incubation with phorbol myristate acetate or 1.3 % dimethylsulfoxide, respectively. Effect of urate on immune cells viability and phagocytosis was evaluated by flow cytometry. The interference in hypochlorous acid (HOCl) production was measured by taurine chloroamine-DTNB assay. Effect of urate (0.5 mM) in the microbicidal activity of a cell free system containing myeloperoxidase (MPO, 100 nM), hydrogen peroxide (H2O2, 0.5 mM) and chloride (0.9 %) was evaluated against PA14 (107 cells). Activation of NF-jB was measured by Western blot. Urate did not affect immune cells viability or phagocytosis. Incubation of PA14 with neutrophils did not induce a significant increase in HOCl formation (3.8 ± 0.6 in neutrophils and 4.8 ± 1.9 lM in neutrophils plus PA14). No difference was found in cells incubated with urate. Urate did not interfere in the bacterial killing capability of the MPO/H2O2/Cl- system since no growth was observed in this system against 0.2 9 105 ± 0.0 CFU in controls. Urate significantly increased HOCl formation in this cell free system from 13.11 ± 6.1 to 38 ± 14 lM in absence or presence of urate, respectively. Incubation of immune cells with PA14 activated p65 NF-jB phosphorylation. The ratios for phospho-p65/b-actin and for the inhibitor IjB-a/b-actin were 0.207, 0.38 and 0.056 O.D. and 0.408, 0.315 and 0.591 O.D in macrophages alone, macrophages plus PA14 and macrophages plus PA14 plus urate. Conclusion:: These results present a new role for uric acid in immune cell response in bacterial killing. The inhibitory effect of urate upon NF-jB would explain the decrease in TNF-a and IL-1b. The anti-microbicidal effect of urate is not related to a decrease in immune cell viability, phagocytosis and oxidants production. The modulation NF-jB pathway by urate might be the responsible by the alteration in the bactericidal activity of the immune cells. Financial support FAPESP (2011/18106-4, 2014/01936-2, 2013/07937-8), CNPq and USP.
B142 MYCOBACTERIUM BOVIS BCG ENHANCES PPARc EXPRESSION/ACTIVATION THROUGH MTOR DEPENDENT PATHWAYS AND COMPARTMENTALIZES IMPORTANT CELL SIGNALING ENZYMES WITHIN LIPID DROPLETS Alan B. Carneiro1, Henrique NS Carvalho1, Tamyres S. Salles1, Giovana H. Carvalho1, Miriam B. Werneck2, Joa˜o P. Viola2, Heloı´sa D’A´vila3, Patrı´cia T. Bozza1 1 Laborato´rio de Imunofarmacologia, Instituto Oswaldo Cruz, FIOCRUZ-RJ, Brazil.; 2Instituto Nacional do Caˆncer, RJ, Brazil.; 3 Universidade Federal de Juiz de Fora, MG, Brazil
Introduction: Lipid droplet (LD) is considered a dynamic cellular organelle that can play several functions on cells. LD biogenesis, composition and function may vary according to the cell and stimuli. Recent studies indicate a remarkable action of this organelle during host responses against pathogens infection. Our group already demonstrated that Mycobacterium bovis BCG infection induces LD biogenesis in macrophages through mechanisms dependent on TLR2 and induction of PPARc expression (Almeida et al. 2009). PPARc is a nuclear receptor that plays a substantial role in the regulation of cellular differentiation, metabolism and inflammation. Furthermore, the PPARc inhibition leads to LD biogenesis reduction together to increased mycobacterium killing. So, the objectives of this work are to evaluate the signaling pathways induced by M. bovis BCG involved in PPARc expression/activation and to analyze the protein
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Inflamm. Res. composition of LD with particularly attention for presence of phosphorylated kinases. Methods and Results: For this study, HEK 293T cells were transfected with different constructs containing TLRs, co-receptors and PPARc genes. BCG infection was able to induce NF-kB activation, as observed by luciferase activity, and IL-8 production in TLR2-transfected cells in the presence or absence of PPARc; whereas only in PPARc-transfected HEK 293T LD biogenesis was induced by BCG. By western blot analysis, there was an increase expression of PPARc and FABP4, a target gene of this nuclear receptor, in response to BCG infection. Moreover, BCG activated the mTOR pathway in TLR2transfected HEK 293T cells, inducing the phosphorylation of mTOR, S6 kinase and 4E-BP1. BCG infection-induced mTOR pathway activation was also observed in mouse bone marrow-derived macrophages (BMDM). Treatment with rapamycin inhibited BCG-induced PPARc expression. Our next goal was to study the LD composition following BCG infection in BMDM. LDs were purified by ultracentrifugation in sucrose gradient; non-stimulated cells or oleic acid, a well-known LD inducer, were used respectively as negative and positive control. Phosphokinases were identified by a membrane-based phosphokinase antibody array, followed by confirmation by western blot. BCG was able to trigger the compartmentalization within LD of activated members of MAPK, mTOR and Src signaling pathways. Conclusion: The above set of results shows that the Mycobacterium bovis BCG can play a key role in manipulating the intracellular signaling system of host cells, stimulating the PPARc expression/ activation and LD formation in a mTOR-dependent mechanism and leading to the compartmentalization of important cell signaling pathways within host LD. These effects may contribute to the escape mechanism of the parasites on host responses. Financial Support CNPq, FAPERJ and CAPES.
B143 PLATELETS MEDIATE MONOCYTE AND ENDOTHELIAL CELL ACTIVATION IN DENGUE THROUGH PLATELET-CELL INTERACTION AND CYTOKINE SIGNALING Eugenio D. Hottz1,2, Juliana F. Lopes1, Gisele Barbosa-Lima1, Isabel Medeiros-de-Moraes1, Andrew S. Weyrich3, Guy A. Zimmerman3, Fernando A. Bozza2, Patrı´cia T. Bozza1 1
Laborato´rio de Imunofarmacologia, IOC, Fiocruz, Brazil; 2Instituto Nacional de Infectologia, Fiocruz, Brazil; 3University of Utah, Salt Lake City, USA Dengue is the most prevalent human arbovirus disease worldwide. Dengue virus (DENV) infection may be asymptomatic or cause disease which intensity may vary from self-limiting febrile illness (mild dengue) to life-threatening disease of bleeding and shock (severe dengue). The pathogenesis of severe dengue is not completely understood, however it is known that progression to severe dengue is associated to increased immune activation with overproduction of inflammatory mediators. Although thrombocytopenia and increased vascular permeability are hallmarks of severe dengue, the role played by platelets in dengue pathogenesis is not completely understood. Platelets, classically known as essential effectors of hemostatic responses, are now increasingly recognized by their role in inflammation. Platelets can mediate immune and inflammatory responses through different mechanisms including secretion of immune mediators and interaction with leucocytes. Here we investigate the potential contribution of platelet activation in dengue pathogenesis. We evaluate the levels of inflammatory mediators secreted by DENV-
Inflamm. Res. activated platelets and the ability of activated platelets to modulate monocyte and endothelial responses through cytokine signaling and/ or platelet-cell interaction. We observed that platelets from dengueinfected patients and platelets exposed to DENV in vitro became activated. Platelets activated by DENV in vitro secreted pro-inflammatory mediators including IL-1b, RANTES, VEGF and MIF. During dengue infection in patients, activated platelets formed circulating platelet-monocyte aggregates. Using a model of platelet-monocyte interaction ex vivo, we demonstrated that dengue-activated platelets lead to monocyte activation with secretion of IL-1b, IL-8 and IL-10, expression of CD80, and accumulation of lipid droplets in monocytes. We showed that cytokine secretion by platelet-monocyte aggregates depended on P-selectin on activated platelet. In addition, IL-10 was specifically secreted in response to phosphatidylserine on apoptotic platelets. Blocking of MIF with the MIF antagonist Iso-1 prevented platelet-induced lipid accumulation in monocytes. We also observed that MIF and IL-1b in conditioned medium from DENV- or thrombinstimulated platelets were able to increase endothelial cell permeability in vitro. Our data provide new evidence that platelet driven cytokine and cell–cell signaling contribute to inflammatory responses in dengue. Financial Support CNPq, FAPERJ, CAPES, PRONEX
Inflammatory Mediators B144 INSULARIN REDUCES INFLAMMATION IN AN EXPERIMENTAL MODEL OF RENAL ISCHEMIA AND REPERFUSION Reinaldo C. Silva1, Raphael J.F Felizardo2, Marcos A. Cenedeze2, A´lvaro Pacheo-Silva2, Maisa Della-Casa3 1 University of Sa˜o Paulo, Sa˜o Paulo, Brazil; 2Federal University of Sa˜o Paulo, Sa˜o Paulo, Brazil; 3Institute Butantan, Sa˜o Paulo, Brazil
Introduction: Platelets are circulating cells that respond to vascular disorders, adhering to endothelial substructures and also physically interact with other platelet, leukocytes and endothelial cells by adhesion receptors expressed on their surfaces. Platelets secrete chemotactic agents, growth factors and fibrinogen, stimulating tissue remodeling after injury through mechanisms involving cell migration, proliferation and matrix synthesis. In renal pathologies, the role of platelets is still restricted to observations and associations without exposing its real mechanism of action. Therefore, this study aims to evaluate that platelets participate in the process of tissue remodeling after injury, because of its role in adhesion and cell signaling mechanisms. Methods and Results: For this purpose, C57BL/6 mice were subjected to ischemia and reperfusion (I/R) and treated or not with insularin 50 lg/kg, one via the bifunctional inhibitor of integrin. The animals were sacrificed 24 h after ischemia, corresponding to the stabilization of renal injury. Renal dysfunction was analyzed by biochemical testing using blood samples. Serum creatinine, urea, renal tissue histology and gene expression of cytokines were evaluated. Animals subjected to I/R showed an increase in renal dysfunction when compared to control group, while the I/R group treated with insularin shows a significant reduction in serum creatinine and urea. Increased gene expression of proinflammatory cytokines was also observed, as IL1b, TNF and IL-6 in the I/R group, with significant reduction in animals treated with insularina. Histological analysis showed less tissue damage in I/R group treated with insularina and lower platelet adhesion in renal tissue.
S159 Conclusion: Here, we demonstrate that insularin has an important effect on ischemia and reperfusion, with improvement of renal function, reducing inflammation and tissue injury. Financial support Fundac¸a˜o de Amparo a Pesquisa do Estado de Sa˜o Paulo (FAPESP 2013/11712-1), CAPES, CNPq.
B145 EARLY IDENTIFICATION OF SECONDARY BACTERIAL INFECTIONS Praveen Papareddy, Gopinath Kasetty, Erik Malmstro¨m, Johan Malmstro¨m, Heiko Herwald Lund University, Lund, Sweden Background: Bacterial infections are major causes of sepsis, leading to millions of deaths worldwide. Particularly secondary infections are emerging clinical problem that patients surviving the early phase of sepsis are not able to eradicate the secondary infection. This group of patients is at great risk to die from subsequent secondary nosocomial infections because their hyper-inflammatory state may lead to an immunosuppressive condition and a subsequent impaired immune response. The host response involves hundreds of mediators and they keep changing in various stages of sepsis. It is therefore unlikely that one single biomarker is able to satisfy all the needs and expectations for sepsis research and management. Aim: To identify a new sepsis biomarker that can aid in early therapeutic decision-making and add information about screening, diagnosis, risk stratification, and monitoring of the response to therapy. Methods: For better understanding the molecular mechanisms behind secondary infection processes, mice were infected intraperitoneally with S. aureus, followed by an intranasal challenge with P. aeruginosa. Plasma samples were analyzed for cytokine profiling and lung morphology was analyzed by histology and electron microscopy (SEM). Results: The primary infection in mice caused either by bacteria S. aureus or P. aeruginosa had no significant effect on survival. Whereas in mice with established primary S. aureus infection followed by secondary P. aeruginosa challenge, dramatically decrease their survival rate. Analysis of cytokines in plasma samples of primary infected mice displayed high levels of pro-inflammatory cytokines IL-6, TNF-a, MCP-1 and anti-inflammatory cytokine IL-10. SEM and histology of lung tissues of primary and secondary infected mice demonstrated more pulmonary leakage of proteins and damage of lung morphology in mice with secondary infection compared to primary infected mice. Conclusions: Here we show the host immune responses at various stages in severe bacterial infectious. Advances gained by this approach, will lead to a better understanding of the molecular mechanisms and help in developing novel antimicrobial treatments and tools for diagnosis.
B146 DUAL FUNCTIONALITY OF ALPHA1ANTITRYPSIN: AN INFLAMMATORY S-NITROSYLATED FORM STIMULATES MACROPHAGES DURING BACTERIAL INFECTIONS Ziv Kaner1, Galit Shahaf1, Shahar Dotan1, Rotem Engelman2, Yaffa Mizrachi Nebenzahl1, Moran Benhar2, Eli C. Lewis1
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Ben-Gurion University of the Negev, Beer Sheva, Israel; 2Technion Israel Institute of Technology, Haifa, Israel Background: Human alpha1-antitrypsin (hAAT) is a circulating serine-protease inhibitor that rises during acute phase responses and possesses immune-modulating and tissue-protective activities. hAAT reduces the levels of inflammatory cytokines (e.g., TNFa, IL-1b and IL-6) and chemokines (KC and MCP-1) while increasing anti-inflammatory proteins, such as IL-10 and IL-1Ra; nonetheless, although highly consistent, these outcomes are observed primarily during sterile immune responses. We recently reported that hAAT is responsible for an unexpected early and temporary inflammatory peak in responding innate cells and actually reduces bacterial load during peritonitis. While hAAT has no direct anti-bacterial properties, it has been shown to be S-nitrosylated (S-NO-hAAT) upon excess nitric oxide and then to directly block bacterial growth. Yet there is no data regarding the effect of S-NO-hAAT on innate cell responses to live bacteria. Aim: Investigate innate cell activation in the presence of S-NOhAAT. Methods: Mice transgenic for lung-specific hAAT or strain-matched wild-type mice were inoculated intranasally with lethal and sublethal doses of Streptococcus pneumonia (strain WU2) and animal survival and bacterial burden were determined, respectively. In vitro, clinicalgrade hAAT underwent chemical S-nitrosylation and then introduced to cultures of peritoneal macrophages, either alone or in the presence of LPS (10 ng/ml); for nitric oxide control, equimolar concentrations of the nitric oxide donor, S-nitroso-glutathione (GSNO), were used. Subsequently, macrophage activation was evaluated. To assess whether general physical properties had changed between hAAT and S-NO-hAAT, their thermal stability was measured. Results: hAAT-transgenic mice displayed significantly reduced mortality rates and lower bacterial load. In vitro, in the absence of added LPS, hAAT reduced TNFa levels 1.9-fold, but S-NO-hAAT increased TNFa levels 4.3-fold, compared to untreated cells. In the presence of LPS, hAAT and GSNO significantly reduced TNFa levels while S-NO-hAAT did not. After 6 h of incubation, S-NO-hAAT induced transcript levels of IL-1b, TNFa, KC and IL-6, as well as iNOS, TLR2 and CD14 (ranging 1.3- to 3.6-fold relative to untreated cells); no significant changes were observed in IL-10 and IL-1Ra. Structurally, S-NO-hAAT was found to be significantly more heatlabile than hAAT. Conclusions: hAAT has a context-specific dual function upon S-nitrosylation that may play a role in enhancing bacterial clearance, in part by acting as a macrophage-stimulating agent. Its anti-inflammatory properties are thus postulated to occur distal to the site of infection. The mechanism behind this profound duality is unknown; it is possible that nitrosylation-induced structural changes affect its interaction with binding partners, modulating immune signaling to, ideally, minimize tissue vulnerability and maximize the performance of innate cells towards a more effective host defense.
B147 MATERNAL OBESITY AND MARKERS OF INFLAMMATION IN BLOOD AND HUMAN MILK Mahmi Fujimori1, Vanessa Fiorin2, Tassiane C. Morais1, Eduardo L. Franc¸a3, Adenilda C. Hono´rio-Franc¸a3, Luiz C. Abreu1,2 1
University of Sa˜o Paulo, Sa˜o Paulo, Brazil; 2School of Medicine of ABC, Spokane, USA; 3Federal University of Mato Grosso, Cuiaba´, Brazil
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Inflamm. Res. Objectives: Recent evidence suggests an excessive inflammatory response in maternal obesity during pregnancy and it is associated with adverse pregnancy outcome. Whether this imbalance can be transferred from mother to breast milk remains to be established. Methods: 15 lean, 15 overweight and 15 obese pregnant woman were recruited in this study. Maternal blood was collected from each woman before delivery and colostrum samples were collected postpartum in the first 48 h. Samples were analyzed for interleukin (IL)-2, IL-6 and tumor necrosis factor (TNF)-a by flow cytometer and the data were analyzed using the software FCAP Array 1.0. Leptin was measured using a high-sensitivity enzyme-linked immunosorbent assay method and C-reactive protein (CRP) were determined by turbidimetric method. Results: No significant variations were detected in the IL-2 concentrations between the different groups studied (p [ 0.05). Maternal serum levels of IL-6, TNF-a, leptin and CRP were significantly different between the groups and obese mothers presented the highest levels (p \ 0.05). Colostrum leptin levels were higher in the obese group versus lean group (p \ 0.05). No differences in colostrum cytokines and CRP concentration were detected among the groups (p [ 0.05). Conclusions: Maternal obesity in pregnancy is associated with changes in cytokines and acute phase proteins of maternal blood in the third trimester, with an increase in IL-6, TNF-a and CRP in the obese category, but these changes were not observed in breast milk. Obese woman show elevations in serum leptin relative to normal weight woman during pregnancy and in human milk in postpartum. Further investigation is needed to determine the extent to which obesity-induced changes affects maternal and infant health. Table 1 Laboratory measurements according to Body Mass Index Category Cytokines Samples
Concentration Lean
IL-2 Colostrum 3.35 ± 1.25 (pg/mL) Maternal 1.97 ± 0.71 Blood
Overweight
Obese
1.59 ± 0.23
1.65 ± 0.14
2.0 ± 0.35
2.05 ± 0.25
IL-6 Colostrum 34.73 ± 7.68# 38.20 ± 7.07# 44.04 ± 10.28# (pg/mL) Maternal 1.73 ± 0.18 2.87 ± 0.67 4.03 ± 0.76* Blood TNF-a Colostrum 10.58 ± 1.77# 11.67 ± 2.1# (pg/mL) Maternal 5.49 ± 0.70 5.86 ± 0.64 Blood
12.91 ± 2.44#
Leptin Colostrum 1.70 ± 0.32# (ng/mL) Maternal 21.60 ± 3.10 Blood
3.24 ± 0.56*#
CRP (mg/dl)
1.94 ± 0.33#
8.63 ± 0.61*
32.81 ± 3.77* 36.84 ± 1.92
Colostrum 0.38 ± 0.12#
0.47 ± 0.13#
0.56 ± 0.18#
Maternal Blood
1.50 ± 0.37
8.50 ± 2.01*
0.91 ± 0.33
The results reepresent the mean and standard error of cytokines concentration of the 15 samples of colostrum and maternal blood. * Indicates intergroup differences within each sample; and # indicates differences between sample (colostrum and blood) considering the same cytokines.
Inflamm. Res.
B148 MAS RECEPTOR DEFICIENCY EXACERBATES LIPOPOLYSACCHARIDE-INDUCED INFLAMMATION IN THE MOUSE BRAIN One´sia Cristina Oliveira Lima1, Mauro Cunha Xavier Pinto1, Johan Duchene2, Michael Bader2, Natalia Alenina2, Robson Augusto Souza Santos1, Juliana Carvalho Tavares1 1
Federal University of Minas Gerais, Belo Horizonte, Brazil; 2Max Delbru¨ck Center for Molecular Medicine, Berlin, Germany
The G protein-coupled receptor Mas is a functional binding site for the angiotensin-(1–7). In the brain, the Mas receptor is expressed on vascular endothelium, neurons and microglia. Several evidences supports the involvement of Mas receptor in inflammatory response, however it’s role in neuroimmunological mechanisms remains to be elucidated. The aim of this work was to evaluate the role of Mas receptor on brain inflammation induced by Lipopolysaccharide. C57Bl/6 wild type (WT) and Mas deficient mice (Mas-/-), 8–12 weeks-old, were challenged by intra-peritoneal (i.p.) injection of Lipopolysaccharide (LPS Escherichia coli O111:B4, 5 mg/Kg). The experiments were performed at 3 and 24 h after LPS injection. The leukocyte endothelial interaction in the brain microvasculature was evaluated by intravital microscopy. The phenotype of leukocyte recruited to the brain microvasculature, as well the microglial activation were evaluated by immunofluorescence. Brain expression of MCP-1; CXCL-1; CXCL2 and IL-1b were evaluated by ELISA. The CD11b expression on bone marrow neutrophils was evaluated by Flow cytometry. Mas-/- mice presented significant increase in leukocyte adhesion to the brain microvasculature compared to WT, as well as, a higher number of monocytes and neutrophils recruited to the pia-mater. The higher number of adherent leukocytes to the brain in Mas-/- mice was associated with increased brain expression of CXCL1, CXCL2 and MCP-1 24 h after LPS injection, as well as, increased expression of CD11b in bone marrow neutrophils from Mas-/- mice. The leukocyte adhesion to brain microvasculature 3 h after LPS injection was not associated with microglial activation, which occurred 24 h after LPS in both groups. In conclusion, this study suggest a potential role for the Mas receptor as regulator of brain inflammatory response induced by LPS in mice, once Mas receptor deficiency seems to be related to exacerbated inflammation in LPS-challenged mice and delay the cerebral process of resolution of inflammation.
B149 PERITONITIS AS A KEY TOOL FOR EVALUATION OF RESOLUTION PHARMACOLOGY Vincent Baillif, Ge´rald Cheˆne, Charlotte Guigne´, Estelle Wanecq, Emeline Van Goethem, Marc Dubourdeau Ambiotis SAS, Toulouse, France Introduction: Inflammation is a natural process for host defense. It starts, increases and stops. The end signaling is a key and active process, which allows the come back to homeostasis. Return to tissue equilibrium involves many cellular and molecular factors. Immunoinflammatory cells issued and recruited from the blood stream are key components for the control of inflammation. They are notably involved in the secretion of bioactive mediators derived from polyunsaturated fatty acids that participate and orchestrate resolution.
S161 Thus, the cellular recruitment and its kinetic is a process to calibrate before evaluating any drugs. In this work, we characterized the effect of different doses of zymosan and of TNF-a on the kinetic of recruitment of cells in the peritoneal cavity. We determined the functional lipidomic signature of TNF-a-induced peritonitis. Methods: Experiments were carried out by injecting 0.3; 1 and 3 mg of zymosan or 0.001 mg of TNF-a in the peritoneal cavity. Inflammatory parameters were assessed by counting the cells in the peritoneal lavage and by identification of the leukocyte subpopulations. SPMs and other mediators issued from fatty acids were analyzed using a liquid chromatography-tandem mass spectrometry (LC–MS/MS) methodology to quantitatively evaluate their production. Results: In these experiments, we initially studied the inflammatory kinetics by counting cells and identifying leukocyte subpopulations over time in the peritoneal lavage after zymosan or TNF-a injection in the peritoneal cavity. Kinetic, inflammatory peaks and the resolution intervals were calculated and have been shown to be dependent on the dose of zymosan. These data may vary according to the batch of zymosan. Concerning the TNF-a-induced peritonitis, we have shown a lower and sustainable recruitment from 2 to 18 h at around 2 millions of leukocytes compared to the zymosan-induced peritonitis. We evaluated also the SPMs signature in the TNF-a-induced peritonitis. We have shown that TNF-a is able to induce production of 14-HDOHE and 17-HDOHE as well as 7-Mar1. Conclusions: Taken together those results suggest that the kinetic and the cellular events controlling peritonitis are key components to evaluate before doing any experiments on inflammation. Zymosan- or TNF-a-induced peritonitis are two different models of cell recruitment in kinetic and intensity that allows evaluation of different type of compounds with different mechanisms of action.
B150 DECIPHERING THE SIGNALS REQUIRED FOR FOLLICULAR HELPER T CELL GENERATION IN THE ABSENCE OF EXOGENOUS MICROBIAL STIMULI Nicolas Riteau, Sandy D. Oland, Dragana Jankovic, Alan Sher National Institute of Allergy and Infectious Diseases, Bethesda, MD, USA Follicular helper T (Tfh) cells are specialized providers of T cell help to B cells by delivering essential signals for germinal center formation and affinity maturation. Therefore, a more detailed understanding of Tfh biology could aid in the design of improved vaccines based on the induction of humoral immunity. While the triggers required for Th1, Th2 or Th17 polarization are well defined, those responsible for Tfh differentiation are still only partially delineated. In order to identify signals that could be used to promote Tfh responses, we have utilized an experimental model in which C57BL/6 or gene KO mice are immunized with either peptide or protein antigen emulsified in the oil and surfactant-only containing adjuvant IFA (Incomplete Freund’s Adjuvant). Using this immunization protocol in wild-type mice we find that at the peak of the response in the draining lymph nodes, half of the antigen specific CD4+ T cells have differentiated into Tfh based on their expression of the chemokine receptor CXCR5 and the lineage defining transcription factor Bcl6 while little or no increase in Th1 or Th17 cells is observed. Similar results were observed with other oil and surfactant-only adjuvants. Interestingly, both the overall number and the frequency of antigen specific Tfh were substantially decreased in IFA immunized MyD88-/- mice, but not in animals lacking TRIF, TLR4 or IL-1R signaling. In addition, while gut microbiota-derived TLR5 triggering has recently been reported to be required for the Tfh
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S162 response to unadjuvanted vaccines, our results show that TLR5-/mice or orally antibiotic treated immunized mice have no impairment in Tfh differentiation. Furthermore, although STAT3 signaling is known to be critical for the Tfh response to infections, we found only a minor effect on IFA triggered Tfh differentiation of conditional STAT3 deficiency in CD4+ T cells. Importantly, a much larger defect in Tfh polarization was observed in the absence of type I IFN signaling. The nature of this type I IFN requirement is currently under investigation but does not appear to reflect TLR3, MDA5 or STING dependent production of the cytokine. Together, our results show that Tfh responses can be promoted by adjuvants that lack exogenous PAMPs through a pathway involving type I IFN and, paradoxically, the microbial sensing related adaptor MyD88.
B151 CTX-4430, A POTENTIAL FIRST-IN-CLASS LEUKOTRIENE A4 HYDROLASE INHIBITOR FOR TREATMENT OF INFLAMMATORY DISEASES E. Springman, R. Grosswald, L. Bhatt, T. Van, S. Ahuja Celtaxsys, Inc., Atlanta, United States The Leukotriene B4 (LTB4) pathway has seen a resurgence of interest for pharmaceutical development stemming from recent discoveries linking LTB4 to a diversity of inflammatory processes, including neutrophil swarming behavior, NALP3 inflammasome activation, and adipose-related inflammation. Additionally, the role of LTB4 in stimulating immune response is no longer thought to be limited to innate immunity, but rather to also stimulate certain facets of adaptive response. The balance between LTB4 and its anti-inflammatory counterpart Lipoxin A4 (LXA4) appears critical for restoring immune homeostasis during bouts of infection or inflammation. The LTB4 and LXA4 pathways share a common precursor in Leukotriene A4 (LTA4) and their production is, thus, linked in an opposing manner. For example, when LTB4 is overproduced, LXA4 production is reduced. LTAA4 Hydrolase (LTA4H) is the enzyme responsible for catalyzing the rate limiting step in formation of LTB4 and, therefore, serves as an interesting target for both reducing LTB4 production and potentially restoring LXA4 balance. Here we report the mechanism of action and pharmacodynamics of CTX-4430, a potent and orally active inhibitor of LTA4H that is currently undergoing clinical trials. Orally administered CTX-4430 rapidly reduces production of LTB4 in the blood and airways in animals and humans and has demonstrated therapeutic effects in animal models of skin, lung, vascular and CNS inflammation. Its pharmacodynamic and pharmacokinetic profile in humans suggests an optimal dose range of 50–200 mg for once daily administration. The rationale for ongoing and planned proof-of-concept clinical trials will be briefly discussed.
B152 CATHELICIDIN RELATED ANTIMICROBIAL PEPTIDE CONTRIBUTES TO BACTERIAL CLEARANCE AT EARLY STAGE OF PULMONARY INFECTION WITH ACINETOBACTER BAUMANNII Seong Gak Jeon1, Jun-Young Lee2, Yu-Jin Jeong1, Min-Jung Kang2, Jong-Hwan Park2
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Inflamm. Res. Konyang University, Nonsan, South Korea; 2Chonnam National University, Gwangju, South Korea
1
Cathelicidin-related antimicrobial peptide (CRAMP) is a murine antimicrobial peptide that has various biological functions such as immune regulation, chemotaxis, and direct bacteria killing effect. In the present study, we sought to determine whether CRAMP has antimicrobial effect against multidrug resistant Acinetobacter baumannii. The gene and protein expression of CRAMP was increased in the lung of mice infected with A. baumannii at 6 h after infection. Such CRAMP expression was impaired in the lungs of TLR2/4 double-deficient mice, as compared with WT mice. In vitro assay revealed that neutrophils are a major source of CRAMP expression. In addition, the bacterial growth was inhibited by the addition of recombinant CRAMP in a dose-dependent manner. In vivo study showed that bacterial clearance was impaired in the lungs of CRAMP-deficient mice at 1 day after infection, as compared with WT mice. These results suggest that CRAMP has antimicrobial effect against A. baumannii and can be a candidate for therapeutic agents.
Inflammatory Pain and Analgesia B153 COLONY-STIMULATING FACTOR- 1 (CSF-1) AND TUMOR NECROSIS FACTOR- a (TNF- a) IN ARTHRITIC PAIN AND DISEASE Reem A. Saleh, Derek C. Lacey, John Hamilton, Andrew Cook Department of Medicine, Royal Melbourne Hospital, Melbourne, Victoria, The University of Melbourne, Parkville, Victoria, Australia Colony-stimulating factor-1 (CSF-1) is a key cytokine that has been associated with the development of arthritis in animal models. Its role in neuropathic pain has been studied by several laboratories; however, its potential involvement in arthritic pain has not received attention. Tumor necrosis factor-alpha (TNF-a) is a pro-inflammatory cytokine which has been implicated in the pathogenesis and progression of RA, as well as the development of arthritic pain. Thus, the objectives of the current study were as follows: (1) to examine the role of CSF-1 in arthritic pain using an acute mono-articular methylated bovine serum albumin (mBSA)-induced arthritis model; (2) to compare the TNFmediated pain to the CSF-1-induced pain; and (3) to elucidate the mechanisms by which CSF-1 can mediate arthritic pain. Results from this study showed that systemic administration of CSF-1 can induce pain in the mBSA model and exacerbate arthritis. Data showed that early CSF-1-induced pain was not reversed following indomethacin administration, suggesting that CSF-1-mediated pain is cyclooxygenase-independent. Systemic administration of TNF-a was also able to induce pain in the novel mBSA model and exacerbate arthritis severity. Unlike CSF-1, early TNF-mediated pain was abolished following the administration of indomethacin. This indicated that the TNF-induced pain is mediated through the production of eicosanoids. In addition, indomethacin treatment had no anti-inflammatory effects on the CSF-1-mediated arthritis, whereas, TNF-mediated arthritis was relatively reduced following indomethacin administration. In conclusion, both cytokines were able to induce pain; however, each cytokine induced arthritic pain via a different pathway. Further insight into the mechanisms by which CSF-1 mediates pain could determine its contribution to arthritic pain and may provide novel therapeutic targets or strategies for joint pain in inflammatory diseases such as rheumatoid arthritis (RA).
Inflamm. Res.
B154 LIMONENE REDUCES HYPERALGESIA INDUCED BY GP120 AND CYTOKINES BY MODULATION OF NF-JB, SOD, IL-1 b IN MICE Candida A. L. Kassuya1, Ana Claudia Piccinelli1, Priscila N. Morato1, Elisabete C. Konkiewitz1, Edward B. Ziff2, Julio Croda3,1 1
Federal University of Grande Dourados, College of Health Science, Dourados, MS, Brazil; 2Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, USA; 3Oswaldo Cruz Foundation, Campo Grande, Brazil The envelope glycoprotein gp120 of HIV can play an important role in the generation of pain. Intrathecal administration gp120 induces the production of cytokines, including IL-1, IL-6 and TNF, which in turn induces neuroinflammation and hyperalgesia. A previous study from our group demonstrated antihyperalgesic and antidepressive actions of limonene administered orally in a neuropathic pain model. The present work has investigated the antihyperalgesic effects of (R)-(+)-limonene in mice that received intrathecal gp120 by analyzing the roles of cytokines involved in these processes as well as the mechanisms. Male Swiss mice (n = 6) received gp120 (50–500 ng) intrathecally or sterile saline as a control. Intrathecal administration of gp120 increased mechanical sensitivity measured with an electronic Von Frey apparatus, but not cold hypersensitivity (measured by the acetone test), at 2 and 3 h after the injections. Limonene significantly decreased this mechanical sensitivity at 3 h after of the injection. In addition, intrathecal injection of gp120 increased IL-1b (measured by the ELISA test) in the serum of mice, and limonene prevented the ability of gp120 to increase this cytokine. Limonene also inhibited TNF and IL-1b-induced mechanical hyperalgesia and IL-1b-induced cold hypersensitivity. Western blot assay demonstrated limonene was capable of significantly decreasing the expression of NF-kB while limonene increased SOD expression in cytoplasm of cells from spinal cord at 4 h after intrathecal IL-1b injection. These results demonstrate that gp120 administered intrathecally causes mechanical hyperalgesia and a peripheral increase in IL-1b, and that limonene inhibits this change. Also limonene modulates the activation of NF-kB and SOD expression in the spinal cord after spinal IL-1b application. The ability of limonene to inhibit the mechanical hyperalgesia induced by TNF and IL-1b emphasizes the anti-inflammatory action of limonene, specifically its ability to inhibit cytokine production.
B155 ANTI-INFLAMMATORY AND ANTI-OXIDANT EFFECTS OF STANDARDIZED EXTRACT OF OCIMUM GRATISSIMUM L LEAVES IN CARRAGEENAN/KAOLIN—INDUCED MONOARTHRITIS IN RATS Abayomi M. Ajayi1, Benneth Ben-Azu1, Samuel E. Okhale3, Olusegun G. Ademowo1 1
Department of Pharmacology and Therapeutics, Faculty of Basic Medical Sciences, College of Medicine, University of Ibadan, Ibadan, Nigeria; 2Department of Pharmacology and Therapeutics, Faculty of Basic Medical Sciences, College of Medicine, University of Ibadan, Ibadan, Nigeria; 2Department of Pharmacology and Therapeutics, Faculty of Basic Medical Sciences, College of Medicine, University of Ibadan, Ibadan, Nigeria; 3Department of Medicinal Plant Research
S163 and Traditional Medicine, National Institute for Pharmaceutical Research and Development, P.M.B. 21, Abuja, Nigeria Introduction: Arthritis is a major inflammatory disorder usually affecting the joints and characterized by inflammation of the synovial membrane, pain and restricted joint movement. Ocimum gratissimum leaves contain polyphenolics that may be responsible for protection against chronic disease due to their anti-inflammatory and anti-oxidant activities. In this study, the anti-inflammatory and anti-oxidant effects of standardized methanol extract of O gratissimum leaves was tested using a model of carrageenan/kaolin—induced monoarthritis. Methods and results: The methanol extract (ME) was obtained by successive maceration of O gratissimum dried leaves first with n-hexane and chloroform and then methanol to obtain a polyphenolic enriched extract. Fingerprint of the extract was obtained using a High Performance Liquid Chromatographic method. Female Wistar rats (180–250 g) were subjected to 3 % carrageenan/3 % kaolin induced monoarthritis model. Groups were divided into vehicle, ME (100, 200 and 400 mg/kg, p.o for 3 days) and Indomethacin (2 mg/kg, p.o for 3 days). One h after the last treatment, 0.1 mL of a mix of 3 % carrageenan and 3 % kaolin was injected into the knee joint cavity and the leg was flexed and extended for about 5 min. The following parameters were then assessed; Paw volume and knee circumference, mechanical hyperalgesia withdrawal threshold, locomotor activity, joint histopathology and plasma anti-oxidant levels (reduced glutathione, superoxide dismutase and thiobarbituric reacting substances). O gratissimum extract dose-dependently and significantly (p \ 0.05) reduced swelling (as assessed by paw volume and knee thickness), and inflammatory pain (as assessed by mechanical hyperalgesia and locomotor activity). Rats injected with KC and treated with vehicle alone had higher TBARS and reduced GSH and SOD levels as compared to groups treated with ME (100, 200 and 400 mg/kg) and Indomethacin (2 mg/kg). Similarly, the extract and Indomethacin reduced histological signs of inflammation in the knee joint cavity. Conclusion: The standardized extract of O gratissimum leaves exerted a beneficial effect in carrageenan/kaolin—induced monoarthritis model in rats and is probably related to its anti-inflammatory and antioxidant properties, which may be due to its polyphenolic content.
B156 ANTI-INFLAMMATORY AND ANTINOCICEPTIVE EFFECTS OF NEW N-PHENYL-PYRAZOLE COMPOUND Daiany P. B. Silva1, Iziara F. Florentino1, Daniela C. Vinhal2, Luiz C. Cunha3, Ricardo Menegatti2, Elson A. Costa1 1 Laboratory of Pharmacology of Natural Products, Institute of Biological Sciences, Federal University of Goias, Goiaˆnia, GO, Brazil; 2Laboratory of Medicinal Pharmaceutical Chemistry, Faculty of Pharmacy, Federal University of Goias, Goiaˆnia, GO, Brazil; 3 Nucleus of Studies and Research Toxic Pharmacological, Faculty of Pharmacy, Federal University of Goias, Goiaˆnia, GO, Brazil
Introduction: LQFM-102 was projected from the molecular hybridization strategy using the analgesic acetaminophen and antiinflammatory Clopirac, aims to maintain the therapeutic activities profile of both in the new compound created and to promote the reduction of its side effects. The aim of this study is to evaluate the anti-inflammatory and antinociceptive effects of LQFM-102, as well as clarify the mechanisms involved.
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Methods: Experiments were performed using male Swiss albino mice [35–40 g (n = 9). The anti-inflammatory and antinociceptive effects of LQFM-102 were evaluated by methods of formalin-induced pain, hot plate test, paw edema and pleurisy induced by carrageenan. All the experimental protocols were approved by the Research Ethics Committee of the UFG (Protocol No. 182/10]. Results and discussion: In the first phase of formalin-induced pain test (0–5 min.), the treatment with LQFM-102 (75, 150 or 300 mg/kg p.o.) reduced the time of reactivity to pain by 36, 48 or 43 % as compared to the control group treated with vehicle 10 mL/kg (81 s). In the second phase (15–30 min.), the treatments with LQFM-102 (75, 150 or 300 mg/kg) reduced the time of reactivity to pain by 30, 35 or 70 %, respectively, as compared to the control group (207 s). In the test of paw edema induced by carrageenan, treatments with LQFM-102 75, 150 or 300 mg/kg (p.o.) reduced the edema by 12, 21 or 22 %, respectively in the first hour, 20, 28 or 33 %, respectively in the second hour, 16, 35 or 32 % respectively in the third hour and finally in the fourth hour treatments with LQFM-102 150 or 300 mg/ kg reduced the edema by 29 or 31 % as compared to the control group (Difference between the paws 136, 130, 123 and 108 lL, first, second, third and fourth respectively). In the test of pleurisy induced by carrageenan, treatment with LQFM-102 150 mg/kg p.o. reduced cell migration by 39 % compared to the control group (8.8 9 106 Leukocytes/mL) and also reduced the proteic exudation by 77 % as compared to the control group (Blue Evan’s concentration 18 lg/ mL). In the hot plate test, treatment with LQFM-102 150 mg/kg p.o. did not increase the latency to thermal stimulus. The results obtained in the formalin test suggest an antinociceptive activity that might be dependent on an anti-inflammatory activity. The edematogenic effect in the paw edema and reduced cell migration and proteic exudation suggest that LQFM-102 has anti-inflammatory effect. The result in the hot plate test suggest that this compound doesn’t has a central analgesic action. Financial support CAPES ans CNPq.
articular injection of zymosan (Zy) (2 mg, 40 lL) in the left TMJ. In another series of experiments rats were treated with ZnPP-IX (3 mg/ kg), a specific HO-1 inhibitor, or with aminoguanidine (30 mg/kg) (i.p.),a selective inhibitor of nitric oxide synthase (iNOS), before AEL (1 mg/kg). Von Frey test was used to evaluate hypernociception (g) at 4 h after Zy. 6 h after Zy injection it was collected synovial lavage for leukocyte counting and myeloperoxidase (MPO) measurement, and TMJ tissue for histopathological analysis (H&E) and immunohistochemistry for TNF-a, IL-1b, HO-1. Also TMJ periarticular tissue and trigeminal ganglion were removed for TNF-a and IL-1b dosage (ELISA). Vascular permeability was evaluated by Evans Blue extravasation measurement. Results: AEL (0.01, 0.1 or 1 mg/kg) increased (p \ 0.05) the nociceptive threshold (56.7 ± 1.1; 69.1 ± 2.1 or 81 ± 1.7, respectively), when compared to Zy group (43.8 ± 2.2). AEL (1 mg/kg) reduced (p \ 0.05) cell influx (1183 ± 219.3), MPO activity (22.1 ± 10.7), inflammatory cell influx in the synovial membrane (0.5 ± 0.2), and Evans Blue extravasation measurement (131.7 ± 3.1), compared to Zy group (37844 ± 6203; 127.5 ± 27.6; 3 ± 0.4; 166.3 ± 6.7, respectively). AEL (1 mg/kg) also reduced TNF-a and IL-1b levels in both TMJ tissue (2.52 ± 0.07) (3.11 ± 0.49) and trigeminal ganglion (2.52 ± 0.09) (1.55 ± 0.34), when compared to Zy group (7.94 ± 0.49, 11.01 ± 0.83 and 7.12 ± 0.40, 4.72 ± 0.47, respectively). TMJ immunohistochemical analyses showed that AEL (1 mg/ kg) increased HO-1 and decreased TNF-a and IL-1b expression was observed in the AEL-treated group compared to the zymosan. These effects of AEL were not observed in the presence of ZnPP-IX, but they were maintained with aminoguanidine. Conclusions: AEL is effective in zymosan-induced TMJ inflammatory hypernociception in rats, and its efficacy, at least in part, depends on TNF-a and IL-1b inhibition and the HO-1 pathway integrity. AEL may represent a potential therapeutic to ameliorate the inflammatory TMJ painful condition. Funding sources FUNCAP, CNPq, CAPES, and INCT-IBISAB.
B157 LECTIN FROM ABELMOSCHUS ESCULENTUS DECREASES LEVELS OF TNF-a AND IL-1b AND DOWN-REGULATES HEME OXYGENASE-1 ON ZYMOSAN-INDUCED TEMPOROMANDIBULAR JOINT INFLAMMATORY HYPERNOCICEPTION IN RATS
B158 A SEMI-SYNTHETIC DERIVED FROM NATURALLY OCCURRING TEPHROSIA TOXICARIA PERS. REDUCES TNFa AND IL-1b LEVELS AND ACTIVATES L, D, AND KAPPA RECEPTORS IN THE MODEL OF FORMALININDUCED TEMPOROMANDIBULAR JOINT INFLAMMATORY HYPERNOCICEPTION IN RATS
Mirna MB Brayner1, Raul S. Freitas1, Jose´ Thalles JGD Lacerda3, Tatiane Santi-Gadelha3, Carlos Alberto DA Gadelha3, Vicente de Paulo T. Pinto1, Gerardo C. Filho1, Karuza MA Pereira1, Gerly Anne DC Brito4, Hermany C. Freitas1, Renata FDC Leita˜o4, Ellen LD Assis1, Danielle RD Val2, Lorena V. Vieira1, Hellı´ada V. Chaves1 Federal University of Ceara´, Sobral, Brazil; 2Federal University of Pernambuco, Recife, Brazil; 3Federal University of Paraı´ba, Paraı´ba, Brazil; 4Federal University of Ceara´, Fortaleza, Brazil 1
The antinociceptive and anti-inflammatory effects of a newly-discovered lectin named AEL, isolated from organic Okra seeds (Abelmoschus esculentus L Moench) (Okra), were investigated in the zymosan-induced temporomandibular joint (TMJ) inflammatory hypernociception in rats. Methods: Experiments were approved by the Institutional Animal Care and Use Committee of the Federal University of Ceara´, Fortaleza, Brazil (74/2013). Rats were pretreated i.v. with AEL (0.01, 0.1 or 1 mg/kg) or saline 30 min before the intra-
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Mirna MB Brayner1, Danielle RD Val2, Alice RD Freitas1, Rodrigo DS Santos1, Fernanda MLDS Lopes1, Raul S. Freitas1, Maria VS Teixiera1, Aldeˆnia RD Santos1, Francisca RLD Silva1, Angela MC Arriaga1, Cristina GD Macedo3, Simone MS Lamana3, Juliana TC Napimoga3, Maria BDS Maia2, Hellı´ada V. Chaves1 1
Federal University of Ceara´, Ceara´, Brazil; 2Federal University of Pernambuco, Recife, Brazil; 3State University of Campinas, Campinas, Brazil Temporomandibular joint (TMJ) disorders are a group of conditions associated with high levels of inflammatory pain-related disability. The activation of opioid receptors (l, d, and j) may suppress the production of inflammatory neurotransmitters. Tephrosia toxicaria Pers (Tt). is a shrub that has been used in Amazonian countries traditional medicine to alleviate inflammatory pain. Recently our group demonstrated the effect of Tt in a model of inflammatory hyperalgesia
Inflamm. Res. induced by zymosan in rats. Considering these results we developed a semi-synthetic derived from naturally occurring Tt identified by the acronym PHO. Thus, the present study was aimed at investigating the antinociceptive and anti-inflammatory efficacy of PHO in the model of formalin-induced TMJ inflammatory hypernociception in rats. Additionally, we also investigated whether PHO efficacy involves activation of the central opioid system. Experiments were approved by the Institutional Animal Care and Use Committee of the Federal University of Ceara´, Fortaleza, Brazil (57/2010) Rats were pretreated (per os) with PHO (0.01, 0.1 or 1 mg/kg) 60 min before formalin (1.5 %) or serotonin (225 lg) injection (intra-TMJ). Non-treated group received formalin (1.5 %) or serotonin (225 lg) injection (intra-TMJ) and saline (per os). Behavioral nociception response was evaluated for a 45 min observation period. Periarticular tissues were collected 45 min after formalin injection to TNFa and IL-1b assays (ELISA). In other series of experiments rats were pretreated with an intrathecal injection of naloxone or CTOP [antagonist of Mu (l) opioid receptor], naltrindole hydrochloride [antagonist of Delta (d) opioid receptor], or Nor-Binaltorphimine [antagonist of Kappa (j) opioid receptor], before PHO (0.01 mg/kg; per os) injection, and followed by formalin (1.5 %) intra-TMJ. PHO (0.01, 0.1 or 1 mg/kg) reduced (p \ 0.05) formalin-induced hypernociception (82.4 ± 5.3; 89.7 ± 6.6; and 110.8 ± 8.4, respectively), when compared to nontreated group (245.0 ± 14.2). PHO (0.01 mg/kg) also reduced serotonin-induced hypernociception (22.2 ± 7.2), when compared to nontreated group (99.0 ± 5.1). PHO (0.01 mg/kg) decreased (p \ 0.05) both TNFa (3.3 ± 0.1) and IL-1b (5.2 ± 0.2) levels in the periarticular tissue, when compared to non-treated group (6.3 ± 0.3 and 15.1 ± 2.2, respectively). PHO was not able to reduce formalin-induced hypernociception when opioid receptors antagonists were administered. PHO effectively decreased formalin-induced TMJ inflammatory hypernociception dependent, at least in part, upon TNFa and IL-1b inhibition and opioid receptors activation. Taking into account the well-demonstrated antinociceptive and anti-inflammatory efficacy of PHO, the designing of alternative compounds to classical anti-inflammatory and analgesic agents is very encouraged to define new pharmacological targets for the inflammatory TMJ painful condition treatment. Funding sources FACEPE, RENORBIO, CNPq, CAPES, and INCTIBISAB.
B159 ANTI-INFLAMMATORY AND ANTINOCICEPTIVE EFFECTS OF GYY-4137, A SLOWRELEASING HYDROGEN SULFIDE (H2S) DONOR, ON TEMPOROMANDIBULAR JOINT SYNOVITIS INDUCED BY CARRAGEENAN IN RATS Flavia B. Lira1, Marcos A. de Paula1, Simone A. Teixeira1, Mark Wood2, Matt Whiteman2, Soraia K. Costa1, Marcelo N. Muscara1 1 Institute of Biomedical Sciences, University of Sa˜o Paulo, Sa˜o Paulo, Brazil; 2University of Exeter Medical School, Exeter, USA
Temporomandibular joint (TMJ) disorders are usually associated with inflammation and pain. We have previously demonstrated that hydrogen sulfide (H2S) exerts beneficial effects on nociception and inflammation secondary to carrageenan (CGN)-induced knee joint synovitis in rats. GYY-4137 is a slow H2S-releasing compound that has shown promising results as anti-inflammatory agent, although the mechanisms involved have not been yet completely defined. We thus decided to investigate the effects of GYY-4137 on the nociception
S165 and inflammation induced by CGN when injected into the TMJ of rats, and to pharmacologically characterize the mechanisms involved. The protocol was approved by the local Ethics Committee for Animal Experimentation (CEUA-ICB 46, book 2/85, 2010). Under anesthesia with inhalatory isofluorane (3 % in O2), male Wistar rats (7 week old) received an intra-articular (i.art.) injection of 500 lg of CGN. Four hours later, mechanical allodynia was evaluated by measuring the force threshold necessary forhead withdrawal with the aid of an electronic analgesimeter based on the Von Frey filaments principle. Myeloperoxidase (MPO) activity was measured in the TMJ capsule tissue as a marker of neutrophil infiltration. GYY-4137 (1.25–20 lg/joint), glibenclamide (a KATP channel blocker, at 10 and 30 lg/joint) and ODQ [1H-(1,2,4) oxadiazolo [4,3-a] quinoxalin-1one, a specific soluble guanylate cyclase inhibitor, at 0.8 and 8 lg/ joint] were co-injected with CGN. The results were analysed by unpaired Student t-test or ANOVA followed by the Dunnett’s test, when applicable. In comparison with saline (control group), the intra-articular injection of CGN into the rat TMJ evoked mechanical allodynia, as evidenced by the significant decrease in the force threshold (-31.6 ± 3.5 vs. 0.3 ± 4.0 g; p \ 0.001) and increased MPO activity (26.8 ± 5.2 vs. 1.3 ± 0.3 U/joint; p \ 0.001). GYY-4137 significantly reduced CGN-induced mechanical allodynia in a dosedependent manner (between 2.5 and 20 lg/joint, p \ 0.01) as well as MPO activity (12.9 ± 4.5 vs. 26.8 ± 5.2 U/joint, p \ 0.05) at the 2.5 lg/joint dose. Glibenclamide (30 lg/joint) prevented the antinociceptive effect of GYY-4137 (22.3 ± 2.9 vs. 8.8 ± 0.8 g; p \ 0.05) while ODQ did not alter these effects. These data provide evidence on the anti-inflammatory and antinociceptive effects of GYY-4137 on the CGN-induced TMJ synovitis in rats. Pathways involving KATP channels but not cGMP seem to be involved in the antinociceptive effects of GYY-4137. Financial support FAPESP (Grant #2014/24518-1), CNPq and CAPES.
B160 THERAPEUTIC EFFICACY OF FISH OIL CONCENTRATE ON EXPERIMENTAL MODEL OF NEUROPATHIC PAIN Ana Luisa P. Miranda, Rafaela V. Silva, Cleverton Kleiton F. Lima, Ewerton P A Santos, Bianca W. Lobo Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil Introduction: Neuropathic pain (NP) is a multifactorial condition arising from injury or malfunction of peripheral or central nervous system. The pathophysiology is characterized by strong neuro-immune interaction. Omega 3 polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are well known for their immunomodulatory activity displayed by their endogenous conversion to resolvins and protectins as lipid mediators (Serhan 2002). Aim: To evaluate therapeutic efficacy of fish oil concentrate (FOC), rich in EPA and DHA, for the treatment of partial sciatic nerve ligation- induced (PSNL) NP in mice. Methodology: NP was induced by PSNL on the left paw (Seltzer 1990). Two treatment protocols were employed: daily oral treatment initiated at 5th day after surgery (therapeutic protocol) and daily oral treatment initiated prior to surgery (preventive protocol). Animals were treated with vehicle, FOC (4.6 and 2.3 g/kg) or Gabapentin (100 mg/kg). Thermal hypernociception and mechanical allodynia
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S166 were assed up to 24 h following first administration on 5th day and also at 7th and 9th days after surgery. Ethics committee protocol number FARMACIA04 (CEUA-UFRJ). Results: The highest dose of FOC increased mechanical and thermal withdrawal threshold in animals receiving daily oral administration in a therapeutic protocol initiated on 5th day after NP induction. In the first 24 h after FOC administration it was observed a decrease in mechanical allodynia, but not in thermal hypernociception. A statistically significant reduction in mechanical allodynia was observed at 7th and 9th days post-surgery whereas thermal hypernociception was totally reversed at 9th day after PSNL, showing the therapeutic efficacy of FOC. In a preventive treatment protocol, beginning at the day of the surgery, the highest dose of FOC prevented mechanical allodynia behavior induced by PSNL. Conclusions: Our results indicate that FOC oral treatment reverses mechanical and thermal hypersensitivity after peripheral nerve injury in mice in preventive and therapeutic treatment protocol. Therefore, it might arise as an alternative treatment for neuropathic pain. Financial support CAPES, CNPq, FAPERJ.
Inflammatory Processes in Cardiovascular Diseases B161 EVALUATION OF THE EFFECTS ON CARDIOVASCULAR PARAMETERS BY CHRONIC TREATMENT WITH SELECTIVE CYCLOOXYGENASE 2 INHIBITORS IN NORMAL RATS Janetti N. Francischi, Regina MM Turchetti-Maia, Ligia F. Brenck, Taissa IC Frade, Geovanne D. Cassali, Andrea S. Haibara Federal University of Minas Gerais, Belo Horizonte, MG, Brazil Introduction: Use of selective cyclooxgenase (COX) 2 inhibitors (ICOX-2) to treat inflammation and pain may be associated with an increase in blood pressure following chronic administration. The aim of the present study was to evaluate whether celecoxib or nimesulide would affect the blood pressure and the heart rate of normotensive rats following chronic administration. Materials and methods: Holtzman male rats weighing (165–200 g) were randomly assigned to one of the three groups: (1) control (n = 5), (2) nimesulide (NS; n = 8), (3) Celecoxib (CX; n = 8). After 5 days of adaptation, all the animals were anesthetized and implanted with a probe in the abdominal aorta, for collection of continuous blood pressure and heart rate data by telemetry. Within 10 days of recovery, control and drug-treated animals received saline, a dose of 30 mg/kg (CX) or 5 mg/kg (NS) by gavage (in 0.1 mL/ 100 g), divided in two halves at 8:00 and 17:00 h of the same day, starting at the day zero. Experiments were developed during 30 days in two turns (April and August 2014). Data were plotted and analyzed using SigmaPlot and Sigma Stat software, respectively. Results: Animals didn’t present any variation in blood pressure or the heart rate all through the study. However, two animals in each druggroup died during the experiments. Conclusions: Chronic treatment with selective COX-2 inhibitors is not, by itself, sufficient to alter the blood pressure and the heart rate in normal rats. However, death may happen in a parcel of the individuals (25 %) in the chronically treated ICOX-2 groups. Support: CNPq and Capes.
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B162 PERSISTENCE OF INFLAMMATION ACCORDING TO ENDOMYOCARDIAL BIOPSY AND CARDIAC MAGNETIC RESONANCE AND THE LEVEL OF CIRCULATING AUTOANTIBODIES AND CYTOKINES IN PATIENTS WITH DILATED CARDIOMYOPATHY AND CONDUCTION DISTURBANCES Elena M. Gupalo, Natalia A. Mironova, Liudmila I. Buryachkovkaya, Olga Stukalova, Tatiana Malkina, Tatiana Sharf, Evgeniy Efremov, Petr V. Chumachenko, Sergei Golitsyn Russian Cardiology Research and Production Complex, Moscow, Russia Purpose: To evaluate the level of circulating autoantibodies and cardiovascular magnetic resonance (CMR) data in dilated cardiomyopathy (DCM) patients (pts) and pts with conduction disturbances and structurally normal heart (CD). Methods: 30 pts with CD and structurally normal heart (11 male, mean age 39.3 ± 11.0) and 40 pts with DCM (18 male, mean age 40.5 ± 10.5) underwent Holter ECG monitoring with subsequent CMR with assessment of myocardial edema (ME), early gadolinium enhancement (EGE) and late gadolinium enhancement (LGE) All DCM pts underwent EMB with PCR analyses for virus persistence. In serum of all pts persistence of autoantibodies to beta1-adrenergic receptor (b1-AAbs) and M2 muscarinic receptor (M2-AAbs) as well as cytokines TNFa, IL6, TGFb1 was evaluated by ELISA. Results: EMB revealed active inflammation 24 (60 %) DCM patients resolved myocarditis with huge areas of fibrosis in 8 (20 %) pts and no inflammation in 8 cases (20 %). In 16 (40 %) samples parvovirus B19 was detected. Virus persistence in EMB samples was not associated with inflammatory cell infiltration. CMR found EGE in 10 (33.3 %) pts and ME in 6 (20 %) pts CD pts. None of CD pts revealed LGE at CMR. 4 (13.3 %) pts had both EGE and ME which matched topographically in ventricular septum. The most frequent finding in DCM pts was LGE—in 19 pts (48 %), ME was found in 11 pts (40 %), sighs of EGE in 16 pts (40 %). Totally C2 positive MRI criteria indicating myocarditis [4] were detected in 13 (32.5 %) DCM pts. TNFa was detected in 62.5 % DCM and 23.3 % CD pts (p = 0.024), IL6 in 27.5 % DCM and 5.3 % CD pts (p = 0.016). The increased level of TNFa and IL6 didn’t correlate with the activeness of inflammation according to EMB in DCM pts. In some CD pts increased level of these cytokines can indicate a systemic inflammation. CD pts demonstrated increased level of TGF-b1 (in 44.7 % pts compared to 17.5 % in DCM group, p = 0.012). Increased level of the main profibrotic cytokine did not correlated with fibrosis volume in DCM pts (r = 0.15). 31 (77.5 %) of DCM pts have shown elevated level of b1-AAbs IgG. 18 (60 %) CD pts revealed enhanced level of IgM to M2-AAbs, while in DCM group it was positive only in 14 (35 %) pts. The titer of M2-AAbs was higher in CD group than in DCM pts (0.68 [0.49; 1.24] vs 0.55 [0.43; 0.86], p = 0.04). Thelevel of IgM to M2-AAbs correlated with the number of CD3+, CD4+, CD8+ and CD68+ cells in EMB samples of DCM pts (r = 0.54, 0.72, 0,67 and 0,75). The amount of b1-AAbs in DCM pts correlated with number of non-sustained ventricular tachycardia (VT), and volume of myocardium with LGE (r = 0.71, 0.61, respectively). LGE positive pts were characterized by higher number of VPC (3643 [255; 8500] vs 659 [64; 2403] p = 0.04) and higher level of b1-AAbs IgG (1.16 [0.71; 2.05] vs 0.69 [0.54; 0.93], p = 0.019).
Inflamm. Res.
B163 A COMPLEX ATHEROSCLEROTIC PHENOTYPE OF MIF GENE-DEFICIENT MICE IN AN ATHEROGENIC APOE2/2 BACKGROUND UNCOVERS A NOVEL CONNECTION BETWEEN MIF AND B CELLS Corinna Schmitz, Omar El Bounkari, Christina Klasen, Heidi Noels, Ju¨rgen Bernhagen RWTH Aachen University, Aachen, Germany Macrophage migration inhibitory factor (MIF) is an evolutionarily conserved pleiotropic cytokine. Originally identified as an inhibitor of random macrophage migration, MIF was recently found to act as a major regulator of inflammation and atherogenesis by displaying chemokine-like functions. This was shown in various experimental disease models including Mif gene knockout in Ldlr-KO mice, antibody blockade of MIF in ApoEKO mice, and more recently by adenoviral knock-down of Mif in ApoE-KO mice. Pro-atherogenic effects of MIF involve amplification of plaque inflammation and destabilization, but most important are effects on leukocyte recruitment. Upon engaging the non-cognate chemokine receptors CXCR2 and CXCR4, MIF mediates monocyte and T lymphocyte recruitment, respectively, into atherosclerotic plaques. Its blockade in the atherosclerotic prone ApoE-KO mouse was associated with diminished amounts of intimal immune cells and inflammatory mediators. Thus its broadly implication in inflammatory diseases is kind of clear-cut, however the role of MIF in cardiovascular diseases (CVD) seems to be more complex as MIF-effects in acute phases of myocardial infarctions even include cardioprotective mechanisms like metabolic activation, apoptosis suppression and antioxidative stress. In addition peripheral neutralization of MIF by local applications of a blocking monoclonal antibody in ApoE-KO mice showed marked impact on inflammatory markers but did not reduce lesion size. Here we studied the functional role of MIF in experimental atherosclerosis in the ApoE-KO mouse model by comparing mice genetically depleted in Mif (Mif-/- ApoE-/-) with MIF-expressing ApoE-/- animals. As expected, Mif-/- ApoE-/- mice fed a western type diet showed atheroprotective properties towards equally treated MIFexpressing mice. Interestingly, significant reductions in plaque sizes were observed in brachiocephalic arteries and in abdominal aortas of Mif-/- ApoE-/- mice whereas different atherosclerotic prone regions like aortic roots or aortic arch were not affected. Analysis on plaque composition revealed no differences in infiltrated cell quantities but changes in systemic immune responses could be observed. Considering the more recent recognition on the role of B cells and adaptive immune responses by artery tertiary lymphoid organ formation in atherosclerosis, we assume that a previously unrecognized effect of MIF on B cells may account for the complex atherosclerotic phenotype seen upon genetic Mif deletion in the ApoE-/-atherosclerosis model. At the conference, we will present data, suggesting effects of MIF on B cell maturation and subset differentiation that may underlie this phenotype and that are suggestive of a novel role for MIF-mediated B cell regulation in atherogenesis.
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B164 MACROPHAGE LIPID LOADING LEADS TO TYPE I INTERFERON HYPORESPONSIVENESS Marieke C.S. Boshuizen, Marten A. Hoeksema, Annette E. Neele, Saskia van der Velden, Jan Van den Bossche, Menno P.J. de Winther Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Macrophage-derived foam cells are critical components of atherosclerotic lesions and the ways in which the inflammatory response of foam cells influences atherogenesis is of great interest. Previously we demonstrated that interferon-beta (IFN-b) promotes atherogenesis. But how IFN-b influences foam cell inflammatory responses is not understood yet. IPA analysis indicated a downregulated interferon signaling upon monocyte and macrophage lipid loading. Hence we wanted to assess whether macrophage lipid loading also results in a decreased interferon response. To do so, we loaded bone marrow-derived macrophages overnight with acLDL followed by 6 h IFN-b treatment. Surprisingly, lipid loading impaired the induction of IFN-b target genes like CCL5 and CXCL10. To validate these findings in vivo, LDLR-/- mice were put on normal chow (NC) or a high cholesterol diet (HCD) for 10 weeks. Peritoneal macrophages (PEMs) were collected 4 days after intraperitoneal thioglycollate administration, combined with IFN-b, IFN-c (both 5000 U/mL) or PBS administration 24 and 8 h before sacrifice. This diet-induced lipid loading also resulted in PEM IFN-b-hyporesponsiveness, since several IFN-b target genes were again less expressed compared to NC PEMs. In addition, ex vivo culturing of PEMs from IFN-b-treated animals on HCD versus NC showed an overall decreased inflammatory activity, as gene expression of inflammatory markers was reduced and secretion of IL-6, TNF and NO was decreased. This hyporesponsiveness was specific for IFN-b, as IFN-c target genes were still induced upon IFN-c treatment, and an adequate inflammatory response could also still be observed ex vivo. Interestingly, similar effects were observed in human primary macrophages. The mechanism behind this hypercholesterolemia-induced hyporesponsive state remains to be discovered. Currently we assess whether the IFN-binduced activation of STAT2 and IRF9 is affected under lipid conditions, as we found STAT1 protein expression to be unaffected. Altogether, we observed that macrophage lipid loading results in hyporesponsiveness to IFN-b stimulation, in different models of foam cell formation. Future research will clarify whether this hyporesponsiveness also affects IFN-b-mediated antiviral activity, which might have implications for obesity related disorders. This work was supported by the Dutch Heart Foundation, Grant #2010B022.
B165 HIGHER MYOCARDIAL INFLAMMATION IN DCM PATIENTS SUBMITTED TO HEART TRANSPLANT WAS ASSOCIATED WITH GOOD PULSE THERAPY RESPONSE WITH DECREASE IN BACTERIA ANTIGEN LEVELS IN THE MYOCARDIUM Joyce T. Kawakami, Maria de Lourdes Higuchi, Renata n. Ikegami, Marcia M. Reis, Pablo Pomerantzeff, Sandrigo Mangini, Fernando Bacal, Edmar Bocchi
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S168 Heart Institute (InCor), University of Sa˜o Paulo Clinics Hospital, Sa˜o Paulo, Brazil Purpose: Dilated Cardiomyopathy (DCM) has been related to bacterial and viral infection accompanied or not by myocardial inflammation. In this paper we studied if Borrelia burgdorferi (Bb), Mycoplasma pneumoniae (Mp) and myocarditis are present in DCM receptor hearts (RH), and related to outcome after heart transplantation. Methods: Endomyocardial biopsies (EMBs) were studied regarding Bb and Mp bacterial antigens in RH, donor heart (zero time); moderate rejection (MR) and persistent rejection (PR) by immunohistochemistry. GI (n = 5)—patients having one episode of MR and GII (n = 5)—patients presenting persistent MR. In RHs, we also evaluated the CD3 Tcells/mm2. Results: The mean % area of Bb and Mp antigens in RH of GI were: 7.5 and 26.6, and in GII were 9.3 and 22.6 without significant difference. There was significant increased numbers of CD3T cells/mm2 in GI (18.6 ± 9.8) than in GII (4.3 ± 1.2), p \ 0.01. In GI, a positive correlation was observed between Bb vs Mp (r = 0.76, p = 0.23) without statistical significance (meaning a symbiotic association) and absence of correlation in GII (r = 0.07, p = 0.93). The mean % area of Bb and MP in EMBs of zero time—donor, MR and after PT in GI and GII are shown in the figure. In zero time of both groups there were a large amount of Mp and few Bb, which increased during MR episodes. In group GII, Bb remained increasing after pulse therapy (PT), and decreased in GI. Conclusion: Higher myocardial inflammation in DCM pts submitted to HT was associated with MR regression after PT and low myocardial inflammation with persistent MR. Bb in symbiotic association with Mp may be related to a good PT response during episodes of MR in GI.
Inflamm. Res. found in archaea. Now we searched if circulating MPS are associated with archaeal elements in the serum of HF chagasic pts. Methods: 2 groups of sera from chagasic pts, IF (n = 8) and HF (n = 7), were studied by electron microscopy (EM). Sera were centrifuged obtaining a pellet, which was processed for electron microscopy exam embedding in EPON/Araldite resin. Immunolectron technique was used with primary anti-archaemetzincin-1 antibody (AMZ1, Novus Biologicals) and in situ hybridization (ARCH 915 probe), both linked with 10 nm colloidal gold. The mean number of MPs and positive dots/photo inside and outside MP, of 50,0009 magnification of each case was obtained. Results: The mean numbers of MPs in HF versus IF groups did not differ. HF had higher numbers of AMZ1 immunogold positive dots outside MPs than IF group (Table 1). The mean numbers of MPs exhibited a positive correlation with numbers of archaeal DNA outside MPs in HF sera but not in IF. Conclusion: The increased amount of AMZ1 collagenase in the sera differentiated HF chagasic patients from IF pts. The correlation between number of MPs vs archaea DNA extracellular in HF group suggests that these MPs are pathogenic archaea, releasing collagenases. Further studies may reveal if the increased amount of archaeal AMZ1 in the serum may be biomarkers of HF. Table 1 Mean number/EM photo of Microparticles (MP), AMZ1 positive dots extra MPs, and Archaeal DNA positive dots (Arch DNA) extra MPs, in the pellet of sera from Heart Failure (HF) and Indetermiante Form (IF) of chagasic patients
MPs
HF
IF
p
7.5
7.3
0.48
AMZ1
79.2
15.2
0.01
Arch DNA
15.4
11.3
0.35
Correl no MPs 9 Arch DNA-r (P) 0.95 (0.001)
-0.370 (0.76)
B167 INFLAMMATORY MARKERS IN PATIENTS WITH UNSTABLE ANGINA AFTER CORONARY ANGIOPLASTY AND STENTING B166 HEART FAILURE IN CHAGAS’ DISEASE MAY BE RELATED TO MICROPARTICLES AND ARCHAEAL ELEMENTS IN THE SERUM Joyce T. Kawakami, Maria de Lourdes Higuchi, Jaqueline J. Pereira, Barbara Ianni, Sandrigo Mangini, Renata N. Ikegami, Marcia M. Reis, Edmar Bocchi Heart Institute (InCor), University of Sa˜o Paulo Clinics Hospital, Sa˜o Paulo, Brazil Introduction: In Chagas’ disease, approximately 30 % of infected individuals develop myocarditis and heart failure (HF). Antigens and T. cruzi DNA are scarce, not explaining the intensity of myocarditis. The individuals remaining asymptomatic, are called indeterminate form (IF). Circulating microparticles (MPs) in the serum have been associated with heart failure in dilated cardiomyopathy patients (pts). Previously we found archaeal-like MPs in the myocardium and serum of HF chagasic pts. Collagenases are activated in HF, and are widely
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Ludmila I. Gapon1, Natalia A. Musikhina1, Tatiana I. Petelina1, Irina V. Osipova1, Vadim A. Kuznetsov1 1
Tyumen Cardiology Center, Tyumen, Russia
Aim: To study inflammatory markers and lipid profile in patients with unstable angina (UA) after coronary stenting. Methods: A total of 95 patients (mean age 60.5 ± 9.5 years) with UA and significant coronary artery stenosis [75 % undergoing percutaneous coronary intervention (PCI) with drug-eluting stent placement were examined. The parameters were evaluated at baseline and 3, 6 and 12 months after PCI. All patients received optimal medical treatment that included statins and dual antiplatelet therapy. Lipid profile parameters, inflammatory markers (hs-CRP, TNF-alpha, homocysteine, interleukin 1b, 6, 8, 16; sCD40 L, MMP-9, TIMP-1); endothelial dysfunction markers (endothelin-1, nitrites) were measured. Results: High levels of atherogenic index, hs-CRP, TNF-alpha, MMP9, CD40, sCD40L, homocysteine, endothelin-1 were found initially as well as the following positive correlations were detected: between homocysteine and APO B/A-1, CD 40, MMP-9. After 3 months significantly elevated levels of homocysteine, MMP-9 and TIMP-1
Inflamm. Res. and valid reduction of TNF-alpha, CD40, sCD40L, endothelin-1 were revealed. In 6 month MMP-9, TNF-alpha, hs-CRP remained high, however level of sCD40L was elevated. After 12 months of follow-up we detected reduction in atherogenic index, total cholesterol, triglycerides, MMP-9, TNF-alpha, and significant increase in the concentration CD40. Endothelial dysfunction was also observed: the level of endothelin-1 remained high. Conclusions: The study showed that prolonged endothelial inflammatory process is an initiating factor of atherosclerotic process destabilization in patients with UA after coronary angioplasty and stenting.
B168 SMALL INTESTINE MODULATES SYSTEMIC INFLAMMATION AND CARDIOVASCULAR FUNCTION INDUCED BY DIETARY OXIDIZED FATTY ACIDS THROUGH LYSOPHOSPHATIDYLCHOLINE PATHWAY Azadeh Esmaeili David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA Background: Systemic inflammation induced by oxidized lipids affects cardiovascular function. Our group has demonstrated that dietary lipids can undergo oxidative modification in the GI tract and result in the elevation of circulating factors including serum amyloid A (SAA). Objective: In the current study we sought to determine the role of the small intestine in modulating systemic inflammation stimulated by dietary lipids. Methods, Male LDL receptor deficient mice (groups with n = 23) were treated with (a) Laboratory rodent chow, or (b) high fat high cholesterol diet (Western diet) for 2 weeks. At this time the mice were fasted overnight, blood was removed from the retroorbital sinus, the mice were perfused with cold saline, the intestine was removed, washed with cold saline and kept at -80 C. Lipids were extracted from plasma and from jejunum prepared for liquid chromatographyelectrospray ionization mass spectrometry to determine the level of lysophosphatidylcholine (LPC) and its oxidative derivatives. Results: the level s of LPC with stearic acid at the sn-1 position did not differ significantly in the plasma or in jejunum between the two groups. Plasma and intestinal levels of LPC with oleic, linoleic or arachidonic acid at the sn-1 position showed significantly higher level in the Western diet treated group (p \ 0.01, 0.02 and 0.01 respectively). Conclusion: Dietary fat can convert to the potent growth promoter LPC in the intestine resulting in elevated levels of SAA that readily has deleterious inflammatory effects on cardiovascular function.
B169 TO NON-INVASIVELY DETECT AND CHARACTERIZE MYOCARDIAL INFLAMMATION IN AN EXPERIMENTAL RAT MODEL OF MYOCARDITIS Priyadarshini A. Panjwani1, Vale´rie Boivin-Jahns1,2, Xavier Helluy4, Takahiro Higuchi2,3, Yu-Xiang Ye4, Franz Kaiser3,2, Karin Klingel5, Karl-Heinz Hiller4, Peter Jakob4, Martin Lohse1, Roland Jahns1,2,6 1 Institute of Pharmacology and Toxicology, University of Wu¨rzburg, Wu¨rzburg, Germany; 2Comprehensive Heart Failure Center (CHFC), University Hospital of Wu¨rzburg, Wu¨rzburg, Germany; 3Department
S169 of Nuclear Medicine, University Hospital of Wu¨rzburg, Wu¨rzburg, Germany; 4Research Center Magnetic Resonance Bavaria (MRB), Wu¨rzburg; 5Department of Molecular Pathology, University Hospital of Tu¨bingen, Tu¨bingen, Germany; 6Interdisciplinary Bank of Biomaterials and Data Wu¨rzburg (ibdw), University Hospital Wu¨rzburg, Germany Objective: To non-invasively detect and characterize myocardial inflammation in an experimental rat model of myocarditis. Background: Myocarditis is characterized by inflammation, myocyte necrosis/apoptosis and subsequent fibrotic replacement of heart muscle. In the human, about 30 % of myocarditis-patients develop DCM. Because the clinical picture of myocarditis is multi-faceted, its diagnosis is difficult. Methods: n = 150 female Lewis rats were immunized with (pig) cardiac myosin (CM) emulsified in CFA, injected with heat killed B. Pertussis on days 0 and 3, and were antigen-boosted on days 7, 14, and 28. Development of anti-myosin-antibodies was followed by ELISA and cardiac function was surveyed by echocardiography and cardiac magnetic resonance imaging (cMRI). The development of myocardial effusion (day 18–21) was visualized by cMRI. On day 21, the rat hearts were removed and consecutive 3 lm heart sections were stained with Hematoxylin/ Eosine (HE), Masson Goldner trichrome (MG), or with anti-CD68 (immune-staining of mononuclear cells), and then compared with MRI findings (T2 and T2*/Flash sequences). On top, we then assayed nuclear tracers such as gallium 67 citrate, gallium 68 linked to somatostatin, and gallium 68 linked to integrin peptide (RGD) to detect cellular infiltrates in our model. In parallel, apical cardiac tissues were analyzed in-depth for the expression of pro-inflammatory and pro-fibrotic markers. Results: Sera from immunized rats strongly reacted with cardiac myosin. In immunized rats, echocardiography and subsequent MRI revealed large pericardial effusion (days 18–21). Analysis of the kinetics of myocardial infiltrates revealed maximal macrophage invasion between day 14 and 28. Histological findings correlated well with MRI-findings but strikingly better with autoradiograms showing an high uptake of gallium 67 citrate and gallium 68 linked to somatostatin in strongly infiltrated myocardial areas. Disappearance of macrophages resulted in replacement-fibrosis in formerly invaded myocardial areas. This finding was confirmed by the time-dependent differential expression of corresponding cytokines in the myocardium. Immunized animals reacted either with an early or a late pattern of post-inflammation fibrosis. Conclusion: By applying a broad panel of biochemical, histological, molecular and imaging methods here we revealed that different patterns of reactivity may occur upon induction of myocarditis using one and the same rat strain. In particular, immunized Lewis rats may react either with an early or a late pattern of macrophage invasion and subsequent post-inflammation fibrosis. As compared to cMRI, promising results achieved in the acute inflammatory phase of the rat hearts with gallium 67 citrate and gallium 68 linked to somatostatin will, this will stimulate further development of nuclear strategies for the non-invasive detection of acute myocarditis, in the near future perhaps in patients.
B170 CP-3(IV), A NOVEL AZAPEPTIDE THAT BINDS SCAVENGER RECEPTOR CD36, ATTENUATES INFARCT SIZE AND OXIDATIVE STRESS AND PRESERVES CARDIAC FUNCTION IN A MURINE MODEL OF TRANSIENT MYOCARDIAL ISCHEMIA ˆ me David N. Huynh1, Vale´rie L. Bessi1, Liliane Me´nard1, Je´ro Piquereau1, Caroline Proulx1, Maria Febbraio2, William D. Lubell1, Andre´ C. Carpentier3, Yan Burelle1, Huy Ong1, Sylvie Marleau1
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S170 1
Universite de Montreal, Montreal, Canada; 2University of Alberta, Edmonton, Alberta; 3Universite de Sherbrooke, Sherbrooke, Que´bec
Aims: The CD36 receptor plays a pivotal role in the regulation of energy metabolism in the heart, where it facilitates fatty acid transport to the myocardium. The present study aimed to investigate the cardioprotective effect of a novel azapeptide (azaPhe4) derivative, CP3(iv) (Ala-D-Trp-Ala-AzaPhe-D-Phe-Lys-NH2), as a potent and selective CD36 ligand in a model of transient occlusion of the left anterior descending coronary artery in mice. Methods: C57BL/6 mice (CD36+/+) and CD36-deficient (CD36-/-) mice were pretreated with a daily subcutaneous injection of CP-3(iv) (289 nmol/kg) or 0.9 % NaCl vehicle for 14 days before undergoing myocardial ischaemia (30 min) and reperfusion (MI/R) (6 or 48 h) elicited by the temporary ligation of the left anterior descending coronary artery, or underwent sham surgery. Myocardial infarction size was determined by the Evans blue/TTC double staining method and planimetry of digital photographs. Left ventricular (LV) function was assessed using pressure–volume conductance catheter technique. LV and mitochondrial reactive oxygen species (ROS) were assessed using lucigenin-enhanced chemiluminescence and saponin-permeabilized ghost muscle fibers with the fluorescent probe Amplex Red, respectively. Results: MI/R was associated with a consistently large mean AAR to total LV area of 53 ± 3 and 47 ± 2 % in vehicle-treated CD36+/ + and CD36-/- mice, respectively. Whereas CP-3(iv) reduced infarct area (IA) by 54 % (p \ 0.001) in CD36+/+ mice, no effect of the azapeptide was observed in CD36-/- mice. CP-3(iv) treatment improved myocardial haemodynamics as shown by a significant increase in stroke volume (SV), cardiac output (CO), and of the loadindependent contractility parameter, PRSW, by 36 % (p \ 0.05), 40 % (p \ 0.01), and 47 % (p \ 0.001), respectively, while decreasing total peripheral resistance (TPR) by 26 % (p \ 0.05) when compared to vehicle-treated MI/R group. Stimulated ROS production levels in heart and mitochondrial LV were decreased by 51 % (p \ 0.01) and 20 % (p \ 0.05), respectively, whereas aconitase activity was increased by 33 % (p \ 0.05) at 6 h of reperfusion of ischaemic hearts in CP-3(iv)-treated CD36+/+ mice. Conclusion: Our results show that a pretreatment with the CP-3(iv) azapeptide analog exerts a CD36-dependent cardioprotective effect in part through attenuating myocardial oxidative stress. Our results support the potential application of azapeptide derivatives targeting CD36 for cardioprotection.
B171 THE LEVEL OF CIRCULATING LARGE RETICULATED PLATELETS AND INFLAMMATION IN PATIENTS WITH ACUTE CORONARY SYNDROME TREATED WITH PRIMARY PERCUTANEOUS INTERVENTION Nikita V. Lomakin1, Ludmila I. Buryachkovskaya2, Alexander B. Sumarokov2, Irina A. Uchitel2 1 Central Clinical Hospital of the Presidential Administration of Russian Federation, Moscow, Russia; 2Russian Cardiology Research Complex, Moscow, Russia
Background: Platelets represent an important link between inflammation, thrombosis and atherogenesis. Mediators of inflammation affect megakaryocytes, thereby causing generation and release into blood of large reticulated platelets (LRPs) with high thrombogenic activity. Antiplatelet drugs are widely used to reduce the risk of
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Inflamm. Res. thrombotic complications, but their effect on the subpopulation of large platelets is not as yet well-known. Methods: Sixty patients (pts) with acute coronary syndrome treated with primary percutaneous intervention (PCI) and dual antiplatelet therapy with clopidogrel were tested for platelet reactivity using light transmission aggregometry (spontaneous and 1.0 lM ADP aggregation). Platelet morphology and leukocyte-platelet aggregates (LPA) were assessed by scanning electron microscopy. Mean platelet volume (MPV) was estimated by thrombocytocrit method, serum high sensitive CRP (hs-CRP) level was analysed by nephelometric method. Tests were performed 30 days and then 1 year post-PCI. Results: In all pts 30 days after PCI the levels of LPA and LRP were 62.7 ± 12.4 and 6.8 ± 1.7 %, respectively, which are normally not observed in healthy subjects (HS). The presence of LPA and LRP is usual in pts with inflammation. In 52 % of pts the number of LRP was higher than 4 %, and that correlated with the level of hs-CRP (1.3 ± 0.2 lg/l in pts with \4 % of LRP and 3.1 ± 0.4 lg/l in pts with LRP [4 %). MPV was 12.6 ± 2.7 vs 8.4 ± 0.7 fl in HS and correlated with the LRP level. Spontaneous and 1.0 lM ADP aggregation increased. At 1 year post-PCI all parameters went back to normal with 91.1 % of pts. Spontaneous aggregation was retained in 4 patients, LPA went down to 12.4 ± 1.6 %, the level of LRP did not exceed 1.2 %, MPV was 8.9 ± 0.8 fl. hs-CRP level returned to normal and constituted 0.6 ± 0.1 lg/l. Conclusion: An inflammatory process in pts with acute coronary syndrome treated with PCI is related to an increased level of superactive platelet production. Dual antiplatelet therapy, in addition to its antithrombotic action, may also have anti-inflammatory potential.
B172 MESENTERIC ARTERY RELAXATION TO EXOGENOUS NITRIC OXIDE ‘‘IN VITRO’’ IS IMPAIRED IN RATS WITH LIGATURE-INDUCED PERIODONTITIS Flavia N. de Jesus1, Elly S. do Amaral Neto1, Camila F. Wenceslau2, Simone A. Teixeira1, Aline Maia-Dantas1, Gisele K. Couto2, Luciana V. Rossoni2, Luis C. Spolidorio3, Soraia K. Costa1, Marcelo N. Muscara1 1 Department of Pharmacology, ICB University of Sa˜o Paulo, Sa˜o Paulo, Brazil; 2University of Sa˜o Paulo, Sa˜o Paulo, Brazil; 3 State University of Sa˜o Paulo, Sa˜o Paulo, Brazil
We have previously shown that periodontal disease has some nitric oxide (NO)-mediated remote effects on rat heart and kidney, as well as aorta endothelial dysfunction and decreased adrenergic contractililty. We thus decided to study the effects of periodontitis on the mesenteric resistance artery. The protocol was approved by the Ethics Committee for Animal Experimentation (CEUA-ICB 170, book 2/113, 2011). Cotton ligatures were subgingivally placed around both lower first molars of male adult Wistar rats; sham animals had the ligatures immediately removed. Seven days later, the animals were euthanized and the vessels (3rd. order mesenteric artery branches) were isolated and mounted on a myograph for evaluation of the in vitro responses to acetylcholine (Ach), phenylephrine (Phe), sodium nitroprusside (SNP) and sildenafil (Sil). From the concentration–response curves, potency (pD2) and maximal response (Emax) values were calculated. The vessels were also analysed for NOS isoenzyme gene expression and activity of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT). The results were analysed by unpaired Student t-test.
Inflamm. Res. Alveolar bone loss was observed in the animals with ligature, as bilaterally measured at the first and second molar levels. No differences were observed between the groups in terms of vascular responses to Phe or ACh. However, SNP-induced relaxation was less potent in the ligature group (pD2: 6.4 ± 0.2 vs. 7.1 ± 0.1; p \ 0.01), similarly to Sil (pD2: 8.6 ± 0.2 vs. 10.3 ± 0.2; p \ 0.001), but efficacies (Emax) were unaltered. Decreased SOD activity (0.7 ± 0.05 vs. 0.4 ± 0.05; p \ 0.01) and increased iNOS mRNA (1.0 ± 0.1 vs. 20.9 ± 5.9; p \ 0.01) and CAT activity (2.3 ± 0.2 vs.3.2 ± 0.3; p \ 0.05) were found in the arteries from rats with periodontitis. With basis on the results above, we conclude that during the early phase of ligature-induced periodontitis in rats, functional changes related to the NO-cyclic GMP pathway occur in the mesenteric artery smooth muscle, which may be secondary to the excessive production of iNOS-derived NO, superoxide anion or their combination. Financial support FAPESP (Grant #2011/17800-4), CAPES, CNPQ.
B173 THE INDUCIBLE NUCLEAR PROTEIN IjBf REGULATES THE TRANSITION FROM ADAPTIVE CARDIAC HYPERTROPHY TO HEART FAILURE DURING PRESSURE OVERLOAD Yasutomi Higashikuni1, Daiju Fukuda2, Masataka Sata3, Issei Komuro1 1
Department of Cardiovascular Medicine, The University of Tokyo, Tokyo, Japan; 2Department of Cardio Metabolic Medicine, The University of Tokushima, Tokushima, Japan; 3Department of Cardiovascular Medicine, The University of Tokushima, Tokushima, Japan Background: Inflammation has been implicated in the pathophysiology of heart disease. However, the roles and the regulatory mechanisms of inflammation in the heart remain largely unknown. It has been reported that the NF-jB system contributes to inflammatory responses in the heart and pathological cardiac hypertrophy during pressure overload. The inducible nuclear protein IjBf, encoded by the NFKBIZ gene, regulates proinflammatory cytokine expression pattern by modulating the NF-jB system. This study was performed to clarify the role of IjBf in heart disease. Methods and results: Pressure overload was induced in mice by transverse aortic constriction (TAC). TAC-operated Nfkbiz+/+ hearts showed adaptive cardiac hypertrophy until 2 weeks after the operation, whereas at 4 weeks after TAC, Nfkbiz+/+ mice fell into heart failure with impaired systolic function and left ventricular dilatation. The failing hearts showed higher mRNA expression levels of IjBf with higher interleukin-6 (IL-6) to IL-1b expression ratios compared to the hearts with adaptive hypertrophy. Nfkbiz+/- mice showed similar adaptive cardiac hypertrophy to Nfkbiz+/+ mice until 2 weeks after TAC. However, at 4 weeks after TAC, Nfkbiz+/- hearts showed preserved systolic function with attenuated LV dilatation compared with Nfkbiz+/+ hearts. The bone marrow transplantation experiment revealed that IjBf expressed in the heart, but not in bone marrowderived cells, contributes to cardiac phenotype during pressure overload. Histological assessment showed attenuated interstitial fibrosis and macrophage infiltration and higher capillary density in Nfkbiz+/- hearts compared with those in Nfkbiz+/+ hearts at 4 weeks after TAC, whereas cardiomyocyte hypertrophy in Nfkbiz+/+ and Nfkbiz+/-- mice was comparable. Real-time PCR revealed that IL-6 to IL-1b expression ratios remained lower in Nfkbiz+/- hearts compared
S171 to Nfkbiz+/+ hearts at 4 weeks after TAC. Western blot analysis showed that the phosphorylation levels of Akt, which can be activated by IL-1b and contributes to cardiac adaptive mechanisms, were higher in Nfkbiz+/- hearts than in Nfkbiz+/+ hearts at 4 weeks after TAC. Consistently, mRNA expression levels of suppressor of cytokine signaling 3 (SOCS3), that is induced by chronic gp130 signaling and suppresses Akt phosphorylation, were higher in Nfkbiz+/+ hearts that in Nfkbiz+/- hearts. In vitro experiments demonstrated that pharmacological activation of the NF-jB system induced apoptosis of cardiomyocytes and proliferation of cardiac fibroblasts, which were suppressed by IjBf knockdown. Conclusions: Our results suggest that IjBf regulates the transition from adaptive cardiac hypertrophy to heart failure during pressure overload, possibly in part, through the modulation of proinflammatory cytokine expression pattern in the heart, which may regulate the balance between adaptive and maladaptive signaling pathways. Thus, IjBf may be a novel therapeutic target for treating heart failure.
B174 DRUG A SUPPRESSES INFLAMMATORY RESPONSES INDUCED BY 27-HYDROXYCHOLESTREOL Bo Young Kim, Koanhoi Kim Pusan National University, Busan, South Korea Atherosclerosis is an inflammatory disease that is characterized by the infiltration of immune cells into the artery wall, followed by the development of atherosclerotic plaques. We reported that 27-hydroxycholesterol (27OHChol) detected in abundance in atherosclerotic lesions could induce inflammation by activating monocytic cells. In the current study, we sought to find drugs or chemicals that influence inflammatory responses induced by 27OHChol. Treatment of THP-1 cells with 27OHChol resulted in significant increase in expression of inflammatory chemokines like CCL2, CCL3, and CCL4, and the increase was does-dependently inhibited in the presence of drug A, but not of aspirin. Also, drug A attenuated the expression of MMP-9 induced by 27OHChol. Conditioned media isolated from 27OHChol-treated THP-1 cells, which contained a high amount of the ligands, enhanced migration of monocytic cells and Jurkat T cells expressing CCR5, a characteristic chemokine receptor of Th1 subtype. Addition of drug A resulted in complete inhibition of the migration of both types of cells. We propose that drug A can be used for the treatment of inflammatory responses in atherosclerotic lesions.
Inflammatory Processes in Central Nervous System Diseases B175 MODULATION OF IMMUNE-INDUCED AND TRAUMATIC CNS INJURY BY DENDRITIC CELLS’ EXPRESSION OF CEBPD, A NOVEL THERAPEUTIC TARGET Masoud Hassanpour Golakani1, Mohammad G. Mohammad1, Vicky W. Tsai1, Hui Li1, Marc Ruitenberg2, Samuel N. Breit1, Paul E. Sawchenko3, David A. Brown1
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Laboratory of Neuroinflammation, St Vincent’s Centre for Applied Medical Research and University of New South Wales, Sydney, NSW, Australia; 2Queensland Brain Institute, The University of Queensland, Brisbane, Queensland, Australia; 3Laboratory of Neuronal Structure and Function, The Salk Institute for Biological Studies, La Jolla, CA, USA CCAAT/enhancer binding protein delta (CEBPD) is a transcription factor that modulates many biological processes including central nervous system (CNS) injuries. However, the cellular machanisms remain largely unknown. We aimed to determine the mechanism of action of CEBPD in immune-mediated and traumatic spinal cord injury (SCI). For immune-induced SCI we used a mouse model of multiple sclerosis (MS), experimental autoimmune encephalomylitis (EAE), which is actively induced with myelin oligodendrocyte glycoprotein (MOG). Our findings indicate that mice deficient in CEBPD had less sever EAE. This was associated with a significant reduction of ratios of pro-inflammatory T helper (Th) 17 cells to anti-inflammatory regulatory T-cells (Tregs) in an interleukin-10 (IL-10)dependant manner. We have showed that this is mediated by CD11c+ dendritic cells (DCs). Bone marrow chimeric mouse experiments showed that peripherally-derived immune cells mediated the beneficial effects of reduced CEBPD expression in EAE. Examination of CD11c+ DCs mediated Th-cell development in vitro and in vivo suggested that CEBPD modulated interleukin-10 (IL-10) expression in DCs. Direct examination confirmed that the changes mediated by reduced CEBPD expression was mediated by IL-10 secretion both in vitro and in vivo. The inhibition of IL-10 actions by a specific antiIL-10 receptor antibody treatment reversed the effect of absent DCCEBPD both in vitro and in vivo. Extending these findings to a model of traumatic spinal cord injury, we found that CEBPD knockout mice display significantly augmented locomotor recovery after contusive spinal cord injury compared to wild-type mice. These findings identify CEBPD as an important DC transcription factor modulating CNS injury from a variety of causes and suggest it as a novel potential therapeutic target in CNS injuries.
B176 ANNEXIN A1 MODULATES THE LPS INDUCED TSPO EXPRESSION IN BV2 CELLS Lorena N. Pantaleao1, Rodrigo Azevedo1, Erick Barioni1, Mauro Perretti2, Egle Solito2, Sandra P. Farsky1 1
FCF-USP, Department de Ana´lises Clı´:nicas e Toxicolo´gicas (Av. Prof. Lineu Prestes, 580 Bloco 13B, Cid. Universita´ria, SP CEP: 05508-000); 2William Harvey Research Institute, QMUL, London, UK Introduction: The translocator protein (18 kDa) (TSPO) is a benzodiazepine receptor localized in the outer mitochondrial membrane. It is as a biomarker in brain inflammation and modulates the development of inflammatory diseases. Annexin A1 (ANXA1) is 37 kDa protein produced by different cell types and inhibits and induces the development and resolution of innate inflammation, respectively. Gene and protein expression of ANXA1 is induced by glucocorticoids and TSPO modulation on inflammation is dependent on glucocorticoids. Objective: To investigate the connection of ANXA1 and TSPO in BV2 in basal or inflammatory conditions. Method: Murine BV2 microglial cells were seed in culture plates, exposed to different concentration of an inflammatory stimuli (LPS, 10 or 100 ng/mL), and co-incubated or not with ANXA1 (10 or100 ng/mL) for 4 or 12 h. TSPO expression was evaluated by
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Inflamm. Res. FACS, and cytokines (IL1b, TGF) secreted in the culture media was measured by ELISA. Control cells were incubated with cultured media. Results: Incubation of BV2 cells with LPS increased TSPO expression (70 % vs control); ANXA1 per se did not alter the TSPO expression. Co-incubation with ANXA1 (10 or 100 ng/mL) inhibited the LPS-induced (10 ng/mL) expression of TSPO after 4 and 12 h (53.5 % or 50.42 %); effect was not detected with 100 ng/mL LPS. Moreover, ANXA1 treatment reduced the LPS-induced IL-1b (86,9 %) and inhibited reduction of TGF secretion evoked by LPS. Discussion: TSPO is expressed by LPS stimulation in BV2 cells and this effect is modulated by ANXA1. In addition, ANXA1 seems to inhibit the BV2 LPS-induced polarization. The role of TSPO in this latter effect has been investigated. Agency: FAPESP (2013/25903-3 fellowship grant; Financial support 2014/07328-4)
B177 EFFECTS OF GP120 GLYCOPROTEIN INJECTION AT NUCLEUS ACCUMBENS IN RODENT Elisabete C. Konkiewitz1, Ana C. Piccinelli1, Priscila N. Morato1, Jose´ A. Pochapski2, Edmar Miyoshi2, Edward B. Ziff2, Caˆndida L. Kassuya1 1
Universidade Federal da Grande Dourados (UFGD), Dourados, Brazil; 2New York University, New York City, NY, USA; 2 Universidade Estadual de Ponta Grossa (UEPG), Ponta Grossa, Brazil
HIV infection affects the central nervous system causing depression and behavioral and motor disorders. The gp120 protein plays a role in these effects of HIV infection with its ability to induce neuroinflammation, which may lead to apoptotic cell death. The Nucleus accumbens is a brain region that functions in behavioral and emotional regulation. Lesions to this brain region may result in prominent symptoms of apathy as well as behavioral changes. Therefore, the present work has investigated histological parameters and neuronal apoptosis following stereotaxic injection of gp120 into the Nucleus accumbens in rats. The behavioral effects were also evaluated. Male Wistar rats (n = 7) were divided into groups: control, gp120-injected (400 ng) and TNF-alpha-injected (100 ng). Cannulas were implanted in rats through stereotaxic surgery, and after 5 days the rats received a bilateral injection in the nucleus accumbens of saline, gp120 or TNFalpha, as noted. After 2 days, the open field and sucrose preference tests were conducted to evaluate locomotor activity and depression. After sacrifice, brains were collected, fixed in 4 % of paraformaldehyde and then embedded in paraffin. Sections were stained for immunohistochemistry analysis. NeuN primary antibody (Millipore MAB377, 1:200) was used to stain for neuronal apoptosis. The immunohistochemistry results showed that TNFa decreased NeuN levels 18.70 %, and gp120 decrease the NeuN expression 16.01 % compared the control group. Gp120 and TNFa however did not induce significant changes in depression in sucrose preference test, or in rearing and locomotor activity in the open field test when compared to the control group. The results suggest that although gp120 and TNF-alpha did not cause behavioral changes, injection into the Nucleus accumbens resulted in loss of neurons. References Nasirinezhad F, Jergova S, Pearson JP, Sagen J. Attenuation of persistent pain-related behavior by fatty acid amide hydrolase (FAAH) inhibitors in a rat model of HIV sensory neuropathy. Neuropharmacology. 2014 Dec 5. S0028-3908(14) 00440-7. doi:10.1016/ j.neuropharm.2014.11.024.
Inflamm. Res. Reddy PV, Gandhi N, Samikkannu T, Saiyed Z, Agudelo M, Yndart A, Khatavkar P, Nair MP. HIV-1 gp120 induces antioxidant response element-mediated expression in primary astrocytes: role in HIV associated neurocognitive disorder. Neurochem Int. 2012 Oct; 61(5):807–14. doi:10.1016/j.neuint.2011.06.011.
B178 DYNAMICS OF INFLAMMATORY IMMUNE RESPONSE IN A MOUSE MODEL OF TRAUMATIC SPINAL CORD INJURY Masoud Hassanpour Golakani1, Mohammad G. Mohammad1, Hui Li1, Marc Ruitenberg2, Samuel N. Breit1, David A. Brown1 1
Laboratory of Neuroinflammation, St Vincent’s Centre for Applied Medical Research and University of New South Wales, Sydney, NSW, Australia; 2Queensland Brain Institute, The University of Queensland, Brisbane, Queensland, Australia Aim: Inflammatory responses post spinal cord injury (SCI) may be detrimental or beneficial. However, the dynamics of this inflammatory response are largely unknown. The aim of this study was, using a mouse model of traumatic SCI, to quantify and characterise immune cells in and around the spinal cord injury site and their relationship with locomotor functional recovery after SCI. Methods: Mice had a severe, 70 kilo dyne force, contusive SCI induced using the Horizon impactor after laminectomy. Locomotor function was assessed, using the Baso Mouse Scale (BMS) scoring system. At days 7, 14, 21, and 28 post injury, the entire spinal cord was removed and mononuclear cells associated with SCI were isolated, prior to fluorescent labeling for multiparameter flow cytometry to characterize and quantify 15 subtypes of immune cells. Results were validated using both immunohistochemistry (IHC) and immunofluorescence (IF) staining and correlated with histological assessment of injury parameters and injury course. Results and discussion: Post SCI mice had complete paralysis, with functional recovery to score 3–4 on BMS at 28 days post injury (dpi). Smaller mice had significantly less recovery. Peripherally derived immune cells progressively increased over the course of recovery from SCI, most of which were T-cells. Both CD4 and CD8 T-cells increased by four- to five-fold at day 28 compared to day 7 dpi. The CD4 T-cell population were predominantly INFg (Th1) expressing, with FoxP3+ (T-regulatory) and IL17+ (Th17) cells being less frequent. Myeloid DCs (mDCs) were the predominant subtype of DCs present and increased significantly from day 21 to 28 dpi. On the other hand, CD8a DCs were the least frequent DC subtypes and significantly decreased by about threefold at day 21 and then increased at 28 dpi. Plasmacytoid DCs (pDCs) were mostly unchanged. There were also no significant changes in macrophages and B cells over days 7–28 dpi, indicating early recruitment. Immunohistology revealed CD11c+ cells and CD3+ T-cells were predominantly confined to the injury core. While GFAP+ astrocytes surrounded the core. Iba-1+ microglia were dispersed throughout the entire spinal cord. White mater surrounding the injury core was substantially demyelinated and myelin was observed in injury core. Functional recovery was significantly correlated with the total numbers of injury associated macrophages and their percentage of total CD45+ cells at 28 dpi. There was also a significant correlation with CD11c+ DCs’ percentage of total CD45+ cells, which were mostly mDCs. On the other hand, pDC numbers were negatively correlated with functional recovery. The changes for T-cells and their subtypes were not significantly related to improvement. These data
S173 suggest a role for DCs and macrophages in the resolution of SCI and identify them as a potential therapeutic target.
B179 MATERNAL IL17 PATHWAY PROMOTES AUTISM–LIKE PHENOTYPES IN OFFSPRING Jun Huh University of Massachusetts Medical School, Worcester, USA Accumulating evidence points to a central role for immune dysregulation in utero as a risk factor in Autism Spectrum Disorder (ASD). Human studies suggest that maternal viral infections early in pregnancy correlate with an increased frequency of ASD in their offspring. This observation, coined maternal immune activation (MIA), has been modeled in rodents by inducing inflammation in pregnant dams. MIA requires the pro-inflammatory effector cytokine, interleukin 6 (IL-6), to produce ASD-like phenotypes in the offspring. However, the specific immune cell population(s) that induces MIA phenotypes has not been identified. We have found that pro–inflammatory CD4+ T helper cells expressing RORat are required in mothers for MIA to induce ASD–like phenotypes in affected offspring. These data suggest that the Th17 cell lineage, a major contributor to autoimmune inflammation, may serve as a therapeutic target in susceptible pregnant mothers to reduce the likelihood of bearing children with inflammation–induced ASD phenotypes.
B180 IL-33 SIGNALLING IS ESSENTIAL TO ATTENUATE THE DEVELOPMENT OF VIRAL INDUCED ENCEPHALITIS BY DOWNREGULATING INOS EXPRESSION IN THE CENTRAL NERVOUS SYSTEM Rafael FO Franca1, Renata S. Costa2, Jaqueline R. Silva1, Raphael S. Peres2, David F. Colon2, Fernando Q. Cunha2 1
Oswaldo Cruz Foundation FIOCRUZ, Rio de Janeiro, Brazil; University of Sa˜o Paulo, USP, Sa˜o Paulo, Brazil
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Interleukin-33 (IL-33) is an immunomodulatory cytokine, member of the IL-1 family that binds to IL-1 receptor ST2, leading to Th2associated cytokines production. Despite a few studies, the role of IL33 signaling on viral infections is still poorly explored. Viral encephalitis are a common cause of lethal infections in humans and several different viruses are documented to be responsible. However, the immunopathogenic process of these different infections is still poorly understood. Rocio virus (ROCV) is a flavivirus that causes a severe lethal encephalitis syndrome in humans and also mice, providing an interesting model to study the central nervous system (CNS) compartmentalized immune response. Therefore, we aimed to explore how the IL-33/ST2 axis regulates the local immune response during ROCV infection. We have shown that ST2 receptor is expressed in the CNS of ROCV infected mice and that T1/ST2(-/-) mice presented increased susceptibility to infection. ST2 deficiency were correlated with increased tissue pathology, higher viral load, cellular infiltration and increased CNS levels of TNF-a and IFN- c transcripts, compared to with wild-type (WT) mice. As consequence, increased CNS Th1 cytokines level acted on infiltrating macrophages inducing the expression of inducible nitric oxide synthase (iNOS), contributing
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S174 to brain injury. Moreover, iNOS-/- mice were more resistant to ROCV encephalitis, presenting a lower clinical score and reduced mortality rate, despite the increased tissue pathology. In conclusion, we provide evidences of a specific role for IL-33 receptor signaling in NO induction through local IFN-c modulation, suggesting that NO overproduction might have an important role in progression of experimental viral encephalitis.
Inflammatory Processes in Shock and Trauma B181 ROLE OF HMGB1 IN NUCLEIC ACID-MEDIATED INNATE IMMUNE RESPONSES AND INFLAMMATORY DISEASES Hideyuki Yanai Department of Molecular Immunology, Institute of Industrial Science, University of Tokyo, Japan High-mobility group box protein 1 (HMGB1) is a nuclear protein conserved among almost all eukaryotic cells. HMGB1 is thought to be a danger signal mediator when it is released both actively from activated immune cells and passively from dead cells into extracellular milieu upon pathogen infections or sterile inflammations. Released HMGB1 act as an inflammatory cytokine and promote inflammatory disorders. In addition to released HMGB1, recently we have found that the role of cytosolic HMGB1 in innate immune responses. We found that HMGB1, 2 and 3 bind to immunogenic nucleic acids and are involved in nucleic acid-mediated innate immune responses. We hypothesized that selective activation of nucleic acid-sensing cytosolic and Toll-like receptors is contingent on the promiscuous sensing of nucleic acids by HMGB proteins. Consistent with this notion, we also found that non-immunogenic nucleotides with high affinity for HMGB proteins can strongly suppress the activation of innate immune responses induced by cytosolic nucleic acid receptors including RIG-I-like receptors and Toll-like receptors. In addition, in vivo function of HMGB1 is still elusive since Hmgb1 gene-deficient mice are lethal and we established HMGB1 conditional knockout (cKO) mice recently. In this poster presentation, we want to discuss our findings in relation to the critical role of HMGBs in initiating immune responses and the possible use of these non-immunogenic nucleotides in therapeutic interventions. Further, we would like to discuss our findings in HMGB1 cKO mice.
B182 THE ROLE OF INFLAMMATION IN FORMATION OF MULTIPLE ORGAN DYSFUNCTION SYNDROME AT CRITICAL STATES Anatoliy V. Kubyshkin, Ludmila Anisimova, Michail Fedosov, Irina Fomochkina, Vladimir Kharchenko, Viacheslav Mikchailichenko, Olga Malchenko V.I. Vernadsky Crimea Federal University, Simferopol, Republic of Crimea One of the most dangerous complications of critical states, which lead to the death of patients, is the multiple organ dysfunction syndrome (MODS). At present, the role of inflammation in pathogenesis of
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Inflamm. Res. critical states is still one of the most debatable problems. It is known, that massive injuries of tissues are accompanied by discharge of big amount of biologically active substances, which are supposed to be the key factor for formation of systemic inflammatory response syndrome. However, the question, in what measure can the mentioned changes be the starting factor for development of complications and organs pathology formation, is still unstudied. We studied the organs pathology development by using the model of reperfusion injury by applying the tourniquets on both posterior limbs of rats at the level of inguinal fold for 6 h. Revascularization was provided 6 h after applying the tourniquets. The activity of proteinase-inhibitory system’s components was determined using enzymatic methods with spectrophotometer Biomat 5. The concentration of cytokines IL-1b, IL-6 and TNF-a was determined by ELISA method (RayBio). The morphological changes in the lungs, liver, kidneys, heart and intestine were studied using immunohistochemical and electron-microscopic methods. The provided experimental researches have shown, that under reperfusion injury the activation of nonspecific proteinases occurs within 24 h on the local (injured muscular tissue), systemic (blood serum) and organ levels (bronchoalveolar lavage, peritoneal fluid, tissues of kidney and liver). At the same time, the content of proinflammatory cytokines considerably grows. We have found more than tenfold grow of IL-1b and more than forty-fold grow of IL-6 and TNF-a during 12–24 h after the reperfusion. At the same time we have found the signs of inflammatory changes in the target organs; these signs manifested by activation of the inflammation’s and necrosis’ markers and by ultrastructural changes on the cellular level. The obtained results allow to conclude that excessive systemic activation of pro-inflammatory cytokines and nonspecific proteinases under decrease of inhibitor control may play an important role in activation of injury processes in the target organs by formation of numerous inflammatory foci and all these changes can leads to development of multiple organ dysfunction syndrome.
B183 PHOSPHATASE AND TENSIN HOMOLOG CONTROLS MACROPHAGE ENDOTOXIN TOLERANCE Nicole L. Glosson-Byers, Soujuan Wang, C. Henrique Serezani Indiana University School of Medicine, Indianapolis, IN, USA The host immune response to sepsis is characterized by an initial dominant hyper-inflammatory phase followed by a persistent immunosuppressive phase. A signature of sepsis-induced immunosuppression is macrophage tolerance, which might contribute to increased susceptibility to secondary infections and mortality in sepsis. Phosphatase and tensin homolog (PTEN) is a dual phosphatase recognized as a tumor suppressor, but we and others have also shown that PTEN inhibits many macrophage antimicrobial effector functions. We hypothesize that PTEN regulates macrophage tolerance and sepsis outcome. Using the cecal ligation and puncture (CLP) mouse model of polymicrobial sepsis, we found that post-septic mice are more susceptible to secondary pulmonary infection with methicillin-resistant Staphylococcus aureus (MRSA), a pathogen commonly found in ICUs, in comparison to sham-treated mice. Interestingly, Pten expression is significantly increased in both the peritoneal cavity and the lung of mice 3–4 days after CLP. Enhanced Pten expression is accompanied by increased expression of immunosuppressive mediators such as Il10, Socs3 and Irak3. Using an in vitro endotoxin tolerance model, we found that Pten expression is increased in tolerant macrophages and that pharmacologic inhibition of PTEN impairs tolerance in both
Inflamm. Res. murine macrophages and human monocytes. In addition, peritoneal cells isolated from CLP-treated mice display PTEN-mediated tolerance upon in vitro stimulation with endotoxin. Together, these data suggest that PTEN could be an important mediator of the immunosuppressive phase of sepsis by controlling macrophage endotoxin tolerance. Thus, PTEN might be a therapeutic target to control sepsisinduced immunosuppression and susceptibility to secondary infections, resulting in increased survival of septic patients.
B184 ESTRADIOL MODULATES LEUCOCYTES MOBILIZATION AFTER INTESTINAL ISCHEMIA/ REPERFUSION IN FEMALE RATS Evelyn T. Fantozzi1, Fernanda Y. Ricardo da Silva1, Sara Rodrigues Garbin1, Ricardo M. Oliveira Filho1, Bernardo B. Vargaftig1, Ana Cristina Breithaupt Faloppa2, Wothan Tavares de Lima1 1
Department of Pharmacology, ICB/USP, Sa˜o Paulo, Brazil; Cardiovascular Surgery and Circulation Pathophysiology Laboratory, Heart Institute (Incor), Sa˜o Paulo University Medical School, Sa˜o Paulo, Brazil
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Objectives: Experimental evidence shows that female sex hormones may exert protective effect on organ injury caused by trauma-hemorrhagic shock (T-HS). The intestinal ischemia/reperfusion (i-I/R) causes local and remote injuries similar to those found in the T-HS. Since estradiol appears as a mediator of protection against the organ injury after T-HS, in the present study we sought to analyze the role of estradiol on the control of leucocytes migration into gut after i-I/R. In parallel, the involvement of estradiol on the magnitude of bone marrow cells and blood leukocytes was also studied. Methods: The studies were performed in accordance to IACUC from the Institute of Biomedical Sciences, University of Sao Paulo. After 7 days of ovaries removal (OVx), Wistar rats (60 days old) were submitted to 45 min occlusion of the superior mesenteric artery, followed by 2 h reperfusion. A group of OVx rats received one single dose (280 mg/kg, s.c.) of estradiol 24 h before induction of i-I/R (i-I/R + E). OVx-sham i-I/ R rats were used as controls. Circulating leukocytes were quantified in blood samples using a hematological analyzer. Neutrophil recruitment to the gut was evaluated by myeloperoxidase (MPO) activity assay. During i-I/R the intestine of rats was packed in a plastic bag in order to collect the intestinal fluid to quantify the total and differential leucocytes by optical microscopy. Comparisons between groups were made by one-way ANOVA followed by Bonferroni post test. Results: Estradiol treatment reduced the percentage of increase (before and after i-I/R) of total white blood cell relative to non-treated i-I/R. In contrast, estradiol did not alter the neutrophil number in blood, whereas that of monocytes was increased compared to non-treated i-I/R group (iI/R = 522.7 ± 70.23; i-I/R + E=864.3 ± 102.5 monocytes/mm3, p = 0.0027). Bone marrow cell number was not altered by i-I/R. Interestingly, estradiol increased MPO activity in the intestine after i-I/R (i-I/R = 0.23 ± 0.024; i-I/R + E=0.4 ± 0.09; p = 0.0024). In comparison to non treated i-I/R group, estradiol reduced the number of mononuclear cells (i-I/R = 72 ± 4.9; i-I/R + E=55.56 ± 2.84 %, p = 0.0062) and increased those of neutrophils in intestinal fluid (OVx = 22.86 ± 2.95; E = 35.75 ± 2.33 %, p = 0,012). Conclusion: Our data suggest that during i-I/R, estradiol modulates differently the traffic of circulating leukocytes into gut and to intestinal cavity, likely controlling the local inflammatory response and, as consequence the remote organ injury observed after i-I/R. Ethics Committee number: 111/10/03—CEUA—ICB/USP. Financial support CAPES, CNPq and Fapesp (2013/15291-0).
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B185 ESTRADIOL AFFECTS THE LEUKOCYTE MOBILIZATION CAUSED BY INTESTINAL ISCHEMIA AND REPERFUSION IN MALE RATS Evelyn T. Fantozzi1, Fernanda Y Ricardo da Silva1, Sara Rodrigues Garbin1, Ricardo M. Oliveira Filho1, Bernardo B. Vargaftig1, Ana Cristina Breithaupt Faloppa2, Wothan Tavares de Lima1 1
Department of Pharmacology, ICB/USP, Sa˜o Paulo, Brazil; Cardiovascular Surgery and Circulation Pathophysiology Laboratory, Heart Institute (Incor), Sa˜o Paulo University Medical School, Sa˜o Paulo, Brazil
2
Objects: Intestinal ischemia and reperfusion (i-IR) induces local and remote organ injury characterized by leukocyte mobilization, increased microvascular permeability and systemic inflammation. Studies show female resistance to local repercussions of i-IR over male, which is attributed to sex hormones mechanisms, notably estradiol. However the mechanisms underlying the effects on male are not totally explored. Methods: The studies were performed in accordance to IACUC from the Institute of Biomedical Sciences, University of Sao Paulo. Anesthetized male rats (Wistar, 60 days old) were submitted to superior mesenteric artery occlusion (45 min), followed by reperfusion (2 h), during this period the rats had the intestine packed in a plastic bag. As control, sham operated animals (Sham) were used. Estradiol (17b) was given (280 mg/kg, s.c.) 24 h before induction of i-IR (E24). White blood cell (WBC) and bone marrow cell (BMC) count were assessed. Also, the intestinal fluid collected from the intestinal bag was analyzed for total and differential leucocyte counts (optical microscopy) and chemokines were quantified in the serum (Multiplex). Results: Comparisons between groups (n = 6–11), were made by one-way ANOVA followed by Bonferroni post test (p \ 0.05). WBC count did not differ between the groups. However, after i-IR BMC count decreased in comparison to Sham and E24 treatment increased the number of cells to similar levels found in Sham group (Sham = 51.83 ± 2.35; i-IR = 31.19 ± 4.08; E24 = 47.72 ± 4.91 9 106 cells/mL). The intestinal fluid analysis showed increased leucocytes count in i-IR group and E24 group decreased the cells (Sham = 30.31 ± 7.9; i-IR = 143.1 ± 24.34; E24 = 46.74 ± 12.97 9 104 cells/mL). Intestinal fluid in Sham group was predominantly neutrophilic, whereas in i-IR the number of mononuclear cells was predominant. Yet, E24 group increased the neutrophils (Mononuclear cells: Sham = 13.5 ± 1.55 vs i-IR = 72.6 ± 7.27 %; E24 = 58.83 ± 8.006; Neutrophils: Sham i-I/R = 87.4 ± 2.82; i-IR = 24.4 ± 6.4 %; E24 = 54.50 ± 9.021). Chemokines increased after i-IR, and were decreased in E24 group: IP-10 (Sham = 370.1 ± 19.43; i-IR = 988.3 ± 125.7; E24 = 598.1 ± 59.80 pg/mL), GMCSF (Sham = 0.89 ± 0.09; i-IR = 3.22 ± 0.75; E24 = 1.25 ± 0.18 pg/mL), GRO/KC (Sham = 203.9 ± 37.74; i-IR = 2248 ± 422.8; E24 = 855.6 ± 137.9 pg/mL), MIP-1a (Sham = 16.55 ± 1.3; i-IR = 234.7 ± 52.17; E24 = 97.20 ± 31.69 pg/mL), VEGF (Sham = 63.37 ± 12.45; i-IR = 119.6 ± 17.61; E24 = 68.96 ± 6.97 pg/mL). Conclusion: Our data suggest that i-IR mobilizes leukocytes from the bone marrow and increases the levels of chemokines in serum by a mechanism under control of estradiol. Thus, the previous treatment with estradiol may be of interest to control the local and systemic effects of ischemic events. Ethics Committee number: 111/10/03—CEUA—ICB/USP. Supported by CAPES, CNPq and FAPESP 2013/15291-0.
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B186 EXTRACELLULAR MICRORNA PROMOTES COMPLEMENT FACTOR B EXPRESSION VIA TLR7-MYD88 SIGNALING IN BACTERIAL SEPSIS Lin Zou, Ganqiong Xu, Yan Feng, Wenling Jian, Wei Chao Massachusetts General Hospital, Boston, MA, USA, Harvard Medical School, Boston, MA, USA Objective: Bacterial sepsis induces massive activation of the complement system including the alternative pathway (AP). Complement factor B (cfB) is an essential component of AP activation. We have recently demonstrated that cfB is the downstream effector of TLRs and plays a pivotal role in the pathogenesis of sepsis. However, the upstream mediator leading to cfB production in sepsis remains largely unclear. microRNA (miRNA) is a group of small non-coding RNAs that negatively regulate target gene translation. Host miRNAs are released into the extracellular space during bacterial sepsis. Whether or not miRNAs induce cfB production is unknown. Methods: Bacterial sepsis was created by cecum ligation and puncture (CLP) in mice. Plasma miRNAs were analyzed using microRNA array 24 h after sham or CLP surgery. Bone marrow-derived macrophages (M/) from WT or TLR3-/-, TLR7-/-, MyD88-/- mice were treated with 50 nM of miRNA mimics in the presence of lipofectamine for 18 h. Medium cfB, phospho-ERK1/2 and P38 were tested with Western blot. Three mg RNase was injected i.p. 1 h before and 12 h after surgery. Cardiac complement mRNA was tested by qRT-PCR. Results: Plasma miRNA array demonstrated that among the 68 miRNAs tested, six miRNAs had more than twofold increase in septic mice as compared with sham mice with fluorescence counts [100, namely miR-145, miR-146a, miR-122, miR-34a, miR-192 and miR210. Treatment with miRNA mimics miR-145, -146a, -34a, -122, but not miR-192 or -210, induced significant increase in cfB production in M/. This effect appeared to be specific because their UA mutants did not induce any increase in cfB expression. In M/ deficient of TLR7 or MyD88, these miRNA mimics failed to induce cfB production. In contrast, TLR3-deficiency had no impact on the effect of these miRNAs. In addition, miR-146a treatment led to phosphorylation of ERK1/2 and P38. TLR7-deficiency completely blocked this activation. To test the role of extracellular RNA in cfB production in animal model of sepsis, we tested cfB gene expression in the heart following sepsis. CLP led to a significant increase in cfB gene expression in the heart but had no impact on other complements tested, such as C3 and C5. Eliminating extracellular and circulating RNA by RNase administration led to a 46 % reduction in cfB gene expression, suggesting that extracellular RNA including miRNA may in part mediate cardiac cfB production during sepsis. Conclusion: We demonstrate that miRNAs induce cfB production in M/ via TLR7-MyD88-dependent mechanism. Polymicrobial infection leads to specific cardiac cfB gene expression and extracellular RNA in part mediates the cfB production in vivo.
B187 LUNG INFLAMMATION AND INDUCIBLE NITRIC OXIDE SYNTHASE AFTER BRAIN DEATH: PROESTRUS FEMALE VERSUS MALE DONORS Ana Cristina Breithaupt-Faloppa1, Sueli G. Ferreira1, Guilherme K. Kudo1, Roberto Armstrong Junior1, Wothan Tavares de Lima2, Paulina Sannomiya1, Luiz Felipe P. Moreira1
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Cardiovascular Surgery and Circulation Pathophysiology Laboratory, Heart Institute, Sao Paulo University Medical School (HC-FMUSP), Sao Paulo, Brazil; 2Department of Pharmacology, ICB/USP, Sao Paulo, Brazil
Objectives: Lung transplantation depends on heartbeating donors after brain death. Brain death (BD) is associated with inflammation, release of mediators and generalized ischemia–reperfusion injury, which is accompanied by upregulation of inducible nitric oxide synthase (iNOS) expression. Female sex hormones influence the lung inflammatory and immune responses after trauma and this raises questions relative to their influence on donor lung state after BD. In this study, we investigated the differences between male and female rats on the lung inflammation after brain death in rats. Methods: Groups of male (M), proestrus female (PF) (high estradiol secretion period) and ovariectomized (OVx) Wistar rats were submitted to BD by intracranial balloon catheter sudden inflation. BD was confirmed by maximally dilated and fixed pupils, apnea, absence of reflexes, and a drop in mean arterial pressure (MAP). After 6 h, lung samples were collected and the iNOS gene expression was analysed. Lung sections were analysed by histology and the iNOS expression by immunohistochemistry. Lung myeloperoxidase activity (MPO) was determined and vascular permeability (VP) assessed using the Evans blue dye extravasation method. Estradiol (E) and progesterone (P) serum levels were quantified. Results: After 6 h of BD, E and P concentrations in serum of proestrus female rats were significantly reduced (E initial: 60.51 ± 1.74, 6 h: 0.89 ± 0.5 pg/mL, p \ 0.0001; P initial: 1362 ± 379, 6 h: 231 ± 104 ng/mL, p = 0.0017). Proestrus female rats presented augmented MPO (PF = 0.837 ± 0.029 versus M = 0.724 ± 0.017; p = 0.028) and VP (PF = 171.9 ± 10.27 versus M = 104.9 ± 8.35; p = 0.0017). iNOS relative gene expression was significantly higher in the proestrus female in comparison to other groups (PF = 701.2 ± 223.3, OVx = 37.68 ± 5.05, M = 191.4 ± 23.77; p = 0.0046). In relation to leukocyte infiltration to the lung, we did not find significant differences among the groups, but the iNOS protein expression was higher in the lungs of the proestrus female group compared with other groups. Conclusion: The data evidenced important differences between genders after BD with higher lung inflammatory compromise in proestrus female rats, which have the hormone levels acutely reduced. There is evidence that estradiol limits the induction of iNOS, thus we suggest that the estradiol reduction might lead to higher lung iNOS expression and inflammation. The results are also consistent with the notion that female sex hormones could influence the result of lung transplant and should be considered as important elements for the maintenance of donor lung status. Financial support Sao Paulo Research Foundation (FAPESP 2013/20282-0).
B188 EFFECTS OF Co-ultraPEALut ON INFLAMMATORY EVENTS ASSOCIATED WITH TRAUMATIC BRAIN INJURY Rosalia Crupi1, Giuseppe Bruschetta1, Irene Paterniti1, Rosalba Siracusa1, Marika Cordaro1, Emanuela Esposito1, Salvatore Cuzzocrea1,2 1
Department of Biological and Environmental Sciences, University of Messina, Viale Ferdinando Stagno D’Alcontres 31-98166 Messina, Italy; 2Manchester Biomedical Research Centre, Manchester Royal Infirmary, School of Medicine, University of Manchester, UK
Inflamm. Res. Introduction: Traumatic brain injury (TBI) is considered important burdens to society and remain challenging to diagnose, manage, and treat. His diagnosis includes a broad range of short- and long-term physical, cognitive, and emotional impairments. It is characterized by neurological dysfunction, due to the progressive destruction of local and distal neuronal networks, resulting from injury-induced tissue damage and subsequent local, cellular, and biochemical reactions. The neuroinflammatory cascade contributes to neuronal damage and behavioral impairment. N-palmitoylethanolamide (PEA) is an endogenous fatty acid amide belonging to the family of the N-acylethanolamines (NAEs). PEA is an important analgesic, antiinflammatory and neuroprotective mediator, acting at several molecular targets in both central and sensory nervous systems as well as immune cells. However, PEA lacks a direct antioxidant capacity to prevent the formation of free radicals, and to counteract the damage of DNA, lipids and proteins. Luteolin (Lut), a common flavonoid present in many plants, has strong antioxidant and pharmacological activities, including a memory-improving effect. It displays excellent radical scavenging and cytoprotective properties, particularly when tested in complex biological systems where it can interact with other antioxidants, such as vitamins. Lut displays specific anti-inflammatory effects, which are only partly explained by its antioxidant capacities. Aim: In the present study, we performed a widely-used model of TBI to determine the neuroprotective propriety of palmitoylethanolamide (PEA) and the antioxidant effect of a flavonoid luteolin (Lut), given as a co-ultramicronized compound Co-ultraPEALut. Methods: TBI was induced in mice by controlled cortical impactor. Co-ultraPEALut (1 mg/kg, soluble 10 % ethanol, i.p.) were administered 1 h after craniotomy. At 24 h after TBI, the brains were collected. Results: We demonstrated that the treatment with Co-ultraPEALut resulted in a significant improvement of motor and cognitive recovery after controlled cortical impact (CCI), as well as markedly reducing lesion volumes. Moreover, our results revealed the ability of Co-ultraPEALut, to reduce brain trauma through modulation of NF-jB activation. In addition, treatment with Co-ultraPEALut significantly enhanced the post-TBI expression of the neuroprotective neurotrophins GDNF compared to vehicle. Co-ultraPEALut at the dose of 1 mg/kg, also modulated apoptosis, the release of cytokine and ROS, the activation of chymase, tryptase and nitrotyrosine. Conclusions: Thus, our data demonstrated that Co-ultraPEALut at a lower dose compared to PEA alone, can exert neuroprotective effects and the combination of both could improve their ability to counteract the neurodegeneration and neuroinflammation induced by TBI.
Inflammatory Responses, Stem Cells and Tissue Regeneration B189 FACTORS SECRETED FROM DENTAL PULP STEM CELLS SHOW MULTIFACETED BENEFITS FOR TREATING ACUTE LUNG INJURY IN MICE Hirotaka Wakayama1, Naozumi Hashimoto2, Yoshihiro Matsushita1, Noriyuki Yamamoto1, Masaya Nishikawa1, Yoshinori Hasegawa2, Akihito Yamamoto1 1 Department of Oral and Maxillofacial Surgery of Nagoya University, Nagoya, Japan; 2Department of Respiratory Medicine of Nagoya University, Nagoya, Japan
S177 Background and objectives: Acute respiratory distress syndrome (ARDS) is a severe inflammatory disorder characterized by acute respiratory failure, resulting from severe, destructive lung inflammation and irreversible lung fibrosis. Here, we evaluated the use of stem cells derived from human exfoliated deciduous teeth (SHEDs) or SHED-derived serum-free conditioned medium (SHED-CM) as treatments for bleomycin (BLM)-induced mice acute lung injury (ALI), exhibiting several pathogenic features associated with the human disease ARDS. Methods: Mice with BLM-induced ALI with or without SHED or SHED-CM treatment were examined for weight loss and survival. The lung tissue was characterized by histological and real-time quantitative PCR analysis. The effects of SHED-CM on macrophage differentiation in vitro were also assessed. Results: A single intravenous administration of either SHEDs or SHED-CM attenuated the lung injury and weight loss in BLM-treated mice, and improved their survival rate. Similar recovery levels were seen in the SHEDs- and SHED-CM-treatment groups, suggesting that SHED improves ALI by paracrine mechanisms. SHED-CM contained multiple therapeutic factors involved in lung-regenerative mechanisms. Importantly, SHED-CM attenuated the BLM-induced proinflammatory response and generated an anti-inflammatory/tissueregenerating environment, accompanied by the induction of anti-inflammatory M2-like lung macrophages. Furthermore, SHED-CM promoted the in vitro differentiation of bone marrow-derived macrophages into M2-like cells, which expressed high levels of Arginase1, CD206, and Ym-1. Discussion and conclusions: Taken together, our results suggest that SHED-secreted factors provide multifaceted therapeutic effects, including astrong M2-inducing activity, for treating BLM-induced ALI. This work may open new avenues for research on stem-cellbased ARDS therapies.
B190 PROTECTIVE EFFECT OF HEMATOPOIETIC STEM CELLS IN STROKE Felicity NE Gavins, Helen K. Smith, Shantel Vital, D.Neil Granger LSUHSC-S, Shreveport, LA, USA Increasing evidence suggests that stem cells may be beneficial as a treatment for stroke. We investigated the effects of hematopoietic stem cells (precursors to cells of the blood) in a mouse model of cerebral ischemia and reperfusion (I/R). Mice underwent surgical middle cerebral artery occlusion (MCAo) for 30 min using a 6-0 nylon filament, which was then removed to induce cerebral I/R. 24 h later, mice were injected with 1 million lineage negative bone marrow cells (Lin- BMCs, hematopoietic cell precursors which expressed no markers of differentiation), derived from donor mice. After the following 24 h (i.e. 48 h post cerebral I/R) and up to 2 weeks, various improvements in Lin- BMC-treated mice versus vehicle-treated mice were assessed through outcome parameters including: mortality rates, infarct volume, stroke score (behavioral assessment) and levels of inflammation (as indicated by leukocyte-endothelial interactions). All of these were significantly reduced in Lin- BMC versus vehicle-treated mice. In addition to a large drop in mortality (from 50 to 7 % in Lin- BMC and vehicle-treated mice, respectively), results showed a clear improvement in infarct volume, leukocyte activity and stroke score, which were all reduced by reduced by at least 50 % at 48 h reperfusion and \95 % by 1 week (p \ 0.05). After establishing their efficacy, it is important to investigate the differentiation of transplanted cells and/or their release of protective
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S178 substances after stroke; we therefore aimed to identify the mechanism by which the cells were of benefit. The observed reduction in leukocyte activity indicated a possible immunomodulatory mechanism. As such, brain slices were stained for Iba-1 to indicate the presence of activated microglia—previously identified to be of significance in mediating damage after stroke. These images showed a 50 % reduction in activated microglia, both contra- and ipsilaterally, in mice treated with Lin- BMCs versus vehicle. Microglia activation and an excessive inflammatory response have consistently been associated with poor outcome following stroke, therefore there results suggest a way in which hematopoietic stem cells may be beneficial as a treatment. In conclusion, not only have we provided significant and broad evidence of the therapeutic effects of hematopoietic stem cells in stroke (including improved mortality, infarct volume, levels of inflammation and functional recovery), but we have given a primary insight into their immunomodulatory potential by demonstrating a down-regulation of microglial activation. We believe our data will contribute to the development of an optimized stroke therapy based on the use of stem cells.
Inflamm. Res. appeared to be at 25 mg/mL. When urticaria was induced with 10 mg/ mL histamine, Allegra slightly inhibited both dermal irritation and wheal and flare, whereas Cortaid inhibited the wheal and flare only. When urticaria was induced with 25 and 50 mg/mL histamine, Allegra as well as Cortaid inhibited both dermal irritation and wheal and flare. The inhibitory effects of Allegra and Cortaid were observed at 25 and 45 min post dose. Histamine at 50 mg/mL was shown to induce urticaria with sustained dermal irritation and wheal and flare in Hanford miniature swine. Conclusion: A histamine induced urticaria model has been established with the female Hanford miniature swine and can be used for the testing of topical treatments for dermal irritation and inflammation.
B192 DOES SUBSTANCE P PLAY A ROLE IN ALLERGIC CONTACT DERMATITIS? Francis FY Lam, Ethel SK Ng, Nick H. Ng The Chinese University of Hong Kong, Hong Kong, China
Inflammatory Skin Disorders B191 DEVELOPMENT OF A MODEL OF DERMAL INFLAMMATION AND IRRITATION (URTICARIA) IN THE MINIATURE SWINE Miao Zhong, Alain Stricker-Krongrad, Jason Liu, Guy Bouchard Sinclair Research Center, LLC, Auxvasse, USA Background: Urticaria is dermal edema that results from vascular dilation and leakage of fluid into the skin in response to molecules released from mast cells. Histamine, the major mediator of mast cells, is known to induce a short lived urticaria when applied intra-dermally in human. Unlike other animals, the swine has a fixed skin tightly attached to the subcutaneous tissues similar to that in humans, which makes it a preferable model for dermal studies. The effect of histamine to induce urticaria in pig has not been investigated. Objectives: To develop a reproducible dermal urticaria model in miniature swine. Methods: Three female Hanford (3–18 months old) were used in the study. The animals were pricked on their back skins with a skin test device (Lincoln Diagnostics Inc, Decatur, IN) that were loaded with vehicle or histamine in vehicle. The irritation and wheal and flare responses of the dermis were evaluated with Draize scoring and with wheal size measurement. To investigate the time and dose responses of histamine, dermal reactions were evaluated at 10 and 20 min post prick. To evaluate the abilities of cortaid (1 % hydrocortisone) and allegra (2 % diphenhydramine) creams to block the histamine induced dermal urticaria, animals were challenged with histamine for 15 min before being treated with test formulations. The biological reactions of skin were assessed at 10, 25, and 45 min after test formulation application. Results: Histamine dose-dependently induced skin irritation at both 10 and 20 min after treatment. The most prominent erythema and edema (Draize score) responses were observed at 10 min after treatment, which were slightly diminished at 20 min after treatment. Histamine also caused skin wheal that ranged between 4 and 7 mm in diameter. Although wheal sizes increased over time following treatment, this change in wheal size appeared not related to histamine effect. The optimal concentration of histamine to induce urticaria
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Objective: Allergic contact dermatitis (ACD) is a common inflammatory skin disease. It is a hypersensitive reaction to allergens that causes rash or skin lesion at the site of exposure. Substance P(SP) is a sensory neuropeptide known to be involved in neurogenic inflammation. In this study, the importance of SP in the development of ACD was investigated. Methods: Male 6-week-old Balb/c mice were used in the present studies. Test drugs were applied topically on to one ear of eachmouse for 6 times over a course of 10 days to induce ACD. The contralateral ear received parallel vehicle application to serve as an internal control. Agents tested as inducers of ACD include the standard ACD inducer dinitrofluorobenzene (DNFB), SP, and capsaicin (anagent that can deplete sensory neuropeptides). Agents tested as inhibitors of ACD include capsaicin, RP67590 (a NK1 receptor antagonist), and dexamethasone (an anti-inflammatory glucocorticoid). Results: Topical application of DNFB increased the thickness and weight of the mice ears, indicating successful induction of ACD. Microscopic examination confirmed spongiosis and inflammatory cells in the affected ears. The same treatment with SPor capsaicin did not produce ACD. The symptoms of ACD induced by DNFB were suppressed by dexamethasone and capsaicin, but not by SP or the NK1 receptor antagonist RP67580. Conclusions: The present findings do not support a role for SP in the development of ACD in mice. However, similar to dexamethasone, capsaicin produced marked inhibition on the ACD symptoms induced by DNFB. This is unexpected since SP is not involved in the development of ACD. The mechanisms of the inhibitory effects of capsaicin on ACD remain to be elucidated.
B193 AGGREGATION OF THROMBIN C-TERMINAL FRAGMENTS—A NOVEL HOST DEFENSE MECHANISM Jitka Petrlova, Finja Hansen Division of Dermatology and Venereology, Departments of Clinical Sciences, Lund University, Lund, Sweden
Inflamm. Res. Host defense peptides (HDPs) have important roles in the first line of defense against invading microorganisms in blood and at epithelial surfaces. Thrombin is a key enzyme in the coagulation cascade, a fundamental host defense system, activated during injury or infection. We have previously reported that human neutrophil elastase (HNE) cleaves thrombin, generating an 11 kDa major fragment, denoted thrombin-derived C-terminal peptide (TCP), with antibacterial effects. Here, we generated recombinant TCP (rTCP) and applied biophysical and microbiological techniques to determine its mode of action and interaction with lipopolysaccharide (LPS). Our novel data demonstrate that LPS induces aggregation of rTCP. Amorphous rTCP containing aggregates were detected by negative stain electron microscopy, and an increase of b-sheet structure was identified by circular dichroism spectroscopy and a thioflavin T1 assay. Similar results were obtained using intact thrombin digested by HNE. Furthermore, antimicrobial effects of rTCP against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa were demonstrated, and found to be mediated by rTCP-induced bacterial aggregation. In vivo, a colocalization of C-terminal thrombin fragments and LPS was detected in aggregates present in fibrin sloughs, obtained from patients with infected wounds, indicating the presence of LPS-TCP-aggregates at physiological conditions. Furthermore, generation of C-terminal thrombin fragments of similar molecular size as TCP, were detected in acute wound fluid. Taken together, our in vitro and in vivo data disclose previously unknown host defense mechanism of TCPs, involving LPS induced aggregation/scavenging and microbial killing, thus providing a protection against endotoxins and pathogens at the wound site.
S179 Results: In the RAW cells, KJ extract inhibited LPS-induced inflammatory cytokines (TNF-a, IL-6 and IL-1b) production. Furthermore, KJ extract reduced skin hyperplasia and expressions of IL6, IL-1b and MCP-1 on dorsal skin of mice models. Conclusion: It was proved that the KJ extract inhibited the secretions of pro-inflammatory cytokines in LPS-stimulated RAW cells. Moreover, KJ extract applications on the dorsal skins of the DNCBinduced CD mice reduced the thickness of the epidermis and dermis by decreasing the secretions of inflammatory cytokines and chemokines. These findings not only indicate that applications of KJ extract may lessen skin thickening of CD by inhibiting the expressions of inflammatory mediators, but also suggest that it is a natural option for the treatment of skin inflammation.
B195 BEYOND THE ADRENAL GLAND: THE ESSENTIAL ROLE OF LOCALLY SYNTHESISED GLUCOCORTICOIDS IN REGULATING INFLAMMATION IN HEALTHY SKIN AND PSORIASIS Rosalind F. Hannen1, Chinedu Udeh-Momoh2, Mike Wright3, David Halsall3, Rod Flower1, Lisa Sevilla4, Stafford Lightman2, Paloma Perez4, Mike Philpott1 Queen Mary University of London, London, UK; 2University of Bristol, Bristol, UK; 3Addenbrookes Hospital, Cambridge, UK; 4 Instituto de Biomedicina de Valencia, Valencia, Spain 1
B194 KORTHALSELLA JAPONICA ALLEVIATES THE DERMAL AND EPIDERMAL HYPERPLASIA IN A CONTACT DERMATITIS MICE MODEL Jinju Kim Department of Korean Physiology, College of Pharmacy, Kyung Hee University, Seoul, South Korea Aim of the study: Contact dermatitis (CD) is skin inflammation that is characterized by redness, swelling, heat, and itching. During the occurrence of skin inflammation, macrophages accumulate at the specific skin sites and secrete many inflammatory mediators, including cytokines and chemokines. These inflammatory mediators promote a wide range of inflammatory responses, such as itchiness and skin thickening. Korthalsella japonica (KJ) is a traditional medicinal plant, which has various biological and pharmacological activities. In this study, we investigated anti-inflammatory effect of (KJ) ethanol extract in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and 2, 4-dinitrochlorobenzene (DNCB) induced skin inflammation mice models. Materials and methods: RAW 264.7 cells were pretreated with KJ extract for 1 h, and then stimulated with LPS for 24 h. The antiinflammatory activity of KJ extract was determined by measuring production of tumor necrosis factor-a (TNF-a), interleukin (IL)-6 and IL-1b. In the in vivo study, BALB/c mice were sensitized by topically applying 100 ll of 1 % DNCB (in acetone: olive oil = 4:1) on shaved dorsal skin on days 1–3. Four days later, the mice were resensitized by applying dorsal skin with 100 ll of 0.5 % DNCB for 2 weeks. KJ extract was treated on before 2 h of DNCB application. Thickness of the epidermis and dermis were determined by skin histological examination. IL-6, IL-1b and monocyte chemoattractant protein-1 (MCP-1) synthesis were analyzed using ELISA.
Endogenous glucocorticoids (GCs) are essential for maintaining the epidermal skin barrier and their powerful anti-inflammatory effects are exploited therapeutically to treat inflammatory skin conditions such as psoriasis. Skin contains the enzymatic machinery required for synthesising GCs from cholesterol and local cortisol production has been observed by healthy human skin (1–4). However, little is known about the interplay between local and systemic GC production and how this impacts on skin pathologies. Here we show that both de novo cortisol synthesis and GC receptor expression are severely compromised in psoriatic skin. Specifically, de novo cortisol synthesis was reduced to less than 10 % production in both non-lesional and lesional psoriatic tissue compared to healthy skin controls (healthy skin 809.6 ± 120.4 ng/mL, psoriatic non-lesional 63.1 ± 7.9 ng/mL, psoriatic lesional 67.7 ± 11.8 ng/ mL, n = 8, p = 0.001 as measured by LC–MS/MS). In addition, radiometric assay demonstrated that cultured primary psoriatic keratinocytes also exhibited dysfunctional cortisol synthesis (healthy keratinocytes, 38.8 ± 6.1 %, uninvolved keratinocytes 8.7 ± 0.4 %, lesional keratinocytes 8.6 ± 0.2 % of cortisol formed from [3H]pregnenolone after 24 h, n = 4, p = 0.01). This suggests that defective GC synthesis is an inherent defect of psoriatic skin. Expression of the GC receptor was reduced in non-lesional psoriatic tissue and was further down-regulated in lesional psoriatic skin. Experiments using adrenalectomised wild type (WT) and GR epidermal knockout (GREKO) mice highlighted the significance of systemic vs local GC production in experimental model of skin inflammation induced by topical PMA. Remarkably, adult GREKO mice compensated for loss of GR by up-regulating localized GC production and this protected the animals from PMA induced IL-1b/ IL-6/TNF-a generation, even post-adrenalectomy. These studies demonstrate that skin-derived GC production is capable of protecting the tissue from inflammation in the absence of systemic GCs. Crucially this pathway is defective in psoriatic skin
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S180 and therefore presents a critical pathway to target in this inflammatory skin condition. References 1. Hannen, R.F., Michael, A.E., Jaulim, A., Bhogal, R., Burrin, J.M., and Philpott, M.P. 2011. Steroid synthesis by primary human keratinocytes; implications for skin disease. Biochem Biophys Res Commun 404:62–67. 2. Vukelic, S., Stojadinovic, O., Pastar, I., Rabach, M., Krzyzanowska, A., Lebrun, E., Davis, S.C., Resnik, S., Brem, H., and TomicCanic, M. 2011. Cortisol Synthesis in Epidermis Is Induced by IL-1 and Tissue Injury. J Biol Chem 286:10265–10275. 3. Cirillo, N., and Prime, S.S. 2011. Keratinocytes synthesize and activate cortisol. J Cell Biochem 112:1499–1505. 4. Slominski, A., Wortsman, J., Tuckey, R.C., and Paus, R. 2007. Differential expression of HPA axis homolog in the skin. Mol Cell Endocrinol 265–266:143–149.
B196 PHARMACOLOGICAL VALIDATION OF THE MOUSE IMIQUIMOD (IMQ)-INDUCED PSORIASIS MODEL Harunor Rashid, Fraser McIntosh, Agathe Bedard, Luc Chouinard, Joe Cornicelli, Rana Samadfam Charles River Labs, Wilmington, USA Background: Psoriasis is an immune-mediated inflammatory disease that mainly affects skin and is characterized by thickening of epidermis and red scaly patches. A recent article (van der Fits et. al. J Immunology 2009; 182:5836–5845) suggested development of psoriasis-like skin lesions in mice by application of TLR7/8 ligand, imiquimod (IMQ). Objective: The aim of the present study was to further characterize the IMQ-induced psoriasis model in mice using both pharmacological and histological approaches. Methods: Male BALB/c mice were purchased from Charles River Canada and acclimated for 7–8 days. On the Day 1 of the study, skin was shaved and baseline thickness measurements were taken before randomizing animals into different treatment groups. IMQ cream (5 %, 62.5 or 47 mg) or control Vaseline cream was then applied on back skin and/or ear from Day 1 to Day 5. From Day 2 to Day 6, back skin and/or ear thickness was measured using an engineering caliper and visual scoring was done for extent of erythema and scales. On Day 6, animals were euthanized and back skin and/or ear tissues were collected for histopathological evaluation. For assessing pharmacology with commonly used anti-psoriatic agents, effects of the following drugs were examined: dexamethasone (0.3 and 3 mg/kg, oral gavage, Day 1–6, QD), clobetasol (0.05 % cream, topical, Day 1–6, QD), etanercept (10 mg/kg, SC, Day 1 and 3) and IL17A antibody (100 ug per mouse, SC, Day -1, 2 and 4). Results: Application of 5 % IMQ cream caused erythema, scaling and thickening of skin starting from Day 2 and these effects peaked at Day 6. Effects on back skin seemed more pronounced than on the ear, and thus only back skin was chosen for IMQ cream application for subsequent studies. In histopathological evaluation of skin samples collected terminally, epidermal thickness was measured using Aperio or Image-Pro system, and IMQ cream was found to increase epidermal thickness (acanthosis) very similar to the in-life results. 5 % IMQ cream also caused parakeratosis and inflammatory infiltration in the skin tissues. Among the reference drugs, 0.05 % clobetasol cream almost completely blocked IMQ-induced skin lesions while dexamethasone had moderate effects at 3 mg/kg dose. Etanercept and antimouse IL17 antibody had no effects at the doses tested.
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Inflamm. Res. Conclusions: Overall, 5 % IMQ was found to consistently cause psoriasis-like skin lesions in mice as evaluated using both in-life and histopathological methods. Steroidal agents clobetasol propionate, which is the most commonly used drug for skin disorders including psoriasis, and dexamethasone significantly reduced IMQ-induced skin changes in mice. Antibodies against TNF-alpha and IL17A were barely effective although only a single dose of each was tested and thus further studies with higher doses of these clinically used antipsoriatic agents are warranted.
B197 RIP KINASE SIGNALING IN KERATINOCYTE CONTROLS NECROPTOSIS—MEDIATED SKIN INFLAMMATION Snehlata Kumari, Manolis Pasparakis CECAD Research Centre, Institute for Genetics, University of Cologne, Germany Receptor interacting kinase 1 (RIPK1) is involved in cell death and inflammation. We used conditional targeting to investigate the role of RIPK1 in epidermal keratinocytes. Mice with epidermis-specific RIPK1 deletion (RIPK1E-KO) develop a progressive inflammatory skin disease starting after six to seven postnatal days resulting in severe cutaneous inflammation by the age of 3–4 weeks. The skin lesions of RIPK1E-KO mice are characterized by non-cell autonomous epidermal hyperproliferation, infiltration of immune cells and upregulation of pro-inflammatory cytokines such as IL-1b, IL-33 and TNF. We identified increased numbers of apoptotic and necrotic keratinocytes in the epidermis of RIPK1E-KO mice and reasoned that keratinocyte death could trigger inflammation. To specifically inhibit necroptosis, we generated RIPK1E-KO mice lacking Receptor interacting kinase 1 (RIPK3) or Mixed lineage kinase domain-like (MLKL) gene, which are essential for necroptosis. We found that the RIPKE-KO; Ripk3-/- or RIPKE-KO; Mlkl-/- double deficient mice, did not show keratinocyte necrosis and did not develop inflammatory skin lesions, demonstrating that inhibition of necroptosis prevented skin inflammation. Furthermore, deficiency of TNFR1, partially protected RIPK1E-KO mice from keratinocyte death and inflammation showing that TNFR1 signaling contributes to the induction of necroptosis. Our study identified a unique role of epithelial RIPK1 signaling in the maintenance of skin homeostasis and the prevention of skin inflammation. Importantly, knock-in mice expressing a kinase inactive RIPK1 allele did not develop skin lesions showing that kinase independent RIPK1 functions regulate keratinocyte survival and skin inflammation. Collectively, these results identified RIPK1-medited inhibition of necroptosis as a key mechanism for the maintenance of skin homeostasis and the prevention of inflammation, suggesting that keratinocyte necroptosis could be relevant for the pathogenesis of human inflammatory skin diseases.
B198 IL-1b PRODUCING MONOCYTE DERIVED DENDRITIC CELLS ARE CRITICAL FOR IL-23INDUCED SKIN INFLAMMATION Tej Pratap Singh1, Howard H. Zhang1, Michael N. Hedrick1, Brian L. Kelsall1, Bjorn E. Clausen2, Joshua M. Farber1 1
Laboratory of Molecular Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD,
Inflamm. Res. USA; 2Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, 55131 Mainz, Germany Human studies have strongly implicated IL-23 in psoriasis, and injecting IL-23 in mouse skin produces psoriasis-like inflammation. IL-23 injection leads to accumulation of dendritic cells (DCs), which are also abundant in psoriatic skin. In analyzing myeloid cells, we found monocyte-derived langerin-positive cells (moLCs) appearing in the epidermis, and increased numbers of other monocyte-derived cells, including monocyte-derived dermal DCs (moDDCs) and macrophages, plus non-monocyte derived DCs (cDCs) in the dermis after IL-23 injection. Depletion of all CD11c+ cells in diphtheria toxintreated CD11c-DTR mice blocked IL-23-induced IL-22, IL-19, IL-17, IL-36, IL-1b and s100A, as well as the psoriasis-like changes. IL-22 and IL-17 are known to be important mediators of pathology in this model. Using Il1r1-/- mice, we also identified a major role for IL-1, which we found was produced by moLCs and moDDCs but not cDCs. Despite the dramatic effects of depleting CD11c+ cells, Batf3-/-, diphtheria toxin-treated Bdca2-DTR, and Flt3l-/- mice, which lack CD103+ cDCs, pDCs, and all cDCs and pDCs, respectively, each showed little protection against IL-23-induced changes. Although depleting conventional LCs alone had no effect, inflammation was much diminished in langerin-DTR mice also depleted of moLCs. Furthermore, depleting Ly6C+ monocytes but not neutrophils significantly reduced the moLC and moDDC accumulation in skin, IL-22, IL-17A/F and IL-1b production, and skin inflammation. Together, these data suggest that CD11c+ moDDCs and moLCs contribute significantly to pathology. Previously, we have reported that Ccr6-/mice are resistant to IL-23-induced skin inflammation, and the skin of Ccr6-/- mice showed no changes in numbers of CD11c+ cells in dermis or epidermis after IL-23 injection. Repeated and selective depletion of Ccr6+/+ CD11c+ cells using mice reconstituted with a mixture of bone marrows from Ccr6-/- and (Ccr6+/+) CD11c-DTR mice revealed that CCR6 was required for the recruitment and/or activity of the pathologic CD11c+ cells or their precursors. Using Ccr6-EGFP mice, we identified expression of Ccr6 in blood monocytes and we demonstrated that monocytes migrate to the CCR6 ligand, CCL20. Importantly, we found that although both Ccr6+/+ and Ccr6-/- monocytes restored inflammation when injected, together with IL-23, into ears of Ccr6-/- mice, only the Ccr6+/+ monocytes were effective if injected intravenously. Together, our data suggest that blood monocytes are recruited to inflamed skin using CCR6 and give rise to moDDCs and moLCs, which produce IL-1b and are critical for IL-23-induced psoriasis-like pathology.
Inhibitory Receptors and Immune Checkpoints B199 THE ANTI-INFLAMMATORY ACTIVITY OF THE NUCLEAR RECEPTOR PXR AND THE MINOR ISOFORM PXR3 Jerusalem Alleyne, Andrew Bennett FRAME laboratory, School of Biomedical Sciences, University of Nottingham, UK Objective and design: The major isoform of the Pregnane X receptor (PXR1) controls the expression of genes involved in liver detoxification. Recent findings have revealed that PXR1 represses inflammatory pathways. The minor isoform PXR3 is transcriptionally inactive and its role is unknown. It is believed that PXR1 undertakes
S181 an atypical association with regulatory proteins to repress gene expression. This mechanism is termed transrepression and is thought to be the consequence of the post-translational modification (PTM) of PXR1 by SUMO (Small Ubiquitin-Like Modifier). This work sought to investigate the link between SUMO and the anti-inflammatory role of the PXRs. Additionally, the possible anti-inflammatory function of PXR3 was examined. This is important since PXR gene mutations are linked to the onset of inflammatory bowel disease. Materials and methods: Site-directed mutagenesis was used to create PXR1/PXR3 Lys to Arg mutants at predicted sumoylation sites. The human Il-8 and mouse iNOS reporters, possessing NF-jB response elements were induced by lipopolysaccharide (LPS) in RAW264 macrophages. The activity of these reporters in the presence of the PXR1/PXR3 mutants was then measured using luciferase reporter assays. Next, each wild type PXR1/PXR3 or putative SUMO site mutant was transiently co-transfected into HELA cells with His-tagged SUMO1, SUMO2 or SUMO3. Nickel pull down assays and immunoblotting were then performed to probe for the presence of PXR. Results and discussion: In the presence of wild type PXR1/PXR3, LPSmediated induction of the human Il-8 (Interleukin-8) and mouse iNOS (Inducible nitric oxide synthase) reporters were repressed close to basal levels of promoter activity. PXR1 SUMO mutants repressed LPS-induced IL-8 activity but to a lesser extent than wt PXR1. However, repression of this activity by the PXR3 SUMO mutants was comparable to that of wt PXR3. This may indicate the occurrence of sumoylation events at multiple sites within PXR and that the mutation of one single site is insufficient to prevent sumoylation. Nickel pull down assays confirmed previous findings that PXR1 was conjugated by each of the SUMO proteins. This was also true for PXR3. Conjugation of proteins with SUMO like other PTMs, alters the functions of proteins. The sumoylation of both PXR1 and PXR3 could help elucidate how PXR acquires this anti-inflammatory ability in addition to its transactivation function. Importantly, the repressive activity and sumoylation of PXR3 are novel findings and may specify a role for PXR3 in inflammation. In fact, PXR1 and PXR3 are expressed in many of the same tissues, therefore the anti-inflammatory activity attributed to PXR1 may actually be attributed to both PXR1 and PXR3.
Innate Immunity—Macrophages, Dendritic Cells, Neutrophills, Basophils, Mast Cells, Eosinophils B200 ROLE OF PROSTAGLANDIN D2 IN EOSINOPHIL ACTIVATION INDUCED BY LEPTIN Nata´lia Tasmo-Amorim1, Ludmilla Dellatorre-Teixeira1, Tatiana Luna-Gomes1, Patricia Bozza2, Clarissa Maya-Monteiro2, Christianne Bandeira-Melo1 1
Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; Fundac¸a˜o Oswaldo Cruz, Rio de Janeiro, Brazil
2
Eosinophils are classically associated with allergic diseases and helminth infections. Recently, immunomodulatory roles of eosinophils have been described, such as the ability of adipose tissue-resident eosinophils to regulate adipose macrophage functions and tissue hemostasis. Leptin, a hormone/cytokine produced by adipocytes, is a survival factor for eosinophils, which are known to express its receptor. Inasmuch as eosinophils are known to be cell sources of an autocrine/paracrine functionally active prostaglandin D2 (PGD2), the aim of this study was to evaluate the role of eosinophil-derived PGD2 in leptin-induced eosinophil activation. For in vivo assays, mouse
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S182 pleurisy was triggered by intrapleural injection of leptin. Within 24 h, in parallel to eosinophil influx and activation (characterized by eosinophil lipid body biogenesis and increased levels of pleural LTC4), we found increased levels of PGD2 in the pleural cavity of leptin-stimulated mice. PGD2 appeared to be a key mediator of leptin-induced eosinophil activation in vivo, since the pre-treatment with HQL-79, an inhibitor of hematopoetic PGD synthase, inhibited both lipid body biogenesis within eosinophils and LTC4 synthesis. Accordinlgy, using either human eosinophils purified from healthy donors or bone marrow-differentiated mouse eosinophils, leptin was also able to trigger PGD2 synthesis in vitro as detected in eosinophil supernatants within 1 h of stimulation. In vitro HQL-79 pre-treatment inhibited both eosinophil lipid body biogenesis and LTC4 synthesis elicited by leptin. Our data clearly uncover the ability of leptin to activate PGD2 synthesizing machinery in both mouse and human eosinophils. Moreover, such leptin-driven eosinophil-derived PGD2 displays autocrine/paracrine regulatory role in leptin-induced eosinophil activation. Inasmuch as PGD2 is now emerging as a immunomodulatory molecule, our findings may indicate potential functions on obesity and other inflammatory disorders.
B201 INTERNALIZED CRYPTOCOCCUS NEOFORMANS ACTIVATES NOT ONLY THE CANONICAL CASPASE-1 BUT ALSO THE NONCANONICAL CASPASE-8 INFLAMMASOMES Guangxun Meng1, Mingkuan Chen1, Yue Xing1, Ailing Lu1, Wei Fang2, Wanqing Liao2 1
Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China; 2Shanghai Key Laboratory of Molecular Medical Mycology, Changzheng Hospital, Shanghai, China Cryptococcus neoformans (C. neoformans) is an opportunistic fungal pathogen which causes Cryptococccosis in immunocompromised patients as well as immunocompetent individuals. Host cell surface receptors that recognize C. neoformans have been widely studied. However, intracellular sensing of this pathogen is still poorly understood. Our previous studies have demonstrated that both biofilm and acapsular mutant of C. neoformans are able to activate the NLRP3 inflammasome. In the current study, it was found that opsonization mediated internalization of encapular C. neoformans also activated the canonical NLRP3-ASC-caspase-1 inflammasome. In addition, the internalized C. neoformans activated the noncanonical NLRP3-ASCcaspase-8 inflammasome as well, which resulted in robust IL-1b secretion and cell death from caspase-1 deficient dendritic cells. Interestingly, we found that caspase-1 was inhibitory for the activation of caspase-8 in dendritic cells upon C. neorformans challenge. Further mechanistic studies showed that both the phagolysosome membrane permeabilization and potassium efflux were responsible for C. neoformans induced activation of either the canonical NLRP3-ASCcaspase-1 inflammasome or the noncanonical NLRP3-ASC-caspase-8 inflammasome. Moreover, infection with Candida albicans or challenge with the fungal PAMP zymosan also led to the activation of the noncanonical NLRP3-ASC-caspase-8 inflammasome in cells absent for caspase-1. Collectively, these findings uncover a number of novel signaling pathways for the innate immune response of host cells to C. neoformans infection, and suggest that manipulating NLRP3 signaling may help to control fungal challenge.
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B202 PLATELET ACTIVATING ACETYLHYDROLASE INVOLVED IN INFLAMMATION RESOLUTION IN MACROPHAGES AND NEUTROPHILS WHICH HAVE BEEN STIMULATED WITH URATE CRYSTALS Darshna Yagnik Middlesex University, Department of Natural Sciences, London, UK Background: A model of gout which consists of human blood derived in vitro differentiated monocytes/macrophages and neutrophils are immune cells which have been investigated over the years to determine the role these cells play in the resolution phase of gout. Macrophages and neutrophils are able to phagocytose monosodium urate monohydrate (MSU) crystals without releasing inflammatory factors. This study analysed platelet activating acetylhydrolase (PAFAH) secretion by human macrophages and neutrophils derived from whole blood upon phagocytosis of MSU crystals. Method: Monocytes and neutrophils were isolated from whole blood taken from human blood donations collected from the National Blood NHS Service using standard isolation procedures using percoll and dextran sedimentation. Isolated monocytes were differentiated for a period of 7 days in vitro into macrophages. Day 1 monocytes or day 7 in vitro differentiated macrophages were then stimulated with MSU crystals (0.5 mg/mL), or LPS (10 lg/mL) and signalling inhibitors for a period of 17 h. Cultured supernatants were then collected and assayed for Tumour necrosis (TNF) alpha, PAF acetyl hydrolase (PAF-AH) secretion by ELISA. Results: Analysis of supernatants from in vitro differentiated macrophages stimulated with MSU crystals revealedPAF-AH secretion whereas monocytes did not secreted PAF-AH. Release of macrophage PAF-AH (0.6 pg/mL ± 0.01) was inhibited by a protein kinase inhibitor (SB203580 at a range of 50–300 nM) when added to the assay at the same time as MSU crystals. Similar results were obtained with neutrophils. Conclusion: This study identifies a role for neutrophil and macrophage derived PAF-AH in inflammation resolution through PKC signalling in the pathway by which immune cells ingest MSU crystals and resolve the concomitant inflammation. The PAF-AH enzyme could be used therapeutically for treatment of patients with recurrent or treatment resistant gout.
B203 BACTERIAL METABOLITES, SHORT CHAIN FATTY ACIDS, ATTENUATE THE IMMUNE RESPONSE TO AGGREGATIBACTER ACTINOMYCETEMCOMITANS Renan Oliveira Correa, Aline Vieira, Erica Moraes Sernaglia, Marco Aurelio Ramirez Vinolo State University of Campinas (UNICAMP), Campinas, Brazil Short chain fatty acids (SCFAs) including butyrate, propionate, and acetate are products of bacterial fermentation. These metabolites attenuate inflammation and may be useful in the treatment of conditions such as inflammatory bowel disease and obesity. However, their role in the immune response during infectious diseases is still unknown. The aim of this study was to test the effect of SCFAs in leukocytes recruitment and effector function in response to Aggregatibacter
Inflamm. Res. actinomycetemcomitans (Aa), a bacteria implicated in periodontal disease. For this, we have used the in vivo subcutaneous chamber model in C57BL/6 mice. Ten days after subcutaneous implantation of a stainless coil chamber, a solution containing Aa with or without different concentrations of SCFAs was inoculated. After 4 or 24 h, the animals were sacrificed and the inflammatory exsudates were collected for measurement of cytokines (TNF-a, IL-1b, IL-6, IL-10, IL-12) and chemokines (Cxcl1, Cxcl2), evaluation of leukocyte recruitment, bacterial killing capacity and phagocytic activity. The presence of the SCFAs did not modulate leukocyte recruitment in response to bacteria. However, the production of TNF-a, IL-10, and Cxcl2 decreased in the group inoculated with SCFAs at 24-h. In addition, the killing of Aa by the immune cells was 10 times less effective for the SCFAs-treated group. Furthermore, the phagocytic activity of the immune cells was also reduced when treated with SCFAs for 4 h. In vitro experiments have confirmed these in vivo observed patterns. By incubating neutrophils and Aa (or LPS) together with non-cytotoxic concentration of SCFAs separately for 6 h, the production of TNF-a and IL-10 decreased when treated with some concentrations of propionate, butyrate, and acetate (this last one only for IL-10 in the LPS group). However, an increase in the production of IL-1b was observed for similar conditions. Moreover, the SCFAs also reduced the phagocytic activity of neutrophils in vitro after 2 h of incubation. The results of this study are still preliminary, but they suggest that SCFAs reduce the immune response to Aa and may play act as a bacterial mechanism of evasion. This may be relevant for periodontitis development but also for other types of infections caused by anaerobic bacteria.
B204 RECOGNITION OF PRODUCTS FROM NEUTROPHIL DEGRANULATION BY TOLL LIKE RECEPTORS MEDIATE KILLING OF LEISHMANIA AMAZONENSIS IN MACROPHAGES Natalia M. Tavares1, Lilian Afonso1, Martha Suarez1, Mariana R. Ampuero1,3, Stella d´Areˆde1,3, Aldina Barral1,3, George dos Reis2, Vale´ria M. Borges1, Cla´udia Brodskyn1,3 1 CPqGM/FIOCRUZ, Salvador, Brazil; 2Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; 3Universidade Federal da Bahia, Salvador, Brazil
Neutrophils mediate early responses against pathogens and they become activated during endothelial transmigration towards the inflammatory site. In the present study, human neutrophils were activated in vitro with immobilized extracellular matrix (ECM) proteins. Neutrophil activation by fibronectin (FN), but not other ECM proteins, induces the release of granules content, measured by matrix metalloproteinase 9 (MMP-9) and neutrophil elastase (NE) activity in culture supernatant, as well as reactive oxygen species (ROS) production. Upon contact with L. amazonensis-infected macrophages, these FN-activated neutrophils reduce the parasite burden through a mechanism independent of cell contact. The release of granules content (NE, MMP-9) and production of inflammatory mediators, TNF-a and leukotriene B4 (LTB4), participate in parasite killing by infected macrophages. Besides, inhibition of degranulation from FN-activated neutrophilsabrogatedLTB4 production and reverted parasite killing. Moreover, the addition of exogenous granules proteases increased leishmanicidal activity of infected macrophages and this effect was dependent on Toll like receptor (TLR). Together, these results points out the role of degranulation from FNactivated neutrophils in the induction of protective mechanisms of macrophages that may contribute to the control of Leishmania infection, but also to the local inflammatory response.
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B205 PGD2 MEDIATES EOSINOPHILIC INFLAMMATION DURING S. MANSONI INFECTION Laı´s C. Agra1, Camila R.R. Pa˜o1, Isaac Bellas1, Tatiana LunaGomes1, Valdirene S. Muniz2, Ludmilla Dellatorre-Teixeira1, Natalia R. Amorim1, Ligia A. Paiva3, Christianne Bandeira-Melo1 1 Instituto de Biofı´sica Carlos Chagas Filho, UFRJ, Rio de Janeiro, Brazil; 2Instituto de Cieˆncias Biome´dicas, UFRJ, Rio de Janeiro, Brazil; 3Instituto Oswaldo Cruz, Fiocruz/RJ, Instituto Oswaldo Cruz, Fiocruz/RJ, Rio de Janeiro, Brazil
Background: Prostaglandin D2 (PGD2) is a lipid mediator mainly produced by mast cells in chronic allergic asthma and other inflammatory disorders. Recently, it has been demonstrated that eosinophils are also cell sources of PGD2, which in turn is recognized as a potent stimulus of eosinophil activation. During S. mansoni infection, schistosoma-derived PGD2 has emerged as a key parasite regulator of immune defense evasion, controlling cutaneous immune response through inhibition of Langherhans cells migration. However, the role of host PGD2 during S. mansoni infection is not established. Aim: In this work, we investigated the role of host-derived PGD2 during the progression of experimental S. mansoni infection. Methods: C57/Bl6 mice were infected by transcutaneous penetration of 60–70 cercariae of Schistosoma mansoni (BH strain, Institute Oswaldo Cruz, RJ, Brazil). At 24th day, osmotic pumps (Alzet pump; 100 ll) containing HQL-79 (deliver rate: 24 lg/day), a specific inhibitor of hematopoeitic PGD synthase (H-PGDS), were implanted subcutaneously in infected and non-infected mice for 4 weeks, when animals were sacrificed. Besides, infection parameters, eosinophilia, lipid mediator production and fibrosis were analyzed in bone marrow, blood, peritoneal fluid, and livers. Results: First, we learned that increased levels of PGD2 are found in liver and peritoneal cavity of S. mansoni-infected mice and that such systemic production of PGD2 was impaired by HQL-79 treatment. HQL-79 decreased eosinophilia in peripheral blood, peritoneal fluid and in liver granulomas of S. mansoni-infected mice, while no alteration was observed in bone marrow eosinophilia, indicating that inhibition of PGD2 synthesis affects only peripheral eosinophilia. Whereas the size of hepatic granulomas was not modified by HQL-79, inhibition of PGD2 synthesis reduced the number of schistossomal granulomas in the liver compared to non-treated mice. Moreover, HQL-79 treatment decreased hepatic amounts of leukotriene C4, indicating that infectionrelated endogenous PGD2 induces lipoxygenase activity during infection. However, we did not observe any difference in infection-induced collagen deposition between HQL-79-treated and non-treated mice. Conclusion: Our results indicate that hematopoeitic cells-derived endogenous PGD2 is a key lipid mediator of schistosomiasis, controlling granuloma formation and systemic eosinophilia installation during infection.
B206 INTERFERON REGULATORY FACTOR 8 REGULATES THE MICROGLIAL CELL RESPONSE TO STERILE INJURY IN THE BRAIN Iain L. Campbell1, Rue Dan Xie1, Na`dia Villacampa2, Beatriz Almolda2, Berta Gonza´lez2 1
University of Sydney, Sydney, Australia; 2Autonomous University of Barcelona, Barcelona, Spain
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The transcription factor interferon regulatory factor (IRF) 8 regulates myeloid cell differentiation and function. We identified IRF8 to be a constitutive nuclear factor that regulates the homeostatic properties of microglia—the tissue-resident macrophages of the brain. Here we asked if IRF8 modulates the microglial response to sterile neuronal injury. Facial nerve axotomy (FNA) was performed in wild type (WT) and IRF8-/-(KO) mice and the brains removed at different times post-lesion. A subset of mice was injected with bromodeoxyuridine (BrdU) prior to retrieval of the brain. Changes in the facial nucleus (FN) were examined by immunohistochemistry and histochemistry. In brains from IRF8 KO mice, nucleoside diphosphatase (NDPase) histochemistry and lectin staining revealed gross alterations in the morphology of microglia, which were stunted and hypertrophied. After FNA in WT mice, a progressive increase in microglial activation was observed in the lesioned FN peaking at day 7 and was accompanied by dense staining for Iba1, lectin, NDPase and CD11b. By contrast, in IRF8 KO mice, the microglial response to FNA was markedly attenuated with little staining for Iba1, while the density of staining for lectin, NDPase and CD11b was reduced significantly. The attenuated microglial response in IRF8 KO mice was paralleled by a significant decrease at day 3 post-lesion in proliferation of these cells when compared with WT. Furthermore, a decrease in the number of PU.1-positive cells was observed in the FN of IRF8 KO mice compared with WT. The wrapping of individual motor neuron cell bodies in the axotomised FN by microglia involved in synaptic stripping and phagocytosis was impaired in the absence of IRF8. Finally, in IRF8 KO mice, the degeneration of axotomised motor neurons was significantly increased. These studies demonstrate a crucial requirement for IRF8 in regulating multiple functions of microglia in the innate response of these cells to neuronal injury.
by both agonists. The IL-6 and TNF-a peak responses were reduced only in LPS-stimulated macrophages. Addition of cPAF did not affect macrophages response to the MyD88-independent Poly(I:C). Aditionally, cPAF reduced the LPS-induced COX-2 expression and PGE2 production, but did not affect the iNOS expression and nitrite formation. The down regulation of cytokine production induced by cPAF was independent on the adaptor molecules, once it did not influence MyD88 and TRIF mRNA expression. However, cPAF decreased the phosphorylation of NF-jB p65 subunit induced by LPS and Pam3Cys in 15 min, without altering IjB-a phosphorylation and p65 translocation to the nucleus. Conclusion: These findings indicate that the down regulation induced by PAF in TLR-triggered responses was caused by impaired transcriptional activity of the p65 subunit. These data unveil a heretofore unrecognized role for the PAF-R in MyD88-dependent activation of NF-jB in macrophages. Financial support FAPESP, CAPES and CNPq
B208 CD39/ENTPD1 EXPRESSION ON MACROPHAGES LIMITS ATP-P2X7 RECEPTOR PROINFLAMMATORY SIGNALING RESPONSES IN SEPSIS Luiz Eduardo B. Savio1,2, Paola A. Mello2,4, Vanessa R. Figliuolo 1, Thiago Fernandes A. Almeida1, Patrı´cia T. Santana1, Suellen D’arc S. Oliveira1,3, Claudia Lucia M. Silva3, Linda Feldbru¨gge 2, Yan Wu2, Simon C. Robson2, Robson Coutinho-Silva1 1
B207 PAF IMPAIRS MYD88-ENHANCED NF-jB P65 TRANSCRIPTIONAL ACTIVITY IN MURINE MACROPHAGES Edson K. Ishizuka1, Carlos H. Serezani2, Luciano R. Filgueiras1, Sonia Jancar1 1
2
University of Sa˜o Paulo, Sa˜o Paulo, Brazil; Indiana University School of Medicine, Indianapolis, IN, USA Introduction: Platelet-activating factor receptor (PAF-R) and Toll-like receptors (TLRs) are receptors highly expressed in macrophages and are important components of homeostatics processes and host defense against invading pathogens. Macrophage responses usually derive from integration of signals transduced by more than one receptor. In this study, we investigated the effect of PAF-R activation in the responsiveness of thioglycollate (TG)-elicited macrophages to MyD88-dependent and independent agonists. Methods: C57BL/6 male mice were intraperitoneally injected with 2 mL of 3 % thioglycollate. After 4 days, peritoneal macrophages (PMs) were collected and stimulated simultaneously with the stable PAF (1-hexadecyl-2-N-methylcarbamyl-glycero-3-phosphocholine; cPAF) 100 nM and the TLR agonists (LPS 100 ng/mL, Pam3Cys 100 ng/mL or [Poly(I:C)] 50 lg/mL). Pro-inflammatory cytokines (IL-6, TNF-a and IL-12p40) and PGE2 were measured by ELISA and the expression of adaptor molecules MyD88 and TRIF by qPCR. Protein samples were subjected to Western blot analysis to investigate the NF-jB pathway. Results: We found that in macrophages stimulated with MyD88-dependent agonists (LPS and Pam3Cys), addition of exogenous cPAF drastically reduced the peak response of IL-12p40 production induced
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Laboratory of Immunophysiology, Biophysics Institute Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; 2Department Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Harvard University, Boston, MA, USA; 3Laboratory of Molecular and Biochemical Pharmacology, Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; 4Faculty of Pharmacy, Federal University of Rio Grande do Sul, Porto Alegre, Brazil Background: Sepsis is a leading cause of death worldwide. Mortality is related to onset of shock with inflammatory responses, which are dependent upon Toll-like receptor (TLR) activation in monocytemacrophages. Upon TLR activation, adenosine triphosphate (ATP) is released into the extracellular milieu. Extracellular ATP (eATP) serves as a novel danger signal to heighten inflammatory responses via activation of the type 2-purinergic receptor P2X7 (P2X7R). We propose that the proinflammatory properties of eATP-P2X7R signaling will be countered by ectonucleotidases, such as CD39 and associated with lipid rafts in plasma membranes. Aim: To investigate the modulatory role of CD39 during ATP-P2X7 signaling in sepsis. Methods: For in vitro assays, peritoneal macrophages (MU) or bone marrow derived MU were primed with 1 lg/mL LPS for 4 h, followed by stimulation with ATP (500 lM) or Bz-ATP (100 lM; a P2X7 agonist) for additional 3 h. Before priming with LPS, cells were also pretreated with select P2X7R antagonists (oxidized ATP (oATP): 300 lM, 2 h; or A740003: 100 nM, 30 min), or with imipramine (acid sphingomyelinase inhibitor; 30 lM, 1 h). Ecto-enzymatic activities were assayed by TLC, protein localization determined by immunostaining, intracellular signaling pathways by Western blot and cytokine release assayed by ELISA. Sepsis was induced by cecal ligation and puncture (CLP) in wild type (WT) and CD39-/- mice. P2X7R function was inhibited pharmacologically in vivo, using the receptor antagonist brilliant blue G (45.5 mg/kg via i.p. 24 h before CLP).
Inflamm. Res. Results: ATP or Bz-ATP boosted CD39 activity in LPS-primed MU. Furthermore, CD39 co-localized with the lipid raft marker Flotillin-2 in the LPS- and ATP-treated cells. Drugs that disrupt cholesterolenriched domains—such as nystatin and methyl-b-ciclodextrin—decreased CD39 enzymatic activity, irrespective of prior stimulation with LPS and ATP. Both oATP and imipramine attenuated ATPinduced increases in CD39 expression and activity as well as concurrent STAT3 activation in LPS-primed cells. LPS- and ATPstimulated CD39-/- cells produced more IL-1b and less IL-10 than WT cells. P2X7 inhibition also decreased LPS-induced cytokine production, as well as ATP-elicited activation of NF-kappaB and AKT/mTOR pathways. CD39-/- septic mice exhibited increased IL1b levels and a reduced production of IL-10 in blood and peritoneal lavage fluid (PLF), when compared to matched WT counterparts. P2X7 blockade decreased blood and PLF levels of IL-1b, IL-6 and IL-10 in both WT and CD39-/- septic mice. Conclusion: This study confirms that eATP signaling through P2X7R contributes to inflammatory responses in sepsis. Further, our findings highlight the crucial role of CD39 in the control of sepsis-related responses and suggest modulatory roles of lipids. CD39 comprises an important purinergic regulatory mechanism crucial in limiting inflammation and restoring homeostasis during sepsis. Financial support NIH, CNPq, CAPES.
B209 ABSENCE OF DIETARY FIBRE OR THE METABOLITE SENSOR GPR43 EXACERBATES NEUTROPHIL RECRUITMENT DURING ACUTE INFLAMMATORY RESPONSES Connie H. Wong1, Marjon Kamp1, Raymond Shim1, Ana Carolina Oliveira2, Linda J. Mason1, Lauren Binge1, Charles R. Mackay1 Monash University, Melbourne, Victoria; 2Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil 1
Fermentation of dietary fibre in the gut yields high amounts of short chain fatty acids (SCFAs). SCFAs can impart immediate biological responses on cells through their engagement of ‘metabolite-sensing’ G protein-coupled receptors (GPCRs). One of the main SCFA receptors, GPR43, is highly expressed by neutrophils, a cell type central to many inflammatory reactions. This suggests that dietary fibre intake and the actions of GPR43 may affect neutrophil recruitment during inflammatory responses in vivo. We examined the inflammatory response of wildtype and Gpr43-/- mice, fed on normal chow, or diets consisting of no fibre or high fibre, in models of acute intestinal inflammation. Under basal conditions, bone marrow neutrophils of no fibre-fed wildtype mice exhibited elevated migratory behaviour towards CXC chemoattractants compared to normal chow-fed wildtype mice. Interestingly, this hyper-migratory behaviour of neutrophils could also be reproduced through simple transfer of a no fibre microbiota into germfree mice, suggesting that the composition and function of microbiota stemming from a no fibre diet mediated the changes in neutrophil migration. Using intravital imaging of the small intestine, we found accelerated intravascular neutrophil rolling and adhesion in Gpr43-/mice in response to LPS at 1 h, with increased numbers of neutrophils found in the lamina propria at 4 h. Similarly, GPR43-deficient leukocytes demonstrated exacerbated migration into the peritoneal cavity following cecal ligation and puncture, or after fMLP injection. Moreover, fMLP-induced leukocyte migration was reduced in wildtype, but not Gpr43-/- mice fed a high fibre diet, or acetate. Therefore, GPR43 and a microbiota composition that allows for SCFA production regulate proper neutrophil recruitment during inflammatory responses.
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B210 NFAT SIGNALING PATHWAY IN INNATE IMMUNE CELLS PRIMES THE INFLAMMATORY PROCESS DURING SKIN FUNGAL INFECTIONS Francesca Granucci1,2, Simona Barresi2, Francesca Mingozzi2, William Santus2, Marina Vai2, Ivan Orlandi2, Ivan Zanoni2,1,3 1
Humanitas Clinical and Research Center, Milan, Italy; 2University of Milano-Bicocca, Milan, Italy; 3Harvard Medical School, Boston, MA, USA Candida (C.) albicansinfections occur frequently in hospitalized patients, especially if they are debilitated or immunocompromized. Candida (C.) albicans infections can develop in different anatomic sites, including skin and subcutaneous tissues. Fungal ligands are among the most potent stimuli able to induce the activation of the NFAT signaling pathway in innate immune cells. The NFAT pathway functions have been best characterized in adaptive immunity while very little is know in innate immune cells. In the present work we have investigated the consequences of NFAT activation in innate immune cells in models of Candida induced inflammation. We found that in vitro NFAT activation in BMDCs in response to curdlanor Candida (C.) albicans stimulation potentiates PGE2 production. When we infected mice intradermally with C. albicans we found NFATc2 nuclear translocation in dermal DCs 1 h post infection. Moreover, inflammatory cell infiltration and ulcer formation were observed in wild type animals infected with Candida. Ulcers normally resolved in 1 week. Diversely, in CD11c+ cells-depleted or NFATc2-deficient mice we observed the formation of a necrotic cyst due to fibroblasts proliferation with no recruitment of inflammatory cells and no ulceration. The necrotic cyst persisted for the entire duration of the experiment. Fibroblast proliferation and cyst formation were induced by the release of active TGFbeta. In wt animals, TGFbeta functions were counteracted by NFAT-dependent PGE2 production. No PGE2 production was, indeed, observed in NFATc2deficient animals and the injection of PGE2 or TGFbeta inhibitors blocked cyst formation and reversed the phenotype. We also observed that both CD11c+ cells and NFATc2 were required to induce C. albicans transport to the draining lymph node and the activation of adaptive T cell responses. In the absence of DCs or NFAT activation C. albicans remained confined to the dermis inside the necrotic cyst. Altogether, our data support the hypothesis that NFAT activation in innate immune cells, particularly in CD11c+ cells, regulates the earliest events of the innate immune response elicited against fungi. Our data demonstrate that NFAT-dependent production of PGE2 orchestrates inflammatory cell recruitment at the infection site and the efficient transport of the pathogen to the draining lymph node thereby controlling adaptive immune system activation.
B211 DUAL ACTION OF PLATELETS AND GLYCOPROTEIN VI IN IMMUNE COMPLEXMEDIATED INFLAMMATION Angele Gros1, Varouna Syvannarath1, Lamia Lamrani1, Ve´ronique Ollivier1, Ste´phane Loyau1, Tobias Goerge2,3, Bernhard Nieswandt4, Martine Jandrot-Perrus1, Benoıˆt Ho-Tin-Noe´1 1 UMRS-1148 Inserm-University Paris Diderot, Laboratory for Vascular Translational Science, Bichat Hospital, Paris, France; 2 Department of Dermatology, University Hospital of Mu¨nster,
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S186 Mu¨nster, Germany; 3Interdisciplinary Center for Clinical Research (IZKF), University of Mu¨nster, Mu¨nster, Germany; 4Chair of Vascular Medicine, University Hospital Wu¨rzburg & Rudolf Virchow Center, University of Wu¨rzburg, Germany Background: Beyond their role in thrombus formation and primary hemostasis, platelets are crucial actors of innate and adaptive immune responses. They maintain vascular integrity during inflammation. Evidence suggests that this protective function is independent of thrombus formation and involves platelet immunoreceptor tyrosine activation motif signalling. However, it remains unclear how platelets prevent inflammatory bleeding. Our hypothesis is that platelets could dampen or repair neutrophil-inflicted vascular damage. Aim: Our objective was to determine how platelets and glycoprotein VI (GPVI) contribute to maintain vascular integrity during inflammation. Methods: In models of immune complex (IC)-mediated dermatitis and peritonitis combined with immunodepletion of platelets and/or neutrophils in wild-type and/or GPVI deficient mice, we investigated the contribution of platelets to the regulation of neutrophil recruitment, infiltration, and injurious activities. Using intravital microscopy, we analyzed the contribution of GPVI to platelet recruitment and the interactions between platelets and neutrophils at the reaction site. Through a transfusion-based experiment of platelets treated with tyrosine kinase inhibitors in GPVI-/- mice, we assessed the consequences of GPVI signaling inhibition on the prevention of neutrophil inflicted vascular damage. Results: Depletion of neutrophils prevented skin bleeding observed in thrombocytopenic and GPVI-deficient mice subjected to dermatitis, indicating that platelets counter the deleterious effect of neutrophils. However, during dermatitis and peritonitis, neutrophil infiltration was reduced in thrombocytopenic mice whereas degranulation, and oxidative stress were reduced in both GPVI-deficient and thrombocytopenic mice as compared to wild-type mice. Intravital microscopy revealed that in inflamed vessels, platelets interacted directly with both neutrophils and the vascular wall. Furthermore, during IC-mediated dermatitis, intravascular binding sites for GPVI were exposed by neutrophils, and GPVI supported the recruitment of platelets to these spots. The platelet secretory response accompanying IC-mediated inflammation was partly mediated by GPVI and blocking of GPVI signalling impaired the vasculoprotective action of platelets. Conclusion: Our results indicate that platelets and GPVI play a dual role in inflammation by enhancing neutrophil recruitment and damaging activities while ensuring sealing of neutrophil-inflicted vascular injury through GPVI-dependent adhesion and activation of single platelets. Our study suggests that platelets should be considered as integral players of immune complex-mediated inflammation that intervene at all phases of the inflammatory response.
B212 THE IMMUNE REGLLATION OF THE NEISSERIA GONORRHOEAE INVOLVED INDOLEAMINE 2,3DIOXYGENASE-1 Sai Li, Wenjing Le, Xiaohong Su Institute of Dermatology, Chinese Academy of Medical Sciences, Chaoyang, China Objective: This study plans to prove the role of the immune regulation of the indoleamine 2,3-dioxygenase-1 in N. gonorrhoeae infection.
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Inflamm. Res. Methods: We firstly detected the expression and the activity of the indoleamine 2,3-dioxygenase-1(IDO-1) in the human monocytederived dendritic cells (moDC) infected with N. gonorrhoeae; Then we tested the effect of the IDO-1 inhibitor, L-1-MT, on the proliferation of the T cells stimulated by the N. gonorrhoeae treated moDC; Finally, neutralizing antibodies against the IFN-c and the TLR4 were added to the coculture system of the N. gonorrhoeae and the moDC, separately, to determined the involvement of the IFN-c or TLR4 in the induction of the active IDO-1. Results: N. gonorrhoeae could induce the active IDO-1 in human moDC, and L-1-MT can enhance the proliferation of the T cells stimulated by the N. gonorrhoeae treated moDC. The induction of IDO-1 by N. gonorrhoeae does not depend on the viability and the intactness of the bacterial cells, but partially mediates by TLR4 and partially depends on the production of IFN-c. Conclusions: N. gonorrhoeae induces the production of IDO-1 in human moDC to down regulate the T cell proliferation, which could be one of the immune regulation mechanisms of the N. gonorrhoeae.
B213 THE ROLE OF MerTK AND ITS CLEAVAGE IN THE RESOLUTION OF ACUTE STERILE INFLAMMATION Bishuang Cai, Gabby Fredman, Ira Tabas Columbia University, New York City, NY, USA Efficient clearance of apoptotic cells (ACs), or efferocytosis, and the generation specialized pro-resolving mediators (SPMs) are required for inflammation resolution. Mer tyrosine kinase (MerTK) is a key efferocytosis receptor in macrophages. Our lab and others have shown that MerTK plays a critical role in clearing ACs in advanced atherosclerosis. The ectodomain of MerTK can be cleaved by the metalloproteinase ADAM17 by pro-inflammatory stimuli, resulting in a decrease of surface MerTK on macrophages. While the anti-inflammatory actions of MerTK have been described, little is currently known about MerTK’s pro-resolving roles. We hypothesize that preventing MerTK cleavage and thus retaining surface MerTK leads to a pro-resolution circuit that involves enhanced efferocytosis and SPM generation. To test this hypothesis, we used a sterile peritonitis model initiated by zymosan A in wild type (WT), Mertk-/- or newly engineered MerTK cleavage resistant (Mertkcr) mice and assessed resolution intervals (Ri), in vivo efferocytosis and SPMs in peritoneal exudates. We found a significant delay in resolution measured by Ri of 13 h in WT vs 23 h in Mertk-/-. Importantly we observed a significant decrease in both in vivo efferocytosis and SPM (i.e. lipoxin A4 and Resolvin D1) synthesis in Mertk-/- mice compared with the control mice, indicating that inflammation resolution was delayed in Mertk-/- mice. In contrast, PMN clearance was enhanced and both in vivo efferocytosis and pro-resolving mediators were increased in Mertkcr mice with a decrease of Ri by 54 %, indicating that blocking MerTK cleavage improved inflammation resolution. To further elucidate the mechanism we observed that stimulating macrophages with a MerTK activating antibody resulted in significantly enhanced LXA4 production compared to the IgG control. Importantly, the MerTK activating antibody was unable to stimulate LXA4 in Mertk-/macrophages. To understand these results in a more physiologic context, we also observed that Mertk-/- macrophages produced less LXA4 during efferocytosis than WT controls. Based on these studies, we conclude that MerTK signaling directly promotes inflammation resolution by enhancing efferocytosis and SPM production.
Inflamm. Res.
B214 MECHANISMS AND CONSEQUENCES OF LIPID BODY-PHAGOSOME INTERACTION IN MYCOBACTERIUM BOVIS, BCG INFECTION Natalia R. Roque1, Silvia L. Lage1, Roberta Navarro1, Clarissa M. Maya-Monteiro1, Jens Rietdorf3, Rossana Melo2, Heloisa D’Avila2, Patricia T. Bozza1 1
Oswaldo Cruz Foundation, Fiocruz, Rio de Janeiro, Brazil; 2Federal University of Juiz de Fora, Juiz de Fora, Brazil; 3Center of Technological Development in Health, Fiocruz, Rio de Janeiro, Brazil Lipid bodies (LB) also named lipid droplets, are lipid rich organelles that have been often associated with inflammatory and infectious conditions. The increase in number and size of these organelles is a well-regulated phenomenon that seems to be involved with bacterial persistence. Here we investigate the mechanisms by which mycobacteria induced—lipid bodies may act in favor of infection. We observed that M. bovis bacillus Calmette-Gue´rin (BCG) and Lipoarabinomannan (LAM), but not nonpathogenic mycobacteria M. smegmatis or noncoated latex beads, was able to induce LB formation in macrophages in vitro. By Transmission electron microscopy (TEM), we showed ADRP-marked LB interacting with phagosomes during BCG infection in vivo. Also, we showed LB in close apposition with phagosomes containing beads coated with LAM from M. tb, but not with non-coated beads. LAM coated beads interacts with LB 30 % more than noncoated beads in macrophages loaded with oleic acid. Moreover, we observed that Rab7, an important endocytic GTPase and late endosome marker, as well as its effector RILP but not Rab5 was co-localized in LB induced by BCG infection at 24 h. By TEM, we observed that Rab7 was co-localized with LB in the site of interaction with an infected phagosome during the experimental BCG infection in vivo. Interestingly, we observed a decrease of LAM coated bead-LB apposition when macrophages were treated with CID1067700, a competitive inhibitor of Rab7. Next, we evaluated the role of LB modulation on mycobacterial infection. Treatment with C75 (fatty acid synthase inhibitor) down-regulated LB formation and PGE2 synthesis induced by BCG. Also, these treatments were able to enhance TNF-a while inhibit the IL-10 production and down-modulation of mycobacterial survival and growth assessed by CFU count. These results suggest that LB-phagosome interaction are well-regulated phenomena modulated by mycobacteria cell wall components and dependent on Rab7. The hijack of active Rab7 to LB may enable the interaction with phagosome, and may favour the exchange of nutrients to mycobacterial survival. In addition, LB modulate pro- and anti- inflammatory mediators in favor of BCG survival and replication. Thus suggesting that inhibition of LB formation and/or function as a promising target for therapeutic interventions in mycobacterial infection. Financial support CNPq, FAPERJ, Fapemig, PAPES-FIOCRUZ, CAPES, CDTS. Animal ethical approval number: # LW32/12.
B215 CRITICAL ROLE OF PLASMACYTOID DENDRITIC CELLS IN IMMUNOREGULATION INITIATED BY A COMMENSAL MICROBIAL POLYSACCHARIDE Suryasarathi Dasgupta, Dennis L. Kasper Harvard Medical School, Boston, MA, USA
S187 The microbiota is critical in shaping the mammalian host’s immune system. Polysaccharide A (PSA), the archetypical immunomodulatory microbial molecule of the gut commensal Bacteroides fragilis, induces regulatory T cells to secrete the antiinflammatory cytokine interleukin 10. We show, in a model of colitis, that PSA requires both innate and adaptive immunity to generate protection. Dendritic cells mediate PSA’s effect on IL-10 production. Unlike conventional DCs, plasmacytoid DCs exposed to PSA do not produce the proinflammatory cytokines tumor necrosis factor-a and IL-12 but PDCs do specifically stimulate IL10 secretion by CD4+ T cells and efficiently mediate PSA-mediated immunoprotection. PSA induces and preferentially ligates Toll-like receptor 2 on PDCs but not on CDCs. Compared with other TLR2 ligands, PSA better enhances PDC expression of costimulatory molecules required for protection against colitis. PDCs orchestrate beneficial immunoregulatory interaction of commensal microbial molecules with CD4+ T cells through both innate and adaptive immunity.
B216 STRESS CONCENTRATIONS OF GLUCOCORTICOIDS CAN ENHANCE IMMUNE AND INFLAMMATORY PATHWAYS Paul M. Guyre, Jane E. Collins, Nick M. Jensen, Patricia A. Pioli, Fiona Barr, Sara Metzler, Brian D. Sites, Mark P. Yeager Geisel School of Medicine at Dartmouth, Hanover, NH, USA Glucocorticoids (GCs) are widely understood to suppress inflammation through regulation of signaling pathways in leukocytes and other effector cells. Until recently, these potent and clinically important effects of GCs have obscured results from early research documenting that GCs can stimulate in vivo defense mechanisms. We and others have shown that a 6-h in vivo exposure of healthy humans to stress-associated concentrations of cortisol induces a substantial increase in the pro-inflammatory response to a subsequent challenge with bacterial endotoxin. In those studies, stress cortisol pretreatment resulted in increased serum TNF-a IL-6, and reduced serum IL-10 during subsequent endotoxemia (Crit Care Med. 2009; 37:2727–32). We have now used gene expression microarray analysis of CD14-selected human monocytes, and additional in vivo studies to begin to identify signaling pathways that are differentially regulated by stress cortisol versus pharmacological concentrations of hydrocortisone. Human monocytes were cultured for 24 h with either 50 nM or cortisol followed by mRNA isolation and analysis using Agilent Technologies 44,000 element DNA microarrays. Several genes exhibiting differential up- or down-regulation by stress versus pharm cortisol were confirmed by qPCR. Our results suggest that time-dependent changes in expression of mRNA for IL1R2, ATF3, DUSP1, DUSP10, TLR2, NFKBIA, CCR2, CX3CR1 and SOCS3 may make important contributions to immune enhancement by stress cortisol. Furthermore, when subjects were pretreated with cortisol or saline and monocyte migration into sterile blisters was assessed 24 h later, mean monocyte density was 3.0 ± 0.6 following control treatment and 7.0 ± 1.9 following cortisol treatment (mean ± S.D.). Flow cytometric studies of cortisol-treated monocytes showed upregulation of TLR2, CCR2 and CD163. These studies have begun to identify permissive and preparatory molecular changes induced by stress cortisol in human monocytes that can enable a more robust response to subsequent challenge by an infectious agent.
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B217 ROLE OF INFLAMMASOME-DEPENDENT AND—INDEPENDENT MECHANISMS IN THE ADJUVANT EFFECT OF FLAGELLIN Silvia L. Lage2,1,3, Carina B. Lima4, Eduardo P. Amaral3, Kelly C. Matteucci1,2,3, Lilian M. Massis4, Marcelo Y. Icimoto5, Adriana K. Carmona5, M. Regina D’Imperio-Lima3, Mauricio M. Rodrigues1,2, Luis C.S. Ferreira4, Gustavo P. Amarante-Mendes3, Karina R. Bortoluci1,2 1 Departamentos de Cieˆncias Biolo´gicas e Microbiologia, Imunologia, e Parasitologia da Universidade Federal de Sa˜o Paulo, Sa˜o Paulo, Brazil; 2Centro de Terapia Celular e Molecular da Universidade Federal de Sa˜o Paul, Sa˜o Paulo, Brazil; 3Departamentos de Imunologia, Universidade de Sa˜o Paulo, Sa˜o Paulo, Brazil; 4 Departamento de Microbiologia, Universidade de Sa˜o Paulo, Sa˜o Paulo, Brazil; 5eDepartamento de Biofı´sica, Universidade Federal de Sa˜o Paulo, Sa˜o Paulo, Brazil
Flagellin is a natural agonist of TLR and NLR receptors that has been extensively investigated as an adjuvant, although the mechanisms involved in its immunomodulatory properties remains controversial. Innate immune recognition of flagellin is shared by transmembranic TLR5 and cytosolic Naip5/NLRC4 inflammasome complex. TLR5 signaling activates a number of pro-inflammatory genes through MyD88 pathway, while inflammasome is responsible for the induction of the proinflammatory cytokines IL-1b, IL-1a and IL-18 and necrotic cell death named pyroptosis through caspase-1 activation. However, the molecular regulation of inflammasome activation in response to flagellin, as well as the mechanisms involved in its imunomodulatory effect are still poorly understood. In this study, we evaluated macrophage activation in response to flagellin from inserted into lipid vesicles (Dotap), which allows flagellin delivery into cell cytosol, as well as, the impact of that stimuli in the adaptative immune response generation. By using this approach, atypical cell death induction was found in peritoneal macrophages deficient in inflammasome components (NLRC4, ASC and caspase-1), besides the absence of IL-1b production. In this context, macrophage death maintains its inflammatory and antimicrobial outcome, being accompanied by IL-1a secretion and early loss of membrane integrity. We also demonstrated that this process is regulated by a lysosomal pathway with a redundant role for cathepsins B and D. Also, cathepsin B regulates the IL-1a and IL-1b secretion, suggesting a cooperation between the inflammasome and lysosomal pathway in response to flagellin associated with Dotap. Furthermore, preliminary results demonstrated that dendritic cells stimulated with flagellin from Salmonella typhimurium inserted into Dotap was able to induce IFN-c and IL-17 production by CD4+ T cells, being dependent of IL-1 signaling. Thus, our data open new perspectives in the generation of vaccine and therapeutic strategies based on the inflammasome activation.
B218 ENGAGEMENT OF THE ARYL HYDROCARBON RECEPTOR IN M. TUBERCULOSIS-INFECTED MACROPHAGES HAS PLEIOTROPIC EFFECTS ON INNATE IMMUNE SIGNALING Babak Memari, Manuella Bouttier, Vassil Dimitrov, John H. White McGill University, Montreal, Que´bec, Canada The innate arm of the immune system detects environmental pathogens and mounts a primary immune response. The aryl hydrocarbon
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Inflamm. Res. receptor (AHR), which is activated by a range of ligands of environmental or dietary origin, has emerged recently as an important regulator of innate immunity. We have been interested in characterizing in detail primary host macrophage transcriptional responses to M.tb. infection. Transcriptome profiling of host macrophages revealed that M. tuberculosis (Mtb) infection induced expression of several enzymes controlling tryptophan (TRP) catabolism. This included indole 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO2), which catalyze the rate-limiting step in the kynurenine pathway, producing ligands for the aryl hydrocarbon receptor (AHR). The AHR and heterodimeric partners AHR nuclear translocator (ARNT) and RelB are robustly expressed in macrophages, and AHR and RelB levels further increased during infection. Infection enhanced AHR/ARNT and AHR/RelB DNA binding, and stimulated expression of AHR target genes, including that encoding the inflammatory cytokine IL1B. AHR target gene expression was further enhanced by exogenous kynurenine, and exogenous TRP, kynurenine or synthetic agonist indirubin reduced mycobacterial viability. Comparative expression profiling revealed that AHR ablation in infected macrophages diminished expression of numerous genes implicated in innate immune responses, including cytokines, antimicrobial peptides and enzymes regulating intracellular signaling. Notably, infected cells depleted for AHR exhibited reduced expression of IL23A and IL12B transcripts, which encode subunits of interleukin 23 (IL23), a macrophage cytokine that stimulates production of IL22 by innate lymphoid cells. The AHR directly induced IL23A transcription in human and mouse macrophages through near-upstream enhancer regions containing extended-consensus AHR binding sites. Taken together, these findings show that AHR signaling is strongly engaged in Mtb-infected macrophages, and has widespread effects on innate immune responses to infection. Moreover, they reveal a cascade of AHR-driven innate immune signaling, as IL1b and IL23 induce expression of the gene encoding IL22, another direct target of AHR transactivation.
B219 RECOMBINANT PENTRAXIN-2 PROTECTS PROXIMAL TUBULAR EPITHELIAL CELLS IN THE KIDNEY DURING CELL STRESS Ivan G. Gomez1,2, Naoki Nakagawa2, Mark Lupher3, Richard Jack3, Jeremy S. Duffield1,2 1 Biogen, Cambridge, MA, USA; 2Renal Division, University of Washington, Seattle, WA, USA; 3Promedior Inc., Lexington, MA, USA
Background: Pentraxin-2 is a naturally produced circulating plasma protein involved in innate immunity, whose level is decreased in chronic human fibrotic diseases. Recent studies indicate that systemic delivery of recombinant PTX-2 inhibits inflammatory diseases associated with fibrosis by blocking pro-fibrotic macrophage activation and promoting anti-inflammatory and regulatory macrophages. Methods: Human PTECs were isolated from discarded human kidneys that were digested using a collagenase-based method followed by magnetic immune affinity separation. To induce cell-stress, PTECs were treated with TGFb (5 ng/mL) or 10 % human plasma with or without rhPTX-2 (25 mg/mL) for 16 h or 24 h and evaluated by immune staining for a mesenchymal marker (Vimentin) and an epithelial marker (E-cadherin), mitochondrial reactive oxygen species (ROS) production and with qPCR for epithelial-mesenchymal-transition (EMT) markers. Additionally, Col4a3 deficient mice were treated with Recombinant human pentraxin-2 (rhPTX-2) twice per week for 5 weeks and functional and histological endpoints were measured. Results: Recombinant human pentraxin-2 (rhPTX-2) therapy attenuates the progression of Alport nephropathy in Col4a3 deficient mice
Inflamm. Res. and rhPTX-2 is distributed not only to macrophages but also to proximal tubular epithelial cells (PTECs). We hypothesized that rhPTX-2 would directly prevent tubular injury in human PTECs. Immunostaining for vimentin was increased and epithelial marker E-cadherin expression was decreased in response to cell stress whereas those changes were dramatically attenuated by rhPTX-2. The mRNA expression levels of vimentin, a-smooth muscle actin and Twist1 were increased by cell stress but significantly suppressed by rhPTX-2 compared to vehicle. In response to cell stress, PTECs also rapidly generated ROS, and lost mitochondrial function but PTECs pretreated with rhPTX-2 produced far less ROS, which was accompanied by decreased caspase-3 activity. rhPTX-2 has a specific binding activity for proximal tubule cells and is internalized via a clathrin-mediated pathway, suggesting an epithelial cell receptor. Conclusions: rhPTX-2 protects proximal tubular epithelium against plasma- or TGFb-mediated EMT changes, mitochondrial dysfunction and cell death. rhPTX-2 is a potential new therapy for human chronic kidney diseases.
Innate Lymphoid Cells B220 PROFILING OF INFLAMMATORY BIOMARKERS FROM STIMULATED NATURAL KILLER CELLS Richard K. Fuerstenberg, Shaya Anderson, Kathy Brumbaugh
S189 Dietary fatty acid balance is recognized as an important factor in inflammation regulation and disease controls. Especially omega-3 polyunsaturated fatty acid (PUFA) including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are widely held to be beneficial in many inflammatory disorders. Also, elevation in tissue omega-3 PUFA levels in omega-3 desaturase (fat-1) transgenic mice that endogenously biosynthesize omega-3 PUFA from omega-6 PUFA exhibits resistance to inflammatory disease models. To elucidate the molecular mechanisms underlying the beneficial effects of omega-3 PUFA, we developed a comprehensive LC–MS/MS-based lipidomics method that can detect and quantify more that 500 of fatty acid metabolites simultaneously. Using a genetic model, namely fat-1 transgenic mice, we examined the impact of enhanced omega-3/ omega-6 ratio in inflammation and tissue homeostasis. Also we demonstrated LC–MS/MS-based lipidomic analyses, and identified potent anti-inflammatory metabolites and key metabolic pathways for omega-3 PUFAs. These metabolites may underlie some of the beneficial actions of omega-3 PUFAs in controlling inflammation and related diseases. References: 1. Endo J et al. 18-HEPE, an n-3 fatty acid metabolite released by macrophages, prevents pressure overload-induced maladaptive cardiac remodeling. J Exp Med 211, 1673–1687 (2014) 2. Kubota T et al. Eicosapentaenoic acid is converted via omega-3 epoxygenation to anti-inflammatory metabolite 12-hydroxy-17,18epoxyeicosatetraenoic acid. FASEB J 28, 586–593 (2014) 3. Kunisawa J et al. Dietary omega-3 fatty acids exerts anti-allergic effect through the conversion to 17,18-epoxyeicosatetraenoic acid in the gut. Sci Rep in press
R&D Systems, Inc, Minneapolis, USA NK (natural killer) cells are immune cells of lymphoid origin but unlike their brothers the T and B cells, they are a component of the innate immune system and thus do convey long-lasting or protective immunity to the host. They instead destroy compromised host cells, such as tumor cells or virus-infected cells, recognizing such cells through a missing self mechanism. Over the past decade, NK cells have come under greater scrutiny as major influencers of the host’s immune response via secretion of cytokines, chemokines and other factors. In addition, studies of the mechanisms of NK cells as antitumor effector cells have advanced our understanding of possible cancer cures. IL-12 is a well-known and potent stimulator of NK cells but is also a key regulator of T cell responses, thereby bridging the gap between innate response and adaptive immunity. In this study, NK cells were enriched from peripheral blood using a negative selection method, grown overnight in tissue culture flasks both with and without IL-12. The cell supernates from both conditions were sampled and assayed for secreted biomarkers as large multiplexes using the R&D Systems, Luminex Screening Assay. The markers that showed a positive response were identified for further analysis, thereby establishing a secretory profile for the study of the actions of NK cells.
Lipids, Their Enzymes and Inflammation B221 EMERGING ROLES OF OMEGA-3 FATTY ACID METABOLITES IN CONTROLLING INFLAMMATION AND TISSUE HOMEOSTASIS Makoto Arita1,2,3 1
RIKEN-IMS, Yokohama, Japan; 2Yokohama City University, Yokohama, Japan; 3PRESTO, JST, New York City, NY, USA
B222 ELUCIDATING THE MOLECULAR MECHANISMS OF INTERACTION BETWEEN MASLINIC ACID AND HUMAN GROUP IIA-SECRETED PHOSPHOLIPASE A2 (HGIIA-SPLA2) AND ITS EFFECT IN REGULATING INFLAMMATORY RESPONSE Wei Hsum Yap1, Nafees Ahemad2, Yang Mooi Lim3 Taylor’s University, Subang Jaya, Malaysia; 2Monash University Malaysia, Bandar Sunway, Malaysia; 3Universiti Tunku Abdul Rahman, Petaling Jaya, Malaysia 1
Maslinic acid (2-a, 3-b-dihydroxyolean-12-en-28-oic acid), a natural pentacyclic triterpenoid found in pomace olive oil has various pharmacological properties which include anti-inflammatory activity. A recent study showed that secretory phospholipase A2 (sPLA2) may be a potential binding target of maslinic acid. Considering sPLA2s contribute to the biosynthesis and release of pro-inflammatory mediators and cytokines in inflammatory cells, it represents a novel target for treating inflammatory diseases. The present study examined the interaction between maslinic acid and human Group IIA (hGIIA)sPLA2 and its effect in monocyte differentiation and migration. In this study, it is demonstrated that maslinic acid inhibit hGIIA-sPLA2 enzyme activity in a concentration-dependent manner, with 75 % inhibition at 100 lM concentration. Molecular docking study using Studio Suite 4.0 software further showed that maslinic acid binds to the calcium binding site and interfacial phospholipid binding site via hydrogen bonding and hydrophobic interactions. These results suggest that maslinic acid inhibit the access of catalytic calcium ion required for enzymatic reaction and substrate binding to membrane
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S190 phospholipid, thereby inhibiting the enzymatic activity of hGIIAsPLA2. We further elucidate the role of maslinic acid in regulating hGIIA-sPLA2-mediated cellular responses inmonocyte THP-1 cells. Our results showed thathGIIA-sPLA2is capable of inducing THP-1 cell differentiation and migration. Incubation of hGIIA-sPLA2-induced THP-1 cells with maslinic acid significantly inhibited cell adhesion at a concentration of 10, 20 and 50 lM and cell migration at a concentration of 20 and 50 lM. In conclusion, these findings provide insight into the interaction between maslinic acid and hGIIAsPLA2 and its effect towards hGIIA-sPLA2-induced THP-1 cell adhesion and migration, an important immune-inflammatory processes occurring in atherosclerosis.
B223 EVALUATION OF ANTI-NOCICEPTIVE AND ANTI-INFLAMMATORY EFFECT OF NEW BUTYLATED HYDROXYTOLUENE COMPOUND Roberta Campos Lino1,2, Larissa de Oliveira Gonc¸alves3, Iziara Ferreira Florentino1, Ricardo Menegatti3, Elson Alves Costa1 1 Laboratory of Pharmacology of Natural Products ICB-UFG. Goiaˆnia-Go, Goiaˆnia, Brazil; 2Department of Pharmacy–State University of Goia´s, Caˆmpus Itumbiara-Go, Itumbiara, Brazil; 3 Laboratory of Pharmaceutical Medicinal Chemistry, FF-UFG. Goiaˆnia-Go, Itumbiara, Brazil
Research seeks the development of new analgesic agents with better pharmacological activity. LQFM-091 or BHT, new butylated hydroxytoluene compound, derivative of nimesulide and BF-389, inhibitors of COX-2 and 15-LIPOX respectively. This worked aimed to evaluate the anti-nociceptive and anti-inflammatory effects of the new compound obtained by hybridizationtechnique. The experiments were conducted as approved by the Animal Ethics Committee of the UFG (no. 37/14). Female albino Swiss mice weighing approximately 30 g were used in this study. Pharmacology: in vitro cyclooxygenases activity and lipoxigenase activity. In vivo: acetic acid-induced abdominal writhing, formalin-induced pain, carrageenan-induced pleurisy, the TNF-a level and myeloperoxidase activity were dosage. LQFM-091 inhibited the activity of lipoxygenase enzyme (IC50 = 38.9 lmol/mL), COX-1 (IC50 = 89.0 lmol/mL) and COX-2 (IC50 = 89.0 lmol/mL). LQFM-091 (70, 140 or 280 lmol/kg, p.o) reduced the number of writhing to 57.1 ± 2.4; 51.5 ± 2.9; 50.5 ± 3.8, respectively when compared to control group (20 % DMSO, p.o.) 84.7 ± 3.2 writhes. The treatment with nimesulide (162 lmol/kg, p.o.) also reduced the writhes to 64.3 ± 5.0. In the first phase of the formalin test, the treatments with LQFM-091 or nimesulide not reduced the licking time, but morphine (13.7 lmol/kg s.c.) reduced the licking time to 3.0 ± 2.5 s when compared to control group: 41.0 ± 5.8 s. In the second phase of the formalin test, the treatments with LQFM-091(140 or 280 lmol/kg, p.o); nimesulide (162 lmol/kg, p.o.) or morphine reduced the licking time to 131.7 ± 15.9; 93.5 ± 10.4; 73.2 ± 14.6 e 8.4 ± 8.4 s, respectively when compared to control group: 181.0 ± 18.9 s. The treatment with LQFM-091 (140, 280 or 560 lmol/kg, p.o.), or dexamethasone reduced the leukocytes migrated to the pleural cavity to 4.4 ± 0.3; 3.1 ± 0.4; 2.8 ± 0.5 9 106/mL (5 lmol/kg, p.o), 2.7 ± 0.2 9 106/ mL when compared to control group: 6.6 ± 0.6 9 106/mL. LQFM091 (280 lmoL/kg, p.o) reduced the levels of: TNF-a to 21.3 ± 9.0 qg/mL, when compared to control group (62.3 ± 5.6 qg/mL), Evan’s blue concentration in inflammatory exudate to 11.3 ± 3.8 e lg/mL, when compared to control group (26.9 ± 3.1 lg/mL), and
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Inflamm. Res. myeloperoxydase activity to 37.8 ± 1.6 mU/mL, when compared to control group (101.1 ± 12.7 mU/mL). In conclusion, the results presented in this study revealed that the new compound exhibited anti-inflammatory activity, by presenting inhibitory effects on different enzymes involved in the inflammatory process. Sources of research support CNPq, FAPEG, UEG and CAPES.
B224 A HOST-MICROBIOME INTERACTION MEDIATES THE OPPOSING EFFECTS OF OMEGA-6 AND OMEGA-3 FATTY ACIDS ON CHRONIC LOW-GRADE INFLAMMATION Jing X. Kang, Kanakaraju Kaliannan Massachusetts General Hospital and Harvard Medical School, Boston, USA Metabolic endotoxemia, commonly derived from gut dysbiosis, is a primary cause of chronic low grade inflammation that underlies many chronic diseases. Here we show that mice fed a diet high in omega-6 fatty acids exhibit higher levels of metabolic endotoxemia and systemic low-grade inflammation, while transgenic conversion of tissue omega-6 to omega-3 fatty acids dramatically reduces endotoxemic and inflammatory status. These opposing effects of tissue omega-6 and omega-3 fatty acids can be eliminated by antibiotic treatment and animal cohousing, suggesting the involvement of the gut microbiota. Analysis of gut microbiota and fecal transfer revealed that elevated tissue omega-3 fatty acids enhance intestinal production and secretion of intestinal alkaline phosphatase (IAP), which induces changes in the gut bacteria composition resulting in decreased lipopolysaccharide production and gut permeability, and ultimately, reduced metabolic endotoxemia and inflammation. Our findings uncover an interaction between host tissue fatty acid composition and gut microbiota as a novel mechanism for the anti-inflammatory effect of omega-3 fatty acids. Given the excess of omega-6 and deficiency of omega-3 in the modern Western diet, the differential effects of tissue omega-6 and omega-3 fatty acids on gut microbiota and metabolic endotoxemia provide insight into the etiology and management of today’s health epidemics.
Microbiome in Health and Disease B225 MESALAMINE PREVENTS TRANSLOCATION OF PLANKTONIC PATHOBIONTS RELEASED IN GUT MICROBIOTA BIOFILMS FROM INFLAMMATORY BOWEL DISEASE PATIENTS, BUT NOT FROM HEALTHY INDIVIDUALS Jean-Paul Motta1, John L. Wallace2, Andre G. Buret1 1
University of Calgary, Department of Biological Sciences, Calgary, Alberta, Canada; 2University of Calgary, Department of Physiology and Pharmacology, Calgary, Alberta, Canada Introduction: Inflammatory bowel disease (IBD) is commonly associated with alterations of gut microbiota composition and structure. In rodents, commensal bacteria live as multispecies biofilms in the healthy gut. We observed that during experimental colitis, these biofilms were fragmented, closely apposed to the epithelial surface,
Inflamm. Res. and associated with severe bacterial translocation. Mesalamine is the first line of treatment for patients with IBD. It has been found to affect bacterial gene expression and reduce the concentration of mucosaadherent bacteria. We hypothesized that (1) human IBD is associated with biofilms that release planktonic bacteria able to break the epithelial barrier, (2) and that mesalamine is able to prevent this abnormality. Methods: Human colon biopsies from healthy donors and Crohn’s Disease (CD) patients (active or inactive sites) were seeded anaerobically into the Calgary Biofilm DeviceTM (CBD) to grow ex vivo into their multispecies biofilm and planktonic counterparts. Live biofilm bacteria were quantified using XTT assay. Biofilms were exposed for 24 h to various concentrations of mesalamine (from 0 to 2 mg/mL). Planktonic bacteria were collected from treated biofilms and were exposed apically to human epithelial monolayers on transwells (Caco-2, 18 h). Translocation of bacteria was quantified by plating aerobically or anaerobically, and was visualized by fluorescent in situ hybridization. Results: At concentrations of 0.1, 1 and 2 mg/mL, mesalamine reduced survival of biofilm bacteria originating from active CD but had no effect biofilms from healthy and inactive CD patients. Aerobic planktonic bacteria from healthy biofilms did not cross the epithelial monolayer, and translocation of anaerobic bacteria was minimal. In contrast, both aerobic and anaerobic planktonic bacteria from active and inactive CD biofilms crossed the epithelial monolayers. Planktonic bacteria collected from active CD translocated significantly more than planktonic bacteria from inactive CD sites. 3D images confirmed translocation through paracellular route. At concentration of 2 mg/mL, mesalamine reduced translocation of aerobic bacteria from active and inactive CD patients, and anaerobic bacteria from active CD. Conclusions: Our results demonstrate that host-microbiota biofilm interactions differ between the healthy and the IBD intestine. Planktonic bacteria released from IBD microbiota biofilms are able to cross the epithelial barrier, increasing the likelihood to trigger inflammatory flares. The findings also demonstrate that mesalamine targets pathobionts evading from IBD microbiota biofilms, paving the way towards a novel therapeutic paradigm using this drug.
microRNAs and lncRNAs in Inflammation (microRNAs 5 miRs; Long Non-Coding RNAs 5 lncRNAs) B226 EXOSOMAL APOC3-TRANSFERRED NCRNA PROMOTE OSTEOCLASTS DIFFERENTIATION Chi-Yuan Li1, Kuo-Ting Sun1 1
China Medical University Hospital, Taiwan, China; 2China Medical University Hospital, Taiwan, China Background: Bone homeostasis requires an orchestrated balance between osteoblasts and osteoclasts during bone formation and resorption. Exosomes containing bioactive proteins and genetic materials that may be regulates cell development and differentiation. ncRNA occurs in response to the cellular program to mediate differentiation. Exosomal APOC3-transferred ncRNA may be a crucial function in osteoclasts differentiation. However, the mechanism is remains unclear. The aim of this study was to investigate how Exosome-transferred APOC3-ncRNA adapts to osteoclasts differentiation. Methods: Using Proteomics, RNA-sequencing and lncRNA Q-PCR array, we demonstrated expression of exosomal APOC3-transferred
S191 ncRNA in plasma of b-thalassemia major patients. Identification of exosomes by dynamic light scattering (DLS), flow cytometry and western blots. TRAP staining and bone resorption assay for osteoclasts number and function. Lentiviral infection, ncRNAs transfection and in vitro knockdown or overexpression obviously increased or ameliorated exosome-induced osteoclasts differentiation. Bioinformatics analysis, luciferase assay and in vitro studies revealed that ncRNAs functioned as a repressing mediator and formed a feedback loop with ncRNAs and target gene to mediate osteoclast differentiation. Results: We observed that APOC3 and ncRNA is differential expressed in the exosomes of b-thalassemia major. We analyzed the effects of ex vivo-derived exosomes for osteoclast differentiation by osteoclast precursor model. In vitro model showed that exosomes of b-thalassemia major were internalized by THP-1/RAW264.7 cells, and increase osteoclasts number and differentiation via up-regulated the p38/pAKT/NF-kB/TRAF6/NFATc1 pathway. In addition, Exosomal APOC3-transferred ncRNA mediates inflammatory cytokine expression. Conclusions: This study reveals the role of extracellular Exosomal APOC3-transferred ncRNA in the regulation of osteoclast differentiation, which may provide the molecular mechanism for a new therapeutic strategy in variety of osteoclast-related disorders.
B227 EXTRACELLULAR RNA INDUCES CELLULAR INFLAMMATION VIA TLR7-MYD88 SIGNALING Yan Feng, Hongliang Chen, Lin Zou, Dan Yan, Ganqiong Xu, Wei Chao Massachusetts General Hospital, Boston, MA, USA Introduction: We have recently reported that extracellular RNA (exRNA) released from necrotic cells induces cytokine production in cardiomyocytes (CMs) and immune cells, and may play a role in a mouse model of myocardial ischemia/reperfusion (I/R) injury. However, the identities of these exRNAs and the molecular mechanism by which exRNAs exhibit the proinflammatory effect are unknown. Methods: I/R model mice were subjected to sham procedure or coronary occlusion for 45 min followed by reperfusion. exRNA RNA in media and sera was extracted using Trizol LS and quantified using Quant-iTTM RNA assay kit. miRNA array 68 miRNAs in plasma were quantified using miRNA assay kit (Firefly, Cambridge, MA). Cell treatment and cytokine measurement CMs, neutrophils, and bone marrow-derived macrophages (BMDMs) were treated with exRNA mixed with lipofectamine for 18 h. Cytokines in media were measured by ELISA. miRNA uptake Uptake of fluorescent FAM-labeled miRNA in BMDMs was measured by fluorescent microscopy and flow cytometry . Results: Twenty-four h after hypoxia/serum-deprivation, RNA in CM culture medium rose from 12 ng/mL to approx. 600 ng/mL. In vivo, 24 h after I/R, serum RNA level was increased from 247 ± 46 to 464 ± 92 ng/mL. Unbiased plasma miRNA array data indicate that 31 out of 68 miRNAs tested were significantly increased by [ twofold at 4 h following I/R compared to the sham mice. To test the ability of exRNA to induce inflammatory response, CMs and immune cells (neutrophils and BMDMs) were treated with cardiac RNA (2.5–10 lg/mL) or 8 miRNA mimics (10–1500 nM) selected from the miRNA array data. Both RNA and 6 miRNA mimics (miR-34a, -122, -133a, 142a, -146a, -208a) were found to induce MIP-2 production in a dose-dependent manner. The effects were abolished by pre-treatment of RNase, but not DNase. Moreover, RNA-induced cytokine production was significantly inhibited by a specific TLR7 inhibitor in
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S192 both CMs and immune cells, or diminished in TLR7-/- or MyD88-/-, but not TLR3-/- or Trif-/- BMDMs. Similarly, miRNAinduced cytokine response was completely blocked in TLR7-/- or MyD88-/-, but not TLR3-/- or Trif-/- BMDMs. To test whether TLR7 mediates miRNA trans-membrane uptake, WT and TLR7-/BMDMs were incubated with FAM-miRNA-133a for 4 h. There was no difference in cellular fluorescent miRNA-133a between WT and TLR7-/- BMDMs as determined by fluorescent microscopy and flow cytometry. Finally, i.p. administration of cardiac RNA or miRNA mimics induced acute peritoneal inflammation as evidenced by IL-6 production and neutrophil activation. Conclusions: Our data demonstrate that (1) RNA, including miRNA, is released from hypoxic CMs in vitro and ischemic myocardium in vivo; (2) exRNA, both cardiac RNA and miRNA mimics, induce cytokine production via TLR7-MyD88; (3) exRNA induces acute peritonitis after i.p. injection. These data suggest that exRNA is a potent proinflammatory ligand that signals through TLR7-MyD88.
Molecular Patterns and Acute Inflammation B228 COMPARATIVE SENSITIVITY AND RELIABILITY OF A MULTI-ANALYTES PLATFORM AND A MULTIPLEX FLOW CYTOMETRY IN THE QUANTITATION OF CIRCULATING CYTOKINES LEVEL IN A MOUSE MODEL OF ENDOTOXEMIA Alain Stricker-Krongrad, Jason Liu, Guy Bouchard, Miao Zhong Sinclair Research Center, LLC, Auxvasse, MO, USA Background: A number of cytokines, such as interleukin 2 (IL-2), interferon c (IFN-c), tumor necrosis factor a (TNF-a), IL-4, IL-6, IL10 and IL-17A, are elevated in response to inflammation and are also key regulators of immune responses. IL-2, IFN-c, and TNF-a are associated with the Th1 response. IL-2, IL-4, IL-6 and IL-10 are associated with the Th2 response. IL-6 and IL-17A are associated with the Th17 response. Hence, measuring expression profiles of these cytokines is important in monitoring the polarization of the immune response and therefore the results should be independent of the quantitation methods. To evaluate this effect, the 7 circulating Th1/Th2/Th17 cytokines were quantified in plasma of lipopolysaccharide (LPS ± dexamethasone versus vehicle) treated mice with two different multiplex platforms (Myriad-RBM and BD Biosciences). Objectives: To compare two different multiplex assay formats to quantify the release of the 7 circulating Th1/Th2/Th17 cytokines in plasma of LPS challenged mice. Methods: Female C57BL6 mice (6–9 months old) were treated with vehicle (0.5 % methyl cellulose in water) or dexamethasone (5 mg/ kg), followed by LPS (0.2 mg/kg) via intravenous injection (IV). At 0.5, 1, 2, 4, and 6 h after LPS challenge, blood samples were analyzed with the Rodent MAP V3.0 Antigens assay, with the Mouse Cytokine Panels A & B assay (Myriad-RBM; Luminex platform) and compared to the Mouse Th1/Th2/Th17 cytokines (Becton–Dickinson: BD CBA) assay on a BD Accuri C6 flow cytometer platform. Results: TNF-a, IL-2, IL-4, IFN-c, IL-6 and IL-10 were shown to be the early phase responders, and IL-17A was shown to be a sustained responder, which was up-regulated through LPS treatment. Pretreatment with dexamethasone reduced expressions of the 7 circulating Th1/Th2/Th17 cytokines. Plasma levels of IL-4 were detected with the Mouse Cytokine Panels A and B assay, but were below the Lower limit of quantification (LLOQ) of the Rodent MAP V3.0
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Inflamm. Res. Antigen assay. In the BD CBA cytokine assay, IL-2 and IL-4 were below the lower limit of detection (LLOD) and IL-17A was below the LLOQ. While TNF-a and IL-6 were the cytokines that were quantified at similar plasma level to those detected with the Myriad-RBM assays, IFN-c and IL-10 were quantified at lower plasma levels. Relatively higher level of IFN-c was detected by Myriad-RBM in the 2-h plasma samples, whereas relatively higher level of IFN-c was detected with BD CBA cytokine assay in the 4-h plasma samples. Conclusion: Reproducible circulating TNF-a and IL-6 levels were obtained for plasma samples of the LPS treated mice with the assays from both Myriad-RBM and BD Biosciences. The BD CBA cytokine assay was not as sensitive as the Myriad-RBM assays in detecting and quantitating circulating IL-2 and IL-4 and IL-17A levels in the LPS treated mice, but was more sensitive and reliable in measuring circulating IFN-c levels. Reliable circulating IL-4 measurements were not achieved by either assay.
B229 COPPER-INDUCED INFLAMMATION LEADS TO CHANGES IN SIRTUIN GENE EXPRESSION AND ALTERED BEHAVIOR IN ZEBRAFISH Talita C B Pereira1, Carlos Eduardo Leite2, Laura R. Nery3, Maria Martha Campos1,2, Mauricio R. Bogo1,3 1
Programa de Po´s-Graduac¸a˜o em Medicina e Cieˆncias da Sau´de, PUCRS, Brazil; 2Instituto de Toxicologia e Farmacologia, PUCRS, Brazil; 3Programa de Po´s-Graduac¸a˜o em Biologia Celular e Molecular, PUCRS, Brazil Copper is an essential micronutrient and key catalytic cofactor in a wide range of enzymes. Under inflammatory conditions, serum copper levels are increased, and when in excess it triggers oxidative stress responses that can finally lead to apoptosis. Copper exposure can be easily modeled in zebrafish, a consolidated model in many research areas. Due to developmental, economical and genetic advantages, it is suitable for large-scale screenings, representing a powerful experimental tool to mechanism-related studies. Toxicological effects of copper have been extensively investigated in zebrafish, and its use as an inflammatory agent has increased. Sirtuins comprises a unique class of evolutionary conserved NAD+ -dependent deacetylases. They play important roles regulating metabolic and enzymatic activity, stress, DNA repair and apoptosis targeting not only histones but also transcriptional factors and structural proteins. Under pathological conditions, sirtuins have also been associated to inflammation, with both pro- and anti-inflammatory effects. Here, we aimed to evaluate the effects of copper-induced inflammation on locomotor behavior in 7dpf zebrafish larva, and mRNA expression levels of all sirtuin family members (SIRT1-7). IL-1b, IL-10, TNF-a and COX-2 mRNA expression was evaluated as indicative of inflammation. The larvae (n = 20/group) were treated for 4 h or 24 h with copper sulfate as inflammatory agent, and immediately submitted to a recorded locomotors behavior assay. Relative mRNA expression levels were determined by qPCR analysis (2-DDCT method), using EF1-a and Rpl13-a as reference genes. Results were statistically compared by T-test, or one-way ANOVA followed by Tukey test, considering p B 0.05 as significant. Larval swimming performance showed a significant decrease on traveled distance, mean speed and number of rotations for both 4 and 24 h exposures. The inflammation mediators IL-1b, IL-10, TNF-a related genes showed a significant activation in all treatments, as well as COX-2 gene for the 24 h treatment, when compared to the control group. SIRTs 2, 3, 4, 6 did not present altered expression; however, we found a decrease on SIRT1 gene expression after 4 h-treatment and an activation of SIRT7 after 24 h exposure.
Inflamm. Res. SIRT3.2, a SIRT3 paralogue, showed increased expression on both treatments. These findings show potential involvement of, at least, SIRT1, SIRT3.2 and SIRT7 in inflammatory events. A tissue-specific approach seems promising to evaluate inflammation effect on sirtuins family members, due their differentiated expression pattern.
Mucosal Immunity B230 ROLE OF NK CELLS IN IFN-E-MEDIATED PROTECTION AGAINST FEMALE REPRODUCTIVE TRACT INFECTION Jemma R. Mayall1, Niamh E. Mangan2, Malcolm R. Starkey1, Richard Y. Kim1, Alexandra C. Brown1, Paul J. Hertzog2, Jay C. Horvat1, Philip M. Hansbro1 University of Newcastle, Newcastle, Australia; 2Monash University, Melbourne, Victoria, Australia
1
Chlamydia trachomatis is the most common bacterial sexually transmitted infection (STI) and infection frequently results in reproductive tract (RT) sequalae such as pelvic inflammatory disease and infertility. However, the immune factors and processes involved in the clearance and immunopathology of Chlamydia infection are not well understood. In previous studies we showed that interferon (IFN)e, a novel type I IFN that is exclusively and constitutively expressed in the female RT, plays an important role in protecting against Chlamydia infections at the earliest stages of infection. Here, we examined the effects of IFN-e on innate cellular responses in the female RT in order to elucidate the potential mechanisms that underpin how IFN-e protects against Chlamydia infections. Female wild type and IFN-e deficient (-/-) C57BL/6 mice were pre-treated with progesterone to synchronise their oestrous cycles and prime for infection. Seven days later, mice were infected intra-vaginally with Chlamydia muridarum or sham-infected. Uterine horns were harvested at 3 days post infection and the effects of IFN-e deficiency on Chlamydia infection, immune factor expression and cellular infiltration were assessed using real-time qPCR and flow cytometry. We show that IFN-e-/- mice have increased Chlamydia 16S expression in the upper RT. This corresponded with fewer NK cells, which are known to play a role in protecting against Chlamydia through the production of IFN-c. IFN-c producing CD45+ cells were decreased in the infected IFN-e-/- mice, of which over 60 % were NK cells. Tissue-resident uterine (u) NKs were also decreased in these mice. The changes in IFN-c+ and NK cells were associated with reduced IL-15, CXCL10, iNOS and STAT1 expression. These findings suggest that IFN-e may protect against Chlamydia RT infections by potentiating the recruitment of protective IFN-c-producing NK cells.
Neutrophils, NETs and PADs B231 HUMAN EOSINOPHILS RELEASE EXTRACELLULAR DNA TRAPS IN RESPONSE TO ASPERGILLUS FUMIGATUS Josiane S. Neves, Valdirene S. Muniz, Mariana SR Bica, Yasmim AV Braga, Rodrigo T. Figueiredo
S193 Federal University of Rio de Janeiro, Rio de Janeiro, Brazil Introduction: Eosinophils mediate the immune response in a number of infectious diseases, including parasitic helminth, bacterial, fungal and viral infections. The process of extracellular release of DNA nets (traps) by leukocytes has been described as an important mechanism of the innate immune response in different infectious diseases including fungal infections. Purified human eosinophils release extracellular DNA traps (EETs) by different stimuli including bacteria and cytokines. Aspergillus fumigatus (AF) is an opportunistic filamentous fungus that may cause invasive aspergillosis, a pathological condition of high morbidity and mortality in immunocompromised patients. In vivo, eosinophils are recruited to the lung after exposure to AF and release cationic proteins, which demonstrate an important role in the elimination of this pathogen. In vitro, eosinophils present potent fungicidal activity against AF. However, the mechanisms that lead to recognition as well as the death of AF by eosinophils remain unknown. In this work we investigated whether eosinophils release EETs in response to AF and the mechanisms involved. Methods and Results: We isolated eosinophils from blood of healthy donors by negative immunomagnetic selection. Cells were then stimulated with AF in the ratios (fungus:cell) 1:1, 10:1, 50:1 and 100:1 being the release of EETs evaluated at different incubation times by a quantitative fluorimetric method and by confocal fluorescence microscopy. We observed that EETs were significantly released after 6, 9 and 12 h of incubation. The incubation time of 6 h and the ratio (fungus:cell) 10:1 were then selected for further studies (control = 84.08 ± 10.71 FU, AF = 281.6 ± 33.82 FU, n = 8, student’s t test p \ 0.05, FU = fluorescence unit). Pretreatment of eosinophils for 30 min with DPI (20 lmol/mL) (AF = 307.2 ± 50.21 FU, DPI = 232.3 ± 45.95 FU, n = 7) or apocynin (100 lmol/mL) (AF = 248.50 ± 38.47 FU, Apo = 229.0 ± 40.57 FU n = 4), both inhibitors of reactive oxygen species (ROS), did not inhibit the AFinduced EETs release. However, the pretreatment with piceatannol (40 lmol/mL) (AF = 214.5 ± 32.47 FU, PCT = 97.08 ± 13.12 FU, n = 6, student’s t test p \ 0.05) or OXSI (2 lmol/mL) (AF = 303.5 ± 59.26 FU, OXSI = 146.9 ± 25.34 FU, n = 6, student’s t test p \ 0.05), both inhibitors of Syk tyrosine kinases, significantly inhibited the AF-induced EETs release. Conclusion: Our results indicate that human eosinophils release EETs in response to AF through a mechanism which involves the Syk tyrosine kinases pathway, but independent of ROS. Financial support FAPERJ, CNPq and Capes.
B232 INTRAVITAL IMAGING OF VASCULATURE REVEALS THAT NEUTROPHIL EXTRACELLULAR TRAP (NET) COMPONENTS ATTACH TO VON WILLEBRAND FACTOR IN A DNASE-INDEPENDENT MANNER Elzbieta Kolaczkowska2,1, Craig N. Jenne1, Bas G. Surewaard1, Ajitha Thanabalasuriar1, Woo-Yong Lee1, Maria-Jesus Sanz1, Kerri Mowen4, Ghislain Opdenakker3, Paul Kubes1 1
University of Calgary, Canada; 2Jagiellonian University, Krakow, Poland; 3University of Leuven, Belgium; 4Scripps Research Institute La Jolla, San Diego, CA, USA Neutrophils form neutrophil extracellular traps (NETs) in response to pathogens and/or during severe inflammation, including sepsis. NETs are composed of extracellular DNA, histones and granular proteins, including proteases such as neutrophil elastase (NE). The structures
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S194 function to trap and immobilize pathogens and subsequently contribute either directly, or indirectly to pathogen elimination. In vivo characteristics of NET formation and removal remain unclear. Therefore we studied NET release, their life-span and consequences of their presence in vasculature by means of intravital imaging, using spinning-disk confocal microscopy, during systemic infection with Staphylococcus aureus. Intravascular S. aureus injection is lethal for Kupffer cell- or platelet-depleted mice, but not those lacking neutrophils suggesting NETs are dispensable. In wild-type mice, neutrophils rapidly infiltrated the liver and formed NETs that persisted in the vasculature for several hours following infection, far beyond the time needed for S. aureus blood clearance. The prolonged presence of NETs led to profound damage to the liver and this damage was associated with proteolytic activity of neutrophil elastase attached to NETs. The prevention of NET formation, as observed in NE- and peptidyl arginine deiminase type IV (PAD4)-deficient mice, eliminated the liver injury. Surprisingly, attempts to remove NETs by DNase were only partially successful. While this treatment did remove all of the extDNA, only some histones and NE detached upon the treatment. NET components were determined to be attached to the glycocalyx via von Willebrand factor (VWF): blockade of VWF before sepsis induction, or VWF shedding during the ongoing sepsis was sufficient to significantly decrease the attachment of NET components to the endothelium. Most recently we observed that although platelets were not involved in NET formation during S. aureus-induced sepsis, platelets were important for the primary interactions between the pathogen and Kupffer cells. In summary, we show that in the vasculature, unlike what is observed under in vitro conditions, NET components attach to a substratum independently of extDNA and this attachment hinders their removal by DNase. Albeit removal of VWF might be a therapeutic option, the consequences on platelet functioning must be better understood before this approach can be used. Due to the difficulty in removing NET components once released, prevention of NET formation proved to be the most effective therapeutic intervention.
B233 RESPIRATORY SYNCYTIAL VIRUS INFECTION OF BOTH ALVEOLAR EPITHELIAL CELLS AND NEUTROPHILS INDUCES THE FORMATION OF NEUTROPHIL EXTRACELLULAR TRAPS Ba´rbara N. Porto1, Ste´fanie P. Muraro1, Giselle A. Funchal1, Nata´lia Jaeger1, Rafael S. Czepielewski1, Gabriela F. Souza1, Renato T. Stein2 1 Laboratory of Clinical and Experimental Immunology, Pontifical Catholic University of Rio Grande do Sul, Porto Alegre, Brazil; 2 Laboratory of Pediatric Respirology, Pontifical Catholic University of Rio Grande do Sul, Porto Alegre, Brazil
Respiratory Syncytial Virus (RSV)-induced acute bronchiolitis is the most prevalent disease in children under 2 years old, which causes a huge impact in hospitalizations and costs to the health system. The alveolar epithelial cells are the initial targets for RSV infection, as well as the first site for activation of the innate immune response. Epithelial cells infected by RSV secrete pro-inflammatory cytokines and neutrophil chemokines, which attract these granulocytes to the site of infection. The excessive neutrophil activation by epithelial cells infected with RSV can be harmful to the host, as neutrophils release neutrophil extracellular traps (NETs), which kill microorganisms but also damage host cells and tissues. NETs are composed by decondensed chromatin and antimicrobial proteins. We have recently
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Inflamm. Res. demonstrated that RSV Fusion protein induce NET formation through activation of TLR-4, NADPH Oxidase-derived reactive oxygen species generation and ERK and p38 MAPK phosphorylation. Thus, we asked whether an active RSV infection would induce NET release. This study aims to investigate the mechanisms underlying the production of NETs induced by alveolar epithelial cells infected with RSV. Human neutrophils were stimulated with different concentrations of RSV (102–104 PFU/mL) for 3 h at 37oC under 5 % CO2. Alternatively, alveolar epithelial cells (A549 cell line -1 9 105/mL) were infected with active RSV or UV-inactivated RSV in RPMI medium without FCS for 2 h at 37 C under 5 % CO2. Afterwards, medium was replaced by RPMI 10 % FCS and the culture was maintained for 48 h. After this, medium was replaced to remove virus particles and neutrophils (5 9 105/mL) were added to the culture. The co-culture was incubated for 150 min. At the end of incubation, NETs were quantified in culture supernatants, using Quant-iT dsDNA HS kit. Here we show that RSV was able to directly induce NET release by human neutrophils in a concentration-dependent fashion. We performed a MTT assay to evaluate the dose- and time-dependent effect of RSV infection on alveolar epithelial cells viability. We found that the RSV concentrations used were not able to affect A549 cell viability until 48 h. In 72 h, RSV (103 and 104 PFU/mL) induced A549 cell death; leading us to infect the cells for 48 h. RSV infection of alveolar epithelial cells was able to induce NET formation in a concentrationdependent manner. This effect was due to an active RSV infection, since UV-inactivated RSV did not stimulate NET release nor promoted a cytopathic effect in A549 cells, as visualized by optical microscopy. The excessive production of NETs induced by RSV could fill the lungs and impair lung function and consequently aggravate the inflammatory symptoms of the infection in young children and babies. We propose that the associated use of DNase could potentially lead to novel therapeutic approaches to help control RSV-induced inflammatory consequences and pathology of viral bronchiolitis.
B234 AUTOPHAGY MEDIATES INTERLEUKIN-1 BETA EXPORTATION FROM HUMAN NEUTROPHILS Analia S. Trevani1,2, Irene Keitelman1, Florencia Sabbione1, Carolina C. Jancic1,2, Leonardo Iula1 1 IMEX-CONICET- Academia Nacional de Medicina, Buenos Aires, Argentina; 2Facultad de Medicina, Universidad de Buenos Aires, Argentina
Introduction: Interleukin-1b (IL-1b) is a pro-inflammatory cytokine synthesized in the cytoplasm as a precursor, pro-IL-1b, which has to be proteolytically processed to acquire biological activity. We have previously demonstrated that human neutrophil IL-1b processing is dependent of caspase-1 and elastase and/or proteinase-3. The release of IL-1b does not follow the conventional ER-Golgi route of secretion, being secreted by poorly-defined non-conventional secretory pathways among which autophagy has been proposed to be involved. However, controversial findings have been reported regarding the role of autophagy in controlling IL-1b secretion in other myeloid cells; with studies indicating either that autophagy targets IL-1b for degradation or that it is involved in its unconventional secretion. Here we tested the hypothesis that an unconventional autophagy mechanism is required for neutrophil IL-1b secretion. Methods and results: We found that autophagy inhibitors like 3-methyadenine markedly reduced IL-1b secretion induced by 5 h stimulation of human neutrophils with LPS or LPS+ ATP evaluated by ELISA, without affecting cell viability. By contrast, these inhibitors did not modulate IL-8 secretion, a cytokine whose release
Inflamm. Res. follows the canonical ER-Golgi pathway. In neutrophil-differentiated PLB985 cells, siRNA knockdown of ATG5, an essential component of the autophagic pathway, abolished IL-1b secretion. In agreement with these findings, stimulation of autophagy by cell starvation promoted IL-1b secretion induced by both agonists. At 4 h poststimulation with LPS+ ATP, intracellular IL-1b showed a vesicular distribution co-localizing with LC3B, a marker of autophagy vesicles, by confocal microscopy. Furthermore, neutrophil starvation increased IL-1b and LC3B colocalization. Conclusion: Taken together, our studies reveal that autophagy is part of the pathway involved in IL-1b exportation from human PMN.
New Models of Inflammatory Mechanisms and Diseases B235 SPONTANEOUS MUTATION IN ZFP-BINDING REGION OF TNF GENE CAUSES CHRONIC POLYARTHRITIS AND HEART VALVE DISEASE Derek C. Lacey1,2, Peter Hickey1,2, Benedicta D. Arhatari3, Lorraine O’Reilly1,2, Leona Rohrbeck1,2, Helen Kiriazis4, Xiao-Jun Du4, Philippe Bouillet1,2 1
The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia; 2Department of Medical Biology, The University of Melbourne, Melbourne, Victoria, Australia; 3ARC Centre of Excellence for Advanced Molecular Imaging, Department of Physics, La Trobe University, Victoria, Australia; 4Baker IDI Heart and Diabetes Institute, and Central Clinical School, Monash University, Melbourne,Victoria, Australia We have studied a new spontaneous mutation that causes severe RA symptoms and heart valve disease in BPSM1 (bone phenotype spontaneous mutation 1) mice. The insertion of a retrotransposon into the 30 untranslated region (UTR) of TNF causes its strong overexpression in myeloid cells. We identified several members of a family of RNA–binding CCCH-containing zinc finger proteins, which can positively or negatively control TNF expression through in the TNF 30 UTR. We have also identified a new regulatory element in TNF 30 UTR, which is essential for the newly identified zinc finger protein’s control of TNF but not Zfp36. The disease in BPSM1 mice is independent of the adaptive immune system, and does not appear to involve inflammatory cytokines other than TNF. In addition to the severe joint destruction, mutant mice also develop aortic root aneurism and aorto-mitral valve disease that can be fatal depending on the genetic background. This is the first animal model showing both RA and heart disease as a direct result of TNF deregulation.
B236 DOWN-REGULATION OF KYNURENINE FORMATION FROM TRYPTOPHAN ATTENUATES INDUCTION OF INSULIN RESISTANCE IN DROSOPHILA MELANOGASTER
S195 Introduction: High sugar diet (HSD) induces insulin resistance (IR) in larvae and hyperglycemia in adults Drosophila flies. HSD-induced IR is characterized by delayed eclosion and decreased body weight of imago [1]. Chronic low grade inflammation is one of the major risk factors for the development of IR and diabetes. Molecular mechanisms mediating the impact of chronic inflammation on the development of IR have not been finalized. We suggested that dysregulation of kynurenine (KYN) pathway of tryptophan (TRP) metabolism is one of the mechanisms of development of inflammation-induced IR. KYN formation from TRP is catalyzed by ratelimiting enzymes: indoleamine 2,3-dioxygenase (IDO) and TRP 2,3dioxygenase (TDO-2) [2]. IDO is activated by pro-inflammatory cytokines while TDO in induced by stress hormones. Rate-limiting enzyme of TRP—KYN metabolism in Drosophila is an evolutionary conserved ortholog of human IDO and TDO that is encoded by vermilion gene. Formation of KYN from TRP is deficient in vermilion mutants of Drosophila melanogaster. Hypothesis: Vermilion flies will be more resistant to inducement of IR by HSD. Aim: To evaluate the effect of HSD on development of IR in vermilion mutants of Drosophila melanogaster. Method: The effect of HSD on eclosion time and body weight of imago was compared in vermilion mutants and wild type (Oregon) flies. Results: HSD delayed eclosion time of Oregon flies by 54 % (151.2 ± 19.2 and 232.5 ± 23.1 h, resp., n = 400, p \ 0.003, Mann–Whitney two tailed test) and decreased body weight of imago by 37 % (0.99 ± 0.004 and 0.62 ± 0.003 mg, resp). In vermilion mutants HSD delayed eclosion time by 18 % (176.5 ± 27.6 and 208.9 ± 22.3 h, resp.) and did not affect body weight of imago (0.93 ± 0.004 and 0.92 ± 0.003 mg, resp). Discussion: Although HSD delayed eclosion in both vermilion mutants and Oregon flies, delay of eclosion was threefold shorter in vermilion mutants (18 vs 54 %) than in Oregon flies suggesting that deficient TRP conversion in KYN attenuates development of IR induced by HSD. Our results are in line with our working hypothesis that deficient formation of KYN from TRP attenuates inducement of IR by HSD. Our data suggest that modulation of inflammation-induced activation of TRP—KYN metabolism might be utilized for prevention and treatment of IR and diabetes. Supported by NIH Grant MH104810 (GO). 1. Pasco MY and P Le´opold. (2012) High Sugar-Induced Insulin Resistance in Drosophila Relies on the Lipocalin Neural Lazarillo PLoS One. 2012; 7(5): e36583. doi:10.1371/journal.pone.0036583 2. Oxenkrug G. (2013) Insulin Resistance and Dysregulation of Tryptophan-Kynurenine and Kynurenine Nicotinamide Adenine Dinucleotide Metabolic Pathways. Mol Neurobiol. 48: 294–301. doi: 10.1007/s12035-013-8497-4
B237 IN VITRO MODELLING ACUTE AND CHRONIC INFLAMMATION BASED ON HUMAN MONOCYTES. ANALYSIS OF THE MODULATION OF IL-1 FAMILY MEMBERS Paola Italiani1, Ettore Mosca 2, Luciano Milanesi2, Diana Boraschi1 1
Valeriya Navrotska1, Lyudmila Vorobyova1, Paul Summergrad2, Gregory Oxenkrug2 1 Karazin National University, Kharkiv, Ukraine; 2Tufts University, Boston, MA, USA
Institute of Protein Biochemistry, National Research Council of Italy, Naples, Italy; 2Institute of Biomedical Technologies, National Research Council of Italy, Naples, Italy The course of an acute physiological inflammatory response can be modelled in vitro by exposing human primary monocytes in culture to
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S196 a sequence of conditions simulating the recruitment from blood into an inflamed tissue, the encounter with inflammatory agents, and eventually the conditions promoting resolution and tissue repair. CD14+ monocytes were exposed sequentially to CCL2, Gram + bacterial vesicles, TNF-a, IFN-c, IL-10, and TGF-b, with concomitant changes in temperature and oxygen levels. A parallel model of chronic inflammation was obtained with a different sequence of stimuli (CCL2, a cocktail of bacterial and viral stimuli plus IFN-c immune complexes, survivin, M/GM-CSF) and maintaining the culture at 39 C in hypoxic conditions. We have profiled by RNAseq the transcriptome of monocytes and analysed the variations of expression of the IL-1 family members. Cytokines and receptors of the IL-1 family members are major players in an inflammatory reaction, taking part in all its phases both in normal defence and in pathological situations. IL-1 family cytokines encompass inflammatory and anti-inflammatory factors, including natural antagonists. IL-1 family receptors can be either activating, accessory or non-signalling ‘‘decoy’’ receptors. Alteration of the qualitative, quantitative and spatio-temporal regulation of the IL-1 family network, as observed during a physiological resolving inflammatory defensive response, may lead to pathological inflammation. In the early phase of both acute and chronic inflammation, expression of six cytokines (IL1B, IL1A, IL1RN, IL18, IL18BP, IL36G) and four receptors (IL1R1, IL1R2, IL1RAP, SIGIRR) is similarly regulated, peaking between during the inflammatory phase and returning to the low basal level during the resolution phase in the acute model (except IL18 that was lower than in basal conditions), while remaining higher than in fresh monocytes in the chronic model. Exceptions are the genes of the inhibitors IL18BP and SIGIRR. SIGIRR decreased during the entire course of the reaction, whereas IL18BP significantly increased in the late phases of chronic inflammation. By GSEA analysis, the expression of all the IL-1 family members is mainly modulated during the first 14 h of stimulation in both models, i.e. in the timeframe in which the largest variation of expression occurs. The analysis of differentially expressed genes between two consecutive time points shows that IL-1 family cytokines and receptors are modulated during the entire course of the inflammatory reactions, except in the late phases of chronic inflammation (in which they remain practically unchanged). These results underline the strong modulation of IL-1 family genes, in particular in the initial phases of inflammation, suggesting their involvement in regulating the inflammation outcomes. Work supported by the EU FP7 project BioCog (GA 602461) and by the Cluster project Medintech of the Italian Ministry of University and Research.
B238 GIARDIA MURIS INFECTION REDUCES THE SEVERITY OF CITROBACTER RODENTIUMINDUCED COLITIS Jean-Paul Motta1, Anna Manko2,1, James A. Cotton1, Bruce A. Vallance3
Inflamm. Res. cause of waterborne diarrheal disease. Giardia infections have been associated with decreased incidence of diarrheal disease and fever, and lower serum inflammatory scores via unknown mechanisms. Furthermore, Giardia infections have been concurrently observed with other pro-inflammatory gastrointestinal pathogens, such as enterohemorrhagic Escherichia coli. Objective: The purpose of this study was to assess whether in vivo Giardia infections modulate host responses or susceptibility to coinfection with intestinal pathogenic bacteria and to identify the mechanisms involved. Methods: Male C57BL/6 mice were co-infected with Giardia muris and Citrobacter rodentium and sacrificed at 14 days post-infection. Animal weights were recorded and stool samples collected to assess for bacterial colony forming units (CFUs). Colonic tissues were stained by hematoxylin-eosin (H&E) and scored for macroscopic damage. Granulocyte infiltration was assessed by myeloperoxidase (MPO) assay. Gut bacteria were stained using fluorescent in situ hybridization (FISH—probe against all bacteria), and Citrobacter rodentium was stained by immunofluorescence (anti-C. rodentium lipopolysaccharide, LPS). The capacity of colonic crypts to kill E.coli was performed by crypt killing assays. Results: At 7 and 14 days post-infection,weight loss was reduced in the G. muris + C. rodentium co-infected mice as compared to mice infected with just C. rodentium. Overall bacteria and C. rodentium CFUs in the stool were reduced in G. muris + C. rodentium animals at 2 and 7 days post-infection, compared to C. rodentium animals. Macroscopic and microscopic damage scores and MPO activity were also reduced in co-infected animals compared to C. rodentium infected mice at 14 days post-infection. In the colon of C. rodentium infected mice, we observed translocation of bacteria into the lamina propria (FISH staining), and mucosal attachment and translocation of C. rodentium via LPS staining. In G. muris + C. rodentium animals, we observed less bacterial translocation, and less mucosal attachment of C. rodentium. Accordingly, aerobic and anaerobic CFUs in liver and spleen homogenates were significantly reduced in co-infected mice as compared to the C. rodentium group. Crypt killing assays demonstrated higher antimicrobial capacity against E. coli, in G. muris and G. muris + C. rodentium groups as compared to C. rodentium group. Conclusion: Giardia muris infection reduces the severity of C. rodentium-induced colitis in mice. These results suggest that Giardia infections are protective against the development of severe gastrointestinal disease induced by a co-infecting gastrointestinal bacterial pathogen, potentially via their ability to enhance the anti-microbial capacity of the intestinal epithelium.
B239 DYNAMIC EQUILIBRIUM OF NEUTROPHIL TRAFFICKING AT SITES OF INFLAMMATION AND INFECTIONS Daniel Irimia1,2,3 Massachusetts General Hospital, Boston, MA, USA; 2Harvard Medical School, Boston, MA, USA; 3Shriners Burns Hospital, Boston, MA, USA
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University of Calgary, Biological Sciences, Calgary, Alberta; University of Calgary, Department of Physiology and Pharmacology, Calgary, Alberta; 3University of British Columbia, Division of Gastroenterology, Vancouver, Canada
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Background: Our understanding of polymicrobial gastrointestinal infections and their effects to host biology remains incomplete. Giardia is a protozoan parasite that infects humans, and is a prevalent
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Appropriate inflammatory responses to wounds and infections require the presence of adequate numbers of neutrophils at injury sites. Both insufficient and excessive neutrophil recruitment can be detrimental, favoring the spread of infection or triggering severe tissue damage, respectively. However, the fundamental rules that regulate the trafficking of neutrophils at sites of inflammation/infection are complex and consequently difficult to study using traditional in vivo models.
Inflamm. Res. To elucidate the neutrophil trafficking rules,we designed devices in which human neutrophils emerge directly from a droplet-size samples of whole blood and migrate towards chambers with chemoattractants and microbe-like particles. Inside these devices, human neutrophils could be monitored in detail, under precise control of the mechanical, biochemical, and microbe interactions conditions. We found that the number of neutrophils recruited by chemotaxis and departing by retrotaxis increases and stabilizes to dynamic equilibrium in the presence of chemoattractants alone. The migration of individual neutrophils ceases immediately after phagocytosis, altering the balance between chemotaxis and retrotaxis and increasing the number of neutrophils accumulating to the site. This number is proportional to the number of microbe-like particles in the chambers. Overall, autonomous neutrophil traffic regulation assures that a continuous supply of fresh neutrophils is available to infection sites, that the number of neutrophils accumulating is appropriate to the number of microbes, and that overpopulation is avoided.
S197 further investigate in an intact living organism the mechanisms that promote hematopoiesis development and related diseases. Funding FONDECYT 1140702/FONDAP 15090007.
Novel and Innovative Platforms for Drug Discovery B241 ASSESSING NK CELL KILLING ACTIVITY TO INFORM DRUG DISCOVERY PROGRAMS ON CLINICAL INFECTION RISK Cristina A. St. Pierre, Laura Engstrom, Alexandra Hicks, Ravisankar A. Ramadas Merck Research Labs, Rahway, NJ, USA
B240 IN VIVO IMAGING OF MAMMALIAN HSCS ENGRAFTMENT AND COLONIZATION INTO THE CHT OF ZEBRAFISH EMBRYOS Margarita M. Parada Kusz1,2, Anne Clatworthy2, Humberto Jijon3, Eduardo J. Villablanca4 1 Centro FONDAP de Regulacio´n del Genoma, Universidad de Chile, Santiago, Chile; 2Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA; 3Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, MA, USA, Harvard Medical School, Boston, MA, USA; 4Translational Immunology Unit, Department of Medicine, Solna, Karolinska Institutet and University Hospital, Stockholm, Sweden
Hematopoietic stem cells (HSCs) generate the full repertoire of blood and immune cell types throughout an organism’s life. They are produced during ontogeny, and actively respond to niche-specific environmental cues to mobilize, expand or differentiate. These nourishing niches dynamically change locations throughout development, beginning in the aorta–gonad–mesonephros (AGM) region and the yolk sac, followed by the placenta, fetal liver, spleen and bone marrow. Similar to mammals, HSCs in zebrafish originate early in the aorta, move transiently to the caudal hematopoietic tissue (CHT) and ultimately mobilize to the thymus and kidney (equivalent to the mammalian bone marrow). Capitalizing on these similarities, we are developing a xenograft model to study HSCs homing and niche-interactions in vivo and in real time using mammalian bone marrow derived HSCs transplanted into fish larvae. To this aim, CD117+ cells where isolated from mice bone marrows by means of negative selection, fluorescently stained, and transplanted into the blastoderm of 5 hpf zebrafish embryos. Live imaging of transplanted embryos throughout development revealed that CD117+ cells in general colocalized with the intermediate cell mass (ICM) at 24 hpf, with the AGM region at 48 hpf, and CHT at 72 hpf. CD117+ cells would evidence in situ proliferation, and in some cases, moved into circulation. The engraftments do not suggest mayor alteration of endogenous hematopoiesis as zebrafish neutrophil development and function is not impaired. Using this approach, murine HSCs could be followed for at least 7 dpf. Engraftment with human promyelocytic HL-60 cells also shows that they are able to colonize the CHT. Collectively, these data reveal the extent of conservation and homology shared on protein cues among vertebrates during hematopoiesis development, positioning the zebrafish as a good model to
Natural killer (NK) cells are critical for the clearance of pathogeninfected cells and tumor cells that lack class I MHC expression. We developed a non-radioactive flow cytometry based assay to evaluate NK cell killing activity in human and mouse tissues. In brief, human PBMC or mouse splenocytes were co-incubated with target cells (MHC class I negative tumor cell lines) under optimized conditions and the percentage of target cell killing was used to evaluate NK cell activity. We found that SYK/ZAP70 antagonists inhibited NK cell killing function in human, but not mouse tissues, suggesting that certain pathways or pathway inhibitors can have species specific effects. We used this assay to compare the impact of clinical immunosuppressants (cyclosporine A, leflunomide, methotrexate, mycophenolate mofetil, prednisolone, rapamycin, and tofacitinib) and pathway specific inhibitors (BTK, IRAK4, JAK, PI3Kd, RORcT, and SYK/ZAP70) on NK cell activity, which has the potential to help inform on dose selection and the relationship between efficacy and safety. This assay could also serve as a phenotypic screen to identify immunostimulators and is amenable to the evaluation of antagonists as well as agonist small molecules and biologics in NK cell responses.
B242 ETANERCEPT, ABATACEPT AND ANAKINRA TREATMENT AMELIORATES INFLAMMATION AND PAIN IN A NOVEL MONO-ARTHRITIC MULTI-FLARE MODEL OF STREPTOCOCCAL CELL WALL INDUCED ARTHRITIS: FURTHER CHARACTERIZATION IN A RODENT MODEL OF COLLAGEN INDUCED ARTHRITIS Kalyan Chakravarthy, Robert Faltus, Anwar Murtaza, Milenko Cicmil Merck Research Laboratories, Rahway, NJ, USA To delineate the role of TNF, T cells, and IL-1 in pathogenesis of SCW-induced arthritis, we investigated the activity of clinical agents, Etanercept, Abatacept and Anakinra ina novel mono-arthritic multiflare Rat Streptococcal Cell Wall (SCW) model. Comparative evaluation of these targeted therapies was also performed in the rat Collagen Induced Arthritis (CIA) model. SCW arthritis was induced in female Lewis rats with an intra-articular injection in the hind ankle joint on day 1 (flare 1) followed by two intravenous challenges on
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S198 days 21 (flare 2) and 42 (flare 3) of SCW extract PG-PS 100p. CIA was induced using methods previously described. Inflammation and pain were monitored by measuring paw swelling and withdrawal threshold respectively. In addition, cytokine profiling, cell phenotyping, bioluminescence/lCT imaging and histopathology were also performed in the local joint. In the SCW model late prophylactic administration of Etanercept, Abatacept and Anakinra significantly inhibited paw swelling by C60 % (p \ 0.001), C60 % (p \ 0.001), 88 % (p \ 0.001) and pain by 37 % (p \ 0.05), C28 % (p \ 0.05) and 64 % respectively in flare 2. Etanercept in flare 3 inhibited paw swelling by 60 % (p \ 0.001) and partially inhibited pain by 27 %. Interestingly, prior treatment with Etanercept in flare 2 followed by a wash out period of 14 days and re-administration in flare 3 led to a loss in efficacy, potentially due to immunogenicity. Abatacept administration in flare 3 had no effect on either paw swelling or pain in rats that were treated in flare 3 alone or in rats that were treated previously in flare 2. In the CIA model, both late prophylactic and therapeutic treatment with Etanercept inhibited paw swelling by 50 % (p \ 0.001). A loss of efficacy with Etanercept was also observed in the CIA model when administered prophylactically possibly due to immunogenicity. Prophylactic, late prophylactic and therapeutic administration of Abatacept in the CIA model significantly inhibited paw swelling by 100 % (p \ 0.001), 42 % (p \ 0.001) and 34 % (p \ 0.001) respectively. The additional biomarkers corroborated with efficacy in both models. We developed a novel multi-flare SCW model that can be used to evaluate clinically relevant parameters of inflammation and pain simultaneously. Using clinical agents Etanercept, Abatacept and Anakinra targeting TNF, T cells and IL-1 respectively we have delineated distinct pathogenic mechanisms of inflammation and pain at different stages of disease in the SCW model. We also show similar profiles of efficacy in late prophylactic and therapeutic regimens in the CIA model. The flaring mechanism in the SCW model allows for drug washout periods in between compound administration. This might provide useful pre-clinical insights on potential immunogenicity mechanisms that may be relevant in a clinical setting. Our novel model can facilitate innovative assessment of anti-rheumatic agents in multiple flares and offers a powerful tool for drug discovery.
Inflamm. Res. readout platform. Optimal adenoviral shRNA delivery conditions were established using reporter constructs in two adenoviral backbones (C01 and C20) and quantified by FACS. Cytotoxicity was assessed using Cell titer blue assay. Negative control (ffluc and eGFP) and putative positive control (IL17RA, NFjB, C/EBPb, CXCL1, IL17RC and MAPK14) shRNAs were used to establish optimal screening conditions and assay window. A set 320 shRNA viruses from the SilenceSelectTM shRNA library was screened at three MOIs to establish final screening characteristics. Results: Recombinant human IL-17A induced a dose-dependent increase in GROa production in all three donors, in both 96- and 384-well format. The signal to background ratio was comparable for AlphaLisa and MSD both in the presence (C259) and absence (C69) of 5 ng/mL TNFa stimulation. The AlphaLisa assay generated more reproducible data. Optimal (100 %) transduction was observed with C20 backbone at MOI *20 in 96 well format. The effect of negative control viruses was \25 % compared to non-transduced cells. Knockdown by shRNA of IL17RA, NFjB, C/EBPb, CXCL1, IL17RC and MAPK14 consistently reduced IL-17A induced GROa production. The assay was successfully automated and screened in duplicate, on two separate occasions, with a pilot screen set. No plate effects observed based on heat maps. Pearson correlation coefficient and Spearman correlation coefficient were [0.4. Kappa statistics [0.2. Conclusions: A reproducible and robust assay was developed in primary human foreskin fibroblasts to identify novel therapeutic targets modulating the IL17 pathway.
Novel Targets and New Drugs in Inflammation B244 THYMOQUINONE INHIBITS INFLAMMATION IN IL-1BETA-STIMULATED SK-N-SH CELLS Olumayokun Olajide1, Ravikanth Velagapudi1, Asit Kumar2, Simon Schindler2, Bernd L. Fiebich2 1
B243 A NOVEL ASSAY FOR HIGH THROUGHPUT shRNA-BASED TARGET DISCOVERY IN THE IL-17 PATHWAY Jeroen DeGroot, Blandine Mille-Baker, David F. Fischer Charles River Laboratories, Leiden, The Netherlands Objectives: The IL-17 pathway is associated with pathology in numerous autoimmune and inflammatory conditions, such as psoriasis, rheumatoid arthritis, multiple sclerosis, Crohn’s Disease and SLE. Consequently, IL-17 and its downstream signaling routes emerge as potential targets for therapy. Monoclonal antibodies against IL-17 (such as secukinumab; recently approved for psoriasis) and IL-17R (e.g. Brodalumab; in phase 2) have been developed. The objective of the current study was to develop and validate a high throughput assay for the identification novel drugable target genes downstream of IL17 receptor A (IL-17RA) using an arrayed adenoviral shRNA library (SilenceSelect). Methods: Human foreskin fibroblasts (3 donors) were cultured in 96 well plates and stimulated with IL-17 in the presence or absence of TNFa. GROa production was assessed by MesoScale Discovery (MSD) or AlphaLisa to confirm cellular functionality and select the
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University of Huddersfield, Huddersfield, England; 2University of Freiburg Medical School, Freiburg, Germany
Thymoquinone is a phytochemical antioxidant compound in the oil obtained from the seeds of Nigella sativa (black cumin seed oil). Studies have suggested that thymoquinone produces anti-inflammatory property. Previously, we have shown that thymoquinone inhibited neuroinflammation in LPS-activated rat priamry microglia. However, nothing is known about its direct effect on neurons. In this study, we have investigated the effects of thymoquinone on inflammation induced in SK–N–SH neuroblastoma cells stimulated with interleukin-1beta (IL-1b). Cultured SK–N–SH cells were treated with thymoquinone (0.5, 1 and 2.5 lM) prior to stimulation with IL-1b. Levels of prostaglandin E2(PGE2) production was measuredusing enzyme immunoassay (EIA), while ELISAs were used to detect levels of pro-inflammatory cytokines tumour necrosis factor-alpha (TNFa) and interleukin-6 (IL-6). Levels of cyclooxygenase-2 (COX-2, microsomal prostaglandin E synthase-1 (mPGES-1) were measured with western blot. Further experiments were carried out on I&B phosphorylation and degradation using western blots. Results showed that thymoquinone (0.5, 1 and 2.5 lM) produced concentration-dependent and significant inhibition of PGE2, TNFa and IL-6. These concentrations of the compound also reduced protein levels of COX-2 and mPGES-1. At 1 and 2.5 lM, there was marked inhibition of I&B phosphorylation and degradation by this compound. Taken together, these results demonstrate that thymoquinone suppressed inflammation
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in neurons, suggesting its therapeutic potential in inflammation-mediated neuronal damage found in neurodegenerative disorders.
inflammatory effect of LQFM-021, shown that the nitric oxide is important to this effect. Sources of research support: CNPq and CAPES.
B245 MECHANISMS INVOLVED IN THE ANTIINFLAMMATORY ACTIVITY OF THE NEW PYRAZOLE COMPOUND 5-(1-(3FLUOROPHENYL)-1H-PYRAZOL-4-YL)-2HTETRAZOLE (LQFM-021)
B246 IDENTIFICATION OF CD44BRIGHT, A NOVEL SUBSET OF UTERINE NK CELLS CORRELATED TO ABORTION IN RECURRENT PREGNANCY LOSS MODEL MICE Jun Tanaka, Yuki Fukunaga
Iziara F. Florentino1, Daiany Priscilla B. Silva1, Roberta C. Lino1, Taciane S. Silva2, Carlos Roge´rio Tonussi2, Ricardo Menegatti1, Elson A. Costa1 Federal University of Goia´s, Goiaˆnia, Brazil; 2Federal University of Santa Catarina, Floriano´polis, Brazil
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Introduction: Inflammation is an adaptive response that is triggered by noxious stimuli and conditions, such as infection and tissue injury. The pyrazole compounds are known to possess anti-inflammatory, analgesic and antipyretic activities. The aim of this work was to evaluate anti-inflammatory effect of the new pyrazole derivative 5-(1(3-fluorophenyl)-1H-pyrazol-4-yl)-2H-tetrazole (LQFM-021) in acute and chronic models of inflammation and investigate mechanisms involved in this effect. Methods: The LQFM-021, pyrazole derivative, was synthesized at the Laboratory of Medicinal Pharmaceutical Chemistry/UFG. In this study were used Swiss albino mice and Wistar rats females. The experimental protocol was approved by the Research Ethics Committee of UFG (number 017/13). The pharmacological tests: Carrageenan-induced paw edema and pleurisy tests and CFA-induced chronic arthritis model. Results: In the paw edema test, the treatments with LQFM-021 30 or 60 mg/kg p.o., began to reduce the edema starting from the 2nd hour by 25.3 and 33.7 %, respectively, at 3rd hour by 25.5 and 33.0 %, respectively, and at 4th hour by 30.4 and 29.0 %, respectively. In the carrageenan-induced pleurisy test, the treatment with LQFM-021 (30 mg/kg p.o.) reduced the leukocyte migration into pleural cavity by 38.1 %, with reduction of polymorphonuclear recruitment, also was observed a reduction of the Evan’s blue concentration in the pleural exudates by 30.9 %, decrease myeloperoxidase activity by 43.0 % and decreased the TNF-a level by 47.5 % and IL-1b levels by 26.8 %. Moreover, was also seen which the pre-treatment with L-name was capable of reversed the effect of the LQFM-021 on leukocytes migration, TNF-a and IL-1b levels. In the CFA-induced chronic arthritis model, the treatment with LQFM-021 (30 mg/kg once/day) promoted a progressive reduction of Paw Lifting Time (PLT) from the 2nd until 6nd day, by 26.3; 31.4; 31.3; 41.8 and 47.0 %, respectively. The formation of edema was reduced from the 1st day of the treatment by 36.9; 23.6; 20.7; 21.7; 24.6 and 18.0 %, respectively. Also, there was a reduction in the number of leukocytes by 34.2 % and polymorphonuclear by 54.3 % to synovial fluid. The treatment with LQFM021 (15 mg/kg twice/day), reduced the PLT from the 2nd until 6nd day, by 29.6; 15.6; 21.2; 29.2 and 29.5 %, respectively. Conclusions: The pyrazole compound LQFM-021 presented anti-inflammatory activity in acute and chronic models of inflammation. In this anti-inflammatory effect involve the reduction leukocyte migration, the TNF-a and IL-1b levels and decreased of the myeloperoxidase activity. In addition, we also found that the inhibition of nitric oxide synthase promoted by L-NAME reduced the anti-
Central Reseaech Laboratory, Japan Blood Products Organization, Izumisawa, China Introduction: Women undergoing three or more consecutive spontaneous abortions are believed to suffer from recurrent pregnancy loss (RPL). Women with RPL display a higher rate of and cytotoxic activity of NK cells in the periphery and endometrium. Conversely, normal uterine NK (uNK) cells play an important role in the establishment and outcome of pregnancy. The triggering mechanism that induces NK cells to attack the fetus remains unexplained; however, most RPL cases have been explained by autoimmune abnormalities. Intravenous immunoglobulin (IVIg) has been utilized in the treatment of several inflammatory and autoimmune disorders. The efficacy of IVIg in the treatment of RPL has been confirmed in several clinical trials; however, its precise mechanism remains unknown, mainly because the mechanism of abortion remains to be elucidated. Therefore, the mechanism of IVIg action, as well as that of abortion in RPL must be explained. Methods: All animal procedures were approved by the Animal Care and Use Committee of our organization. In this study, the CBA/ J 9 DBA/2 N mating mouse model was employed to analyze RPL. The abortion rate was increased by activating maternal immunity via an intraperitoneal LPS injection; the preventive effect of IVIg administration on immune reproductive failure was also examined. The peripheral and uterine NK cells were defined as CD45 + CD3CD49b+ . The percentage of activated peripheral NK (CD69+) cells or the specific uNK cell subset was determined by flow cytometry. Results: IVIg did not affect peripheral immune cell activation; however, it attenuated the rate of abortion in RPL model mice in a dose-dependent manner. Additionally, we confirmed a significant increase in the number of uNK cells in RPL model mice, which was suppressed by IVIg injection. We also demonstrated a novel flow cytometry-based approach for the characterization of uNK subsets by CD44 expression. Specifically, we discovered the presence of two distinct subsets: CD44bright and CD44mid. We observed an increase in the CD44bright uNK subset in RPL model mice, while the CD44mid uNK subset remained unchanged. Furthermore, the CD44bright uNK subset number remained unchanged when the abortion was reduced by IVIg administration, facilitating the differentiation between pathological uNK cells and normal uNK cells, based on CD44 expression. We observed a low expression of the CD94 inhibitory receptor on the CD44bright uNK subset. Moreover, we also confirmed the presence of activated peripheral NK cells belonging to the CD44bright subset in RPL model mice with or without IVIg injection. Conclusion: Based on these results, we proposed CD44 to be the marker of pathological uNK cells, with the CD44bright uNK subset acting as a novel RPL target. Elucidation of the mechanism underlying IVIg activity against RPL may help explain the optimal design of IVIg injection, as well as the mechanisms underlying abortion in RPL.
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B247 RESVERATROL IMPROVES THE PATHOGENESIS OF NONALCOHOLIC STEATOHEPATITIS THROUGH INHIBITION OF ENDOTOXININDUCED LIVER INFLAMATION AND FIBROSIS Takaomi Kessoku1, Koichiro Wada2, Yasushi Honda1, Yuji Ogawa1, Kento Imajo1, Atsushi Nakajima1 1
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Yokohama city university, Yokohama, Japan; Shimane University Faculty of Medicine, Matsue, Japan Introduction: Nonalcoholic fatty liver disease (NAFL) morbidity rate in Asia Pacific region is close to 12–24 %, while in Western countries is about 20–30 %. and NAFLD can progress to nonalcoholic steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma. In spite of its high prevalence, up till now here is no proven effective treatment for NAFLD. Although gut-derived endotoxin (ET), such as lipopolysaccharide (LPS), plays a key role in the pathogenesis of nonalcoholic steatohepatitis (NASH), detailed mechanisms of this pathogenesis becomes clear. We previously reported that overexpression of CD14 via activation of leptin-STAT3 signaling in Kupffer cells induced hyper-inflammatory response to low-dose ET, resulting in progression from simple steatosis to steatohepatitis with liver fibrosis. Therefore, we hypothesized that inhibition of leptin-STAT3 signaling in Kupffer cells may lead to attenuate the progression of steatohepatitis via inhibition of CD14 expression. Aim: The aim of this study was to investigate whether the resveratrol which is known to inhibit activation of STAT3, improves the pathogenesis of steatosis or steatohepatitis in murine model. Methods: 8-week-old male C57BL/6 J mice were randomly distributed into 3 groups of 10 animals each: a high fat diet group (HF), HF supplemented with 2 mg/kg resveratrol daily (HFR2), and HF supplemented with 20 mg/kg resveratrol daily (HFR20). After 12 weeks of dietary treatment, the rats were euthanized and relevant tissues were prepared for subsequent analysis. In this study, E. coliderived LPS (0.25 mg/kg) was used. A.
We investigated whether the resveratrol attenuates HFD-induced steatosis. B. We investigated whether the resveratrol attenuates ET-induced liver damage via inhibition of response to ET. C. We investigated whether the resveratrol improves the pathogenesis of long-term exposed ET-induced steatohepatitis with liver fibrosis. Results: Resveratrol prevented the high fat–induced steatosis assessed by semiquantitative grading, which furthermore corresponded with a complete normalization of the hepatic triglyceride content (p \ 0.001), despite no change in total body fat, and hepatic SREBP1c expression was significantly decreased as compared with HF. HFR showed significant inhibition of hepatic CD14 expression through suppression of STAT3 activity in Kupffer cells, following inhibition of a single low-dose LPS-induced liver damage. Moreover, long-term low-dose LPS-induced liver fibrosis in HFR is significantly decreased as compared with HF. Conclusion: These data indicated that the resveratrol improves not only the pathogenesis of steatosis thorough inhibition of lipogenesis but also steatohepatitis through inhibition of endotoxin-induced liver damage via suppression of STAT3-CD14 signaling in Kupffer cells. The resveratrol may have application for the treatment of NAFLD.
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B248 NEW ANALOGUES FROM IKK-b INHIBITOR LASSBIO-1524 AS POTENT ANTIINFLAMMATORY CANDIDATES Nata´lia M. Cordeiro, Rosana H.C. Freitas, Carlos Alberto M. Fraga, Patricia D. Fernandes Federal University of Rio de Janeiro, Rio de Janeiro, Brazil LASSBio-1524, a new ikB kinase b inhibitor, participates in the activation of NF-kB canonical pathway and demonstrated anti-inflammatory efficacy in vivo (Eur J Med Chem., 46, 1245, 2011). Our objective is to evaluate the anti-inflammatory potential of new substances synthesized from LASSBio-1524: LASSBio-1760, LASSBio-1763, LASSBio-1764. Cell migration was induced by carrageenan injection in the subcutaneous air pouch (SAP), mice (Swiss webster, 22–25 g, n = 6–8) received 1 h before carrageenan injection, treatment with LASSBios (3, 10, 30, 100 lmol/kg, p.o.), IKK-b inhibitor SC-514 (30 lmol/kg p.o.) or dexamethasone (1.5 lmol/kg, i.p.). Air pouch exudate was collected for quantification of TNF-a. Leukocytes from the SAP were pretreated ex vivo with LASSBios (1, 10 and 30 lM) to assess reactive oxygen species (ROS). Results are expressed as mean ± SD and statistical analysis were performed by ANOVA followed by Bonferroni test (*p \ 0.05). The protocol for animal use was approved by CEUA/UFRJ #DFBCICB015-04/16. All substances reduced dose-dependent and significantly leukocyte migration. Animals pretreated orally with vehicle (Polysorbate 80) injected with carrageenan in the SAP presented increase in leukocyte number (78.3 ± 13.5 9 103 cells/lL) when compared to the group injected with PBS in SAP (1.1 ± 0.5 9 103 cells/lL). Treatments with SC-514 (36.2 ± 19.4 9 103 cells/lL), dexamethasone (19.4 ± 8.6 9 103 cells/lL); LASSBio-1524 (3 lmol/kg: 51.8 ± 3.6 9 103 cells/lL, 10 lmol/kg: 42.2* ± 3.9 9 103 cells/lL, 30 lmol/kg: 22* ± 4.1 9 103 cells/lL, 100 lmol/kg: 12.7* ± 4.2 9 103 cells/ lL) or its three analogues LASSBio-1760 (3 lmol/kg: 51.9 ± 32.2* 9 103 cells/lL, 10 lmol/kg: 40.2* ± 22.3 9 103 cells/lL, 30 lmol/kg: 35.7* ± 12.1 9 103 cells/lL, 100 lmol/kg: 11.0* ± 4.2 9 103 cells/lL), LASSBio-1763 (3 lmol/kg: 47.0* ± 18.3 9 103 cells/lL, 10 lmol/kg: 39.3* ± 4.7 9 103 cells/lL, 30 lmol/kg: 27.5* ± 10.0 9 103 cells/lL, 100 lmol/kg: 3.0* ± 1.7 9 103 cells/ lL) and LASSBio-1764 (3 lmol/kg: 40.9* ± 8.8 9 103 cells/lL, 10 lmol/kg: 34.0 * ± 9.6 9 103 cells/lL, 30 lmol/kg: 26.9* ± 10.2 9 103 cells/lL, 100 lmol/kg: 1.7* ± 1.1 9 103 cells/lL) reduced leukocyte migration. All doses also inhibited TNF-a production: LASSBio-1524 reduced in 79, 79, 87 and 94 %; LASSBio1760 reduced in 99, 97, 95 and 93 %; LASSBio-1763 reduced in 92, 96, 93 and 99 % and LASSBio-1764 reduced in 96, 97, 95 and 96 %, respectively. All substances significantly inhibited ROS production: LASSBio-1524 reduced in 30, 41 and 45 %; LASSBio-1760 reduced in 42, 33, 29 %; LASSBio-1763 reduced in 61, 97, 98 % and LASSBio-1764 reduced in 83, 84, 98 %. The new analogues of LASSBio-1524 showed anti-inflammatory potential even better than the original molecule, placing them as new candidates for the development of new anti-inflammatory drugs. Financial support CNPq, CAPES, FAPERJ and Instituto Vital Brazil (donation of animals). Technical support Alan Minho.
Inflamm. Res.
B249 LIPOSOMES LOADED WITH 3-PHENYLCOUMARIN DERIVATIVES AND BACCHARIS DRACUNCULIFOLIA EXTRACT REDUCE THE INFILTRATE OF NEUTROPHILS IN SYNOVIAL FLUID OF ARTICULAR INFLAMMATION INDUCED BY ZYMOSAN Mica´ssio F. Andrade1, Andre´a S.G. Figueiredo-Rinhel2, Ana Elisa ˆ nica T. Pupo3, C.S. Azzolini2, Jairo K. Bastos3, Fla´vio S. Emery3, Mo Yara M. Lucisano-valim2 1
Department of Biochemistry and Immunology, School of Medicine, University of Sa˜o Paulo, Ribeira˜o Preto, Sa˜o Paulo, Brazil; 2 Department of Physics and Chemistry, School of Pharmaceutical Sciences, University of Sa˜o Paulo, Ribeira˜o Preto, Sa˜o Paulo, Brazil; 3 Department of Pharmaceutical Sciences, School of Pharmaceutical Sciences, University of Sa˜o Paulo, Ribeira˜o Preto, Sa˜o Paulo, Brazil Introduction: Neutrophils have an important part in acute inflammatory responses. When they find a stimulus, these cells produce reactive oxygen species (ROS), release lytic compounds of their granules, as well as release of their DNA content. Therefore, these processes can disorders provoked. Natural and synthetic compounds are able to reduce the deleterious effects of neutrophils activation. In this context, 6,7-dihydroxy-3-[30 ,40 -methylenedioxyphenyl]-coumarin (3-PD), a synthetic 3-phenylcoumarin derivative and Baccharis dracunculifolia D.C. (Asteraceae) leaf extracts (BdE) showed, in vitro assays, a negative modulation of neutrophils oxidative metabolism. Thus, the aim of this work is investigate the effect of these two substances, incorporated in liposomes (LPM), in animal model of articular inflammation induced for zymosan. Methods and results: Wistar rats (200 g) were treated i.p., with liposomes loaded with 3-PD (LPM 3PD 1.5 mg/Kg), BdE (LPM BdE 9 mg/ Kg), or with control substances [Quercetin (LPM Quer 1.5 mg/Kg)— phenolic compound pattern, or with dexamethasone (Dexa 4 mg/Kg)— drug pattern, or with dimethyl sulfoxide (LPM DMSO)—positive control]. Liposomes were prepared by ethanol injection method. Vesicles composed of soya phosphatidylcholine and cholesterol (SPC:CHOL 5:1). One h late of treatment, the animals were injected intra-articular with 300 lg of zymosan. Six h late, the animals were sacrificed and back the synovial fluid for total leucocytes and neutrophils recruitment analyze. The diameter of articulation was verified before and after of induction of inflammation. The animals treated with LPM 3-PD and LPM BdE showed reduction significantly in diameter of articulation in relation of control animals (LPM DMSO). Therefore, the treatments reduce significantly the infiltrate of total leucocytes and neutrophils in articulation. This reduction was similar for all treatments. Conclusion: The treatment of animals with LPM 3-PD and LPM BdE reduced all the inflammatory parameters analyzed. Taken together, the dates show these substances, incorporate in this drug carrier system, with potential therapeutic application in the treatment of inflammatory diseases. Support FAPESP, CNPq and CAPES.
B250 LASSBIO-1828: A NEW IKK-b POTENTIAL INHIBITOR Tayna´ S. Valerio, Nata´lia M. Cordeiro, Rosana H.C. Freitas, Carlos Alberto M. Fraga, Patricia D. Fernandes Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
S201 Inflammation is a beneficial host response to injury that has as goal the restoration of tissue structure and function. The LASSBio-1828 is a new n-acilhydrazone derived from LASSBio-1524, an IKK-b inhibitor (Eur J Med Chem., 46, 1245, 2011). Our objective was to evaluate the anti-inflammatory potential of LASSBio-1828 through formalin-induced licking and carrageenaninduced inflammation in the subcutaneous air pouch (SAP). Male Swiss webster mice (22–25 g, n = 4–6) were pre-treated with LASSBio-1828 (10, 30, 100 lmol/kg, p.o.), LASSBio-1524 (30 lmol/ kg, p.o.), SC-514 (IKK-b inhibitor, 30 lmol/kg, p.o.) or vehicle 1 h before formalin injection in mice paws or carrageenan injection in the SAP. Formalin-induced licking response was evaluated during 30 subsequent minutes and 24 h after carrageenan injection in the SAP the exsudate was collected for leukocyte counting, TNF-a and nitric oxide (NO) measurements. Reactive oxygen species (ROS) were measured in leukocytes obtained from exsudate of the carrageenan-induced inflammation in the SAP. Cells were ex vivo incubated with LASSBio1524 (30 lM) or LASSBio-1828 (1, 10, 30 lM) before treatment with phorbol myristate acetate (PMA). Results are expressed as mean ± SD and statistical analysis were performed by ANOVA followed by Bonferroni test (p \ 0,05 %). Protocols for animal use received number #DFBCICB015-04/16. LASSBio-1828 showed any antinociceptive effect in the 1st phase of formalin-induced licking, but all doses inhibited the 2nd phase: 10 lmol/kg = 128 ± 41* s; 30 lmol/kg = 76 ± 26* s; 100 lmol/ kg = 83 ± 32* s when compared to vehicle = 193 ± 32 s. Carrageenan-induced leukocyte migration to SAP was significantly reduced by 100 lmol/kg dose (42 ± 12*cells 9 106/mL, when compared to PBS in SAP = 3±2cells 9 106/mL or carrageenan in SAP = 65 ± 9cells 9 106/mL). All doses of LASSBio-1828 significantly reduced TNF-a level in exudate: 10 lmol/kg = 321 ± 210*ng/mL; 30 lmol/kg = 371 ± 272*ng/mL; 100 lmol/kg = 390 ± 113*ng/mL, when compared with PBS in SAP (20 ± 41 ng/ mL) or carrageenan in SAP (755 ± 84 ng/mL). Results of LASSBio1524 and SC-514 were 99 ± 49*ng/mL and SC-514 = 527 ± 270 ng/mL, respectively. Concentration of NO in the exudate decreased approximately 50 % at all doses of LASSBio1828: 10 lmol/kg = 66 ± 65*lM; 30 lmol/kg = 90 ± 62*lM; 100 lmol/kg = 100 ± 49*lM, when compared to PBS in SAP (26 ± 2 lM) or carrageenan in SAP (279 ± 86 lM). The results of LASSBio-1524 and SC-514 were 65 ± 50 and 64 ± 44*lM, respectively. Reduction in ROS production was: 1 lM = 4 9 105 ± 3 9 104 arbitrary units; 10 lM = 2 9 105 ± 1 9 104*au; 30 lM = 1 9 105 ± 1 9 104*au, when compared to control group (6 9 105 ± 1 9 105au). The result of LASSBio-1524 was 3 9 105 ± 1 9 104 au. These results indicate that the new n-acylhydrazone, LASSBio1828, seems to be, at least in part, better than the original molecule LASSBio-1524 in tested models suggesting this new substance to be evaluated in other models of inflammation. Financial support CAPES, CNPq, FAPERJ, Instituto Vital Brazil (donation of animals). Technical support Alan Minho.
B251 ANTI-INFLAMMATORY ACTIVITY OF FRACTIONS OBTAINED FROM CHOISYA AZTECPEARL, A NEW HYBRID OF CHOISYA TERNATA Patrı´cia Ribeiro de Carvalho1, Denise Ropero2, Fa´bio Boylan 2, Patricia Dias Fernandes1 Federal University of Rio de Janeiro, Rio de Janeiro, Brazil; 2Trinity College Dublin, Dublin, Republic of Ireland
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S202 C. Aztec-Pearl (Rutaceae) is a hybrid of C. Ternata and C. Dumosa var. arizonica. In traditional medicine C. ternata has been used in Mexico. Infusions prepared with the leaves of the latter species are claimed to have antispasmodic and stimulant properties. Our objective was to investigate a possible anti-inflammatory activity from C. Aztec-Pearl’s leaves crude ethanol extract and its fractions. Leaves of C. Aztec-Pearl were collected in Dublin and a voucher specimen (#TCD2,895) was deposited at TCD Herbarium. The crude ethanol extract (E) was submitted to a liquid–liquid extraction yielding the fractions: hexane (H) and ethyl acetate (EA). Anti-inflammatory activities were evaluated by the formalin-induced licking and carrageenan-induced inflammation into the subcutaneous air pouch (SAP) models. Mice (Swiss Webster, 22–25 g, n = 4–6) were orally treated with 10, 30 or 100 mg/kg of each fraction 1 h before formalin or carrageenan injection. After 24 h of carrageenan injection into the SAP, animals were euthanized and exsudate was collected for subsequent dosages of nitric oxide (NO) and protein. The results are presented as mean ± SD and statistical analysis were performed by ANOVA followed by Bonferroni test (*p \ 0.05). Protocols for animal use received number #DFBCICB015-04/16 (COBEA/UFRJ/Brazil). None of the fractions inhibited the first phase (nociceptive) of the response to formalin, although all of them significantly reduced the 2ndphase (inflammatory). The results were: 10 mg/kg: E = 75 ± 18*s; H = 82 ± 10*s; EA = 123 ± 15*s; 30 mg/kg: E = 32 ± 43*s; H = 32 ± 10*s; EA = 68 ± 39*s; 100 mg/kg: E = 70 ± 29*s; H = 13 ± 9*s; EA = 44 ± 13*s, compared to vehicle = 212 ± 22 s. E and EA significantly reduced leukocyte migration into the SAP at doses of 30 mg/kg: E = 13 ± 6*cells 9 106/mL; EA = 27 ± 17*cells 9 106/mL and 100 mg/kg: EA = 24 ± 16*cells 9 106/mL, E = 15 ± 10*cells 9 106/mL; when compared to vehicle-treated group:1.1 ± 0.6 cells 9 106/mL and carrageenan-injected group:70 ± 21 cells 9 106/mL. NO concentration in the exsudate was significantly reduced by E and H fractions at all doses, 10 mg/kg: E = 238 ± 117 lM*; H = 151 ± 35 lM*, 30 mg/kg: E = 413 ± 141 lM*; H = 167 ± 42 lM*; 100 mg/kg: E = 237 ± 142 lM*; H = 246 ± 146 lM*, the EA reduced NO production in more than 60 % only in two doses: 30 mg/kg: 20 ± 14 lM* and 100 mg/kg: 81 ± 44 lM*, when comparing to vehicle-treated: 32 ± 19 lM and carrageenan-treated group: 796 ± 117 lM. All fractions also reduced protein extravasation at doses: 10 mg/kg: E = 165 ± 64 lM*; H = 130 ± 72 lM*; EA = 135 ± 39 lM*; 30 mg/kg: E = 147 ± 112 lM*; H = 33 ± 17 lM*; EA = 51 ± 21 lM*; 100 mg/kg: E = 161 ± 84 lM*; H = 154 ± 60 lM*; EA = 146 ± 97 lM*, when comparing to vehicle-treated: 24 ± 15 lM and carrageenan-treated group: 215 ± 76 lM. Our results indicate a significant anti-inflammatory activity of this plant, and contribute to the studies with the Rutaceae family. Financial support Capes, Cnpq, FAPERJ and Instituto Vital Brazil (donation of animals). Technical support Alan Minho.
Inflamm. Res. Marie-Christine Ceccotti1, Christelle David1, Laetitia Perret1, Martine De Vos3, Reginald Brys2, Rene´ Galien1 1
GALAPAGOS SASU, Romainville, France; 2GALAPAGOS NV, Mechelen, Belgium; 3Ghent University Hospital, Ghent, Belgium Objective: Filgotinib (known as GLPG0634) is a JAK inhibitor that has been shown to be selective for JAK1 over the 3 other family members (JAK2, JAK3 and TYK2) in biochemical and human whole blood assays. It has been shown to be efficacious in arthritis and colitis mouse models. Filgotinib is currently being assessed as a treatment for Crohn’s disease (CD) in a Phase 2 study. Here, we compare signaling mechanisms, in particular JAK1 inhibition-related effects, regulated by filgotinib in mouse and human colon in vivo or ex vivo, respectively. Methods: In vivo efficacy of 30 mg/kg filgotinib QD was evaluated in a mouse chronic DSS (dextran sodium sulphate, 3 cycles)-induced colitis model using macroscopic and histopathological scorings, immunohistochemistry and gene expression analysis by qPCR. Inflamed colon biopsies from IBD patients were cultured for 18 or 24 h in the presence of 5 lM GLPG0634 and gene expression as well as STAT phosphorylation were analyzed using qPCR and 5-Plex STAT kit Luminex, respectively. Results: Filgotinib strongly prevents experimental colitis as demonstrated by reduction of DAI, lower histological disease severity, reduced inflammatory cell infiltration and lower expression of inflammatory genes in colon tissue from filgotinib-treated mice compared to controls. Colons from DSS-treated mice displayed higher STAT3 phosphorylation levels, which were prevented by treatment with filgotinib. Of interest, the expression of genes that activate (IL-6) or are induced by the JAK1 pathway (MX1, MX2) followed the same regulation as pSTAT3, suggesting a relationship between these readouts and a role of the pathway in the disease. Remarkably, the same regulation of IL-6 and MX1 expression and relationship with STAT3 phosphorylation were observed in cultured colon biopsies from IBD patients in presence of filgotinib. Conclusions: These new data provide further mechanistic understanding for the efficacy of JAK1 inhibition in the pre-clinical mouse colitis model, highlighting the importance of JAK1/STAT3 pathway. Similarly a human colon biopsy model further supports the importance of the JAK1 pathway in the etiology of IBD. These data suggest that filgotinib may be beneficial in treating CD patients and support its evaluation in a clinical study.
B253 GPR84 INHIBITION AS A NOVEL THERAPEUTIC APPROACH IN INFLAMMATORY BOWEL DISEASE (IBD): MECHANISTIC AND TRANSLATIONAL STUDIES Sonia Dupont1, Ingrid Arijs3, Roland Blanque´1, Debby Laukens4, Kris Nys3, Marie-Christine Ceccotti1, Didier Merciris1, Steve De Vos2, Mate´ Ongenaert2, Isabelle Parent1, Veerle De Vriendt2, Fre´de´ric Labe´gue`re1, Rene´ Galien1, Martine De Vos4, Paul Rutgeerts3, Nick Vandeghinste2, Se´verine Vermeire3, Reginald Brys2
B252 FILGOTINIB, JAK1-SELECTIVE INHIBITOR, REPRESSES SIMILARLY JAK1/STAT3 PATHWAY IN THE COLON OF MICE WITH DSS-INDUCED COLITIS AND IN CULTURES OF COLON BIOPSIES FROM INFLAMMATORY BOWEL DISEASE PATIENTS
Galapagos SASU, Romainville, France; 2Galapagos NV, Mechelen, Belgium; 3Translational Research Center for Gastrointestinal Disorders (TARGID), KU Leuven, Leuven, Belgium; 4Department of Gastroenterology, University Hospital, Ghent, Belgium
Sonia Dupont1, Carole Delachaume1, Veerle De Vriendt2, Debby Laukens3, Be´atrice Vayssie`re1, Didier Merciris1, Steve De Vos2,
Objective: GPR84 is a GPCR activated by medium chain fatty acids, mainly expressed in white blood cells and regulating cell migration. Recently, we reported that pharmacological GPR84 inhibition reduces
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Inflamm. Res. disease activity in experimental colitis (Dupont et al. UEGW 2014). To gain mechanistic insights, the impact of GPR84 inhibition on colon gene expression was investigated in experimental colitis. In addition, translational studies were engaged, measuring GPR84 expression levels in patient samples. Methods: In vivo efficacy of GLPG1205, a GPR84 antagonist, was evaluated in a mouse chronic DSS (dextran sodium sulphate, 3 cycles)-induced colitis model using macroscopic and histopathological scorings and microarray gene expression analysis. Blood and intestinal samples from ulcerative colitis (UC) and Crohn’s disease (CD) patients were collected (before and after first Infliximab treatment) as well as from healthy controls. GPR84 expression was confirmed by QrtPCR and immunohistochemistry (IHC). Results: Disease activity in a DSS model was strongly reduced by GLPG1205 (10 mg/kg, q.d.) and sulfasalazine (SSZ, 20 mg/kg, q.d.). Microarray expression profiles in DSS-treated mice showed a strong negative correlation between GLPG1205 treatment and disease (R2 = 0.8), comparable to the impact of SSZ (R2 = 0.75). Comparison to human expression datasets showed an impact of GLPG1205 on several gene sets associated with IBD. Microarray and QrtPCR data indicated an increase in GPR84 expression in inflamed UC colon and CD colon/ileum which was more pronounced in patients not responding to infliximab. Moreover IHC showed a positive GPR84 staining in mucosal inflammatory cells which was increased in active IBD mucosa and very pronounced in granuloma and pouchitis samples. Additionally, patient white blood cell samples displayed increased GPR84 expression levels as well. Conclusions: GLPG1205 has profound effects on experimental colitis that were confirmed at gene expression level and were comparable to SSZ. In patient biopsies, an increased GPR84 expression specifically in active disease conditions was observed, which represents an advantage with regard to the safety profile of GPR84 inhibitors. As GPR84 expression is increased in patient blood, GPR84 inhibition may impact disease through systemic effects as well.
B254 FUNCTIONAL RECOVERY OF ALZHEIMER’S DISEASE USING STEM CELLS FROM HUMAN EXFOLIATED DECIDUOUS TOOTH-DERIVED CONDITIONED MEDIUM Tsuneyuki Mita1, Yoko Hibi2, Chiaki Shimojima1, Hisasi Hattori1, Kiyofumi Yamada1, Akihito Yamamoto1 1
Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine, Nagoya, Japan; 2Department of Neuropsychopharmacology and Hospital Pharmacy, Nagoya University, Graduate School of Medicine, Nagoya, Japan Introduction: Alzheimer’s disease (AD), a progressive neurodegenerative disease, is characterized by deterioration of cognitive function accompanying with deposition of b-amyloid (Ab) peptides. Ab peptides are liberated from larger transmembrane amyloid precursor proteins (APP) and generate Ab42/Ab40 peptides. These aberrant Ab peptides form amyloid plaques induce M1-type pro-inflammatory microglia, releasing high levels of cytokines, glutamate, reactive oxygen species, and nitric oxide (NO). This oxidative stress accelerates generation of 3-nitrotyrosine in neuron and consequently suppresses the catecholamine synthesis and depressesneuro-synaptic transmission. Inhibition of the neurotoxic factor production may be a novel AD treatment. We have reported strong immunosuppressive activities of serum free conditioned medium derived from Stem Cells from Human Exfoliated Deciduous Tooth (SHED-CM).
S203 Purpose: In this study, we examined the therapeutic benefits of SHED-CM, Bone marrow mesenchymal stem cells (BMMSC)-CM and skin fibroblast (Fibro)-CM for treatment of the Ab peptide injected mouse AD-like model. Materials and methods: Mouse AD-like model was generated by the i.c.v. injection of oligomerized 5 lg of Ab1-40, or Ab40-1 was into the 9-week-old ICR mice brain. Twenty-four h after the i.c.v. injection, a total of 50 ll of various type CM, were administered to each mice via the olfactory pathway. The therapeutic effects were evaluated by the behavioral, biochemical and histological examination. In vitro, effects of SHED-CM on microglia and neurons were investigated by biochemical examination and TUNEL staining. All experiments were performed in accordance with theGuidelines for Animal Experimentsof Nagoya University Graduate School of Medicine. Result: SHED-CM suppressed the expressions of pro-inflammatory cytokines, iNOS and 3-nitrotyrosine and improved cognitive function of the mouse AD-like model. SHED-CM shifted the M1-type proinflammatory microenvironment associated with mouse AD-like model toward the M2-type anti-inflammatory/neuroprotective one. Conclusion:: Our data demonstrate that SHED-CM exerted multifaceted neuro-repairing activities for the treatment of mouse AD-like model without adverse effects. SHED-CM may provide a novel cellfree neuro-reparative therapy for AD.
B255 NOVEL CARTILAGE-PROTECTIVE MEDIATORS IN PRO-RESOLVING EXUDATES Magdalena K. Kaneva1, Sarah E. Headland1, Prashant Mori2, Adrian Moore2, Mauro Perretti1 1 William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK; 2UCB Pharma, Slough, UK
Introduction: The end-point of the resolution of inflammation is healing, repair and restoring tissue physiology. We set out to search for novel tissue-protective activities produced within the resolution phase of acute inflammation and applied an integrated approach with experimental exudates, proteomic analyses and bio-assays with human chondrocytes and experimental arthritis. Methods: Rat pleural exudates (24 h post-carrageenan injection) were subjected to gel filtration chromatography and run for mass spectrometry using ion-trap mass analyzer (MS/MS LTQ Orbitrap XL). Human C28/I2 chondrocytes were grown in 3D high-density micromass (PMID: 21946086), incubated with the fractions with or without catabolic stimuli like interleukin (IL)-1b (20 ng/mL) or osteoarthritic synovial fluids (OASF) for 48 h. a1-Antitrypsin (AAT) and Gelsolin (GSN) were tested against catabolic stimulation. Sulphated glycosaminoglycans (GAGs) deposition was assessed using Alcian Blue (AB) staining. Gene expression was quantified by qPCR. Data are expressed as 2-DDCt equation–relative amount of target genes, normalized to GAPDH and vehicle, with expression set to 1.0. Experiments were run 3 times. Inflammatory arthritis was induced though K/BxN serum transfer model (PMID: 22562975). Knee joint structure and cartilage integrity were determined 48 h after i.a. injection of AAT or GSN (n = 8) by the GAG-sensitive toluidine blue staining of knee sections. One-way ANOVA was used for statistical analyses. Results: Whilst high molecular weight (HMW) fractions of the exudates inhibited (B60 %) GAG deposition from 3D chondrocyte cultures, low MW fractions (LMW) were anabolic both on their own and even more effective in the presence of IL-1b, causing [60 %
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S204 increase in GAG deposition mirrored by *30 % rise in COL2A1 and ACAN gene expression (p \ 0.05). Proteome analysis identified 66 exudate proteins, including GSN and AAT. Exogenous AAT and GSN abrogated the effect of IL-1b on MMP13 (eightfold and threefold increase over vehicle; p \ 0.05). Incubation of chondrocyte micromasses with OASF augmented IL6 and MMP13 mRNA, with concomitant down-regulation of COL2A1 and ACAN gene products (p \ 0.01): tested at 3–10 lg/mL, both AAT and GSN attenuated these effects (p \ 0.01). GAG deposition confirmed the chondroprotection afforded by these proteins, with *80 % reduction by OASF and reversal to control levels observed following AAT and GSN addition (p \ 0.01). In arthritic mice, knee joint cartilage was eroded by *60 % (GAG content; p \ 0.05). Intra-articular injection of AAT (100 ng) or GSN (30 ng) recovered cartilage integrity by 52 and 34 %, respectively, compared to vehicle-injected contralateral joints (p \ 0.05). Conclusion:: Using resolving exudates as a bio-source, we identified and characterized two novel chondroprotective proteins AAT and GSN, paving the way for new strategies to repair/restore compromised cartilage functions.
Inflamm. Res. significant thinning. Finally, we applied AN5322 topically to the ears of mice stimulated with PMA and examined the ears for the levels of several cytokines; IL-22 mRNA was suppressed more than fifty-fold by AN5322. Similarly AN5322 suppressed calcipotriol-induced production of TSLP protein by more than eighty-fold. New topical therapeutics with novel mechanisms are needed for mild to moderate Ps and AD due to the significant side effects and modest activity of corticosteroids and calcineurin inhibitors. These studies provide evidence that topical PDE4Is may suppress cytokines that promote inflammation, itch and keratinocyte hyperproliferation in Ps and AD.
B257 EVALUATION OF ANTI-INFLAMMATORY ACTIVITY OF ISATIN, N-METHYL ISATIN AND OXOPROPYL ISATIN Thais B.S. Giorno, Ba´rbara V. Silva, Angelo C. Pinto, Patricia D. Fernandes
B256 TOPICALLY ACTIVE PHOSPHODIEASTERASE INHIBITORS SUPPRESS IL-17, IL-23, IL-22 AND TSLP PRODUCTION, INDICATING POTENTIAL APPLICATION IN PSORIASIS AND ATOPIC DERMATITIS Kurt Jarnagin, Chen Dong, Olga Zemska, Charlotte Frates, Shannon Jonesiatauro, Grober Baltazar, Tsutomu Akama Anacor Pharmaceuticals, Palo Alto, CA, USA Psoriasis (Ps) and Atopic Dermatitis (AD) are serious skin diseases affecting the lives of millions of patients. We examined three topical benzoxaborole PDE4 inhibitors (PDE4Is), AN3889, AN5322, and AN6415, for their affinity, selectivity, and cytokine suppression toward: TNFa, IL-23, IL-2, IFNc, IL-17, IL-4, IL-5 and IL-13. Activity was compared to known Ps or AD therapeutics. The crystal structure of AN6415 bound to PDE4 was determined and provides an understanding for the origin of its 0.3 nM affinity and selectivity. A dose-limiting side effect of glucocorticoids is skin thinning; we examined topical AN5322 for its ability to thin the skin of hairless mice. Aberrant growth of keratinocytes mediated by IL-22 is a feature of both diseases. Itch is intense in AD and can be severe in Ps patients, and TSLP is an itch mediator. We also examined the ability of these inhibitors to suppress IL-22 secretion in mouse skin treated with PMA, and TSLP skin treated with calcipotriol. AN3889, AN5322 and AN6415 are potent PDE4Is, with IC50 of 1.8, 3.0 and 0.31 nM, respectively. All three are equally active on the catalytic domain and full length PDE4 enzymes, and thus do not interact with UCR1/2. They are not highly selective among the PDE4 A, B, C or D isoforms. The three candidates are more potent on PDE4 than 15 other cyclic-nucleotide specific phosphodiesterases, exhibiting selectivity ratios from 15 to 300,000-fold. In cultured human leukocytes these compounds are potent suppressors of monocyte cytokine secretion, with IC50’s for TNFa and IL-23 \ 100 nM; they suppress T-cell Th1 cytokines potently with IC50’s \ 50 nM; they suppress IL-17 secretion with IC50’s \1 lM, and Th2 cytokines with IC50’s B 3 lM. These patterns are similar to other PDE4Is and highly distinct from glucocorticoids, or calcineurin inhibitors. AN5322 did not cause skin thinning when applied topically for 16 days, by comparison, dexamethasone and clobetasol caused
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Federal University of Rio de Janeiro, Rio de Janeiro, Brazil Isatin is found in plants of the genus Isatis. Due to the great synthetic versatility there has been growing interest in the synthesis and evaluation of the pharmacological effects of new derivatives. Our objective was to characterize the anti-inflammatory activity of isatin, N-methyl-isatin (MI) and N-methyl-3-(2-oxopropyl)-3-hydroxy-2-oxindole (NMO). Substances (0.1–10 mg/kg, p.o.) were evaluated in the carrageenan-induced cell migration into the subcutaneous air pouch (SAP) to assess cell migration and dosages of nitric oxide (NO), TNFa and IL-1b (by ELISA). To evaluate reactive oxygen species (ROS) production induced by phorbolmyristate acetate (PMA) in leukocytes collected from the SAP after carrageenan injection, the cells were pretreated ex vivo with substances (1–30 lM). The fluorescence was captured in the FL-1 channel of a flow cytometer and expressed as fluorescence intensity. The protocol for the use of animals was approved by CAUAP/UFRJ and received the number DFBCICB01504/16. Statistical analysis was performed by ANOVA and Bonferroni’s test (*p \ 0.05). Pretreatment of animals with different doses of all substances significantly reduced cell migration to the SAP: PBS in SAP = 1.1 ± 0.5 9 106 cells/mL; carrageenan in SAP = 80 ± 8.2 9 106 cells/mL; 0.1 mg/kg: Isatin = 66 ± 2.3 9 106 cells/mL; MI = 69.6 ± 8.5 9 106 cells/mL; NMO = 49.6 ± 1.4 9 106* cells/mL; 1 mg/kg: Isatin = 59 ± 2.5 9 106* cells/mL; MI = 47.5 ± 3.2 9 106* cells/mL; NMO = 45 ± 2.8 9 106* cells/mL; 10 mg/ kg: Isatin = 52.5 ± 7.5 9 106* cells/mL; MI = 48.4 ± 7.2 9 106* cells/mL; NMO = 46.8 ± 7.8 9 106* cells/mL. Isatin (1 mg/ kg = 124.5 ± 9.6* lM; 10 mg/kg = 97.7 ± 21.7* lM) and NMO (0.1 mg/kg = 96.2 ± 10.4* lM; 1 mg/kg = 75.5 ± 13.9* lM; 10 mg/kg = 33.6 ± 20.5* lM) significantly decrease the amount of NO. With respect to quantification of TNF-a and IL-1b, MI (0.1 mg/ kg = 377.1 ± 93,6/1009 ± 48.3 pg/mL; 1 mg/kg = 80.3 ± 15.5*/ 719 ± 73.1* pg/mL; 10 mg/kg = 55.9 ± 28.4*/571.4 ± 124.3* pg/ mL and NMO (0.1 mg/kg = 55.3 ± 13.3*/831 ± 114.6 pg/mL; 1 mg/kg = 35.9 ± 6.5*/576.9 ± 84.9* pg/mL; 10 mg/kg = 32.0 ± 18.5*/539.6 ± 25* pg/mL, respectively) were able to reduce the values when compared with carrageenan in SAP group (529.1 ± 13.3 and 1226.5 ± 139.8 pg/mL, respectively). Isatin, MI and NMO also reduced ROS production (control = 0.04 9 105 ± 0.5 9 104, PMA = 5.6 9 105 ± 3.2 9 104; Isatin: 1 lM = 1.7 9 105 ± 1.5 9 104; 10 lM = 1.9 9 105 ± 1.9 9
Inflamm. Res. 104; 30 lM = 0,4 9 105 ± 0.4 9 104; MI: 1 lM = 2.3 9 105 ± 2.4 9 104; 10 lM = 1,5 9 105 ± 1.0 9 104; 30 lM = 0.83 9 105 ± 1.0 9 104; NMO: 1 lM = 2.2 9 105 ± 2.5 9 104; 10 lM = 1.2 9 105 ± 2.6 9 104; 30 lM = 0.8 9 105 ± 1.2 9 104). With these results we can conclude that: (1) Isatin, MI and NMO inhibited leukocyte migration; (2) Isatin and NMO inhibit NO production; (3) MI and NMO inhibited the production of TNF-a and IL1b; (4) Isatin, MI and NMO decreased ROS production. Data suggest that these substances could be used as new prototype forthe development of new anti-inflammatory drugs. Acknowledgements Alan Minho for technical assistance, Instituto Vital Brazil (Nitero´i, Brazil) for donation of mice. Financial support CAPES, CNPq and FAPERJ.
B258 ANTIULCER AND ANTIOXIDANT ACTIVITY OF LECTIN FROM MUCUNA PRURIENS SEEDS ON ETHANOL-INDUCED GASTROPATHY IN MICE Vicente de Paulo T. Pinto1, Isabela R. Pinto1, Danielle RD Val2, Katia A. Ribeiro4, Raul S. Freitas1, Samuel MP Filho1, Auriana S. Vasconcelos1, Francisca CFD Sousa1, Tatiane Santi-Gadelha3, Jose´ Thalles JGD Lacerda3, Carlos ADA Gadelha3, Gerardo C. Filho1, Karuza MA Pereira1, Ellen LD Assis1, Hellı´ada V. Chaves1, Antonio AR Silva1, Mirna M. Bezerra1 1 Federal University of Ceara´, Ceara´, Brazil; 2Federal University of Pernambuco, Recife, Brazil; 3Federal University of Paraı´bam, Paraı´ba, Brazil; 4Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
Antiulcer and antioxidant activity of lectin from Mucuna pruriens (MpLec) seeds on ethanol-induced gastropathy in mice were studied. After treatment with ethanol 99.9 %, mice were pre-treated with MpLec (0.001; 0.01 or 0.1 mg/kg, i.v.), ranitidine (80 mg/kg, p.o.), or saline. Mice were sacrificed 30 min after ethanol challenge and ` . Hishemorrhagic/ulcerative lesions were measured using ImageJO tological assessment (H&E), iron-induced lipid peroxidation, GSH content, SOD activity and mucosal PGE2 were measured. Yohimbine, indomethacin, naloxone or L-NAME was added in order to clarify MpLec action mechanisms. Mice received MpLec (5 or 10 mg/kg; i.v.) and were observed for toxicity signs and mortality. Ethanol induced gastric damage, edema, and hemorrhagic patch formation, which were reduced by MpLec (2.44 ± 0.8 % versus 21.95 ± 2.96 % injured area %; p \ 0.05). Histopathological assessment show induced a high degree of epithelial cell loss and edema as well as hemorrhagic patch formation on the gastric tissue were significantly reduced (p \ 0.05) in animals administered MpLec (0.1 mg/kg). Also, MpLec decreased lipid peroxidation (163 ± 30.73 versus 341.6 ± 78.83 lg/g of tissue, p \ 0.05), SOD activity (1.55 ± 0.32 versus 0.46 ± 0.09 lSOD/lg protein, p \ 0.05) and increased GSH content (1271 ± 126.8 versus 837.8 ± 37.23 lg/g of tissue, p \ 0.05), Mucosal PGE2 level (2387 ± 133.1 versus 1683 ± 143.6 pg/mL protein, p \ 0.05). MpLec effect was reversed by yohimbine (33.54 versus 2.44 injured area %, p \ 0.05) or indomethacin (36.51 versus 2.44 injured area %, p \ 0.05), suggesting the alpha-2 adrenoceptors and prostaglandin involvement in MpLec efficacy. Nevertheless, MpLec was able to protect mucosa against ethanol gastropathy even in presence of both naloxone (8.809 versus 2.438 %, p [ 0.05) and L-NAME (9.88 versus 2.44 %, p [ 0.05). No symptoms of toxicity or death were observed during the acute toxicity tests. MpLec possesses antiulcer and antioxidant activity through involvement of alpha-2 adrenoceptors activation and prostaglandin
S205 generation in an ethanol-induced gastropathy model in mice, the designing of alternative compounds to classical antisecretory drugs is very encouraged to define new pharmacological targets for preventing mucosal cell damages in clinical trials. Funding sources FUNCAP, CNPq, CAPES, and INCT-IBISAB.
B259 14-3-3 ETA AS A NOVEL RA DRUG TARGET: ANTI-14-3-3 ETA MONOCLONAL ANTIBODY DELAYS THE ONSET AND MITIGATES THE SEVERITY OF ARTHRITIS IN CIA MICE Anthony Marotta1, Abedelnasser Abulrob2, Mario Mercier2, Slavisa Corluka2, Roger MacKenzie2, Shalini Raphael2, Sara Michienzi1, Jane Savill1, Yuan Gui1, Walter P. Maksymowych3 1 Augurex Life Sciences Corp., Vancouver, Canada; 2National Research Council, New York City, NY, USA; 3University of Alberta, Edmonton, Alberta
Background: 14-3-3g is a joint-derived soluble biomarker that is available as a diagnostic test in the US, Canada and Europe. As an extracellular ligand, 14-3-3g potently and concentration-dependently upregulates the expression of multiple factors including TNFa, IL-6, and RANKL and its clinical detection is associated with joint damage progression risk. Several disease modifying agents are available for the treatment of RA with remission efficacy rates around 30 %. No agent has direct companion biomarkers for patient selection and therapy response monitoring. Since RA is driven by multiple factors to varying degrees, within and between patients along the disease course, personalized medicine that enables the specific targeting and measurement of disease potentiators, such as 14-3-3g, is highly desirable. Objectives: To evaluate the in vivo feasibility of targeting 14-3-3g with a monoclonal antibody drug candidate to mitigate the onset and severity of collagen-induced arthritis (CIA) in mice. Methods: 27 DBA/1 mice were randomized to four study groups: noninduced mice (negative control, n = 5), 0.5 mg/kg of dexamethasone group (positive control, n = 6), vehicle (saline) injected mice (placebo group, n = 10), and the treatment arm that was administered 10 mg/kg of 14-3-3g mAb (n = 6). Treatments were initiated 2 days prior to immunization with collagen and administered daily for 6 weeks. A collagen booster was injected on day 18 of the study for all immunized mice. Arthritis scores were determined daily by an established and standardized chart evaluating inflammation and swelling of each paw (0 to 4). All animals were sacrificed 42 days after the beginning of the treatments. Paws were further analysed by x-ray. Student t-test was performed to examine differences (onset CIA, maximum, and end scores, and paw scores) amongst the two groups. Kruskal–Wallis test was used to compare group daily score differences over the course of disease. Results: Non-induced and dexamethasone mice did not develop visible signs of arthritis over the course of disease while 100 % of the mice within the saline arm did. 17 % of the mice in the 14-3-3g mAb group did not develop any signs of arthritis. The CIA score for the 14-3-3g mAb arm had significantly lower CIA onset scores (0.83 ± 0.41 vs 2.7 ± 1.57, p = 0.0119), maximum CIA scores (2.33 ± 1.75 vs 5.3 ± 1.83, p = 0.0052), and end CIA scores (1.83 ± 1.84 vs 4.3 ± 1.7, p = 0.0197) than the saline treated groups. Figure 1 further demonstrates that 14-3-3g mAb treated mice have significantly lower disease over the disease course than animals in the saline group, p \ 0.01, with x-ray paw analysis also demonstrating significance (p = 0.0041).
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Fig. 1 CIA scores by treatment arm over the course of disease. Conclusions: 14-3-3g is a mechanistic joint damage factor involved in the pathogenesis of RA. A research program to exploit modifying the 14-3-3g pathway is underway to develop improved antibody therapeutics for delaying the onset and reducing the severity of this disease.
B260 LASSBIO-1829, A NOVEL ANTI-INFLAMMATORY MOLECULE DERIVED FROM LASSBIO-1524 Tayna´ S. Vale´rio, Thaı´s S. Nascimento, Nata´lia M. Cordeiro, Rosana H.C.N Freitas, Carlos Alberto M. Fraga, Patricia D. Fernandes Federal University of Rio de Janeiro, Rio de Janeiro, Brazil Inflammation is the organism response to infection or tissue injury that leads to restoration of tissue structure and function (LAWRENCE et al. 2002). LASSBio-1829 was developed based on the structure of LASSBio-1524, an IKK-b inhibitor (AVILA et al. 2011). Our aim is to assess the prospective anti-inflammatory effect of LASSBio-1829. Formalin model is characterized by two phases: nociceptive (1st phase) and inflammatory (2nd phase). Formalin was injected in hind paw 1 h after treatment with LASSBio-1829 (30 lmol/kg) and the time that the animal remained licking its paw was recorded. The subcutaneous air pouch model (SAP) was conducted to evaluate cell migration. The animals received 10 mL of sterile air into the intrascapular area and the reinforcement was made after 3 days. At the 6th day, a carrageenan solution (1 %) was injected in the cavity 1 h after treatment with LASSBio-1829 (10, 30, 100 lmol/kg, p.o.) after 24 h the animals were euthanized and the exudate was collected for total leukocyte counting and mediators measurement. To determine reactive species of oxygen production (ROS), cells were treated with LASSBio-1829 (1, 10 and 30 lM) 1 h before stimulation with PMA. Finally, we added the probe DCF-DA. ROS production was assessed by measuring the fluorescence intensity, FL1 of each cell expressed through the flow cytometer. Determination of nitric oxide was performed by nitrite measurement. Controls: LASSBio-1524 (30 lmol/kg, p.o.), SC-514, (an iKK-b inhibitor, 30 lmol/kg, p.o.), dexamethasone (0.5 lmol/kg, i.p.) and acetylsalicylic acid (ASA 20 mg/kg). Mice (n = 6–8) were used. Statistical analysis was performed by ANOVA followed by Bonferroni’s test. Results are expressed as mean ± SD and (p \ 0.05*). In formalin test, treatment with LASSBio-1829 was significant in the inflammatory phase, reducing licking time: 101 ± 35 s compared to vehicle = 197 ± 27 s. The result of ASA was 129 ± 55 s and
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Inflamm. Res. LASSBio-1524 = 120 ± 49 s. In SAP, the 30 lmol/kg dose = 24 ± 14* cells 9 106/mL reduced significantly the migration cell compared to carrageenan group = 70 ± 6 cells 9 106/mL and PBS group = 2.4 ± 1.0 cells 9 106/mL. The results of dexamethasone = 19 ± 7.7 cells 9 106/mL; SC-514 = 36 ± 18 cells 9 106/ mL; LASSBio-1524 = 22 ± 4 cells 9 106/mL. All concentrations of LASSBio-1829 significantly reduced production of ROS: 1 lM = 3 9 105 ± 2 9 104*; 10 lM = 1 9 105 ± 1 9 104*; 30 lM = 1 9 105 ± 3 9 104* compared to group that was stimulated with PMA = 5 9 105 ± 8 9 104. LASSBio-1524: 30 lM = 4 9 104 ± 1 9 104. Only the dose of 10 lmol/kg = 34 ± 26 lM was able to significantly inhibit the production of nitrite compared to group that received SC-514 = 74 ± 43 lM and carrageenan group = 279 ± 17 lM. PBS: 26 ± 5 lM; LASSBio-1829 (30 lmol/kg) = 249 ± 55 lM; dose of 100 lmol/kg = 221 ± 77 lM. The results suggest that LASSBio-1829 has an anti-inflammatory effect due to reduced licking time, cell migration and inflammatory mediators. Financial support Capes, CNPQ, FAPERJ, Instituto Vital Brazil. Technical support Alan Minho.
B261 A PHASE 1 CLINICAL STUDY OF CTX-4430 IN CYSTIC FIBROSIS PATIENTS E. Springman2, R. Grosswald2, E. Philpot2, G. MacGregor3, A. Horsley4, D. Bilton5, J. Stewart6, S. Elborn1 1 Centre for Infection and Immunity, Queens University School of Medicine, Dentistry and Biomedical Sciences, Belfast, UK; 2 Celtaxsys, Inc., Atlanta, GA, USA; 3West of Scotland CF Centre, Gartnavel General Hospital, Glasgow, UK; 4Manchester Adult Cystic Fibrosis Centre, University Hospital of South Manchester, Manchester, UK; 5Royal Brompton Hospital, Department of Respiratory Medicine, London, UK; 6Celerion, Belfast, UK
Introduction: Safe and effective anti-inflammatory treatment (AIT) remains a significant unmet need for CF. Inflammation plays a central role in CF and is responsible for lung injury. Along with infection, inflammation drives acute pulmonary exacerbations and chronic decline in lung function. Therefore, AIT for CF must balance suppression of immune responses leading to inflammation against preservation of immune responses required for microbial control. This need for balance is illustrated by two AIT drugs having divergent outcomes for CF. High-dose ibuprofen has been shown to reduce the rate of FEV1 decline and confer a survival benefit, particularly when started at a young age. Whereas, amelubant (BIIL 284) failed in Phase2 due to an increase in pulmonary exacerbations. Both act via the leukotriene B4 (LTB4) pathway, with ibuprofen acting as a weak inhibitor of LTB4 production and amelubant, a strong receptor antagonist, blocking binding of LTB4 to the receptors BLT1/2. While the reasons for this divergence in outcomes is unclear, it could be due to the different degrees of pathway inhibition achieved between these drugs in the clinic. CTX-4430 is a once daily oral Leukotriene A4 Hydrolase inhibitor that modulates the LTB4 pathway more potently than ibuprofen but less potently than amelubant. It is currently in clinical development as a once-daily oral AIT for CF. Methods: This ascending dose study was conducted at four clinical sites in the United Kingdom (NCT01944735). It included 17 patients with mild to moderate CF. Doses of 50 or 100 mg CTX-4430 or placebo were studied during 15 days of treatment. Safety measures were taken on days 1, 8 and 15, including assessments of adverse events, hematology, blood chemistries and pulmonary function. Experimental measures included serum CRP, sputum DNA, elastase
Inflamm. Res. and microbial counts. Additional assessments of pharmacokinetics and pharmacodynamics in blood are reported separately. Results: Here we report the results of this phase 1 study in CF patients including assessments of safety and tolerability, pulmonary function and inflammatory markers in blood and sputum. In brief, CTX-4430 was safe and well tolerated at both dose levels. No deleterious changes in pulmonary function, sputum microbiology, or circulating neutrophil counts were observed. Positive trends were observed in blood and sputum inflammatory markers (CRP and elastase, respectively) and sputum WBC and neutrophil counts demonstrating potential use of CTX-4430 to preserve CF lung function over time. Conclusion:: These results support further clinical development of CTX-4430 for treatment of CF.
B262 SITE-SPECIFIC IMMUNOMODULATOR (SSI): A NOVEL IMMUNOTHERAPY FOR CANCER David W. Mullins1,2, Shirin Kalyan1, Momir Bosiljcic1, Ashley Westerbeck1, Rebecca Anderson1, Daniel Patton1, Ralf Kleef4, Gauthier Bouche23, Nina Ludwig3, Simon Sutcliffe1, Hal Gunn1 Qu Biologics, Inc., Vancouver, Canada; 2Geisel School of Medicine at Dartmouth, Hanover, USA; 3Reliable Cancer Therapies, Belgium; 4 Institute for Immunotherapy and Integrative Oncology, Vienna, Austria
S207 study in all late-stage cancer patients, those receiving SSI as part of their treatment had a median survival advantage of 12 months compared to those not receiving SSI (n = 43 per group). While this experience comprises uncontrolled, unblinded observations, the data suggest that SSI may induce productive anti-tumour immunity. Qu’s QBKPN SSI product, designed to induce a lung site-specific response, is currently being studied in a Phase 2a clinical trial in patients with non-small cell lung cancer, in collaboration with the BC Cancer Agency (Trial NCT02256852).
Nuclear Hormone Receptors B263 NORETHISTERONE, A PROGESTIN USED IN CONTRACEPTION AND HRT, PROMOTES INFLAMMATION IN THE ECTOCERVICAL ENVIRONMENT VIA THE GLUCOCORTICOID RECEPTOR Donita J. Africander1, Renate Louw-du Toit1, Janet P. Hapgood2
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The immune system is armed with the intrinsic capacity of recognizing and eliminating cells that have undergone malignant transformation. The observation that an intricate relationship exists between immune activation and cancer dates back to the 1700s, when spontaneous tumor remission was observed in some patients experiencing acute microbial infections. Building upon this history, Qu Biologics has discovered that repeated subcutaneous injection of an immunotherapy derived from specific species of killed bacteria known to commonly cause infection in a particular organ or tissue may provide an effective method for the treatment of cancers growing in that organ/body site. We hypothesize Qu’s proprietary platform of immunotherapies, called Site-Specific Immunomodulators (SSI), stimulate the body’s immune system to reverse the immune suppression and dysfunction in the tumor microenvironment, enabling effective anti-cancer immune responses. To test this hypothesis, we evaluated tumor growth and survival in preclinical lung cancer models following repeated subcutaneous administration of Qu’s lung specific SSI, QBKPN (derived from Klebsiella pneumoniae). SSI significantly reduced tumor burden at day 16 post tumor inoculation (p \ 0.0001) and improved median survival by 10 days (p \ 0.005). Similar results were obtained using the B16 model, an aggressive and poorly-immunogenic melanoma cell line growing as metastatic-like lesions in the lungs, demonstrating the site-specific anticancer efficacy is independent of cancer type. Anticancer efficacy was associated with site-specific infiltration of newly recruited monocytes and neutrophils to the lung and mobilization of antigen presenting cells in the lung-draining lymph node. These data provide strong evidence that Qu’s SSI platform may reconstitute effective immunosurveillance in the tumor microenvironment. Therefore, we evaluated SSIs (including QBKPN, QBSAU [derived from Staphylococcus aureus], or QBECO [derived from Escherichia coli]) in compassionate use protocols. 254 patients with advanced cancer were treated with one or more SSIs for up to 3.5 years. In retrospective analysis, patients with metastatic breast cancer receiving SSIs as part of their treatment (the largest patient group) had a 20 months longer median survival than those not treated with SSIs. In a case-matched
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University of Stellenbosch, Stellenbosch, South Africa; 2University of Cape Town, Cape Town, South Africa
Medroxyprogesterone acetate (MPA) and norethindrone enanthate (NET-EN) are widely used as injectable contraceptives in Southern Africa. In addition, for hormone replacement therapy (HRT), MPA is the most commonly used progestin in the U.S.A., while norethindrone-acetate (NET-A) is generally used in Europe. Both MPA and NET are synthetic progestins designed to mimic the actions of the endogenous hormone progesterone (Prog) by binding to the progesterone receptor. However, these progestins also bind to other members of the steroid receptor family such as the glucocorticoid receptor (GR). Studies have indicated that MPA disrupts normal immune responses in the female genital tract which could increase inflammation, and likely affect susceptibility to sexually transmitted infections. Furthermore, we have recently shown that MPA favours a pro-inflammatory milieu in human ectocervical epithelial cells via a GR dependent mechanism. However, not much is known about the effects of NET on local immune function in the female genital tract. This study thus investigated whether NET-A can also regulate the expression of endogenous cytokine genes in a human ectocervical epithelial (Ect1/E6E7) cell line. Using real-time quantitative PCR, we show upregulation of the pro-inflammatory cytokine IL-12p40 gene and downregulation of the anti-inflammatory cytokine gene IL-10 by NET-A. An in-depth investigation into the underlying mechanism of IL-12p40 and IL-10 regulation by NET-A, using a combination of whole cell binding assays, Western blotting, siRNA technology, chromatin immunoprecipitations (ChIP) and re-ChIP assays, show that the GR is mediating these effects. Moreover, the transcription factors CCAAT-enhancer-binding protein (C/EBP)-b and nuclear factor kappa B (NFjB) are needed for the recruitment of NET-Abound GR to the promoter of IL-12p40, while recruitment to the IL10 promoter requires signal transducer and activator of transcription (STAT)-3. Taken together, our biochemical study clearly indicates that NET-A exerts similar GR-mediated pro-inflammatory effects in the human ectocervical epithelial cell line as previously observed with MPA. These results suggest that the use of NET-A in hormonal therapy may modulate inflammation and immune function in the ectocervical environment, possibly leading to enhanced susceptibility to infections such as HIV-1.
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B264 THE BONE MARROW MICROENVIRONMENT PROTECTS PLASMA CELLS FROM THE LYMPHOLYTIC EFFECTS OF GLUCOCORTICOIDS Derek W. Cain, Johnny A. Cidlowski National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA Glucocorticoids exert wide-ranging effects on the immune system, acting primarily through the glucocorticoid receptor (GR). The antiinflammatory actions of glucocorticoids have been attributed to the capacity of liganded GR to inhibit expression of pro-inflammatory genes and to induce lymphocyte apoptosis. Glucocorticoid therapy is associated with reduced serum immunoglobulin concentrations, but it is unclear if glucocorticoids act directly or indirectly on antibody-secreting plasma cells. We have previously shown that B cells in mice express GR from early stages of bone marrow development through maturation in the periphery, and that immature and mature B cells undergo apoptosis in response to the synthetic glucocorticoid dexamethasone. In this study, we investigated GR signaling and effects of dexamethasone on murine plasma cell survival. Splenic and bone marrow plasma cells express GR and are sensitive to dexamethasone-induced cell death in vitro. Dexamethasone treatment of mice reduces plasma cell numbers in the spleen but not in bone marrow, suggesting that the bone marrow microenvironment protects plasma cells from glucocorticoidinduced death. Provision of IL-6, but not IL-5, APRIL, BAFF, or CXCL12, mitigates plasma cell death by glucocorticoid treatment ex vivo. We conclude that peripheral plasma cells are susceptible to glucocorticoid-induced lympholysis, but that specialized niches within bone marrow insulate plasma cells from the apoptotic effects of glucocorticoids.
Ocular Inflammation B265 ROLE OF NOX4-DERIVED ROS PRODUCTION IN 4-HNE-INDUCED DYSFUNCTION AND DEATH OF ARPE-19 ADULT RETINAL PIGMENT EPITHELIAL CELLS Sajita Shah2, Jung-Ae Kim1 Yeungnam University, Gyeongsan, South Korea; 2Yeungnam University, Gyeongsan, South Korea
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Role of Nox4-derived ROS production in 4-HNE-induced dysfunction and death of ARPE-19 adult retinal pigment epithelial cells. Sajita Shah, Jung-Ae Kim* College of pharmacy, Yeungnam University, Gyeongsan 712-749, South Korea Defects of the retinal pigment epithelium (RPE) which constitutes the blood-retina barrier result in photoreceptor damage leading to age-related macular degeneration (AMD). RPE produces large amounts of ROS during phagocytosis of shed outer segments of photoreceptors. During phagocytosis, oxidation of polyunsaturated fatty acids (PUFA), rich components in the outer segments, results in oxidative stress by generating lipid aldehyde radicals including 4-hydroxy-2-nonenal (4-HNE) which contributes to dysfunction and
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Inflamm. Res. death of retinal pigment epithelial cells. In the present study, we investigated the detailed action mechanism by which 4-HNE induces dysfunction of adult retinal pigmented epithelial cells (ARPE-19) in vitro. Treatment of ARPE-19 cells with 4-HNE decreased production of inflammatory cytokine and vascular endothelial growth factor (VEGF) in a time-dependent manner. Also, 4-HNE exerted prominent cytotoxic effects in cultured ARPE-19, manifested by morphological changes and diminished cellular viability. At the same time, 4-HNE enhanced NOX4 expression and activity, resulting in aggravated ROS production in addition to mitochondrial ROS. The 4-HNE-inudced ROS production and cell death were inhibited by apocynin, diphenyleneiodonium (DPI) and VAS2870, while 4-HNE-inudced IL-6 and VEGF decrease was not blocked. The results suggest that 4-HNE-induced NOX4 expression and ROS production plays a major role in cell death but not in cytokine production of ARPE-19 cells.
Proteases B266 A NOVEL ROLE FOR BK CHANNEL IN REGULATING METALLOPROTEASE ACTIVITY Minae Yoshida, Dean Willis Department of Neuroscience, Physiology and Pharmacology, University College London, London, UK Objective: The cells of the immune system expresses an array of different ion channels, yet their roles in these cells are not fully understood. To address the shortfall in this area of inflammation research, we investigated the role of BK channels in LPS mediated activation using a mouse macrophage cell line, RAW264.7 cells. Method: Functional cell membrane expressed BK channel was investigated by whole-cell voltage clamp recordings. A ramp protocol (25 s ranges from -40 to 100 mV) was applied in the presence/ absence of a BK channel selective blocker, 20 nM iberiotoxin (IbTX). The IbTX sensitive current was obtained by subtraction. Released TNF-a was assessed by ELISA and cellular TNF-a protein by Western-blot. mRNA were extracted and TNF-a mRNA levels were determined by qRT-PCR. TACE activity was assessed by incubating cells with FAM fluorescence quenched TACE substrate II, a substrate for ADAM17 and ADAM10. Activity is assessed by fluorescence resulting from the cleavage of substrate. Data are presented as mean ± S.E.M and analysed student t-test. Results: Whole cell voltage clamp recordings indicated the almost absence of BK channel current on the cell membrane in non-stimulated cells. However the BK channel current was present after 24 h LPS (10 ng/mL) stimulation. Subsequently, LPS pre-stimulated (10 ng/mL for 24 h) cells were used in the following experiments to investigate the role for membrane active BK channels in TNF-a synthesis/release. Pre-stimulated RAWs were treated with LPS (150 ng/mL) for 4 h. Application of IbTX (20 nM) during the LPS stimulation significantly enhanced the release of TNF-a. We then investigated whether the TNF-a regulation occurred at pre/posttranscriptional level. Despite the significant difference in TNF-a release, TNF-a mRNA was almost equivalent in both control and IbTX treated group. In comparison, the IbTX treated cells contained significantly less TNF-a within the cells. These results indicated that the BK channel may be involved in the release of TNF-a molecule. TNF-a is known to exist as transmembrane protein and is subsequently released by the action of metalloproteases, e.g. ADAM17 and ADAM10. To investigate the direct link between BK channel and
Inflamm. Res.
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TNF-a release, TACE activity assay was performed. TACE activity was increased to 2.5 fold by IbTX treatment. Conclusion: In this study, we have shown that the membrane active BK channel controls TNF-a release in RAW264.7 cells. We found evidence that BK channel directly regulates proteolytic activity of ADAMs. This study provides a novel insight into the role of ion channel in regulating metalloprotease activity and macrophage reponses.
B267 INTESTINAL EPITHELIAL CELLS SECRETE AN ELASTASE WHICH PARTICIPATES TO MUCOSAL INFLAMMATION IN IBD Ce´line Deraison1,2, Alexandre Denadai-Souza1, Anna Carolina Florence1, Anissa Edir1, Corinne Roland1, Jean-Paul Motta3, Chrystelle Bonnart1, Elena Verdu4, Derek McKay2, Laurent Alric5, Nathalie Vergnolle1 1
2
Inserm, U1043, Toulouse, France; University of Calgary, Faculty of Science, Department Physiology and Pharmacology, Calgary, Canada; 3University of Calgary, Faculty of Science, Department Biological Sciences, Calgary, Canada; 4Farncombe Family Digestive Health Research Institute, McMaster University, Hamilton, Canada; 5CHU Purpan, Gastroenterology department, Toulouse, France
Proteases have a crucial role in the persistence of chronic inflammatory responses of the gastro-intestinal tract. We have previously demonstrated that expression of ELAFIN, an elastase inhibitor, is drastically down-regulated in IBD epithelium. The imbalance between proteases and their inhibitors appears to be crucial to the development of Inflammatory Bowel Diseases (IBD). This study aims at identifying the source of hyperactive elastase released by IBD tissue biopsy and to decipher the consequences of its production on colonic barrier function and inflammatory response. Methods: In situ zymography with FITC-elastin was performed on cryosection of colonic biopsies from healthy (n = 7) and IBD patients (n = 10) taken from inflamed and non-inflamed area. Immunostaining was performed on cryosections from human biopsies. Caco2 epithelial cell line overexpressing elastase (Tg-ELA) was constructed and the potential of elastase hyperactivity to modulate the release of cytokines and permeability changes was evaluated. ELA cDNA-recombinant adenovirus (Ad-ELA) was construct to assess the effect ELA overexpression in vivo. Mice were pre-treated by 2 % DSS for 3 days and then Ad-ELA was administrated intracolonically. At day 7, colons were harvested for measurement of inflammation parameters. Results: Using in situ zymography, we showed a strong elastolytic activity in the enterocytes in healthy human colonic tissue, which was greatly enhanced in inflamed biopsies as well as in non-inflamed biopsies from IBD patients. An elastase’s cDNA was cloned from human enterocytes. Immunostaining of colonic tissues showed that this form of elastase was only expressed in epithelial cells. In IBD, epithelial elastase expression was enhanced. Analysis of elastase expression shows also an up-regulation in colonocytes from DSS-treated mice but absence of protein in colons from germ-free mice. Tg-ELA epithelial cells showed defective barrier function: increase of Dextran-FITC permeability and of bacterial translocation. Western blot analysis revealed that ZO-1 and occluding amounts were decreased in Tg-ELA compared to Caco-2 cells. Thus these proteins of tight junction are targeted by elastase hyperactivity secreted in the
medium. In addition, ELA hyperactivity induced an unbalance of cytokines expression favouring the increase of pro-inflammatory cytokines. Ad-ELA administration in murine colon allowed epithelial ELA expression in tissues and increased elastase activity in epithelial cells. Ad-ELA treated mice presented an increase of macroscopic inflammatory damage score compared to control Ad-null mice. Conclusion:: Our study demonstrates that epithelial cells constitute a major source of elastolytic activity and secretes a form of elastase in the extracellular space. In IBD epithelium, elastase is hyperactive disrupting the epithelial barrier function and maintaining the inflammatory response.
B268 EPITHELIAL CELLS RELEASE TRYPSIN ACTIVITY IN INFLAMMATORY CONDITION: ROLE IN IRRITABLE BOWEL SYNDROME Claire Rolland Fourcade1, Alexandre Denadai-Souza1, Jean-Paul Motta2, Tereza Bautzova1, Nicolas Cenac1, Ian Spreadbury3, Stephen J. Vanners3, Ce´line Deraison1, Nathalie Vergnolle1 Inserm CPTP, Toulouse, France; 2University of Calgary, Calgary, Alberta, Canada; 3Queen’s University, Kingston, Kingston, Canada 1
Trypsin activity is released by human colonic biopsies from IBS patients compared to control patients (Cenac et al. 2007). However, the cellular source of this activity remains controversial. Mesotrypsin or trypsin IV is a form of trypsin, which is expressed in the small intestine of IBS patients (Kerckhoffs et al. 2008), but the cellular origin of this protease, and its functions are not clear. This study aims at determining the cellular source of trypsin activity, its release under inflammatory condition, its effect on neuronal signalling and in inflammatory pain. The general objective of this study was to understand the role of trypsin activity in the context of IBS. Cryosections of biopsies from IBS and control patients were used to perform an in situ zymography of trypsin activity. Intestinal epithelial cells (Caco2) were cultured on transwell, and stimulated with LPS as an inflammatory stimulus. Conditioned media from basolateral and apical sides of epithelial monolayers were used to stimulate primary afferent isolated from mouse Dorsal Root Ganglia. Mesotrypsin protein was detected by western blot and immunohistochemistry and trypsin activity was measured using Tosyl GPR as substrate. Mesotrypsin was administered in mouse colon and nociceptive responses to colorectal distension were recorded. In situ zymography revealed that trypsin activity was more intense in tissues from IBS patients compared to healthy controls, and the strongest trypsin activity was detected in epithelial cells. In culture conditions, epithelial cell monolayers released more trypsin activity in the basolateral compartment after LPS stimulation. This polarised secretion could signal to neuronal terminations. Culture media harvested from basolateral but not apical side of LPSstimulated intestinal epithelial cells induced calcium signalling in cultured sensory neurons, which was inhibited by trypsin inhibitor, suggesting that trypsin activity is responsible for such activation. Expression of cationic, anioc and mesotrypsin was quantified in inflamed epithelial monolayer. Only mesotrypsin expression is increased after LPS stimulation and preferentially released at the basolateral side. Mesotrypsin administration in mouse colonic lumen induces visceral hypersensitivity in a dose dependant manner.
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S210 This study reveals that in IBS patients, trypsin activity is strongly detected in epithelial cells, which are able to produce this activity specifically from the basolateral side. Sensory neurons can be activated by basolateral media of stimulated epithelia, in a trypsin activity-dependent manner. Mesotrypsin was identified as a possible candidate released by intestinal epithelial cells in response to inflammation, which can signal to neurons and further induce visceral hypersensitivity. These data highlight epithelial proteases as important signalling molecules to enteric neurons that might be heavily involved in IBS-associated hypersensitivity symptoms.
B269 EFFECTS OF A PROTEASE INHIBITOR FROM THE TICK RHIPICEPHALUS BOOPHILUS MICROPLUS IN A CIGARETTE SMOKE-INDUCED EMPHYSEMA Juliana D. Lourenc¸o1, Daniela A.B. Cervilha1, Juliana T. Ito1, Davi S. Sales1, Alyne Riani1, Clarice R. Olivo1, Adriana Duran2, Sergio D. Sasaki2, Mı´lton A. Martins1, Fernanda D.T.Q.S. Lopes1
Inflamm. Res. Table 1 ControlVE
ControlrBmTIA
SmokeVE
SmokerBmTIA
50.06 ± 1.10
53.69 ± 2.53
61.57 ± 2.49*
58.89 ± 1.83*
Lm (lm)
42.75 ± 1.51
46.11 ± 2.78
59.68 ± 2.61*
47.18 ± 3.07*,#
Elastic fibers (%)
17.43 ± 0.79
14.25 ± 1.15
23.17 ± 0.39*
19.72 ± 0.87*,#
Collagen fibers (%)
14.01 ± 0.71
11.36 ± 0.94
19.47 ± 1.29*
13.88 ± 0.85*,#
MAC-2 (cells/lm2)
1.65 ± 0.32
1.32 ± 0.14
2.99 ± 0.48*
2.68 ± 0.29*
MMP-12 (cells/lm2)
1.17 ± 0.15
1.17 ± 0.11
3.04 ± 0.43*
2.01 ± 0.17*,#
MMP-9 (cells/lm2)
1.02 ± 0.09
1.01 ± 0.07
1.29 ± 0.06*
1.52 ± 0.11*
Protocol 1 Lm (lm) Protocol 2
Data were expressed as mean ± SE;* p \ 0.05 compared to Control groups; # p \ 0.05 compared to Smoke-VE group. Supported by FAPESP.
University of Sa˜o Paulo, Sa˜o Paulo, Brazil; 2UFABC, Santo Andre´, Brazil 1
Introduction: Considering that tabagism is the main risk factor for the Chronic Obstructive Pulmonary Disease (COPD) development, and the cigarette smoke (CS) exposure models appear to best represent human COPD, it is important to test new therapies to avoid the impairment in parenchymal destruction in such model. Objectives: To verify the effects of rBmTI-A treatment, a protease inhibitor, on the emphysema pathogenesis. Methods: Protocol 1: Mice were exposed to CS (twice daily/30 min/ 5 days a week/12 weeks), and the control animals were exposed to room air. These animals received two doses of rBmTI-A inhibitor or its vehicle (Saline Solution 0.9 %) by nasal instillation after the CS end exposure (Dose 1: 24 h after the end of CS exposure; Dose 2: 7 days after the first dose). Fifteen days after the final of CS exposure, animals were submitted to procedures for respiratory mechanics and the mean linear intercept (Lm) evaluations. Protocol 2: We used the same protocol for CS exposure, but the rBmTI-A treatment was performed during the exposure time (Dose 1: 24 h before the start of CS exposure; Dose 2: 1 month after the exposure start, Dose 3: 2 months after the start). After 24 h of exposure end, we obtained the lung mechanical measurements and the Lm analysis. We also measured the volume proportion of collagen and elastic fibers, and the density of macrophages, MMP-12 and MMP-9 positive cells in parenchyma. Results: For both protocols, there was no difference in respiratory mechanics between the groups. Although Smoke-rBmTIA and Smoke-VE groups showed an increase of Lm compared to Control groups in both protocols, there were a decrease in the Smoke-rBmTIA group compared to Smoke-VE group only in the second one. The same behavior was observed for the volume proportion analysis of elastic and collagen fibers in parenchyma. We also observed an increase in the density of macrophages, MMP-12 e MMP-9 positive cells in the Smoke groups, and the treatment with rBmTI-A only decreased the density of MMP-12 positive cells (Table 1). Conclusion:: These results suggest that rBmTI-A inhibitor attenuated the development of emphysema when administered during the disease induction, probably to a decrease in MMP-12 levels, but there is no treatment effect after the established disease.
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B270 CPM ACTIVITY MODULATION BY KININ B1 RECEPTOR IN ENDOTHELIAL CELL MODELS Paola B. Guimara˜es1, Carolina C. Hoff5,1, Liliam Fernandes2, Jair R. Chagas4,1, Thaysa Paschoalin1, Adriana K. Carmona1, Michael Bader3, Joa˜o B. Pesquero1 1 Federal University of Sa˜o Paulo, Sa˜o Paulo Campus, Sa˜o Paulo, Brazil; 2Federal University of Sa˜o Paulo, Diadema Campus, Sa˜o Paulo, Brazil; 3Max Delbru¨ck Center for Molecular Medicine, Sa˜o Paulo, Brazil; 4Federal University of Sa˜o Paulo, Santos Campus, Sa˜o Paulo, Brazil; 5Paulista University, Santos-Rangel Campus, Sa˜o Paulo, Brazil
Carboxypeptidase M (CPM) is a glycophosphatidylinositol anchored enzyme that plays a role in the kallikrein-kinin system (KKS). CPM catalytic domain hydrolyzes Lys or Arg from C-terminal peptides (i.e., bradykinin and kallidin), generating B1 receptor (B1R) agonist, as des-Arg9-BK. Consequently, both CPM and B1R are important in cardiovascular homeostasis and inflammation. It is known that CPM and kinin B1R are co-localized in plasma membrane microdomains, where they interact with each other, facilitating the receptor signaling. Enzyme-receptor interactions in membrane microdomains are not well understood, especially from the enzymatic point of view. In this context, this study becomes crucial, considering the role of ectoenzymes, like CPM in KKS. The hormonal function of this system depends on circulating levels of its agonists, whose local availability is controlled by the enzymes which are close to their receptors. Therefore, we hypothesized that CPM-kinin B1R interaction might also affect the enzyme activity. Thus, the aim this work was to evaluate the CPM activity and expression in the presence of B1R in endothelial cell models. For this purpose, we studied two different kind of endothelial cell system: (1) a B1R ablation system, namely primary culture of endothelial cells from B1-/- mice; (2) endothelial cells from transgenic rats TGR (Tie2B1) overexpressing the B1R
Inflamm. Res. exclusively in the endothelium. Primary culture was characterized by both immunohistochemistry using anti-von Willebrand factor (vWf) antibody and angiogenesis in vitro on Matrigel. CPM mRNA levels was assayed by real time PCR; CPM protein expression was analyzed by Western blot using anti-CPM primary antibody; and CPM activity was assayed with 200 lM Dansyl-Ala-Arg-OH fluorescent substrate. CPM activity was performed in the presence or absence of 1 lM kinin B1R antagonist R715. Primary culture of endothelial cells presents positive label for vWf and it forms tubular structures on Matrigel, some characteristics of endothelial cell. Results show that cells from B1-/group, in which B1R receptors are absent, have decreased CPM activity, along with reduction in both CPM mRNA levels and protein content. However, when control endothelial cells were pre-treated with B1R antagonist R715, CPM activity did not change. On the other hand, CPM activity is increased in cells with kinin B1R overexpression. In summary, our data show that kinin B1R positively modulates both CPM activity and expression, suggesting that CPM-kinin B1R interaction in membrane microdomains might affect enzyme activity, beyond interfering in receptors signaling. This work highlights the interactions among the different components of the KKS and contributes to a better understanding of its patho-physiological role. Financial support CAPES, CNPq and FAPESP.
Resolution of Inflammation and Tissue Repair B271 RESOLVIN D4 STRUCTURAL CONFIRMATION AND POTENT PRO-RESOLVING ACTIONS IN INFLAMMATION AND INFECTION Jeremy W. Winkler1, Sarah K. Orr1, Romain A. Colas1, Jesmond Dalli1, Nan Chiang1, Nicos A. Petasis2, Charles A. Serhan1 1
Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA; 2University of Southern California, Los Angeles, CA, USA Objective: Resolvins are a family of structurally distinct pro-resolving mediators biosynthesized from polyunsaturated fatty acids that are critical to control acute inflammation. This study presents evidence for the first total organic synthesis and complete stereochemical assignment and confirmation of novel Resolvin D4 (RvD4). Materials and methods: Synthetic compound and endogenous Resolvin D4 were examined by MS–MS metabolo-lipidomic profiling. For in vivo actions and endogenous production, mice received S. aureus infection in dermal pouch. Concomitant with a bacterial challenge, synthetic compound was administered and leukocytic response measured overtime. For in vitro RvD4 was administered to macrophages and phagocytosis carried out. Results: The absolute stereochemical confirmation was established as 4S,5R,17S-trihydroxydocosa-6E,8E,10Z,13Z,15E,19Z-hexaenoic acid by matching authentic endogenous RvD4 with synthetic precursor from total organic synthesis. Our results demonstrate that RvD4 is produced endogenously in a self-limited skin infection model at levels commensurate with its bioactions. We confirmed the potent actions of RvD4 in vivoin reducing neutrophils both in murine peritonitis and dermal infection by 40–60 %. Resolvin D4 promoted the clearance of cellular debris and apoptotic PMN by human macrophage in doses as low as 0.1–10 nM. Conclusion: Total synthesis and matching of RvD4 provides a new tool to explore RvD4 bioactions and opens opportunities for control of unwanted inflammation and infection via Resolution Pharmacology. Keywords: Anti-inflammatory, pro-resolving, infection, mediator
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B272 RESOLUTION FACTORS: WILL THEY BE GOOD CONTROLLERS OF THE INFLAMMATORY RESPONSE? Fla´via CS Fonseca, Janetti N. Francischi Department of Pharmacology, Federal University of Minas Gerais, Brazil Introduction: Detection of resolution factors such as Resolvins, Protectins and Maresins (SERHAN et al. 2015) demarcated a turning point in the understanding of the pathophysiology of the inflammatory process. The aim of the present work was to verify the profile of biological activity presented by Resolvin E1 in the rat paw edema and hyperalgesia test. The used phlogogenic stimuli were: carrageenan (CG), prostaglandin E2 (PGE2), histamine (H), 5-hydroxytriptamine (5-HT) and substance P (SP) in doses known to induce paw edema and/or hyperalgesia (nanogram to microgram range). Paw edema and hyperalgesia were measured at 0, 5, 15, 30 min, 1, 2, 3, 4, 6 and 24 h with an Ugo Basile plethysmometer and the Randall-Selitto analgesimeter (mechanical hyperalgesia), respectively, using male Holtzman rats (weight = 150–180 g). Resolvin E1 (RvE1; in 0.1 mL) diluted in 5 % ethanol was administered intraplantarly by subcutaneous route, 10 min before the phlogogenic stimuli, also given in 0.1 mL at zero time. Initial dose of RvE1 was based on the work of XU et al. 2010. The experiments were approved by UFMG Ethics’s Committee for Animal Use (no. 199/2014). Results: RvE1 reduced edema induced by CG but did not affect SPand PGE2-induced edema. Surprisingly, RvE1 increased H and 5-HT edema. RvE1 also reduced CG-, PGE2-, 5-HT- and SP-induced hyperalgesia as resumed on Table 1 below. Conclusions: Given the predictability of the carrageenan paw edema model in rats to detect potential (non-steroidal) antiinflammatory drugs (MUKHERJEE et al. 1996), RvE1 may represent a good analgesic candidate, but its prolonged use might be associated with adverse effects related with its proinflammatory activities. Support CNPQ and CAPES. Table 1: Profile of anti-inflammatory and analgesic activity of Resolvin E1 in rats Phlogogen stimulus/Dose
EDEMA
Hyperalgesia
Control + RvE1
– –
– –
CG -100 lg
+++
+++
+ RvE1
+
+
PGE2 -200 ng
++
+
+ RvE1
++
–
SP -1 lg
+
++
+ RvE1
+
–
5-HT -1 lg
++
++
+ RvE1
+++
–
H–20 lg
+
–
+ RvE1
++
(hypoalgesia)
+ presence or - absence of the response. Dose of RvE1: 100 ng/paw References 1. SERHAN, C. N.; DALLI, J.; COLAS, R. A.; WINKLER, J. W.; CHIANG, N. Review: Protectins and maresins: New pro-resolving families of mediators in acute inflammation and resolution bioactive
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S212 metabolome. Biochimica et Biophysica Acta 1851, p. 397–413, 2015. 2. XU, Z-Z.; ZHANG, L.; LIU, T.; PARK, J. Y.; BERTA, T.; YANG, R.; SERHAN, C. N.; JI, R-R. Resolvins RvE1 and RvD1attenuate inflammatory pain via central and peripheral actions. Nature Medicine, v.16, n.5, p. 592–597, 2010. 3. MUKHERJEE, A.; HALE, V. G.; BORGA, O.; STEIN, R. Predictability of the clinical potency of NSAIDS from the preclinical pharmacodynamics in rats. Inflammation Research, v. 45, p. 531– 540, 1996.
B273 OXYGENATED N-3 PUFA METABOLITES PRESENT IN THE CIRCULATION AFTER ACUTE CONSUMPTION OF N-3-PUFA CONTAINING OILS REGULATE THE ANGIOGENIC POTENTIAL OF HUMAN ENDOTHELIAL CELLS THROUGH PPARb/D- AND GPCR-DEPENDENT PATHWAYS Caroline PD Wheeler-Jones1, Sally H. Latham1, Anna Nicolaou2, Alexandra Kendall2, Ashton R. Faulkner1, David Bishop-Bailey1, Mauro Perretti3, Thomas Gobbetti3, Kathleen M. Botham1, Wendy L. Hall4, Robert Purcell1 1
Royal Veterinary College, London, UK; 2University of Manchester, Manchester, UK; 3Queen Mary, University of London, London, UK; 4 King’s College London, London, UK Mechanistic understanding of how the nutritionally important n-3 polyunsaturated fatty acids (PUFA) EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) may preserve vascular homeostasis and tissue repair is lacking. In a randomized trial in 16 healthy males we demonstrated divergent postprandial responses to high fat meals containing EPA + DHA-rich oil versus DHA-rich oil, suggestive of differential effects on FA metabolism. Here, we used targeted lipidomics to profile the oxygenated fatty acid metabolites present in baseline and postprandial (6 h) plasma from study participants, and investigated the direct actions of selected mediators and their downstream metabolites on human endothelial cells (ECs). Plasma lipidomics revealed patterns of EPA- and/or DHA-derived metabolites that were largely consistent with the composition of the oils ingested. Significant increases in a range of COX- and LOX-derived mediators were evident following meals containing EPA and/or DHA, with particularly high concentrations of 18-HEPE and 17-HDHA, pathway precursors for the pro-resolving RvE1 and RvD1, respectively. 18-HEPE, 17-HDHA, RvE1 and RvD1, but not the n-6 PUFA-derived metabolites 9- and 13-HODE, increased capillary-like tube formation by HUVEC and adult microvascular ECs in vitro, and these effects were inhibited by the PPARb/d antagonist GSK0660. Responses to 18-HEPE and RvE1 were reduced by treatment with CCX832, a selective ChemR23 antagonist. Quantitative RT-PCR and immunofluorescence in actively tubing cells confirmed expression of FPR2 in ECs. RvD1 elevated intracellular Ca2+, enhanced ERK1/2 phosphorylation and reduced caspase3/7 activity in ECs, responses that were blocked by WRW4 (an FPR2 antagonist) and mimicked by MMK1 (FPR2 agonist). The pro-angiogenic activities of RvD1 and MMK1, but not 17-HDHA, were attenuated by WRW4. VEGF-induced tube formation was abrogated by PPARb/d blockade but was unaffected by antagonists of FPR2 or ChemR32. MMK1, RvD1, 17-HDHA, RvE1 and VEGF increased the tube forming capacity of wild type (WT) embryonic fibroblasts (MEFs) whereas FPR2-/- MEFs failed to undergo tubulogenesis in response to either MMK1 or RvD1 but retained sensitivity to
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Inflamm. Res. 17-HDHA, RvE1 and VEGF equivalent to that of WT MEFs. Collectively, these data support the hypothesis that oxygenated n-3 PUFA metabolites directly target endothelial cells to enhance pro-angiogenic potential in a PPARb/d-dependent manner and demonstrate a key role for FPR2 in mediating the endothelial-directed pro-repair actions of RvD1. These mechanisms likely contribute to explaining the purported beneficial roles of dietary n-3 PUFA as regulators of vascular homeostasis and effective tissue repair.
B274 OLD DRUGS WITH NEW SKILLS: BIASING THE MELANOCORTIN RECEPTORS TO RESOLVE INFLAMMATORY ARTHRITIS Trinidad Montero-Melendez1, Thomas Gobbetti1, Sadani N. Cooray1, Thomas EN Jonassen2, Perretti Mauro1 1 The William Harvey Research Institute, Barts and The London School of Medicine, Queen Mary University of London, London, UK, Charterhouse Square, London, UK; 2Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
Melanocortin receptors (MC1–MC5) are druggable GPCRs that upon activation elicit pro-resolving properties, dampening inflammatory processes. The MC peptide ACTH has been used for over 60 years to treat rheumatoid arthritis, gout and other inflammatory conditions. However, lack of selectivity of ACTH and the synthetic MC drugs developed so far limits the translational delivery of novel MC drugs into patients. A new concept of significant therapeutic interest is the one of biased agonism, which implies ligand-dependent selectivity for certain signal transduction pathways. Here we characterize the molecule AP1189 ((E)-N-[trans-3-{1-(2-nitrophenyl)-1H-pyrrol-2yl} allylidenamino] guanidium acetate), which acts as a biased agonist at MC1 and MC3, since it activates ERK1/2 and Ca2+ signaling but not the canonical cAMP pathway, of relevance as MC1 pro-melanogenic side effects depend on cAMP (see figure below). AP1189 reduces cytokine release by macrophages in vitro, an effect driven by MC1 and MC3, evidenced with knockout cells. It also promotes efferocytosis in an ERK1/2 dependent manner. No melanogenesis was induced by AP1189 in B16-F10 melanocytes. In vivo, oral AP1189 reduces leukocyte trafficking in acute peritonitis, activating the resolution of inflammation three times faster as compared to vehicle-treated mice when the drug was administered at peak of inflammation. Finally, when tested in models of inflammatory arthritis, AP1189 reduced clinical score, paw swelling, disease severity and leukocyte infiltration. In summary, biased agonists at MC receptors offer therapeutic innovation for the development of drugs with improved profile by activating those pathways that are therapeutically relevant and evading those associated with side effects.
B275 HUMAN MILK SPECIALIZED PRORESOLVING LIPID MEDIATORS STIMULATE RESOLUTION OF ACUTE INFLAMMATION Hildur H. Arnardottir, Sarah K. Orr, Jesmond Dalli, Charles N. Serhan Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Perioperative and Pain Medicine,
Inflamm. Res. Harvard Institutes of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA Human milk contains nutrients and bioactive products relevant to infant immune development and protection. Here we investigated the pro-resolving properties of human milk using human milk lipid mediator isolates (HLMI) and determined their impact on resolution programs In vivo and with human macrophages. In murine acute peritonitis, HLMI reduced maximum neutrophil numbers from 14.6 ± 1.2 9 106 cells/exudate to 11.0 ± 1.0 9 106 cells/exudate and shortened the resolution interval (Ri) by 54 % (12 h vs. 26 h) compared to peritonitis plus vehicle mice. Using liquid chromatography tandem mass spectrometry (LC–MS–MS)-based lipid mediator (LM) metabololipidomics, we found that human milk possesses a proresolving LM signature profile, containing specialized proresolving lipid mediators (SPM) at concentrations within their bioactive range (pico-nanomolar concentrations) that enhanced human macrophage efferocytosis and bacterial (E. coli) containment. The SPM identified in human milk included D-series resolvins, [e.g. Resolvin (Rv) D1, RvD2 and RvD3], Protectin D1, Maresin 1, E-series resolvins (e.g. RvE1, RvE2 and RvE3) and lipoxins (LXA4 and LXB4). Milk from inflamed human mammary glands (mastitis) had altered LM profile, with lower SPM and higher proinflammatory LM compared to milk from non-inflamed (healthy) mammary glands. Hence, these results demonstrate that human milk contains proresolving chemical signals that simulate resolution of acute inflammation and infection. Taken together, these findings provide a novel mechanism in maternal-infant biochemical imprinting.
B276 THE SATIATED MACROPHAGE: A KEY PLAYER IN THE RESOLUTION OF INFLAMMATION Amiram Ariel1, Senthil K. Satyanarayanan1, Noa Sher2,1, Sagie Schif-Zuck1 1
Department of Human Biology, University of Haifa, Haifa, Israel; Tauber Bioinformatics Research Center, University of Haifa, Haifa, Israel
2
The engulfment of apoptotic leukocytes (efferocytosis) by macrophages during the resolution of inflammation is essential for tissue homeostasis and results in macrophage reprogramming/immune-silencing. However, a distinct subset of resolution phase macrophages loss their phagocytic potential, and hence were termed satiated macrophages. To characterize the satiation of resolution phase macrophages, apoptotic cells, latex beads (LB) or opsonized LB were injected alongsides the fluorescent dye PKH2-PCL during zymosan A-induced peritonitis, and phagocytosis of the dye as well as the expression of efferocytic receptors, such as CD36, CD206, and Mer, was evaluated. In addition, phagocytic or satiated macrophages were sorted and differences in their gene expression profile were determined by Illumina-based RNA-Seq analysis. Here, we show the engulfment of apoptotic cells, but not other particles, In vivo leads to macrophage satiation and loss of phagocytic capacity. Moreover, satiated macrophages were generated during the resolution of inflammation and maintained proliferative potential exhibited by phagocytic/M2-like macrophages. In addition, we found satiation to be irreversible and associated with reduced surface expression of efferocytic receptors. Furthermore, a differential gene expression analysis revealed that macrophage satiation results in a comprehensive molecular switch that conforms, for the most part, with transition from a reparative/pro-fibrotic phenotype to a pro-resolving/anti-fibrotic one. Thus, satiated macrophages generated during the
S213 resolution of inflammation seem to play an essential role in the termination of tissue repair and the promotion of its return to homeostatic structure and function.
B277 SATIATED MACROPHAGES UPREGULATE CD24 EXPRESSION THROUGH UNCOUPLING PROTEIN 2 Janan Saadi, Senthil K. Satyanarayanan, Nawras Abboud, Sagie Schif-Zuck, Amiram Ariel University of Haifa, Department of Human Biology, Haifa, Israel The engulfment of apoptotic leukocytes (efferocytosis) by macrophages during the resolution of inflammation is essential for tissue homeostasis and results in macrophage reprogramming/immune-silencing. However, a distinct subset of resolution phase macrophages loss their phagocytic potential, and hence were termed satiated macrophages. Here, we used Illumina-based RNA-Seq analysis and Western blotting to identify proteins that are upregulated upon macrophage satiation. We found that CD24 expression is highly upregulated in satiated macrophages, while the expression of Uncoupling Protein 2 (UCP2), a mitochondrial protein that prolongs apoptotic cell clearance, was down-regulated. Moreover, pharmacologic inhibition of UCP2 in resolution phase macrophages resulted in elevated CD24 expression as well as increased TNFa production. Thus, macrophage satiation during the resolution of inflammation seems to be associated with increased CD24 expression and diminished TNFa production through UCP2 down-regulation.
B278 NATURAL KILLER CELLS ARE INDISPENSABLE FOR RESOLUTION OF ANTIGEN-INDUCED INFLAMMATION IN MICE Ingibjorg Hardardottir1, Osk Anuforo1,3,4, Hulda S. Jonasdottir5, Martin Giera5, Jona Freysdottir2,3,4 1
Biochemistry and Molecular Biology, Faculty of Medicine, Biomedical Center, University of Iceland, Reykjavik, Iceland; 2 Department of Immunology, Faculty of Medicine, Biomedical Center, University of Iceland, Reykjavik, Iceland; 3Centre for Rheumatology Research, Landspitali, The National University Hospital of Iceland, Reykjavik, Iceland; 4Department of Immunology, Landspitali, The National University Hospital of Iceland, Reykjavik, Iceland; 5Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands Resolution of inflammation is important in preventing chronic inflammation. Natural killer (NK) cells are known to act as the first line of defense during an infection, but have recently been implicated in resolution of allergic airway inflammation. This study examined the role of NK cells in resolution of inflammation using an antigen-induced peritonitis model, resembling a flare-up in autoimmune diseases. Mice were immunized twice subcutaneously with methylated BSA (mBSA) and peritonitis induced by injecting mBSA into their peritoneum. Mice were injected intravenously with the NK cell depleting antibody, anti-asialo GM1, or a control antibody 24 h prior to peritonitis induction. Prior to and at several time-points following peritonitis induction, peritoneal exudates were collected, cells counted and
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S214 expression of surface molecules determined by flow cytometry. Concentration of cytokines, soluble cytokine receptors and growth factors was determined by ELISA and lipid mediators using LC–MS/MS. The NK cell depleting antibody led to a 50 % decrease in the number of NK cells in the peritoneum 36 h after injection (12 h after induction of the inflammation). Most of the NK cells were mature, CD11b+CD27+ cells. Immature CD11b-CD27+ NK cells increased after induction of inflammation in the control group but not in the NK cell depleted group. The number of peritoneal neutrophils increased after induction of inflammation and peaked in the control group at 6 h, whereas their number in the depleted group continued to increase and peaked at 12 h, being at that time-point twice the number in the control group. By 48 h the number of neutrophils in the control group had almost returned back to baseline but in the depleted group their numbers remained high. NK cell depletion had little effect on other cell types. Peritoneal concentrations of 15-HEPE and PGE2 were lower in the depleted group than in the control group 12 h following induction of inflammation and the concentration of LXA4 had a tendency to be lower. NK cell depletion prevented timely reduction in IL-6, IL-12p40 and G-CSF and increased peritoneal concentrations of IL-1ra and TGF-b at late time-points. These results indicate that NK cells may be important for halting neutrophil recruitment and/or inducing neutrophil apoptosis and are indispensable for resolution of antigen-induced inflammation.
B279 EXUDATE SUCTION BLISTER: A NOVEL IN VIVO MODEL OF ACUTE INFLAMMATION AND RESOLUTION IN HUMANS Madhur Motwani1, Roel De Maeyer1, James Fullerton1, Daniel Marks2, Andrew Smith2, Derek W. Gilroy1 1
Centre for Clinical Pharmacology, Division of Medicine, University College London, London, UK; 2Centre for Molecular Medicine, Division of Medicine, University College London, London, UK Resolution of acute inflammation is an active process and its dysregulation may contribute to chronic inflammatory disorders. While animal models have been used to study resolution their appropriateness has been questioned. We describe a novel model of resolving inflammation in humans triggered by intradermal injection of UV killed E. coli into the forearm of healthy volunteers (n = 30). Inflammatory exudate was drawn into a skin blister induced by negative pressure applied over the injection site. Blister cells were analyzed by flow cytometry while the cell free exudates were assayed for cytokines. To study the different phases of inflammation, blisters were raised at multiple time points post-injection: 4, 8, 14, 24 h, Day 3, Day 5, Day 7, Day 14. Vascular response was monitored by laser Doppler. Onset (4 h) was characterised by peak erythema and neutrophilia (HLA-DR-/ CD16++). By 24 h, neutrophil numbers had fallen by 70 % while monocytes/macrophages (HLA-DR+/CD14+) increased, indicating cellular resolution. From 48 h blood flow declined alongside monocytes/macrophages. Lymphocytes (CD4+, CD8+) dominated the site at day 3 and persisted up to day 14. Levels of TNF-a, IL-1b were maximal during onset phase, IFN-c and IL-8 during resolution, whilst post-resolution phase showed higher levels IL-16 and IL-7. Exudate Suction Blister is safe and reproducible model that allows, for the first time, exploration of cells and soluble mediators in parallel with the clinical signs of resolution in humans. Potential applications include testing efficacy of novel anti-inflammatory and pro-resolving therapies and investigate whether resolution defects underlie pathogenesis of chronic inflammatory disorders.
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B280 SPECIALIZED-PRORESOLVING MEDIATORS AND TRANCRIPTOMIC SIGNATURES IN BLEOMYCIN-INDUCED LUNG FIBROSIS Ge´rald Cheˆne, Vincent Baillif, Guigne´ Charlotte, Belloy Marcy, Wanecq Estelle, Emeline Van Goethem, Marc Dubourdeau Ambiotis SAS, Toulouse, France Introduction: Idiopathic pulmonary fibrosis (IPF) is a disease with unknown origin. IPF is characterized by the apparition of collagen fibers into the lung parenchyma, leading to an excessive and irreversible healing of the tissue, associated with a loss of its function. The clinical course of IPF is dramatic since it is estimated that the 5-year survival is between 20 and 40 %, a higher mortality rate than many cancer types, including colon cancer, myeloma multiple and bladder cancer. Following the description of specialized mediators of inflammation resolution (SPMs) and their potential involvement in the control of fibrosis, their study in animal models of pulmonary fibrosis might help deciphering mechanisms involved in this pathology and thus open new potential therapeutic directions. Methods: For this purpose, mice were inoculated by intranasal challenge of bleomycin. After 2 weeks, mice were euthanized and lungs were collected and prepared for analysis. SPMs and other mediators issued from fatty acids were analyzed using a liquid chromatographytandem mass spectrometry (LC–MS/MS) methodology to quantitatively evaluate their production. RNA expression was evaluated thanks to a Fluidigm dynamic array. Results: During these experiments, we have shown an increase of PGE2, TXB2, PGD2 and 15-HETE, mediators depending on the arachidonic acid pathways. For metabolites linked to EPA, we were also able to demonstrate an increase of PGE3, 15-HEPE and 18-HEPE the precursor of resolvins of type E. Finally, we also observed DHA metabolites with an increase of 13-HDoHE and 17-HDoHE but also of RvD2. Concerning trancriptomic experiments, we evaluated over 90 genes and classified them according to their systemic overexpression, inhibition or no effect over 3 independent experiments that we performed. Interestingly and as an example, we notably noticed an overexpression of inflammatory genes (such as COX1, IL6, Ccl1, MCP-1) and genes involved in the remodeling of the extracellular matrix (Col1, Fn1, Mmp2, Timp1). We also measured an inhibition of Alox12 and no effect on Alox5 mRNAs expression. Conclusions: Taken together those results suggest that fibrosis context is associated with an inflammatory status that did not seem to be counterbalanced by production of SPMs. Indeed, even if RvD2 is increased and the enzymes COX and 15-LOX activated (as seen with production of PGE2 and 15-HETE), none of the other SPMs were detected. These results might thus draw the first evidence of a defect during fibrosis to produce a complete network of SPMs.
Respiratory Disease and Inflammation B281 BLOCKING LTbR SIGNALING IN VIVO SUPPRESSES THE DEVELOPMENT OF SMOOTH MUSCLE HYPERTROPHY IN A CHRONIC MURINE MODEL OF ASTHMA Silke NK Hobbie, Peter Maier, Erb J. Klaus Boehringer Ingelheim Pharma GmbH & Co KG, RhinelandPalatinate, Germany
Inflamm. Res. LTbR receptors are expressed on many different cell types and bind to LIGHT (TNFSF14), which is expressed on activated T cells, and lymphotoxin a and b, expressed on activated T, B and NK cells. Signaling via LTbR receptor has been suggested to play a role in remodeling in chronic airway diseases (Dohertey et al. 2011; Herro et al. 2015). We investigated the effects of blocking LTbR signaling on the development of fibrosis and smooth muscle thickening of the airways in a chronic asthma model in mice. Mice were subjected to an immunization/challenge protocol using a combination of house dust mite extract, cockroach extract and ovalbumin, which leads to a severe asthma remodeling phenotype (Duechs et al. 2014). These animals were either treated with dexamethasone or murLTbR-Fc (which blocks LIGHT and LTab signaling). We found that the treatment of the mice with the murLTbR-Fc but not dexamethasone significantly suppressed the development of smooth muscle thickening and reduced fibrosis without affecting inflammation or lung function. This supports a role of LTbR signaling in remodeling. Since soluble LIGHT but not LTa and LTb protein levels were increased in broncho-alveolar lavage in this model, the LIGHT-induced signaling seems to be mediating these processes. To further investigate the direct effect of LIGHT on lung target cells we performed several in vitro experiments. In normal human lung fibroblasts (NHLF) LIGHT increased the proliferation rate twofold after 24 h stimulation. LIGHT alone had no effect on cytokine release in NHLF, however, in the presence of IFNg, LIGHT increased IL-8 and IP-10 release, 2- and sevenfold respectively. In the broncho-epithelial cell line A549 LIGHT induced EMT (epithelial-mesenchymal fibroblast transition). LIGHT was as efficacious as TGF-b in this assay with a later on-set of action. In human bronchial-smooth muscle cells LIGHT together with IFNg (but neither alone) also induced a significant increase in the proliferation of cells. These data indicate that LIGHT could be involved in remodeling processes in various cell types in vitro also conferring profibrotic properties. Taken together our results show that blocking LTbR signaling reduces remodeling in a severe asthma model in mice. The presence of soluble LIGHT and not LTa and LTb in the lung of the mice together with the stimulating effects on human fibroblasts and smooth muscle cells in vitro point at LIGHT mediating these effects.
B282 NEUTROPHIL PROTEASES ALTER THE INTERLEUKIN-22-RECEPTOR-DEPENDENT LUNG ANTIMICROBIAL DEFENCE Antoine Guillon1,3,2, Youenn Jouan1,3,2, Deborah Brea 1,2, Fabien Gueugnon 1,2, Emilie Dalloneau 1,2, Thomas Baranek 1,2, Cle´mence Henry 1,2, Eric Morello 1,2, Jean-Christophe Renauld 4,5, Muriel Pichavant 7,8,9, Philippe Gosset 7,8,9, Yves Courty 1,2, Patrice Diot1,2, Mustapha Si-Tahar 1,2 1
INSERM, Centre d’Etude des Pathologies Respiratoires, U1100, Tours, France; 2Universite´ Franc¸ois Rabelais de Tours, Tours, France; 3CHRU de Tours, Service de Re´animation Polyvalente, Tours, France; 4Ludwig Institute for Cancer Research, Brussels branch, Brussels, Belgium; 5De Duve Institute, Universite catholique de Louvain, Brussels, Belgium; 6Universite´ Lille Nord de France, Lille, France; 7Institut Pasteur de Lille, Center for Infection and Immunity of Lille, Lille, France; 8CNRS, Unite´ Mixte de Recherche 8204, Lille, France; 9INSERM, U1019, Team 8, Lille, France Acute episodes of exacerbations triggered by respiratory pathogens mark the progression of chronic obstructive pulmonary disease (COPD), leading to substantial morbidity and mortality. Despite the
S215 life-threatening nature of these exacerbations, the underlying mechanisms remain unclear although a high number of neutrophils in the lungs of COPD patients is known to correlate with poor prognosis. We and others previously demonstrated that interleukin (IL)-22 protects and regenerates respiratory epithelial cells upon virus infections and also limits secondary bacterial infections. Indeed, IL-22, through its receptor (IL-22R), is a cytokine that plays a pivotal role in lung antimicrobial defence and tissue protection. Regarding the IL-22/ IL22R antimicrobial pathway, we hypothesized that any alteration of IL-22R in the lungs of COPD patients may compromise innate defence mechanisms and enhance susceptibility to infections. More specifically, we examined whether IL-22R expressed on lung epithelial cells is targeted by the neutrophil proteases present at the surface of lung mucosa. Using in vitro and in vivo approaches as well as RT-qPCR, flow cytometry and/or western-blotting techniques, we first showed that pathogens such as influenza virus promote IL-22R expression in human bronchial epithelial cells whereas Pseudomonas aeruginosa, bacterial LPS do not. We also investigated whether IL-22R lung expression was impaired by cigarette smoke in epithelial cells or in mice chronically exposed to cigarette smoke. Finally, we examined lung tissue from 129 patients (11 % non-smokers, 41 % ‘‘healthy’’ smokers, 48 % COPD patients). IL-22R1 RNA expression was neither associated with the cigarette smoke exposure nor the COPD status. In view of the foregoing data and the evidence that neutrophilia is a pathological hallmark of COPD, we hypothesized that neutrophils induce post translational modifications of IL-22R expression and function. We first exposed human epithelial cells to supernatant from activated neutrophils and observed that IL-22R1 protein expression was strongly decreased under these conditions, whereas a-1 proteinase inhibitor prevented the disappearance of the receptor. Most importantly, neutrophil proteases impaired IL-22-dependent immune signalling and expression of antimicrobial effectors such as b-defensin-2. This proteolysis resulted in the release of a soluble fragment of IL-22R, which was detectable both in cellular and animal models as well as in sputa from COPD patients with acute exacerbations. Hence, our study reveals an unsuspected regulation by the proteolytic action of neutrophil enzymes of IL-22-dependent lung host response. This process likely enhances pathogen replication and ultimately COPD exacerbations.
B283 STEM CELL FACTOR AND INTERLEUKIN 31 EXPRESSION: ASSOCIATION WITH SERUM IgE LEVELS AND DISEASE SEVERITY IN ATOPIC AND NON-ATOPIC BRONCHIAL ASTHMA Mai Moaaz1,2, Salma Aboelnazar1,2 1 Medical Research Institute, Colombo, Sri Lanka; 2University of Alexandria, Alexandria, Egypt
Bronchial asthma is a chronic inflammatory disorder, its prevalence continues to increase over the world and it remains a significant cause of morbidity. The origins of the disease remain elusive. Recently, it has been reported that stem cell factor (SCF) and interleukin-31(IL31) may play a major role in bronchial asthma. The aim of the current study was to validate the obscure associations of these two markers with the susceptibility to asthma and correlate them with serum immunoglobulin E (IgE) level and disease severity. The present study included 160 Egyptian subjects who were divided into 75 atopic asthmatic patients, 35 non-atopic asthmatic patients and 50 normal
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S216 subjects. After measuring disease severity, pulmonary function tests and serum IgE; RNA was isolated from peripheral blood mononuclear cells (PBMCs) to determine gene expression of SCF and IL-31 by real-time PCR. The levels of SCF mRNAs in atopic asthmatic patients PBMCs were significantly higher than those in control subjects (p = 0.0001) and non-atopic asthmatics (p = 0.0001). There was a high statistical significant difference also as regards IL-31 between Atopic asthmatics and controls (p = 0.0001) and between them and non-atopics (p = 0.014). There was a strong significant direct correlation between SCF, IL-31 (r = 0.827 and p = 0.0001**) and between both of them and IgE in asthmatics (r = 0.543 and p = 0.0001**) (r = 0.443 and p = 0.0001**) respectively. A direct correlation between SCF, IL-31 and forced expiratory volumes (FEV1/FVC %),c- reactive proteins (CRP), clinical severity scores and wheezing existed. These findings suggest that both SCF and IL-31 enhances IgE-mediated inflammation in bronchial asthma proposing their role in mediating inflammation and enhancing severity of allergic asthma
B284 FUNCTIONAL DEFECT OF MAIT CELLS IN PATIENTS WITH ACTIVE TUBERCULOSIS IS MEDIATED BY PD-1 SIGNALING Xiaoxing Cheng, Jing Jiang, Hongjuan An Institute of Tuberculosis, Beijing, China Mucosal-associated invariant (MAIT) cells are innate-like T cells that are restricted by the evolutionarily conserved major histocompatibility complex-like molecule MR1. Human MAIT cells display a T cell receptor alpha chain that consists of TRA1-2 gene paired with different TRAJ genes including TRAJ33, TRAJ12 and TRAJ20. MAIT cells are activated by cells infected with bacteria and yeast, such as Mycobacterium tuberculosis, Escherichia coli, Shigella flexneri. Vitamin B metabolites synthesized by microbes can bind MR1 and activate MAIT cells. MAIT cells are abundant in humans, including peripheral blood, liver, gut lamina propria and lungs, and they not only produce cytokines, such as IFN-gamma, TNF-alpha, IL17, but also have cytotoxic effect. Tuberculosis (TB) is the second most common cause of death from infectious disease. Despite high rate of M. tuberculosis infection in humans, especially in developing countries, only 5–10 % of infected people develop activeTB in their life time. Interactions between M. tuberculosis and host largely determine the development and outcome of TB infection. In this study, we compared IFN-gamma and TNF-alpha production of MAIT cells from healthy controls and patients with active TB. To prevent influence of previous antigenexposure and make it comparable in the two groups, we used PMA/ionomycin and E. coli as stimulator. In patients with active TB, defect in IFN-gamma and TNF-alpha production was observed in MAIT cells when E. coli was used for stimulation, while MAIT cell response to nonspecific stimulation with PMA/ionomycin appeared to be normal. To determine whether there is any association between inhibitory receptors and functional defect of MAIT cells, we compared expression of PD-1, PD-L1, PD-L2, Tim-3, CD244, LAG-3, BTLA, CD160 on MAIT cells from patients with active TB and healthy controls. Among the inhibitory receptor examined, PD-1 was the only receptor that had much higher expression on MAIT cellsfrom patients with active TB as compared with healthy controls. Blockade of PD-1 signaling significantly improved IFN-gamma production in MAIT cells. It is concluded that MAIT cells from patients with active TB exhibited functional defect that is mediated by PD-1.
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B285 OSTEOPONTIN MODULATES CYTOTOXIC EFFECTS OF EXTRACELLULAR HISTONES IN COPD Gopinath Kasetty, Ravi Kiran Varma Bhongir, Matthias Morgelin, Arne Egesten Lund University, Lund, Sweden Background: Lung inflammation, cell death and extensive lung tissue remodeling characterize COPD. Regulation of the activity of potentially harmful extracellular histones released from necrotic cells is important for the prevention of excessive tissue damage and disease progression in COPD. Osteopontin (OPN) is an anionic glycoprotein upregulated during a number of physiological and pathological processes has been involved in the regulation of inflammation. In recent studies high levels of OPN have been detected in the airways of patients with COPD. However contribution of OPN to the host response during COPD has not been well characterized. Objective: Investigate if OPN binds to extracellular histones and modulate their cytotoxic effects. Methods: The binding affinity and kinetics of histones with OPN was measured utilizing SPR BIAcore. The cytotoxic effects of extracellular histones in presence and absence of OPN was determined by measuring lysis of erythrocytes, epithelial and endothelial cells and antimicrobial activities of histones in presence of OPN were determined. Results: OPN binds to all subclasses of histones with varying affinities. Functional analysis has revealed that OPN neutralizes the cytotoxic effects of extracellular histones, which is evidenced by low levels of LDH release from epithelial and endothelial cells and complete inhibition of erythrocyte lysis. In addition the antimicrobial activities of histones was suppressed in presence of OPN. Conclusions: These findings suggest a possible protective mechanism of OPN in modulating host immune response by neutralizing extracellular histones, thereby presenting a novel treatment strategy to prevent excessive tissue damage and disease progression in COPD.
B286 THE POLYPHENOL RESVERATROL IMPROVES THE PULMONARY OXIDATIVE STRESS IN ASTHMATIC AND OBESE MICE BY SIRT1 PATHWAY ACTIVATION Diana M. Andre, Marina C. Calixto, Eduardo C. Alexandre, Carolina S. Sollon, Gabriel F. Anhe, Edson Antunes State University of Campinas, Campinas, Brazil Objectives: To evaluate the antioxidant effects of resveratrol via activation of SIRT1 in the lungs of asthmatic and obese mice Methodology: The experimental protocols have been approved by the Ethics Committee of UNICAMP (No. 2709-1). Male C57Bl/6/JUnib mice received high-fat or standard-chow diet for 12 weeks. Lean and obese mice received daily administration of resveratrol (100 mg/kg, gavage, 2 weeks). Mice were sensitized with OVA (100 lg, s.c) and intranasally challenged with this antigen (10 lg) 2 weeks later. At 48 h after OVA challenge, SIRT1, p47 phox and FOXO1 expressions were examined in the lung tissues. Results: SIRT1 expression in lung tissue of obese OVA-challenged mice was reduced by about of 50 % compared to OVA-challenged lean group (p \ 0.05), which was fully restored by resveratrol
Inflamm. Res. treatment. Resveratrol treatment increased by 20 % the p-FOXO1 expression in obese compared with lean mice (p \ 0.05). The p47 phox expression in lung tissue showed no statistical differences between obese and lean groups, but resveratrol reduced by 56 % this protein expression in obese group (p \ 0.05), with no alterations in lean group. Conclusions: Our data show that activation of SIRT1 by resveratrol may lead to increased FOXO1 expression and decreased of p47 phox expression, possibly reducing the pulmonary oxidative stress, thus improving asthma. Financial support CNPq.
B287 LUNG EPITHELIAL TRPA1 TRANSDUCES THE EXTRACELLULAR ROS INTO TRANSCRIPTIONAL REGULATION OF LUNG INFLAMMATION INDUCED BY CIGARETTE SMOKE: THE ROLE OF INFLUXED CA2+ An-Hsuan Lin1, Meng-Han Liu1, Hsin-Kuo Ko1,2, Diahn-Warng Perng2, Tzong-Shyuan Lee1, Yu Ru Kou1 1 Department of Physiology, School of Medicine, National Yang-Ming University, Taipei, Taiwan; 2Department of Chest Medicine, Taipei Veterans General Hospital, Taipei City, Taiwan
The mechanism underlying the inflammatory role of TRPA1 in lung epithelial cells (LECs) remains unclear. Here, we show that cigarette smoke extract (CSE) sequentially induced several events in LECs. The Ca2+ influx was prevented by decreasing extracellular reactive oxygen species (ROS) with the scavenger N-acetyl-cysteine, removing extracellular Ca2+ with the chelator EGTA or treating with the TRPA1 antagonist HC030031. NADPH oxidase activation was abolished by its inhibitor apocynin, EGTA or HC030031. The increased intracellular ROS was halted by apocynin, N-acetyl-cysteine or HC030031. The activation of the MAPKs/NF-kB signaling was suppressed by EGTA, N-acetyl-cysteine or HC030031. IL-8 induction was inhibited by HC030031 or TRPA1 siRNA. Additionally, chronic cigarette smoke (CS) exposure in wild-type mice induced TRPA1 expression in LECs and lung tissues. In CS-exposure trpa1-/- mice, the increased BALF level of ROS was similar to that of CS-exposure wild-type mice, yet lung inflammation was lessened. Thus, in LECs, CSE may initially increased extracellular ROS, which activates TRPA1 leading to an increase in Ca2+ influx. The increased intracellular Ca2+ contributes to activation of NADPH oxidase, resulting in increased intracellular ROS, which activate the MAPKs/ NF-kB signaling leading to IL-8 induction. This mechanism may possibly be at work in mice chronically exposed to CS.
B288 N-ACETYL-DE-O-SULFATED HEPARIN INHIBITS LYPOPOLYSACCHARIDE-INDUCED LEUKOCYTE MIGRATION BY A PLATELET DEPENDENT MECHANISM Yanira Riffo Vasquez1,2, Richard Amison1,2, Simon Pitchford1,2, Clive P. Page1,2 1
King’s College London, London, England; 2Sackler Institute of Pulmonary Pharmacology, London, England
S217 Introduction: Heparin exhibits anti-inflammatory activity independent of its anti-coagulant action. However, the precise mechanism(s) underlying this anti-inflammatory activity is not well established. We have investigated the effect of ex-vivo treatment of platelets with a non-anti coagulant fraction of heparin, N-acetyl-O-Sulfated Heparin (NSH), on neutrophil migration to the lung induced by lipopolysacharide (LPS, E. coli), and expression of P-selectin and PSGL-1 on the surface of platelets. Methods: Platelets were collected from anaesthetized LPS-primed mice (10 lg i.p.) by cardiac puncture and treated ex-vivo with NSH (0.5 mg/mL) for 15 min. Platelets were washed and transfused back into recipient mice rendered thrombocytopenic with Busulfan (20 mg/ kg, i.p.). Lung inflammation was induced by intranasal instillation of 10 lg of LPS and bronchoalveolar lavage and samples of blood undertaken 4 h later. In an independent experiment, mice were treated orally with 20 mg/Kg of NSH just before intra-nasal instillation of LPS. Four h later, blood samples were stained with anti-CD41 FITC and anti-CD62P (P-selectin) or anti-CD162 (PSGL-1) and after 30 min incubation, lysed and prepared for flow cytometry. Results: Mice rendered thrombocytopenic presented a significantly lower neutrophil recruitment in response to LPS (LPS: 29.1 ± 1.3 vs LPS/Busulfan: 3.1 ± 0.9 cells 9 104/mL). Replenishment with LPSactivated platelets, but not platelets pre-treated with NSH, significantly re-established the neutrophilic response to LPS in the lung (LPS/Busulfan/plat.:16.0 ± 1.3 vs LPS/Busulfan/NSH: 6.80 ± 2.3 9 104/mL, p \ 0.0001). LPS significantly increases the expression of P-selectin, but not PSGL-1 in the surface of platelets, in comparison to saline treated mice (saline: 6.6 ± 3.2 vs LPS: 22 ± 1.4 % increase in expression). Pre-treatment with 20 mg/Kg of NSH significantly inhibited the expression of P-selectin, on the surface of platelets in comparison to LPS (NSH: 13.2 ± 1.3 %). Conclusion:: The non-anti coagulant form of heparin, N-acetyl-de-oSulfated heparin, inhibits cell recruitment into the lung by a platelet dependent mechanism.
B289 COMPREHENSIVE ANALYSIS OF RICIN BINDING IN VIVO AND CELLULAR DAMAGE UPON PULMONARY EXPOSURE Anita Sapoznikov, Reut Falach, Ron Alcalay, Nehama Seliger, Yoav Gal, Tamar Sabo, Chanoch Kronman Israel Institute for Biological Research, Ness Ziona, Israel The Departments of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness Ziona, Israel Ricin is one of the most poisonous natural toxins from plants; it belongs to a group of ribosome-inactivating protein type II (RIP II) family, which inhibits protein synthesis. Pulmonary exposure to ricin results in the generation of an acute edematous inflammation, followed by respiratory insufficiency and death. In order to develop a potent therapeutic countermeasure, better understanding of the nature of the interaction between the target host cell and the toxin, and its direct consequences on the whole lung tissue is required. In this study we examined the kinetics of ricin binding and internalization to lung cells in vivo and its effect on the cellular milieu. Interactions between ricin and lung cells were examined concomitantly by fluorescently-labeled-toxin and by specific antiricin antibody binding. We show that although ricin could potentially bind to any of the lung cells, as demonstrated in ex-vivo labeling experiments; In vivo, it bound preferentially to alveolar macrophages (MU), dendritic cells (DCs), pneumocytes type II (PTII) and
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S218 endothelia. Furthermore, the binding to lung cells following pulmonary exposure of mice to ricin occurred at two different kinetics, displaying peak levels at early and late time points. The differential binding pattern could be explained by interactions with cells bearing different classes of receptors, the galactose-containing glycoprotein/ glycolipid receptors or the cellular mannose receptor. MU, DCs and PTII cells, which express the mannose receptor at their surface, displayed early binding, as opposed to endothelial cells which displayed late binding. Although MU, DCs and PTII cells all bound ricin rapidly, elimination of PTII cells from the lungs occurred at a considerably later time-point, in comparison to MU and DCs, which population had been reduced already few hours after intoxication. Interestingly, pneumocytes type I that are known to be very sensitive cells, were not affected by the toxin. Endothelial cells, lacking the mannose receptor, displayed a late binding profile, and their elimination from the lungs occurred after several days. In addition, we could observe, an early reduction in endothelial and epithelial tightjunction protein (VE-Cadherin, Occludin and Claudin5) expression. In contrast to the toxin-binding cells, neutrophils, which were massively recruited to the lungs, did not interact with ricin. Taken together, these results show that differential ricin binding and internalization directly contribute to the loss of maintenance of tissue integrity, which results in functional lung damage.
B290 DOES HYDROGEN SULFIDE INFLUENCE APOPTOSIS PROCESS IN LUNGS FROM ALLERGIC MICE? Heloisa Helena A. Ferreira1, Jackeline A. Mendes2, Matheus C. Ribeiro3, Gislaine Cristina P. Moreira3, Mateus S. Silva3, Natalia H. Dias3, Bruna T. Albaladejo3, Jose´ A. Pereira3, Thalita Rocha3 1 Sa˜o Leopoldo Mandic Institute and Research Center, Campinas, Brazil; 2State University of Campinas/Unicamp, Campinas, Brazil; 3 Sa˜o Francisco University, Campinas, Brazil
Many studies have shown that hydrogen sulfide (H2S) has a relevant role in the pathophysiology of lung diseases. This study aimed to investigate the effect of H2S in modulating apoptosis in lungs from allergic mice. BALB-C mice were sensitized and challenged with ovalbumin (OVA group). Some sensitized mice received sterile saline without OVA at the time of challenge (saline group). Others mice were sensitized but were treated with H2S donor—sodium hydrosulfide (NaHS) 30 min before OVA-challenge (OVA/NaHS group). The euthanasia was performed 48 h after allergen challenge. Bronchoalveolar lavage (BAL) was collected for eosinophils isolation by immunomagnetic method. The right lung lobe was removed and homogenized to study the expression of caspase 3, caspase 9, Bax and Fas-L by western blotting. The left lung lobe was fixed in formalin for 1) histological analysis of inflammatory cell infiltrate using hematoxylin/eosin staining (HE); 2) in situ apoptosis analyses by TUNEL assay and 3) verification of expression of cistationia-c-lyase enzymes (CSE) and cystathionine-b-synthase (CBS) by immunohistochemistry. The histological results showed an inflammatory infiltrate around the bronchi and bronchioles in the OVA group, with a prevalence of eosinophils, which was prevented by NaHS-treatment. The treatment of allergic mice with NaHS also decreased the
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Inflamm. Res. expression of caspase 3 and Fas-L, but not Bax and caspase 9. OVAchallenge or NaHS-treatment was unable to modulate the apoptosis of BAL eosinophils. However, the NaHS avoid the apoptosis increase in bronchial epithelial cells promoted by OVA challenge. An increase in the expression of CSE enzyme in the bronchial epithelium and in the vascular endothelium was observed in the lungs of allergic mice and was amplified by NaHS-treatment. No changes were observed for CBS enzyme. In conclusion our results suggest that NaHS-treatment prevented apoptosis and, consequently, the bronchial epithelium damage, which contributes to the pulmonary inflammation decrease. The CSE enzyme may be involved in this process. Therefore, the H2S can have a protective effect against lung damage caused by allergic reaction, representing a potential therapeutic agent for allergic pulmonary disorders, such as asthma.
B291 MODERATE PHYSICAL EXERCISE DECREASES AIRWAY INFLAMMATION IN A MURINE MODEL OF ASTHMA Paula Fernandes1, Thayse R. Bru¨ggemann1, Clarice R. Olivo1, Fernanda M. Bruni2,3, Mı´lton A. Martins1, Fernanda M. Arantes-Costa1 1
Laboratory of Experimental Therapeutics, LIM20 School of Medicine of University of Sa˜o Paulo, Sa˜o Paulo, Brazil; 2Institute for Investigation in Immunology (iii), INCT, Sa˜o Paulo, Brazil; 3 Laboratory of Clinical Immunology and Allergy, LIM60 Scholl of Medicine of University of Sa˜o Paulo, Sa˜o Paulo, Brazil Introduction: The benefits of moderate physical exercise in allergic asthma are well known, indicating that the physical exercise has a anti-inflammatory effect by reducing the Th2 responses and the pulmonary remodeling, although the mechanisms of this immunoregulation are not very known yet. Therefore, we made this study to investigate the mechanisms of the immunoregulation by moderate physical exercise on asthma inflammation. Methods: Male Balb/c mice (n = 8/group) were sensitized with five intraperitoneal injections of ovalbumin (OVA, 20 lg) and alum (3 mg) on days 0 and 14, 21, 28 and 42 and were challenged with an aerosol of OVA (1 %, 30 min) three times per week, starting on day 21 until day 51 [OVA and OVA+ exercise (OVA + EX) groups]. A maximal exercise capacity test was performed on days 21 and 51. The EX (exercise) and OVA + EX groups practiced moderated exercise on days 21 until 51. EX and Control (SAL) groups received saline and alum intraperitoneal injection and were challenged with saline 0.9 %. Twenty-four h after the last challenge, we evaluated the inflammatory cells influx on the bronchoalveolar lavage fluid, and also the production of immunoglobulin IgG1 and IgG2a in plasma. Results: Physical capacity was increased only in trained groups (p = 0.036). OVA-sensitized mice showed increased influx of macrophages (p = 0.016) and eosinophils (p = 0.05) to the airway and the training reduced this inflammatory infiltrate (OVA + EX) (p = 0.05 and p \ 0.01, respectively). We noticed that IgG1 and IgG2a levels increases in both OVA-sensitized groups (OVA and OVA + EX; p = 0.02 and p \ 0.01, respectively). Conclusion: We conclude that exercise reduces the lung inflammation in asthmatic mice.
Inflamm. Res.
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B292 PROTEINASE-ACTIVATED RECEPTOR 2 PLAYS A PIVOTAL ROLE IN ALLERGEN-INDUCED LEUKOCYTE RECRUITMENT IN EXPERIMENTAL LUNG INFLAMMATION 1
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Natalia A. Matos , Raphael G. Ferreira , Annie R P Alvarez , Karine A. Damasceno3, Geovanni D. Cassali3, Jose´ C. Alves-Filho2, Andre´ Klein1 1 Department of Pharmacology, Federal University of Minas Gerais, Belo Horizonte, Brazil; 2Department of Pharmacology Ribeira˜o Preto Medical School, University of Sa˜o Paulo, Sa˜o Paulo, Brazil; 3 Department of Pathology, Federal University of Minas Gerais, Belo Horizonte, Brazil
Introduction: Proteinase-activated receptors (PARs) are G-proteincoupled receptors comprising a family of four members. We have demonstrated that PAR2 plays a role in eosinophil (E/) recruitment in experimental allergen-induced pleurisy supporting additional evidences for a role for PAR2 in allergic diseases. However, the role of PAR2 in lung inflammation has not been fully understood. In this work, we evaluated the effects of PAR2 activation in allergen-induced lung inflammation. Methods: BALB/c mice (20–25 g) were sensitized intraperitoneally (i.p.) with 100 lg of ovalbumin (OVA) in 2 % Al(OH)3 gel adjuvant on day 0, then challenged intranasally (i.n.) on days 8–10 with 10 lg of OVA or PBS. Bronchoalveolar lavage (BAL) were obtained 4, 24, 48 and 72 h after challenging. In some experiments PAR2 activating peptide SLIGRL-NH2 (SLIG, 30 lg) was coadministered i.n. with OVA challenging on days 8–10 and the BAL was evaluated 72 h after. PAR2 detection on surface of BAL cells was performed through immunohistochemistry on cytospin preparations. Furthermore, mice were treated i.p. with the PAR2 antagonist ENMD1068 (ENMD, 11 lg) 1 h before OVA i.n. administration on days 8-10 and the number of infiltrating leukocytes and cytokine levels present on BAL were analysed by ELISA. Statistical analyses: One-Way ANOVA followed by Newman-Keuls post-test. The experiments were approved by UFMG Ethics’s Committee for Animal Use (certificated number 348/2014). Results: OVA challenging in sensitized mice induces E/ recruitment 4–72 h peaking 48 and 72 h when compared to PBS-treated mice (48 h: PBS, 0.2 ± 0.2; OVA, 9.7 ± 3.7*; 72 h: PBS, 0.0 ± 0.0; OVA, 8.3 ± 3.5* E/s 9 104, *p \ 0.01). SLIG + OVA induced E/ recruitment in sensitized mice 72 h after when compared to PBStreated mice (PBS, 0.0 ± 0.0; OVA + SLIG 10.4 ± 2.6* E/ s 9 104,*p \ 0.001), and PAR2 is localized in both E/s and mononuclear cells 48 h after the OVA challenging. ENMD abolished the neutrophil (N/) recruitment induced by OVA 2, 4, 8, 12 h after challenging (2 h: PBS + OVA, 9.0 ± 1.5; ENMD + OVA, (4 h: PBS + OVA, 1.7 ± 0.1; 4.1 ± 0.3*, N/s 9 104), ENMD + OVA, 0.3 ± 0.04**), (8 h: PBS + OVA, 0.8 ± 0.04; ENMD + OVA, 0.46 ± 0.04**), (12 h: PBS + OVA, 0.7 ± 0.06; ENMD + OVA, 0.27 ± 0.02**) N/s 9 105, *p \ 0.01 **p \ 0.001. ENMD also inhibited E/ recruitment induced by OVA 8, 12, 24 h after (8 h: PBS + OVA, 2.1 ± 0.17; ENMD + OVA 1.2 ± 0.2*, E/ s 9 104) (12 h: PBS + OVA, 0.2 ± 0.03; ENMD + OVA, 0.06 ± 0.01**), (24 h: PBS + OVA, 0.4 ± 0.05; ENMD + OVA, 0.07 ± 0.01**) E/s 9 105 *p \ 0.01 **p \ 0.001. ENMD reduces KC, IL-6, RANTES, AREG levels 2 h after OVA challenging when compared to OVA-treated mice and increased IL-10 4 h after OVA challenging. Conclusion:: This study demonstrates a pivotal role for PAR2 on leukocyte recruitment in allergen-induced lung inflammation, at least in part through the cytokine modulation at the allergic inflammatory
site suggesting an involvement of natural PAR2 ligands such as mast cell tryptase on this process. Financial support CNPq and CAPES.
B293 PROTECTIVE ROLE OF SURFACTANT PROTEIND IN NITROGEN MUSTARD INDUCED LUNG TOXICITY Vasanthi R. Sunil1, Kinal P. Vayas1, Jessica Cervelli1, Michael Goedken1, Rama Malaviya1, Jeffrey D. Laskin2, Debra L. Laskin1 1
Rutgers University, Brunswick, New Jersey; 2Robert Wood Johnson Medical School, Brunswick, New Jersey Nitrogen mustard (NM) and related alkylating agents are potent vesicants that target the lung, causing inflammation and fibrosis. Evidence suggests that alveolar macrophages contribute to the pathogenic response. Surfactant Protein (SP)-D is a pulmonary collectin known to suppress alveolar macrophage activity. In the present studies, we analyzed the effects of loss of SP-D on NM-induced lung toxicity. Wild type (WT) and SP-D-/- mice were treated with PBS or NM (0.08 mg/kg) intratracheally. Bronchoalveolar lavage (BAL) and lung tissue were collected 14 days later. Treatment of WT mice with NM resulted in perivascular inflammation and increases in BAL cell and protein content, indicating alveolar epithelial injury. Loss of SPD resulted in a significant increase in the sensitivity of the mice to NM; thus, perivascular inflammation was more severe and there was evidence of foamy macrophages, perivascular edema, bronchiolar epithelial hypertrophy and hyperplasia, bronchiolar alveolar hyperplasia, bronchiectasis, fibrosis and emphysema. NM-induced oxidative stress, as assessed by hemeoxygenase-1 expression by alveolar macrophages, was also increased. Enlarged and highly vacuolated profibrotic Ym-1+ and mannose receptor-1+ alveolar macrophages were also present; in contrast, cyclooxygenase-2+ proinflammatory macrophages were decreased. These data indicate that SP-D plays an important role in protecting against NM-induced pulmonary toxicity. Elucidating mechanisms regulating macrophage activity following NM exposure may be important in developing therapeutics to treat vesicant-induced lung injury. Support: NIH AR055073 and ES005022.
B294 TRANSCRIPTOMIC AND PROTEOMIC PROFILING OF SPUTUM REVEALS INFLAMMASOME-ASSOCIATED SIGNATURES IN SEVERE ASTHMA Ian M. Adcock1, Christos Rossios1,2, Stelios Pavlides2, Matthew Loza2, Fred Baribaud2, Scott Kuo1,2, Anthony Rowe2, Uraj Hoda1,2, Ratko Djukanovic2, Peter J. Sterk2 NHLI, Imperial College London, London, UK; 2U-BIOPRED Consortia 1
Background: Asthma is a chronic inflammatory disease of the airway driven by Th2-driven eosinophilia. Severe asthma is characterised by poor therapeutic response to corticosteroid therapy and a diverse airway inflammation profile. Aims and objectives: To investigate the relationship between sputum eosinophils and proteomic and transcriptomic profiles in
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S220 sputum from the U-BIOPRED cohort of severe non-smoking asthma (A), severe smoking asthma (B), non-severe asthma (C) and controls (D). Methods and results: Median sputum eosinophil and neutrophil counts were respectively: A: 2.75, 54.0 % (n = 128); B: 4.13, 55.0 % (n = 53); C: 1.05, 44.5 % (n = 47); and D: 0.0, 39.6 % (n = 41). Defining an eosinophil cut-off [1.32 % and neutrophil count [85.7 % (2 SD above mean in cohort D), cohort A had eosinophilic (55.5 %), neutrophilic (9.4 %), mixed granulocytic (7.8 %) and normal granulocytic (27.3 %) patterns of inflammation. Gene expression profiling demonstrated 496 differentially-expressed genes between severe asthma and healthy subjects (FDR \0.05, twofold/healthy cohort), with the most differentially expressed genes associated with the inflammasome: IL18R1, IL1RL1, IL1R2, IL18RAP, IRAK3. Genes for Charcot-Leyden crystal (CLC) protein and IL-33R (IL1RL1) were highly expressed (5.04- and 5.52fold) and were most highly correlated with sputum eosinophil counts (r = 0.77 and 0.76; p \ 10-8). Gene set variation analysis for microarray data (GSVA) of Th2 signatures in Cohorts A and B compared to cohorts C and D. Somalogic SOMAscanTM V3 analysis of sputum showed that 54 analytes were differentially expressed between SA non-smokers (n = 43) and healthy subjects (n = 18) (FDR \0.05 [ twofold/ healthy cohort). Pregnancy-associated plasma protein A (PAPP-A), Serum amyloid P (SAP) and C-reactive protein (CRP) were highly up-regulated in both smoking and non-smoking SA (14.7-, 6.27- and 3.21-fold). PAPP-A (r = 087; p \ 10-10), SAP (r = 0.6; p \ 10-10) and periostin (r = 0.55; p \ 10-10) correlated with sputum eosinophils, while b-endorphin (r = 0.67; p \ 10-10), azurocidin (r = 0.63; p \ 10-10), IL-5 (r = 0.6; p \ 10-10) and IL-8 (r = 0.51; p \ 10-8) with sputum neutrophils. Conclusion:: Severe asthma is associated with acute phase and eosinophil- and neutrophil- associated analytes in sputum. Expression profiling and proteomics in induced sputum of severe asthma provides inflammasome and Th2-high gene signatures that are associated with eosinophilia. Inflammasome activation appears strongest in eosinophilic asthma. The significance of the inflammasome activation in stable disease remains to be determined. Funded by Innovative Medicine Initiative.
B295 A GENE SIGNATURE FROM THE CFA/HDM MODEL OF ASTHMA PREFERENTIALLY MAPS TO HUMAN SEVERE ASTHMA Ian M. Adcock1,2, Kirsty Russell1,2, Stelios Pavlides2, Christos Rossios1,2, Navin Rao2, Kian F. Chung1,2 NHLI, Imperial College London, London, UK; 2U-BIOPRED Consortia 1
Background and aim: A major concern in drug discovery is that animal models of disease do not always translate to human disease. To improve translation, we have used transcriptomic profiles and gene set variation analysis (GSVA) obtained from a novel mouse model of severe asthma to map onto expression profiles of various compartments in human severe asthma. Methods and Results: Male Balb/c mice were exposed to house dust mite (HDM) in Complete Freunds Adjuvant (CFA) for 2 weeks before being challenged with either saline or HDM intranasally. Gene expression analysis was conducted using Affymetrix HT_MG430_PM arrays on lung tissue collected at 1, 3, 4 and 7 days post challenge. A total of 167, 798, 385 and 129 genes were differentially
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Inflamm. Res. up-regulated (FDR \0.01) on days 1, 3, 4 and 7 respectively. In total 26 genes were up-regulated at all time points including genes such as CCL26, POSTN and ITGB2 which are associated with cytokine, chemokine and integrin signalling. These 26 genes were used as a GSVA gene signature to map the CFA/HDM severe asthma model to the U-BIOPRED cohort. Enrichment of this signature in samples collected from blood, biopsy, bronchial brushing, sputum and nasal brushing from all cohorts were analysed. There was a significant enrichment of the signature in severe non-smoking asthma compared to healthy subjects in biopsies, bronchial brushings and sputum samples. This was not significantly different in non-severe asthma. Conclusion: The signature of up-regulated genes in the CFA/HDM model of severe asthma was elevated in human severe non-smoking asthma but not in non-severe asthma. This approach may be used to investigate how other murine models of asthma align with human asthma allowing the selection of distinct mouse models to reflect the specific characteristics of the asthma phenotype to be tested in man. Funded by the Innovative Medicines Initiative.
B296 ENDOGENOUS CHOLINERGIC SYSTEM IS INVOLVED IN SEXUAL DIMORPHISM ON PULMONARY INFLAMMATION IN AN ASTHMA EXPERIMENTAL MODEL Nathalia M. Pinheiro1, Fernanda P.R. Santana2, Francine M. Almeida1, Adenir Perini1, Iolanda F.L.C Tibe´rio1, Milton A. Martins1, ˆ nio M. Prado3, Vaˆnia F. Prado3, Ana Cristina B. Faloppa1, Marco Anto Wothan T. Lima1, Carla M. Prado2 1
University of Sa˜o Paulo, Sa˜o Paulo, Brazil; 2University Federal of Sa˜o Paulo, Sa˜o Paulo, Brazil; 3University of Western Ontario, London, Ontario Introduction: Asthma is an immune disorder characterized by chronic airway inflammation and hyperresponsiveness. Cholinergic anti-inflammatory pathway modulates immune systemic inflammatory responses in different models by release its main neurotransmissor acetylcholine (ACh). The vesicular acetylcholine transporter (VAChT) mediates ACh storage in synaptic vesicles, which is essential for ACh release. It is known that estradiol and progesterone drive cellular recruitment and affect inflammation in asthma. Aim: We tested the relationship between cholinergic deficiency and females hormones. First, male and female VAChT-deficiency (VAChTKD-HOM) or wild type (WT) mice were submitted to ovalbumin (OVA) subcutaneous protocol on days 0, 7, and 14 and then inhaled with OVA or saline protocol. Then, ovariectomized VAChTKD-HOM mice (7 days before the beginning of OVA protocol) and those treated with estradiol, progesterone or a7nicotinic receptor (a7 nACHR) agonist (PNU, 10 mg/kg i.p) were submitted to the same OVA protocol. On day 29th, BAL was collected and inflammatory cells were counted. VAChTKD-HOM male mice exposed to OVA presented an increase in total cells, lymphocyte and eosinophil in BALF compared to WT male mice exposed to OVA. This effect was not observed in female VAChTKD-HOM. The OVX reduced the inflammatory cells, macrophage and eosinophils in female WT but not in VAChTKD-HOM exposed to OVA. Estradiol, treatment maintained the values of inflammatory cells reduced similar to those observed in OVX female, but the progesterone increased the total cells compared to OVX female mice. The stimulus of a7nAChR with PNU in VAChTKDHOM OVX mice reduced the total cells to similar values obtained in WT OVX female mice.
Inflamm. Res. Conclusions: In this model of asthma experimental, cholinergic deficiency worsens lung inflammation only in male mice suggesting that gender dimorphism influence the effects of cholinergic deficiency. However, our data also suggest that the anti-inflammatory effects of ovariectomy in allergic female, which seems to be modulated more by progesterone, depend, at least in part, by intact cholinergic anti-inflammatory system.
B297 SODIUM METABISULFITE CHALLENGE; LITTLE EVIDENCE OF INFLAMMATORY POTENTIAL Daniel E. Maddox, Sharon M. Flynn Mayo Clinic, Phoenix, USA Since the original 1976 report by Prenner and Stevens of metabisulfite sensitivity, there has been controversy surrounding the mechanisms by which patients respond to the compound. The asthmatic responders have been postulated to be converting the metabisulfite in the lung to sulfur dioxide, which is known to activate irritant receptors in the hyperreactive airways of asthmatics. Save for the report by Sokol and Hydick, postulating IgE-mediated sensitivity to the compound being implicated in urticaria and angioedema, other investigators have not been able to demonstrate positive evidence supporting this mechanism. Over the past 24 years, since computerization of the Allergy Clinical Laboratory at Mayo Clinic, we have performed 103 challenges to sodium metabisulfite. The patient population was 29 males and 74 females. All but two of the challenges were orally administered metabisulfite, with two patients (whose reactions being investigated had occurred to injectable drugs preserved with metabisulfite) undergoing skin testing. The symptoms being investigated were anaphylaxis (12 cases), urticaria/angioedema (44 cases), food-associated gastrointestinal symptoms (12 cases), food/drink-associated asthma flares (19 cases), and there was one case each of flushing only associated with food and drink, and one case of food-associated laryngospasm. Shortness of breath with no history of asthma was reported by 20 cases. There were a total of only 3 positive challenges, 2 in urticaria/angioedema patients, and 1 in an asthma patient. One case that was pulled out of the database was pulled because there was 0.5 mg/mL metabisulfite in a specific local anesthetic product used in dentistry, to which the patient was being tested, but that test was incomplete because of emergence of symptoms thought related to the included epinephrine in the solution. There were 18 placeboresponders in this series, or 6 times the number of active material responders. Overall, it appears that there is very low or no pro-inflammatory potential for metabisulfite to trigger these kinds of symptoms in patients with recurrent underlying inflammatory disorders. Our data raises the question of whether this particular investigation is helpful in the clinical evaluation of patients reporting food or drink-associated allergic or pseudoallergic symptoms.
B298 A CORTICOSTEROID-RESISTANT ALLERGIC MOUSE MODEL OF SEVERE ASTHMA WITH CLINICALLY RELEVANT ENDPOINTS: CORRELATING INFLAMMATORY RESPONSES TO ALTERATIONS IN LUNG MECHANICS Paulette W. Andreotta, Dominic R. Beal, Martina Rosado, Samantha Rogers, Stephen T. Sonis, Gregory D. Lyng Biomodels, LLC, Boise, Idaho
S221 Rationale: Resistance to inhaled corticosteroid therapy in conjunction with concomitant neutrophilic and eosinophilic inflammation are hallmarks of patients with severe asthma. Treatment options in this patient population are limited and drug development has been hampered by limited pre-clinical animal models. Here we present a clinically relevant corticosteroid-resistant allergic mouse model of severe asthma characterized by the generation of neutrophilic and eosiniophilic inflammation and Th1/Th2 cytokines which correlate with airway hyperresponsiveness and abnormal changes in lung mechanics. Methods: Balb/c mice were sensitized s.c. on day 0 with House Dust Mite (HDM) combined with Complete Freund’s Adjuvant (CFA) followed by i.n. challenge of HDM on day 14 and methacholine challenge on day 15. A comparator set of animals was sensitized and challenged i.n. with HDM using a classical model to develop an eosinophilic Th2 inflammatory response. In both models, a group of animals were treated with fluticasone propionate (FP). Inflammatory responses were characterized by total and differential cell counts in bronchoalveolar lavage fluid (BALF), analysis of Th1/Th2 panels of cytokines and chemokines by Luminex, lung histopathology and assessment of lung mechanics and airway hyperresponsiveness to methacholine challenge using the flexiVent system (SCIREQ). Results: Mice exposed to HDM/CFA developed an inflammatory profile characterized by Th1 and Th2 responses with the presence of pulmonary neutrophils, eosinophils and Th1 (IL-2) and Th2 (IL-4) cytokines and chemokines. FP failed to inhibit neutrophilic inflammation and moderately inhibited eosinophils in the HDM/CFA animals, but did inhibit eosinophilic inflammation in the classically challenged HDM animals. Airway hyperresponsiveness and lung mechanics were enhanced in the HDM/CFA challenged animals as compared to responses observed in the classically challenged HDM animals. FP- treatment reduced these responses more dramatically in the classically challenged HDM animals with minimal effects in the severe asthmatic mice. Conclusions: We have successfully established a model of severe asthma with matching inflammatory profiles to human severe asthma and for which concurrent clinically relevant functional parameters of airway hyperresponsiveness and lung mechanics measurements were seen. This model will be a useful tool in assessing novel therapies in the treatment of ICS-resistant severe asthma.
B299 EFFECTS OF CHOLINERGIC HYPOFUNCTION IN LUNG MECHANICS AND HISTOPATHOLOGY IN AN EXPERIMENTAL MODEL OF LUNG INFLAMMATION INDUCED BY AIR POLLUTION IN MICE Fernanda PR Santana1,3, Nathalia M. Pinheiro1, Ma´rcis MB Mernak3, Perini Adenir1, Kelly Yoshizaki2, Mariaˆngela Macchione2, Paulo H. Saldiva2, Luciana C. Caperuto3, Milton A. Martins1, ˆ nio M. Prado4,5, Iolanda FLC Tibe´rio1, Marco Anto Vaˆnia F. Prado4,5, Carla M. Prado3,1 1
Department of Medicine School of Medicine, Universidade de Sa˜o Paulo, Sa˜o Paulo, Brazil; 2Department of Pathology School of Medicine, Universidade de Sa˜o Paulo, Sa˜o Paulo, Brazil; 3 Department of Biological Science, Universidade Federal de Sa˜o Paulo, Sa˜o Paulo, Brazil; 4Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario; 5 Department of Anatomy and Cell Biology, University of Western Ontario, London, Ontario
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S222 Introduction: Diesel automotive engines are widely used in urban centers and their exhausts is considered a major environmental and toxic pollutant to human health, mainly because diesel exhaust particulate (DEP) reaches more distal airways and lung parenchyma, inducing an inflammatory response. ACh has been recently described as the main mediator of cholinergic anti-inflammatory pathway since it suppresses cytokine generation which controls the innate immune response. Aim: In the present study, the authors evaluated whether cholinergic dysfunction is involved in DEP-induced lung inflammation. In the present study, the authors evaluated whether cholinergic dysfunction is involved in DEP-induced lung inflammation. Methods: Male mice with VAChT reduction were used, divided according to genotyping for wild-type (WT) and knock-down for VAChT (KD), and submitted to DEP exposure protocol (10 lL of DEP, 3 mg/mL for 30 days) or saline. Pulmonary mechanics, lung inflammation, cytokine expression, extracellular matrix fiber deposition, and mucus production in the airway and nasal epithelial were evaluated. Both in WT and KD mice, DEP instillation induced lung parenchyma responsiveness; increased mononuclear cells in peripheral blood and macrophages in bronchoalveolar lavage fluid (BALF); increased positive cells to TNF-a, IL-4, IL-6 and IL-13 in lung tissue; collagen fiber deposition in alveolar septa; increased mucus production in airway epithelial associated with an increase in secretory cells; and a decrease in ciliary cells. Additionally, DEP exposure in mutant mice also induced an increase in granulocytes in peripheral blood; in neutrophils and lymphocytes in BALF; in elastic fibers content in alveolar septa; and in acid mucus in nasal epithelial. These alterations were not observed in wild-type mice. Moreover, the levels of IL-4 and TNF-a were also higher in mutant mice exposed to DEP compared with wild-type mice exposed to DEP. Conclusion: In conclusion, VAChT-deficient mice seem to be more vulnerable to DEP-induced lung alterations, suggesting that air pollution effects in the respiratory system are likely to be influenced by the cholinergic anti-inflammatory system. Financial support FAPESP, CNPq, LIM-20 HCFMUSP.
B300 microRNA-21 DRIVES SEVERE, STEROIDINSENSITIVE EXPERIMENTAL ASTHMA BY AMPLIFYING PI3K-MEDIATED SUPPRESSION OF HDAC2 Philip M. Hansbro1, Richard Y. Kim1, James W. Pinkerton1, Malcolm R. Starkey1, Ama T. Essilfie1, Jemma R. Mayall1, Bernadette Jones1, Tatt Jhong Haw1, Simon Keely1, Joerg Mattes1, Ian M. Adcock2, Paul S. Foster1, Jay C. Horvat1 1 The University of Newcastle, Callaghan, Australia; 2National Heart and Lung Institute, Imperial College London, London, UK
Background: Severe, steroid-insensitive asthma is a substantial clinical problem accounting for [50 % of asthma-associated health-carecosts. Effective treatments are urgently required, however, their development is hampered by a lack of understanding of the mechanisms that promote the pathogenesis of disease. Steroid-insensitive asthma is associated with respiratory infections, and non-eosinophilic endotypes of disease, including neutrophilic asthma. The mechanisms that underpin infection-induced, severe, neutrophilic, steroid-insensitive asthma may be elucidated using mouse models of disease. Objectives: To develop representative mouse models of this endotype of asthma. To use these models to identify novel pathogenic mechanisms involving microRNA (miR)-21, phosphoinositide-3-kinase
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Inflamm. Res. (PI3K) pathways and histone deacetylase (HDAC)2, and to investigate new treatment approaches. Methods: novel mouse models of chlamydia, and haemophilus, respiratory infection and ovalbumin-induced, severe, neutrophilic, steroid-insensitive allergic airway disease (SSIAAD) in BALB/c mice were developed. The roles and potential for targeting of infectioninduced miR-21 expression and PI3K-dependent signaling in the lung were examined using therapeutic treatments with a specific miR-21 inhibitor (antagomir, Ant-21) and the pan-PI3K inhibitor LY294002. Results: Respiratory infection induced a miR-21-dependent, PI3Kmediated cell-signaling pathway that promoted steroid-insensitive, neutrophilic inflammation and airway hyper-responsiveness (AHR) in AAD. This involved the suppression of nuclear HDAC2 levels. Inhibition of miR-21 or PI3K suppressed nuclear pAkt levels and restored HDAC2 levels. Treatment also restored sensitivity to steroid administration. Conclusions: We have identified a previously unrecognised pathogenic role for a miR-21/PI3K/pAkt/HDAC2 signaling axis in severe, neutrophilic, steroid-insensitive AAD. Our data highlights miR-21 as a novel therapeutic target for the treatment of this form of asthma.
B301 TUMOUR NECROSIS FACTOR-RELATED APOPTOSIS INDUCING LIGAND PROMOTES THE DEVELOPMENT OF EXPERIMENTAL CHRONIC OBSTRUCTIVE PULMONARY DISEASE Philip M. Hansbro1, Tatt J. Haw1,2, Malcolm R. Starkey1,2, Prema M. Nair1, Irwan Hanish3, Duc H. Nguyen1, Gang Liu1, Mark D. Inman4, Richard Y. Kim1, Adam M. Collison1, Darryl A. Knight1, Hideo Yagita5, Joerg Mattes1, Jay C Horvat1 1
Department of Health and Medicine, Faculty of Biomedical Sciences and Pharmacy, Hunter Medical Research Institute, Priority Research Centre for Asthma and Respiratory Diseases, University of Newcastle, New South Wales, Australia; 2Authors contributed equally; 3Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia; 4Department of Medicine, McMaster Immunology Research Centre, Firestone Institute for Respiratory Health, St. Joseph’s Healthcare, McMaster University, Hamilton, Ontario, Canada; 5Department of Immunology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, Japan Background: Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide and causes significant healthcare and economic burden. Cigarette smoking is a major risk factor. There is a lack of effective treatments for COPD due to poor understanding of the underlining mechanisms. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is implicated in respiratory diseases, namely asthma and pulmonary fibrosis. However, the role of TRAIL in the pathogenesis of COPD is unknown. Objectives: To determine the effect of cigarette smoke-induced experimental COPD on production of TRAIL. To elucidate whether genetic deletion or antibody-mediated neutralization of TRAIL influences the pathogenesis of COPD. Methods: Wild-type or TRAIL-deficient mice were exposed to noseonly cigarette smoke inhalation for 8 weeks and key pathological features of disease were assessed. To determine therapeutic potential of targeting TRAIL, wild-type mice were exposed to cigarette smoke inhalation for 12 weeks and given neutralising anti-TRAIL antibody or isotype control intraperitoneally from week 7 to week 12.
Inflamm. Res. Results: TRAIL mRNA expression and/or protein levels in the lung (airway and parenchyma) and serum were increased in a mouse model of cigarette smoke-induced experimental COPD. Genetic deletion of TRAIL significantly reduced cigarette smoke-induced pulmonary inflammation, expression of key pro-inflammatory mediators, emphysema-like alveolar enlargement and improved lung function in experimental COPD. Interestingly, genetic deletion of TRAIL led to spontaneous small airway remodeling characterized by increased airway epithelial cell thickness and collagen deposition. Importantly, antibody-mediated neutralization of TRAIL reduced cigarette smokeinduced pulmonary inflammation, emphysema-like alveolar enlargement and small airway remodeling. Conclusions: Our study is the first to show that TRAIL plays an important role in the pathogenesis of COPD and provides further evidence for TRAIL being a pivotal inflammatory cytokine in respiratory diseases.
B302 RelB-DEFICIENT DENDRITIC CELLS PROMOTE THE DEVELOPMENT OF SPONTANEOUS ALLERGIC AIRWAY INFLAMMATION IN MICE Philip M. Hansbro1,3, Prema M. Nair1, Malcolm R. Starkey1, Tatt J. Haw1, Roland Ruscher2, Muralidhara R. Maradana2, Ranjeny Thomas2,3, Brendan O’Sullivan2,3 1
School of Biomedical Sciences and Pharmacy, Priority Research Centre for Asthma and Respiratory Disease, Faculty of Health and Hunter Medical Research Institute, University of Newcastle, NSW, Australia; 2University of Queensland Diamantina Institute, Translational Research Institute, Princess Alexandra Hospital, Woolloongabba, QLD, Australia; 3Authors contributed equally Background: RelB is a member of the NF-jB family that is essential for dendritic cell (DC) function and inflammatory responses. However, the contribution of RelB to the development of allergic airway inflammation (AAI) is unknown. Objectives: To determine the role of RelB in the spontaneous development of AAI that is independent of allergen exposure and to identify the importance of RelB in DCs. Methods: Two strains of naı¨ve RelB-deficient (-/-) mice were used to assess features of AAI (a targeted knockout and a mutant expressing an MHC transgene). To identify the importance of RelB in DCs, splenic CD11c+ DCs from RelB-sufficient mice were adoptively transferred intravenously into RelB-/- recipients and features of AAI were assessed. Results: Both strains of naı¨ve RelB-/- mice had increased total leukocytes, eosinophils, lymphocytes and neutrophils in bronchoalveolar lavage fluid (BALF) and more severe parenchymal inflammation compared to their respective wild-type (RelB+/+) and heterozygous (RelB+/-) controls. RelB-/- mice also had increased chemokines (CCL-2, -3, -4, -5, -11, -17, CXCL9, CXCL10, CXCL13); Th2-associated cytokines (IL-4 and -5); serum IgE; mucus secreting cells (MSCs); collagen deposition around the airways; and airway epithelial thickening. Transfer of RelB-sufficient DCs to RelB-/- mice decreased total leukocytes and eosinophils in BALF; parenchymal inflammation; chemokines (CCL-2, -3, -4, -5, -11, -17, CXCL9, CXCL10, CXCL13); Th2-associated cytokines (IL-4, -5, 13, -25, -33, TSLP); serum IgE; numbers of type-2 innate lymphoid cells, myeloid DCs and cd T cells; numbers of MSCs; collagen deposition and airway epithelial thickening. Conclusions: These findings demonstrate the importance of RelB in DCs in controlling AAI and a strategy towards DC-targeted therapy for AAI.
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B303 THE ROLE OF PD-1 IN VIRAL PREDISPOSITION TO SECONDARY BACTERIAL PNEUMONIA Philip Hansbro1, Alexandra Brown1, Ama-Tawiah Essilfie1, Emma Beckett1, Alison Thorburn3, Hideo Yagita2, Hideo Yagita2, Paul Foster1, Jay Horvat1 1 University of Newcastle, Callaghan, NSW, Australia; 2Jutendo University, Tokyo, Japan; 3Monash University, Clayton, VIC, Australia
Background: Secondary bacterial pneumonia is a common consequence of respiratory viral infections, and is a debilitating and life threatening disease. Studies have revealed many components of the immune response to be dysfunctional, suggesting the presence of multi-factorial immune suppressants such as the PD-1/PD-L pathway. Aim: We aim to demonstrate that PD-1/PD-L signalling is up-regulated during viral infections and their suppressive function predisposes to secondary bacterial pneumonia. Methods: We employed a mouse model of viral predisposition to bacterial pneumonia by infecting mice with pneumonia virus of mice (PVM), and at the time of viral clearance infected them with a mouseadapted strain of Streptococcus pneumoniae (Spn, D39). PD-1/PD-L expression and immune cell profiles of the lung were analysed by flow cytometry. The role of PD-1 signalling was determined by administration of a PD-1 blocking antibody. Results: Bacterial titres in the lungs of a secondary Spn infection were increased compared to a primary Spn infection alone. Cellular profiles of mice with a prior PVM infection demonstrated elevated PD-1 and PD-L1 expressing cells in response to Spn infection. Blocking PD-1 signalling reduced bacterial titres in the lung during secondary Spn infection. Conclusion:: A prior PVM infection induces an immunosuppressive environment in the lungs during Spn infection that may hamper the hosts’ ability to clear bacterial infection. We have also demonstrated a pivotal role of PD-1 signalling in the clearance of a secondary bacterial infection. This study also highlights a potential role of PD-1 blocking antibody as a novel therapeutic approach for secondary bacterial pneumonia.
B304 ANTIFIBROGENIC EFFECT OF DOMINANTNEGATIVE INHIBITOR OF SOLUBLE TNF XPRO 1595 ON EXPERIMENTAL SILICOSIS IN MICE Patricia MR Silva1, Bianca T. Ciambarella1, Ana Carolina S. Arantes1, Yago AJ Sa1, David E. Szymkowski2, Marco Aurelio Martins1 1
Oswaldo Cruz Foundation, Rio de Janeiro, Brazil; 2Xencor, Monrovia, USA Tumor necrosis factor (TNF) is a multifunctional cytokine known to regulate inflammation, which is presented on the cell surface as transmembrane TNF (tmTNF), acting to promote juxtacrine signaling, and soluble (solTNF), acting in a paracrine fashion. We demonstrated that mice depleted from TNFR1 gene and treatment with thalidomide markedly inhibited lung function alterations and tissue fibrosis in mice stimulated with silica particles, indicating that TNF plays an important role in such dysfunction. A previous report described the invention of a novel class of anti-TNF biologics (DN-TNFs), which selective inhibits the soluble form of TNF (Zalevsky J, J. Immunol,
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S224 179, 1872, 2007). In this study, we aimed to investigate the effect of the XPro 1595 on the experimental model of silicosis in mice. The monoclonal antibody against TNF infliximab was used for comparison. Male Swiss-Webster mice were intranasally instilled with silica particles, and then treated therapeutically with XPro 1595 (1.25 and 10 mg/kg, i.p.) and infliximab (1.25 mg/kg) on days 7, 14 and 21 post-silica. The analyses were performed 24 h after the last dose and included: (1) lung function and airways hyperreactivity to methacholine (invasive plethysmography—Finepointe Buxco System), (2) morphology and morphometry (H&E and Picrus sirius), (3) collagen deposition (Sircol method); iv) cytokine generation (ELISA) and NFkB expression (western blot). We showed that stimulation with intranasal silica led to an increase in the basal levels of lung resistance and elastance as well to airways hyper-reactivity to aerosolized methacholine. A marked tissue leukocyte infiltration accompanied by fibrosis (collagen deposition and granuloma formation) was also noted in the lungs of silicotic mice. Increased levels of cytokines (MIP-1alpha, MIP-2, KC, MCP-1, IL-1beta and TGF-beta) and NFkB activation were detected in lungs of silica-challenged mice. Therapeutic administration of XPro 1595 and infliximab significantly inhibited the decreased lung function and hyper-reactivity as well as fibrosis, including augmentation in collagen deposition and granuloma formation in silica-stimulated mice. Cytokine generation and NF-kB activation were also sensitive to treatment with XPro 1595 and infliximab. Altogether our findings show that treatment with XPro 1595 and infliximab effectively inhibited some critical features of silicosis, including alteration of lung function and fibrogenic response, reinforcing the idea that TNF is implicated in this disease. They also indicate that inflammation in mouse silicosis seems to be primarily driven by sol TNF. Financial support FIOCRUZ/CNPq/FAPERJ (Brazil).
B305 ROLE OF THE ATYPICAL CHEMOKINE RECEPTOR ACKR2 ON CIGARETTE SMOKINGINDUCED COPD IN MICE Diego S. Coutinho1, Tatiana Paula T. Ferreira1, Davidson F. Dias1, Ana Carolina S. Arantes1, Bianca CIAMBARELLA1, Magda F. Serra1, Andrey J. Fernandes1, Antonio Gabriel S. Silva1, Patricia M R Silva1, Massimo Locati2, Marco Aure´lio Martins1 1
Laboratory of Inflammation, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil; 2Humanitas Clinical and Research Center and University of Milan, Milan, Italy Active cigarette smoking is the main risk factor for chronic obstructive pulmonary disease (COPD) development. ACKR2 (previously known as D6) is an atypical chemokine receptor implicated in sequestering and internalization of chemokines and resolution of inflammation. Remarkably, ACKR2 is highly expressed on human alveolar macrophages of COPD patients. Although its non-redundant role in chronic inflammatory conditions is well recognized, the biological role of ACKR2 in COPD remains elusive. This study was undertaken in order to assess the impact of ACKR2 on main pathological changes of COPD triggered by cigarette smoke inhalation in mice. Thirty-two healthy adult female C57Bl/6 mice were equally distributed into 4 groups: (1) WT mice exposed to ambient air (AA), (2) WT mice exposed to cigarette smoke (CS), (3) ACKR2-/- mice exposed to AA and (4) ACKR2-/- mice exposed to CS. Animals were exposed to CS 7 days per week for 12 weeks. The exposure procedure was based on 3 cigarette-smoking cycles per day, 4 cigarettes per cycle and an inhalation time of 6 min per cigarette.
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Inflamm. Res. Animal weight was monitored weekly, inflammatory and anti-oxidative markers as well as the mean linear intercept (Lm), a measure of alveolar space area, were assessed 24 h after the last exposure to CS. Exposure to CS caused a reduction of about 2 g throughout the experiment in both WT and ACKR2-/- mice. Surprisingly, ACKR2-/- mice showed a significant increase in Lm values both in AA condition (from 31.6 ± 0.4 to 35.4 ± 0.7 lm; p \ 0.001) and CS condition (from 41.8 ± 0.5 to 44.5 ± 0.4 lm; mean ± SEM, p \ 0.05). ACKR2-/- mice also exhibited a substantial decrease in catalase levels following AA exposure (from 9.9 ± 0.1 to 1.9 ± 2.1 U/mg ptn; p \ 0.001), suggesting the existence of a COPD predisposing phenotype. However, no significant catalase changes were seen in CS mice despite tendency (from 2.8 ± 0.2 in WT to 1.3 ± 0.1 U/mg ptn in KO mice, mean ± SEM). Following CS exposure, lung MPO levels showed a twofold increased (p \ 0.05) in ACKR2-/mice compared to WT mice. Finally, though the levels of IL-17, TNFa and CCL3L1 were slightly higher in the lung of ACKR2-/- as compared to WT mice grown in ambient air, levels of these inflammatory mediators were similarly increased in the lung of WT and ACKR2-/- mice exposed to CS. These findings suggest that the atypical chemokine receptor ACKR2 prevents the development of pivotal COPD features, including chronic inflammation and emphysema. Financial support UE FP7- 2007-2013—Grant agreement HEALTH-F4-2011-281608.
B306 COMPARATIVE PROTEOMIC ANALYSIS OF SURVIVAL AND NON-SURVIVAL SEPSIS PATIENTS USING iTRAQ-BASED ABSOLUTE QUANTITATION Narendra K. Sharma1, Alexandre K. Tashima1, Milena KC Brunialti1, Flavia R. Machado2, Eliezer Silva3, Otelo Rigato4, Reinaldo Salomao1 1 Escola Paulista de Medicina, Universidade Federal de Sao Paulo; Brazil; 2ICU, Hospital Sa˜o Paulo, Escola Paulista de Medicina, Universidade Federal de Sa˜o Paulo, Sa˜o Paulo, Brazil; 3ICU, Hospital Israelita Albert Einstein, Sa˜o Paulo, Brazil; 4ICU, Hospital Sirio Libanes, Sa˜o Paulo, Brazil
Sepsis is a systemic inflammatory response caused by infection whose biochemical mechanisms are poorly known. The early detection of sepsis remains a great challenge for clinicians because no single biomarker capable of its reliable prediction, hence, delayed diagnosis frequently undermines treatment efforts, thereby contributing to high mortality. There are several source of infection reported for development of sepsis such as pneumonia, abdominal infection and urinary tract infection etc. In the present study, we compared the plasma proteome profile of survival (N-20 at day 0 and N-17 after day 7) and non-survival (N-13 at day 0 and N-9 after day 7) patients of severe sepsis/septic shock secondary to community acquired pneumonia with healthy volunteers (N-23), to understand molecular dynamics of disease. In brief, we depleted high abundant proteins from representative groups and labelled with iTRAQ 8plex kit. Typically 40 fractions were collected by SCX fractionation and analysed by LC– MS/MS (Synapt G2 HDMS, Waters). In this study, we identified total 932 proteins (NCBI database) with 1 % FDR corresponding to several biological pathways such as acute inflammatory response, inflammatory response reactive oxygen species and oxidative stress etc. which are altered at the protein level when compared to healthy volunteers as well as between survival and
Inflamm. Res. non-survival sepsis patients. We also identified some known marker protein such as C-reactive protein (CRP) in corroborating previous studies. We are reporting the first protein level comparison between survival and non-survival sepsis patients in time interval progression of disease with single source of infection. This study may provide insight for diagnosis, prognosis, and novel therapeutic targets in sepsis.
B307 MODULATION OF INFLAMMATORY RESPONSE DURING STREPTOCOCCUS PNEUMONIAE INFECTION PREVENTS LUNG INJURY AND IMPROVES BACTERIA CLEARANCE IN MICE Luciana P. Tavares1,4,3, Cristiana C. Garcia2, Juliana P. Vago1,6, Celso M. Queiroz-Junior1,7, Bruna A. David1,7, Milene A. Rachid1,8, Remo C. Russo1,5, Patrı´cia R. Martins2, Lirlaˆndia P. Sousa1,6,3, Mauro M. Teixeira1,4,3 1
Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; Instituto Oswaldo Cruz, RJ, Brazil; 3Laborato´rio de Imunofarmacologia; 4Departamento de Bioquimica e Imunologia; 5 Departamento de Fisiologia; de Biologia Celular; 8Departamento de Patologia 2
Introduction: Community-acquired pneumonia (CAP), caused mainly by Streptococcus pneumonia, is a major global health problem and a leading cause of morbidity and mortality among children and the elderly. Despite the use of antibiotics, the incidence of pneumococcal infections did not change. Inflammation is crucial to control dissemination of bacteria; however, it could cause tissue damage and high mortality during pneumoccocal infection. Therefore, our question was whether modulating the inflammation by using the PDE4 inhibitor rolipram (ROL) could improve the outcome of the host during infection. Methods and Results: Male Balb/C mice were infected intranasally with S. pneumoniae serotype 3 (ATCC 6303, 104 CFU) and pretreated with ROL (6 mg/kg) or vehicle (V). ROL treatment decreased neutrophil recruitment in lungs and airways, and decreased cytokines levels in bronchoalveolar lavage fluid (BALF). Number of bacteria in BALF and lethality were similar in both groups. However, ROLtreated animals presented better lung pathology scores than V group. Then, immunomodulatory actions of ROL were investigated in combination with the antibiotic ceftriaxone (CFX). Bacterial counts were diminished by CFX, but not by ROL treatment. Combined treatment reduced even more bacteria counts by enhancing macrophage phagocytosis. Delayed treatment with either CFX or ROL did not prevent neutrophil infiltration into lungs or TNF-a, CXCL1 and CCL2 increased levels, whereas combined treatment strikingly reduced neutrophil numbers in the lungs and airways and cytokine levels. ROL or combined treatment markedly improved overall lung injury and dynamic compliance of infected mice. Annexin A1 (AnxA1), is an anti-inflammatory and tissue protective protein biologically active in the intact form. Whereas cleaved (inactive) AnxA1 band was more evident than intact AnxA1 band in V group at 84 h of infection, ROL, but not CFX treatment partially prevented this cleavage. Combined treatment greatly reduced AnxA1 cleavage. In addition, AnxA1 KO mice had a higher lethality than WT mice and a single dose of an active portion of Annexin A1 (Ac2-26–10 mg/kg) could partially rescue mice from lethality and decrease inflammation related to infection. Conclusion: Inflammation was an important determinant of morbidity after infection with S. pneumoniae. Pretreatment with ROL decreased
S225 several parameters of inflammation, ameliorated histological score, without interfering with bacterial load or survival. Partial blockade of pulmonary inflammation may be beneficial for the murine host, mainly when combined to standard antibiotics treatment. Mechanistically, combined treatment improved infection outcome by balancing/reducing inflammation and increasing expression of the pro-resolving and tissue protective protein AnxA1 with bacterial phagocytosis, restoring lung homeostasis. In addition, AnxA1 may play an important role during pneumococcal pneumonia revealing a new treatment strategy.
B308 PHOTOTHERAPY (PHT): ATTENUATION OF CHOLINERGIC HYPERREACTIVITY, b2ADRENERGIC HYPORRESPONSIVENESS AND MITOCHONDRIAL mRNA EXPRESSION IN RAT BRONCHI SEGMENTS AND PULMONARY TISSUE IN E. COLI LIPOPOLYSACCHARIDE-INDUCED AIRWAY INFLAMMATION BY A NF-kB, AP-1 AND MITOCHONDRIAL DEPENDENT MECHANISM Fla´via Mafra de Lima1, Renata Kelly Palma2, Niels Olsen Saraiva Camara1 1
University of Sa˜o Paulo, Av Prof. Lineu Prestes 1730, Cidade Universita´ria, Sa˜o Paulo, Brazil; 2Rehabilitation Sciences Departament, Centro Universita´rio Nove de Julho, Uninove. Rua Vergueiro, SP, Brazil Background and objective: It is unknown if the decreased ability to relax airways smooth muscles in acute respiratory distress syndrome (ARDS), can be influenced by low level laser therapy irradiation. In this context, the present work was developed in order to investigate if PhT could reduce dysfunction in inflamed bronchi smooth muscles (BSM) in rats. Methods: A controlled ex vivo study was developed where bronchi from Wistar rat were dissected and mounted in an organ bath apparatus with or without a TNF-a. In the experimental assays with the participation of TNF-a, the reactivity of BSM to cholinergic agonist or to b2-adrenergic agonist was analyzed. The cAMP level was also investigated in BSM bathed or not with TNF-a and treated or not with PhT. In other series of experiment, the BSM from rat were incubated with LPS in order to investigate the PhT effect on TNF-a mRNA expression in BSM. Mechanical properties of lungs were measured (QTAS HORAS) after LPS injection. Lung resistance (RL) and elastance (EL) were computed by linear regression fitting of recorded signals during mechanical ventilation (tracheal pressure, flow and volume). Static (Est) and dynamic (Edyn) elastances were obtained by the end-inspiratory occlusion method. Results: TNF-a caused cholinergic hyperreactivity and b2-adrenergic hyporesponsiveness when compared to respective controls. PhT administered perpendicularly to a point in the middle of the dissected bronchi with a wavelength of 655 nm and a fluence of 2.6 J/cm2, partially decreased BSM hyperreactivity to cholinergic agonist, restored BSM relaxation to isoproterenol and reduced the TNF-a mRNA expression. The cAMP level diminished by TNF-a in comparison with isoproterenol was restored by PhT. An NF-kB antagonist blocked the PhT effect on dysfunction and cAMP level in inflamed BSM. The RL and EL were higher after LPS injection in compared to control and laser treatment reduces these values. Similar increases were induced by LPS injection in Est and Edyn.
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S226 Conclusion:: We show that low level laser therapy (LLLT), can decrease hyperreactivity in isolated organ and improve the pulmonary ventilation (in vivo) after QTAS HORAS ventilation. Our results shown that this effect is mediated by MyD88, IRAK1, and this treatment is efficient to activate Cyt c, TFAM, NFR2 and increase mitochondrial membrane potential.
Small Molecule Therapeutics for Inflammatory Diseases B309 GEDUNIN BINDS TO MD-2 AND IMPAIRS LPSINDUCED TLR4 SIGNALING IN MACROPHAGES Perla V. Borges1, Katelim H. Moret1, Clarissa M. Monteiro3, Franklin S. Silva4, Carlos R. Alves4, Ernesto Caffarena5, Paulo R. Batista5, Maria das Grac¸as Henriques1,6, Patrı´cia Pacheco1, Carmen Penido1,6 1 Laborato´rio de Farmacologia Aplicada, Farmanguinhos, Fundac¸a˜o Oswaldo Cruz, Rio de Janeiro, Brazil; 3Laborato´rio de Imunofarmacologia, Instituto Oswaldo Cruz, Fundac¸a˜o Oswaldo Cruz, Rio de Janeiro, Brazil; 4Laborato´rio de Biologia Molecular de Doenc¸as Endeˆmicas, Instituto Oswaldo Cruz, Fundac¸a˜o Oswaldo Cruz, Rio de Janeiro, Brazil; 5Programa de Computac¸a˜o Cientı´fica, Grupo de Biofı´sica Computacional e Modelagem Molecular, Fundac¸a˜o Oswaldo Cruz, Rio de Janeiro, Brazil; 6Centro de Desenvolvimento Tecnolo´gico em Sau´de, CDTS/INCT, Fundac¸a˜o Oswaldo Cruz, Rio de Janeiro, Brazil
Recognition of bacterial lipopolysaccharide (LPS) by innate immune system is mediated by the CD14/TLR4/MD-2 complex. In the present study, we have investigated the modulatory effect of gedunin, a limonoid from species of the Meliaceae family described as Hsp90 inhibitor, on LPS-induced response in immortalized murine macrophages. The pretreatment of wild type (WT) macrophages with gedunin (0.01–10 mM, non-cytotoxic concentrations) inhibited LPS (50 ng/mL)-induced calcium influx, TNF-a and nitric oxide (NO) production, in a concentration-dependent manner. The selective effect of gedunin onMAL/MyD88- and TRAM/TRIF-dependent signaling pathways was further investigated. The pretreatment of WT, TRIF KO and MAL KO macrophages with gedunin (10 lM) significantly inhibited LPS (50 ng/mL)-induced TNF-a, IL-1b and IL-6 production, within 6 h and 24 h, suggesting that gedunin modulates a common event between both signaling pathways. Furthermore, gedunin (10 lM) inhibited LPS-induced PGE2 production, cyclooxygenase-2 (COX-2) expression and NFjB translocation into the nucleus of WT macrophages, demonstrating a wide range effect of this chemical compound. In addition to the ability to inhibit LPSinduced pro-inflammatory mediators, gedunin also triggered antiinflammatory factors. The pretreatment with gedunin induced the production of IL-10 and the expression of heme oxigenase-1 (HO-1) and heat shock protein (Hsp)70 in macrophages stimulated or not with LPS. In silicomodeling studies revealed that gedunin efficiently docked into the MD-2 LPS binding site, a phenomenon further con-
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Inflamm. Res. firmed by surface plasmon resonance. Our results reveal that, in addition to Hsp90 modulation, gedunin acts as a competitive inhibitor of LPS, blocking the formation of TLR4/MD-2/LPS complex.
B310 VENETOCLAX (ABT-199), A POTENT AND SELECTIVE BCL-2 INHIBITOR, IS EFFICACIOUS IN MOUSE MODELS OF LUPUS NEPHRITIS AND REDUCES HUMAN LYMPHOCYTE LIFESPAN IN VITRO Stephen Clarke1, Stuart Perper1, Kristie Grebe1, Annette Schwartz1, Christian Goess1, Liz O’Connor1, Dawna Hartman1, Susan Westmorland1, Candace Graff1, Andrew Souers1, Joel Leverson1, Steven Elmore1, Lisa Olson1, Li Chun Wang1 1
AbbVie Bioresearch Center Worcester, MA, USA1; AbbVie, Inc. North Chicago, IL, USA Venetoclax (ABT-199), a Potent and Selective BCL-2 Inhibitor, is Efficacious in Mouse Models of Lupus Nephritis and Reduces Human Lymphocyte Lifespan in vitro Stephen Clarke1, Stuart Perper1, Kristie Grebe1, Annette Schwartz1, Christian Goess1, Liz O’Connor1, Dawna Hartman1, Susan Westmoreland1, Candace Graff1, Andrew Souers2, Joel Leverson2, Steven Elmore2, Lisa Olson1 and Li Chun Wang1 1 Abbvie Bioresearch Center, Worcester, MA, United States; 2 AbbVie, Inc. North Chicago, IL, United States Proteins in the BCL-2 family are key regulators of apoptosis, or programmed cell death. We have evaluated the effects of venetoclax (ABT-199), a highly potent and orally available BCL-2 selective inhibitor, in two mouse models of lupus nephritis and in human cells in vitro. In the mouse models (spontaneous and IFN alpha-induced NZBW F1), venetoclax treatment dose-dependently reduced the incidence of severe proteinuria and prolonged survival in both models compared to vehicle controls and attenuated glomerulonephritis, tubular dilatation, immune cell infiltrates and IgG deposition in the kidney. Venetoclax mediated a significant reduction in the numbers of splenic T cells but conferred a preferential reduction in select B cell subsets. Interestingly, venetoclax did not impair the number of CD138+ long-lived plasma cells in the bone marrow, which was consistent with unaltered circulating antidsDNA titers in these animals. Venetoclax efficacy also correlated with a dose-dependent reduction of lymphocytes in peripheral blood of NZBWF1 mice. Consistent with these findings, human lymphocytes from both healthy donors and systemic lupus erythematosus (SLE) patients treated with venetoclax in vitro have reduced lifespan. Taken together, these data underscore the essential role of BCL-2 in the pathogenesis of lupus and support further exploration of selective BCL-2 inhibition in autoimmune diseases such as SLE/ Lupus Nephritis. Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.
Inflamm. Res.
B311 CHARACTERIZATION OF A SMALL MOLECULE IRAK4 KINASE INHIBITOR FOR THE TREATMENT OF AUTOIMMUNE AND INFLAMMATORY DISEASES Chrystelle Lamagna, Meagan Chan, Sothy Yi, Chi Young, Roy Frances, Stacey Siu, Sylvia Braselmann, Don Payan, Darren McMurtrie, Ryan Kelley, Thilo Heckrodt, Rose Yen, Yan Chen, Hui Li, Rajinder Singh, Gary Park, Esteban Masuda, Vanessa Taylor Rigel Pharmaceuticals, South San Francisco, CA, USA IRAK4 is a key protein kinase in immune cells. It is responsible for initiating MyD88-dependent signaling from most Toll-Like Receptors (TLR) and Interleukin-1 Receptors (IL-1R), resulting in downstream production of pro-inflammatory cytokines. Hence, inhibitors of IRAK4 kinase activity represent valuable therapeutic tools to treat cytokine-driven autoimmune and inflammatory diseases. Through cell-based screening, we identified potent small molecule IRAK4 kinase inhibitors that block TLR4- and IL-1R-induced cytokine production with potencies \100 nM. Our inhibitors exhibit good selectivity against a broad panel of kinases. In vivo, our lead compound decreases serum IL-6 in an acute mouse model of IL-1betainduced cytokine release, demonstrating excellent pharmacokinetics properties. In addition, our IRAK4 inhibitor reduces the paw swelling in the chronic rat model of collagen-induced arthritis, as well as the clinical score and overall inflammatory phenotype of mice in the imiquimod-induced psoriasis model. The in vitro and in vivo characterization of our lead candidate is promising and confirms IRAK4 as an attracting therapeutic target for the management of cytokinedriven diseases.
B312 b-CARYOPHYLLENE MODULATES NEUTROPHIL MIGRATION IN INFLAMMATORY REACTION INDUCED BY MYCOBACTERIUM BOVIS-BCG Magaiver Andrade-Silva1, Luana B. Correa1, Andre´ Cande´a1, Simone C. Cavalher-Machado1, Elaine C. Rosas1, Maria das Grac¸as Henriques1,2
1 Laboratory of Applied Pharmacology, Farmanguinhos, Fundac¸a˜o Oswaldo Cruz, FIOCRUZ, RJ, Brazil; 2National Institute for Science and Technology on Innovation on Neglected Diseases (INCT/IDN), Center for Technological Development in Health (CDTS); Fundac¸a˜o Oswaldo Cruz, FIOCRUZ, RJ, Brazil
b-caryophyllene (BCP) is a natural sesquiterpene found in essential oils from a variety of plant species and it was described as a natural agonist of cannabinoid 2 (CB2) receptor. Previous studies have demonstrated an anti-inflammatory effect of BCP in different inflammation models. However, there are no reports of its action on inflammatory process induced by mycobacteria. The aim of this study was to evaluate the effect of BCP on neutrophils migration in an inflammatory reaction induced by M. bovis-BCG. To evaluate the effects of BCP on pleurisy, mice received an intrathoracic injection (i.t.) of BCG (4x105 CFU/cavity) 1 h after the oral pretreatment (p.o) with BCP (50 mg/kg), dexamethasone (by intraperitoneal injection, i.p.;Dex, 2 mg/kg) or synthetic selective CB2 agonist, GP1a (i.p.;10 mg/kg). After 24 h of the injection, the pleural wash (PW)
S227 was collected. For pulmonary model of infection, mice were pretreated with BCP (p.o.; 50 mg/kg) 1 h before and 24 h after intranasal instillation (i.n.) of BCG (1x106 CFU/lung). The effects were evaluated 48 h after infection in the bronchoalveolar lavage (BAL). The total and differential leukocyte migration were analyzed in both models (PW and BAL) using May Grunwald-Giemsa staining or by flow cytometry. Cytokine production was assessed by ELISA. All protocols were approved by the Fundac¸a˜o Oswaldo Cruz Animal Welfare Committee (LW-43/14.). Adhesion and chemotaxis were performed in vitro. The stimulus with BCG induced an increase of total leukocytes, predominantly neutrophil influx in both models. The oral pretreatment with BCP (50 mg/kg) decreased the numbers of total leukocyte in the pleural cavity and in the lungs, showing a remarkable effect on neutrophil migration, 45 and 97 % of inhibition, respectively. In pleurisy, BCP was able to reduce the levels of IL-12 (21 %) and NO (54 %). In the lungs, BCP inhibited IL-10 (99 %), MCP-1 (100 %), IFN-c (98 %) and IL-12 (97 %) release. GP1a and Dex also reduced the neutrophil migration and NO production in PW. Flow cytometry analysis revealed that BCP reduced integrin CD11b/ CD18 expression on Ly6G positive cells in PW (neutrophils). In addition, neutrophils pretreated with BCP (10 lM) reduced adhesion to endothelial cells previously stimulated with mTNF-a (10 ng/mL) and chemotaxis toward LTB4 (10–7 M). These data suggests that BCP modulates early inflammatory events in BCG infection by inhibition of neutrophil’s migration to inflamed site. Financial support CAPES; Faperj; CNPq.
B313 DISCOVERY OF SELECTIVE RORc INVERSE AGONISTS AND DEMONSTRATION OF EFFICACY IN INFLAMMATORY DISEASE MODELS Ravi K. Ujjinamatada, Ravi D. Krishna Babu, Kavitha Nellore, Samiulla S. Dodheri, Charamanna K B, Subhendu Mukherjee, Kancharla BabuRao, Wesley Roy Balasubramanian, Sanjay B. Rathod, Anirudha Lakshminarasimhan, Narasimha K. Rao, Anuradha Ramanathan, Natarajan Mahalingam, Girish A R, Shekar Chelur, Susanta Samajdar, Chetan Pandit, Murali Ramachandra Aurigene Discovery Technologies Limited, Bengaluru, Karnataka Objective: Th17 cells play a key pro-inflammatory role in a variety of autoimmune diseases including psoriasis, arthritis, lupus, multiple sclerosis and asthma. The nuclear hormone receptor RORc controls the differentiation of Th17 cells and expression of IL-17. Aurigene’s objective is to develop RORc inverse agonists for treatment of autoimmune disorders. Methods: RORc inverse agonists were designed using a combination of approaches including structure and knowledge based methods. Compounds were screened in a RORc radio-ligand binding assay using 3H 25-Hydroxycholesterol, as well as in a cell based reporter assay to demonstrate inverse agonism. Selected compounds were screened against RORa as well as other nuclear receptors to evaluate selectivity. Crystal structure of RORc in complex with known inverse agonists as well as novel compounds, were solved. Th17 differentiation assay was developed using primary mouse/human CD4+ ve T-cells to determine functional effect of the compounds. DMPK profile of potent compounds was determined to support evaluation in animal models. Efficacy of lead compounds was evaluated in LPS induced IL-17 model as well as in collagen induced arthritis model. Results: Lead compounds have demonstrated good activity (\100 nM) in reporter assay. Co-crystal structure of RORc with compounds clearly showed the mode of binding. Selected compounds
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S228 demonstrated [100 fold selectivity against RORa as well as other nuclear receptors. Compounds from multiple series have shown significant inhibition (\100 nM) of IL-17 release from differentiated Th17 cells. Lead compounds have shown optimal physicochemical profile including good solubility and high free fraction, leading to excellent pharmacokinetic profile in mice. Efficacy of lead compounds has been demonstrated in LPS induced IL-17 model as well as in collagen induced arthritis model. Conclusions: We have identified structurally diverse small molecule inverse agonists of RORc and have demonstrated efficacy in relevant disease models.
T Regulatory Cells B314 THE FOXP3 T REGULATORY CELLS IN OBSTRUCTIVE AND NON OBSTRUCTIVE SMOKERS: A DESCRIPTIVE STUDY IN AIRWAYS AND LYMPHOID FOLLICLES Davi S. Sales, Ivy A. Zanchetta, Juliana T. Ito, Raquel Annoni, Thais Mauad, Milton A. Martins, Fernanda DTQS Lopes University of Sa˜o Paulo, Sa˜o Paulo, Brazil Background: The COPD progression has been associated with a persistent inflammatory process into the airways walls characterized by an innate and adaptive inflammatory immune cells infiltration which can be organized into bronchus-associated lymphoid tissue (BALT) or may be dispersed as lymphoid clusters lacking organization (iBALT) in parenchyma. Smoking is configured as the main risk factor for COPD development; however, not all smokers develop clinically significant COPD, which suggests that there are individual intrinsic factors involved in such disease progression. Although lymphoid follicles are a feature of the adaptive immune response and have been described in COPD patients, there are few studies describing the inflammatory cells types in such structures and its differences between obstructive and non obstructive smokers. Aims: To compare the inflammatory profile associated to the regulatory adaptive immune response in obstructive and non obstructive smokers. Methods: Small and large airways, BALTs and iBALTs were assessed in surgically resected lung tissue from patients, divided into three groups: Control (n = 21), Obstructive Smokers (COPD) (n = 17) and Non Obstructive Smokers (FNO) (n = 22). CD4+ and CD8+ T cells, FOXP3 regulatory T (Treg) cells, and the expression of IL-10 and IL17 interleukins were evaluated by immunohistochemistry and morphometric analysis. Results: We observed an inflammatory process characterized by an increase in CD4+ and CD8+ lymphocytes in small and large airways, BALT and iBALT in smokers; however the higher values were detected in small airways of COPD individuals (p \ 0,001). In addition, it was observed a decrease in regulatory T cells density only in small airways of this group (p \ 0,001), with a consequent decrease in IL-10 expression in small and large airways (p \ 0,001). However in BALT we observed a different response, with an increase in Treg cells density in COPD group, and in iBALT we observed an increase in Treg cells only in FNO group without differences between the groups in IL-10 expression in both structures. Regarding the IL17 expression, there was an increase in small and large airways (p \ 0,001), and iBALTs (p \ 0,001) as the airflow obstruction progressed.
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Inflamm. Res. Conclusions: Our results suggest that the obstruction progression in smokers was related to a decrease in inflammation regulatory activity on the small airways, mediated by T regulatory cells, leading to less expression of interleukin 10 (IL-10) and increased interleukin 17 (IL17) production. Also, we verified that such inflammation regulatory activity showed differences between BALTs and small airways. Supported by FAPESP and Instituto de Laborato´rios de Investigac¸a˜o Me´dica-HC/FMUSP
B315 IMPACT OF REGULATORY T CELLS ON CHEMOKINE RESPONSE IN TUBERCULOSIS Divya Kamboj, Dipendra Kumar Mitra, Anant Mohan All India Institute of Medical Sciences, New Delhi, India Purpose: Regulatory T cells dampen the inflammatory immune response in various diseases including tuberculosis. In tuberculosis, local immune response is an important determinant in shaping the disease manifestation. A robust response at the disease site, results in effective granuloma formation containing the infection thus preventing its dissemination. Utilizing various mechanisms, regulatory T cells suppress protective effector T cell response. Granuloma formation requires local production of Th1 associated chemokines facilitating the effector T cells at the pathological site. We previously reported higher levels of Treg cells and IL-10 at the local disease site in tuberculosis. Here, we envisage that locally enriched regulatory T cells may alter the tissue chemokine response thereby influencing the effector T cell influx. This may be a mechanism underlying the local immune deficit observed in tuberculosis patients. Materials and methods: In this study, we expanded regulatory T cells in vitro and examined the effect of Mycobacterium tuberculosis specific T reg expansion on chemokines like MIP1-a and MIP1-b using polychromatic flow cytometry and in vitro cell culture techniques. Results: We observed that T reg cells from tuberculosis patients could be expanded using All Trans Retenoic Acid (ATRA) in vitro. After T reg cells expansion, it was found that there was decreased production of MIP1-a and MIP1-b by both monocytes and lymphocytes. Presence of ATRA also led to increased proliferation of T reg cells subsequently resulting in heightened IL-10 and TGF-b production. Blocking of IL-10 and TGF-b in culture could rescue these chemokines which influence the Th1 cytokines. Conclusion: Here, we show that T reg cells derived from tuberculosis patients can inhibit the production of Mycobacterium tuberculosis specific chemokines required for the recruitment of Th1 effector T cells. We propose that this may lead to failure of effector T cell homing and elicitation of optimal local immunity in tuberculosis. Our results indicate a novel role of in vitro expanded T reg cells in modulating the chemokine response resulting in weakened recruitment of effector T cells. This impedes the granuloma formation leading to failure of immune containment and dissemination of disease.
B316 RESTORATION OF TREG CELL FUNCTION WITH PD-L1 FC PROTEIN IN RHEUMATOID ARTHRITIS Kaustav Chowdhury1, Dipendra Kumar Mitra1, Uma Kumar1, Soumabha Das1, Prabin Kumar1, Uma Kanga1, Jaydeep Chaudhuri2, Santu Bandyopadhaya2, Parasar Ghosh3, Ravi Kiran Basyal1, Maumita Kanjilal1
Inflamm. Res. 1
All India Institute of Medical Sciences, New Delhi, India; 2Indian Institute of Chemical Biology, Kolkata, West Bengal; 3Rheumatology Center, IPGMER, Kolkata, West Bengal Programmed death receptor -1 (PD-1) and its ligands (PD-L1, PD-L2) interaction is known to dampen effector T cell response. Rheumatoid arthritis (RA) is a chronic autoimmune disease of joints resulting in joint inflammation and bone erosion. Here, we investigated the importance of PD-1/PD-L1 interaction in modulating inflammatory pathogenic T cell response at the disease site of RA. We attempted to study the cause of failure of locally enriched Treg cells in controlling the inflammatory cytokine producing cells. We demonstrate that lack of PD-L1 on synovial fluid derived machorphages (CD14+ cells) incapacitates functionality of Treg cells and upon engaging PD-L1 can functionally resuscitate the Treg cells. We recruited active RA patients (n = 15) for the study and isolated mononuclear cells from peripheral blood (PBL) and synovial fluid (SF) of the same individual patients. PD-1 and its ligand expression (PD-L1) were seen on Treg cells and macrophages (CD14+) by the use of flowcytometry. Anti inflammatory cytokine IL-10, TGF-b in Treg (CD4+ FoxP3+) and proinflammatory cytokines IFN-c, IL-17A, TNF-a in effector T cells (CD4+) were identified with FACS based intracellular staining after treatment with PD-L1 Fussion chimeric protein (PD-L1 Fc) in presence of TCR engagement with anti-CD3 & anti-CD28. Our result shows that synovial Treg cells are compromised in their IL-10, TGF-b production in-spite of local enrichment of PD-1+ Treg (CD4+ FOXP3+) cells. Engaging PD-1-PD-L1 with PD-L1 Fc, Treg cells could produce higher level of IL-10 & TGF-b. On the other hand such engagement decreased the frequency of synovial effector T cells producing inflammatory cytokines (IFN-c+, TNF-a+, IL-17A+) and proliferation of these cells (Ki67+). Interestingly blocking IL-10, TGF-b with specific antibody in similar condition(s) failed to reduce the number of proinflammatory cytokine producing T cells. This suggests a critical role of PD-1-PD-L1 pathway in controlling inflammation in RA to get resolution.
Tissue Damage/Repair B317 CRITICAL ROLE OF CXCR7 IN MODULATING INFLAMMATION, ANGIOGENESIS AND FIBROGENESIS IN SKIN INJURY IN MICE Alan S. Barbosa1,2, Leandro C. Freitas-Limas1,2, Camila P. Almeida1,2, Luiza D.C. Lima1,2, Maria C.C. Canesso1,2, Ange´lica T. Vieira1,3, Robson A.S. Santos2, Daniela V. Rosa4, Erika K. Sacramento4, Marco A. Romano-Silva4, Mauro M. Teixeira1,3, Charles C. Mackay5, Lucı´ola S. Barcelos1,2 1 Immunopharmacology group; 2Department of Physiology and Biophysics, Federal University of Minas Gerais, Belo Horizonte, Brazil; 3Department of Biochemistry and Immunology, Federal University of Minas Gerais, Belo Horizonte, Brazil; 4School of Medicine, Federal University of Minas Gerais, Belo Horizonte, Brazil; 5Faculty of Medicine, Nursing and Health Sciences, Monash University, Melbourne, Victoria, Australia
CXCL12/SDF-1a, a chemokine expressed in both homeostatic and pathological conditions, and its main known receptor, CXCR4, have been implicated in skin wound healing, although with controversial roles. The disparity may be related to a recently identified second high affinity receptor, CXCR7; however, its role in wound healing was never evaluated. Our aim was to elucidate the role of CXCR7 on
S229 excisional wound healing. 8 weeks-old C57Bl/6 mice (n = 5–7 animals/group) received either IgG2 isotype control or anti-CXCR7 blocking antibodies (1.7 mg/mL, i.p.) 24 h before creation of dorsal excisional wounds and once a week till euthanasia. Wound area was calculated after digital caliper measures and results were expressed as closure percentage from original size. Treatment with anti-CXCR7 impaired the wound closure when compared to isotype control. Accordantly, wound epithelization, assessed in H&E-stained histological sections, was reduced in anti-CXCR7-treated animals. Interestingly, myeloperoxidase and N-acetyl-b-D-glucosaminidase activities, used to assess accumulation of neutrophils and macrophages, respectively, were marked increased in wounds of antiCXCR7-treated mice. However, levels of the chemokines CCL2/ MCP-1, a macrophage recruiter, or CXCL1/KC, a neutrophil recruiter, were lower in wounds of anti-CXCR7-treated mice. Instead, levels of CXCL12 and TNF-a were higher in wounds of anti-CXCR7treated mice than in control group. Similarly, and offering insight on the evolutionary conservation of CXCR7 pathway, we observed that anti-CXCR7-treated 3-day-old zebrafish larvae (1.7 mg/mL in culture medium) presented increased cell recruitment to wound site. Regarding vascularization, anti-CXCR7-treated mice showed reduced blood supply and angiogenesis in the wounded area, as evaluated by laser Doppler perfusion imaging and histology. Conversely, wounds of anti-CXCR7-treated mice exhibited greater accumulation of collagen (Sirius Red histological staining) and scar tissue area, which were associated with increased levels of TGF-b1 in wounds when compared to control group. Therefore, we may point the impaired wound healing is associated with impaired epithelization and persistent accumulation of inflammatory cells and CXCL12 and TNF-a in the injured area, suggesting CXCR7 absent activity may be linked to chronicity. This scenery is also characterized by reduced angiogenesis and increased scarring. Overall, our findings suggest CXCR7 is essential for controlling skin wound healing by modulating critical features such as inflammation, angiogenesis, and fibrogenesis after injury.
B318 THE DEVELOPMENT OF NEW THERAPY FACIAL NERVE INJURY USING THE M2 MACROPHAGE INDUCTION FACTORS Fumiya Kano, Kohki Matsubara, Akihito Yamamoto Nagoya University Graduate School of Medicine, Nagoya, Japan Backgrounds: The divergent activation states of monocyte/macrophage linages play central roles in the inflammatory responses of the traumatic nerve injury. M1-like cells initiate inflammation by releasing high levels of pro-inflammatory cytokines, glutamate, reactive oxygen species, and nitric oxide (NO). These neurotoxic factors accelerate glial scar formation, neuronal cell death and the retraction of damaged dystrophic axons. In contrast, M2-like cells counteract the pro-inflammatory M1 conditions and promote tissue repair by secreting anti-inflammatory cytokines, phagocytizing cellular debris, enhancing axonal elongation, and promoting the proliferation and differentiation of oligodendrocyte progenitor cells. We have identified a novel set of set of inducers for anti-inflammatory M2-like macrophages: monocyte chemoattractant protein-1 (MCP-1) and the secreted ectodomain of sialic acid-binding Ig-like lectin-9 (ED-Siglec-9). MCP-1 and ED-Siglec-9 administration into the injured spinal cord induced M2-like macrophages and led to a marked recovery of hindlimb locomotor function after SCI (Matsubara et al. The Journal of neuroscience, 35, 2452–2464, 2015).
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S230 Objective: We examined the therapeutic effects of MCP-1 and EDSiglec-9 in the treatment of the peripheral facial nerve injury. Method: The rat facial nerve was resected 5 mm. Collagen scaffolds infiltrated with MCP-1 and ED-Siglec-9 were placed in the resected site. The facial nerve recovery was evaluated by whiskers’ behavior, histological analysis with electron microscope and the tracer analysis for the restoration of the nerve circuit. Results: MCP-1/ED-Siglec-9 treatment restored the whiskers’ behavior, regenerated resected facial nerve and restored the nerve circuit. Conclusion:: MCP-1/ED-Siglec-9 may provide a novel strategy to treat the peripheral nerve injury.
B319 ASSESSMENT OF PORCINE DERMIS SCAFFOLDS AND THEIR BIOCOMPATIBILITY USING A MODEL OF XENOGENEIC IMPLANTATION Sonia M. Oliani1, Kallyne KO Mimura1, Andreia R. Moraes1, Tahera Ansari2, Karin V. Greco2
1
University of Sa˜o Paulo State (UNESP), Sa˜o Paulo, Brazil; University College London (UCL), London, UK
2
Off-the-shelf availability of tissue-engineered skin constructs, tailored by different combinations of reagents to produce a highly preserved biological matrix is often the only means to help patients suffering skin damage. The challenge facing tissue engineering, however, is to combine different reagents to preserve the structure and function of a complex mixture of proteins that forms the extracellular matrix (ECM) and keep their functions to promote tissue regeneration. Therefore, this study assessed the composition of porcine dermal scaffolds and their biocompatibility in rats. The two types of methods (1 and 2) used to produce dermal scaffolds showed to be efficient not only in removing cellular material and debris from dermal scaffolds but also in preserving the ECM components (glycosaminoglycans and collagen). Histological examinations and analysis by SDS-PAGE revealed that these scaffolds retained structural properties comparable to a native dermal matrix. Biocompatibility (integration, cell density, mineralization, and collagen quantification), ED-1 macrophages and blood vessels of the scaffolds were assessed in rats on the 3, 14 and 21 days after implantation. Blood was collected for leukocytes quantification and plasma for TNF-a, IL-6 and IL-1b analysis. Histopathological analysis showed a gradual increase in the integration and cell density of both scaffolds (mainly on scaffold 1) in comparison to Permacol (used as control). No mineralization was observed on the assessed time-points. Under polarized light a decrease of collagen was observed on the 14 day in comparison to the 3. However, on the 21 day we observed deposition of new collagen fibers, suggesting initiation of the tissue remodeling process. Our results showed an initial increase on leukocytes (day 3) with a significant reduction in the subsequent time-points. However, no significant difference was observed in the inflammatory mediators. Immunohistochemical techniques revealed significant increase in ED1 positive macrophages inside the scaffolds after day 14, and blood vessels positive to vWF after 21 days of implantation. Altogether our findings suggest that both scaffolds were biologically compatible, permitting cells recruitment, vasculogenesis and gradual substitution of the acellular matrix; and therefore supporting tissue remodeling and regeneration. The experimental procedure were approved by the Research Ethics Committee (0182/12).
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Inflamm. Res. Financial support FAPESP (2012/21603-2) and CNPq (308144/20147).
B320 THE WORSENING ASTHMA PROCESS DRIVEN BY BRONCHOALVEOLAR EPITHELIUM WITH CO-EXPOSITION TO CIGARETTE SMOKE Thayse R. Bru¨ggemann1, Paula Fernandes1, Fernanda M. Bruni2,3, Mı´lton A. Martins1, Fernanda M. Arantes-Costa1
1 Laboratory of Experimental Therapeutics, LIM20 School of Medicine of University of Sa˜o Paulo, Sa˜o Paulo, Brazil; 2 Laboratory of Clinical Immunology and Allergy, LIM60 School of Medicine of University of Sa˜o Paulo, Sa˜o Paulo, Brazil; 3Institute for Investigation in Immunology (iii), INCT, Sa˜o Paulo, Brazil
Introduction: Recent genetic, structural and functional studies have identified the airway and lung epithelium as a key orchestrator of the immune response. The inability of the airway epithelium of asthmatics to effectively defend the lung against normally innocuous inhaled agents suggests that the airway epithelial barrier of asthmatics is compromised. We designed this study to investigate the role of bronchial epithelium against the exposition to cigarette smoke in a model of allergic asthma. Methods: Male Balb/c mice (n = 8/group) of the OVA and OVA+ cigarette smoke (CS) groups were sensitized with two intraperitoneal injections of ovalbumin (OVA, 20 lg) and alum (3 mg) on days 0 and 14, and were challenged with an aerosol of OVA (1 %, 30 min) on days 21, 23, 25 and 27. The CS and OVA + CS groups were exposed to cigarette smoke once a day (7 cigarettes/session) for twelve consecutive days (from 16 to 27). CS and Control (SAL) groups received saline and alum intraperitoneal injection and were challenged with saline 0.9 %. Twenty-four h after the last challenge, we evaluated the production of cytokines (IFN-c, IL-5 and IL-13) in bronchoalveolar lavage fluid by cytometric bead array (CBA), GM-CSF, TSLP and TGF-b in lung homogenate by ELISA. Epithelium remodeling was measured by lung stained slides with Periodic acid-Schiff alcian blue (PAS-AB) for epithelium area and mucus production. Results: OVA-sensitized mice showed increased production of cytokines as IL-5 and IL-13 that was even enhanced when there was co-exposition to cigarette smoke. We noticed that TSLP levels increases with exposition to cigarette smoke, but when combined to OVA-sensitization it reduces significantly but not until OVA levels. The levels of TGF-b increased on CS and OVA groups. Comparing OVA + CS to OVA groups, we noted that the levels of TFG-b showed maintained, but decreased when compared to CS group. The OVA-sensitization process per se was capable to increase IFN-c that was not observed when there was exposition to cigarette smoke combined or alone. We also observed a remodeling process of the epithelium on sensitized group by increased epithelium area and mucus production that was intensified after co-exposition to cigarette smoke. We observed no changes in GM-CSF levels among all groups. Conclusion: Based on this data, we conclude that co-exposure of sensitized mice to cigarette smoke once a day worse the lung inflammation induced by the antigen OVA. It is clear that epithelium in involved in this process by the remodeling presented on it as well as the enhanced production of cytokine such as TSLP that is mainly produced by the epithelium cells.
Inflamm. Res.
B321 NERVE REGENERATION IN INJURED TOOTH IS MEDIATED BY THE INTERACTION BETWEEN THE COMPLEMENT FRAGMENT C5A AND PULP FIBROBLASTS Fanny Chmilewsky1, Imad About2, Warda Ayaz1, Seung H. Chung1 1
University of Illinois at Chicago, USA; 2Aix-Marseille universite´, Marseille, France Dentin-pulp regeneration is closely linked to the presence of nerve fibers in injured teeth, since there is an amplification of healing mechanism by the sprouting of the nerve fiber’s terminal branches in the pulp beneath injury (Kumazawa et al. 1996; Arai, 1991). Although this process seems to be associated with a local enhancement of several growth factors such as Nerve Growth Factor (NGF) (Byers et al. 1992a), little is known about initial mechanisms that regulate the dental pulp nerve sprouting beneath the injured site. It has been recently demonstrated that the complement system activation, which occurs during carious decay (Chmilewsky et al. 2013), contributes to tissue regeneration through the local production of anaphylatoxin C5a (Chmilewsky et al. 2014). The aim of this study is to examine the roles of the active fragment C5a in dental nerve regeneration following tooth damage and further identify the underlying mechanisms. Immunofluorescence staining was performed in vivo on intact and carious tooth sections to localize the C5a receptor (C5aR). The C5aR expression was then examined in vitro using western blotting and double immunofluorescence staining with untreated or lipoteichoic acid (LTA)–stimulated (carious condition) human pulp cells. The neurotrophin production by untreated, or LTA–stimulated human pulp cells, treated or not with a C5aR specific antagonist (W54011), was quantified by ELISA assay. Finally, the neurite outgrowth from human neurons toward untreated, or LTA–stimulated human pulp cells, treated with or without W54011, was examined with the AXIS Axon isolation device. Our results show that pulp cells, localized beneath the carious injury, express the C5aR. This observation is consistent with our in vitro results that showed an ectopic expression of C5aR in pulp fibroblasts stimulated with LTA, a complex component of Grampositive bacteria cell walls. Finally, the treatment with specific C5aR antagonist significantly inhibited both the NGF and Brain-Derived Neurotrophic Factor (BDNF) expressions in LTA-stimulated pulp fibroblasts, and the neurite outgrowth from human neurons toward the direction of LTA stimulated fibroblasts. These findings suggest that upon bacterial infection, the C5a, through its interaction with pulp fibroblast C5aR, contributes to the tissue regeneration by mediating nerve outgrowth on the injured site. This effect seems to be due to the significant C5a involvement in neurotrophin secretions by pulp fibroblasts. These data may provide a useful therapeutic tool targeting pulp fibroblasts in dentin-pulp regeneration process.
B322 MOLECULAR IMAGING TECHNIQUES TO EVALUATE NITROGEN MUSTARD INDUCED LUNG INJURY Edward J. Yurkow1, Justin Schumacher2, Alessandro Venosa2, Derek Adler1, Jeffrey Laskin2, Debra L. Laskin2
S231 1
Rutgers Molecular Imaging Center, Rutgers University, Piscataway NJ 08854; 2Department of Pharmacology and Toxicology, Rutgers University, Piscataway, NJ 08854 Nitrogen mustard (NM, mechlorethamine) is a cytotoxic alkylating agent that induces acute lung injury followed by delayed fibrosis. We previously characterized the effects of NM on lung histology, inflammation and function. In this study we used molecular imaging techniques to periodically track the development of NM-induced alterations in the lungs of live animals throughout a 28-day period. Male Wistar rats (225–250 g; n = 3/group) were treated with 0.125 mg/kg NM or PBS control by intratracheal instillation. Magnetic Resonance Imaging (MRI), consisting of gradient spin echo (GRE) and fast spin echo (FSE) sequences, as well as Computed Tomography (CT) were performed on each animal on days 1, 3, 7, 14, and 28. VivoQuantTM image processing software was used to analyze MRI and CT scans. Three-dimensional images of lungs were rendered from MRI scans and the volumes of the injured and normal lung tissue were quantified. Following NM exposure, the volume of injured tissue increased significantly within 1 day, a response that persisted for 28 days. Lung volumes of air, dynamic lung tissue, and consolidated lung tissue were determined via segmentation of CT scans based on voxel intensity. The dynamic lung volume was significantly decreased 3 days after NM administration. Conversely, the volume of consolidated lung tissue was significantly increased on both day 14 and day 28, when compared to day 1 and day 3. These data show that MRI and CT imaging are useful methods to detect and track the progression of NM-induced lung injury in rats. Since these molecular imaging techniques can be conducted on live animals, each animal can serve as its own control, fewer animals can be used, and paired data analysis can be conducted, thus reducing variability. Supported by NIH AR055073 and ES005022.
Toll Receptors B323 A NOVEL CELL SURFACE PROTEIN THAT IMPARTS CYTOKINE SPECIFICITY TO TLR4 SIGNALLING Nilesh J. Bokil1,2, Lin Luo1,2, Jennifer L. Stow1,2, Matthew J. Sweet1,2 1
Institute for Molecular Bioscience, Brisbane, Australia; 2University of Queensland, Brisbane, Australia Toll-like receptors (TLRs) are a family of pattern recognition receptors that sense both pathogen-associated molecular patterns as well as endogenous host factors to drive pro-inflammatory responses. For example, the archetypal TLR, TLR4, recognises both lipopolysaccharide (LPS) from Gram-negative bacteria, as well as a range of endogenous host factors including amyloid-beta peptide and oxidized low-density lipoprotein. Upon ligand recognition, TLR4 dimerises and recruits the Toll/Interleukin-1 Receptor (TIR) domaincontaining proteins MAL (MyD88 adaptor-like protein), MyD88 (Myeloid differentiation primary response gene 88), TRAM (TRIFrelated adapter molecule) and TRIF (TIR-domain-containing adapterinducing interferon-b) through homotypic TIR-TIR domain interactions, ultimately enabling the activation of specific serine/threonine kinases to relay downstream signalling. TLR4 is also tyrosine phosphorylated upon activation, however the mechanisms controlling this process are much less understood. Here we report a novel cell surface protein that is required for LPS-induced TLR4 phosphorylation. Using siRNA-mediated gene knock-down in primary mouse
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S232 macrophages we also show that this protein is required for primary LPS signalling responses (activation of NF-kappaB, p38 and JNK), as well as LPS-inducible secretion of IL-6 and IL-12. Interestingly, this pathway had no role in regulating TNF production. These differential effects were also apparent at the level of gene expression. Conversely, gain-of-function studies by retroviral over-expression in primary murine macrophages revealed selective upregulation of IL-6 and IL12, with no effect on TNF production. Our findings thus reveal a new level of specificity emanating from the cell surface to mediate the TLR4-inducible production of a sub-set of pro-inflammatory cytokines.
B324 ALTERED TOLL-LIKE RECEPTOR SIGNALING IN INDIVIDUALS WITH TYPE 1 BIPOLAR DISORDER Andrea Wieck1,2,3, Carine Hartmann do Prado1,2, Laura Petersen1, Ba´rbara Nery Porto4, Rodrigo Grassi-Oliveira2,1, Moise´s Evandro Bauer1,5,3 1 Laboratory of Immunosenescence, Institute of Biomedical Research, Pontifical Catholic University of the Rio Grande do Sul, PUCR, Porto Alegre, Brazil; 2Cognitive Neuroscience Research Group (GNCD), Centre of Studies and Research in Traumatic Stress (NEPTE), Postgraduate Program in Psychology, PUCRS, Porto Alegre, Brazil; 3 Postgraduate Program in Biomedical Gerontolgy, PUCRS, Porto Alegre, Brazil; 4Centro Infant, Pontifical Catholic University of Rio Grande do Sul, PUCRS, Porto Alegre, Brazil; 5Faculty of Biosciences, PUCRS, Porto Alegre, Brazil
Bipolar disorder (BD) has been associated with immune imbalance, characterized with chronic low-grade inflammation profile. Toll-like receptors (TLR) are pivotal receptors in microbial recognition, initiating innate immune responses and providing a link between innate and adaptive immune responses. Given the importance of TLR in coordinating inflammatory immune responses, the main objective of this study is to immunophenotype circulating monocytes for TLR expression (TLR1, TLR2, TLR4, TLR5 and TLR6) and signaling. Thirteen euthymic participants with type 1 BD and 15 healthy controls (HC) took part in this study. Peripheral blood mononuclear cells (PBMCs) were isolated, differentiated into monocytes and stimulated in vitro with specific TLR agonists (flagelin, LPS, LTA, BLP and PGN) to assess inflammatory cytokines (IL-8, IL1-b, IL-6, IL-10, TNF-a and IL-12p70). TLRs and cytokines were assessed by multicolor flow cytometry. Increased percentages of TLR1+ and TLR2+ monocytes were observed in BD (p = 0.007 and p = 0.005 respectively). In contrast, reduced expression of TLR5 in monocytes from patients was also observed (p = 0.034). Following stimulation with specific TLR agonists, BD patients had higher percentages of TLR1 + and TLR2 + monocytes (p = 0.018 and p = 0.023 respectively), as well as TLR6 (p = 0.010) as compared with HC. Increased levels of IL-8, IL12-p70 and TNF-a (p = 0.0001; p = 0.006 and p = 0.0001, respectively) were observed following stimulation with specific TLR1, TLR2 and TLR6 ligands, further indicating increased signaling via those receptors in BD patients. Our data indicates altered signaling via TLR1 and TLR2 in BD patients, which was corroborated by increased levels of cytokines when stimulated with specific agonists. Given the importance of TLR2/1 in triggering immune responses, our data suggests an important role for innate immunity in development/maintenance of the immune imbalance observed in BD.
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Inflamm. Res.
B325 NEURONAL TLR SIGNALING CONTROLS NEURONAL EXCITABILITY, NEUROINFLAMMATION, PAIN, AND ITCH Ru-Rong Ji, Chul-Kyu Park, Tong Liu, Zhen-Zhong Xu, Qingjian Han, Yong Ho Kim, Temugin Berta Duke University Medical Center, Durham, NC, USA It is generally believed that Toll-like receptors (TLRs) are expressed by immune cells to regulate innate immunity. However, we found functional TLR3 and TLR7 are also expressed by small-sized primary sensory neurons in dorsal root ganglion (DRG) that are known to regulate pain and itch. Interestingly, TLR3 agonist PIC and TLR7 agonist immiquimod and loxoribine can cause immediate activation (within seconds) of sensory neurons via a novel non-canonical signaling pathway that requires TLR and ion channel (e.g., TRPA1) but not Myd88. Immiquimod induces robust scratching (itch-like behavior) in wild-type mice, which is reduced in Tlr7-deficient mice. Strikingly, we found that specific miRNAs containing GUUGUGU motif such as Let-7b can act as endogenous ligands of TLR7. Extracellular application of let-7b to dissociated DRG neurons is sufficient to induce rapid inward currents and action potentials in DRG neurons. These electrophysiological responses are abolished in mice lacking Tlr7 or Trpa1. Injection of let-7b into a hind paw of mouse elicits rapid spontaneous pain via TLR7 and TRPA1 in wildtype mice. Although let-7b-induced acute pain does not require MyD88, Myd88-mediated canonical signaling is essential for let-7binduced chronic pain. Specific deletion of Myd88 in nociceptive sensory neurons (Nav1.8-expressing neurons) lead to a reduction in chemotherapy-induced neuropathic pain. Furthermore, chemotherapy-induced innate immunity and adaptive immunity in the peripheral nervous system (DRG) is impaired after conditional deletion of Myd88 in nociceptive neurons. Finally, we found that TLR7 is also co-expressed with TRPA1 in human DRG neurons from non-diseased donors, and activation of TLR7 by let-7b is also sufficient to excitate human DRG neurons via TRPA1. Together, our findings have demonstrated that TLR signaling in primary sensory neurons controls acute pain, acute itch, chronic pain, and neuroinflammation via both non-conical and canonical signaling pathways. Our study also suggests that RNAs (such as total RNAs and miRNAs) can serve as extracellular signaling molecules to activate TLR3 and TLR7 in sensory neurons. This work is supported by NIH RO1 Grants NS67686, DE17794, DE22743, and NS87988 to R.R.J.
Translational Medicine and Biomarkers B326 MOLECULAR DETECTION OF INFLAMMATION IN CELL AND ANIMAL MODELS USING HYPERPOLARIZED 13C-PYRUVATE Julia Nguyen, Jeffrey T. Gu, Linda Nguyen, Renuka Sriram, Dave Korenchan, John Kurhanewicz, John D. MacKenzie Department of Radiology and Biomedical Imaging at UCSF, San Francisco, CA, USA
Inflamm. Res. Purpose: To test and validate hyperpolarized 13C-pyruvate and alterations in its conversion to 13C-lactate as an imaging biomarker for disease severity and treatment response in inflammation. Methods: With IACUC approval, inflammation was induced with Freund’s complete adjuvant (0.4 mL/kg) in healthy Balb/c mice. At the peak of inflammation (7d post-induction), animals were anesthetized and positioned in the NMR scanner. Proton (1H) and carbon-13 (13C) imaging was performed on a 14.1-T Varian 600WB animal NMR scanner through the area of inflammation (paw) and control paw. 13C spectra were obtained immediately after the intravenous injection of 300 ll of hyperpolarized 13C1pyruvate using DNP (15–20 % liquid-state polarization). Tissues were processed and inspected for histological changes of inflammation. J774A.1 mouse macrophage cells were cultured, stimulated with lipopolysaccharide (LPS) into their activated inflammatory state, and biomarkers were studied to evaluate the cells response to treatment (mRNA levels of lactate transporters MCT1/4 & lactate dehydrogenase (LDH) A/B using qRT-PCR and nitric oxide (NO) production with Griess assay). Cells were also pre-treated with indomethacin (IND) (10 lM) 4 days prior to stimulation and were also administered IND at the time of LPS stimulation. To measure 13C-lactate production, J774A.1 cells were encapsulated into alginate beads and either left as control or activated with 100 ng/mL lipopolysaccharide (LPS) for 24 h. The cells were then placed in a bioreactor, and the production of 13C-lactate measured with NMR after incubation with hyperpolarized 13C-pyruvate. Imaging data were processed with VNMRJ and SIVIC, and ACD/ NMR imaging software. Results: Elevated levels of lactate were detected in areas of inflammation in mice, using 13C-MRSI. LPS-stimulated cells had a substantial increase in the production of mRNA of MCT4 lactate transporter (4.7 ± 1.8 SD fold increase) whereas LDHA/B and MCT1 remained at control levels. Indomethacin inhibited NO production by LPS-stimulated cells. Nine-fold elevation in the production of hyperpolarized 13C-lactate was observed after stimulation in a macrophage cell model of inflammation. Discussion: We have identified a robust method of studying inflammation with 13C-MRSI. Our experiments indicate that lactate production is elevated in inflammation, which can be readily detected by 13C-MRSI with hyperpolarized 13C-pyruvate. Unlike other molecular imaging techniques that are still experimental, this technique has IND approval and should translate rapidly to clinical usage for inflammatory disorders such as adult rheumatoid and juvenile idiopathic arthritis.
B327 OMEGA-3 NUTRITIONAL SUPPLEMENTATION IMPROVES AMYLOID-BETA IMMUNITY THROUGH SPECIALIZED PRO-RESOLVING MEDIATORS Milan Fiala, Sam Famenini, Rachel Rubattino, Bien Sagong, Jessica Leung, Ramesh Halder
S233 mediators (SPMs) resolvins, protectins and maresins on phagocytosis, inflammation and cognition. Methods: Phagocytosis of FITC- Abeta1-42 was measured by flow cytometry [expressed as mean fluorescence intensity (MFI)] and fluorescence microscopy; resolvin D1 (RvD1) by ELISA; inflammation by RNA-Seq of PBMCs; cognition by Minimental state examination (MMSE) score; and macrophage phenotype by the M1 markers CD54 (ICAM-1) and CD80 (costimulatory protein) and the M2 markers CD163 (scavenger receptor) and CD206 (mannose receptor). Results: 12 MCI patients and 2 pre-MCI patients have been followed on daily nutritional supplementation with omega-3 fatty acids (DHA 1 gm and EPA 1 gm in the Smartfish drink, Oslo Norway) for 5–18 months (the first visit was without supplementation and subsequent visits on supplementation). MFI increased from 530 on the first visit to 1306 on the last visit (p \ 0.016). RvD1 (n = 6) increased in 3 and did not change in 3 patients. MMSE score (n = 8) improved in 7 but later declined in 4 (related to disease or noncompliance) and declined in one ApoE4 patient. Most patients had mixed macrophage phenotype with highest expression of CD54 and CD206; two had an inflammatory type but one switched to a mixed type on a subsequent visit. On the initial visit, FITC- Abeta1-42 phagocytosis by macrophages of most patients was improved more by in vitro treatment with resolvin D1, resolvin D2 or maresin D1 than by omega-3 emulsion (Fig. 1a), whereas on later visits omega-3 was generally more effective (Fig. 1b). RNA sequencing (n = 4) showed inflammatory signature in 2 and non-inflammatory signature in 2. Conclusions: In MCI patients, omega-3 fatty acids have positive effects on immunity against Abeta1-42 through SPMs.
B328 TRANSLATIONAL PROPERTIES OF A FOLATEAMINOPTERIN SMALL MOLECULE DRUG CONJUGATE WITH A METABOLICALLY ACTIVATED LINKER SYSTEM Yingjuan Lu1, Satish Rao1, Paul Kleindl1, Fei You1, Michael Pugh1, Vicky Cross1, Elaine Westrick1, Alex Lloyd1, Leroy Wheeler II1, Patrick Klein1, Iontcho P. Vlahov1, Philip S. Low2, Christopher P. Leamon1 Endocyte, Inc., West Lafayette, IN, USA; 2Purdue University, West Lafayette, IN, USA
Department of Surgery, UCLA School of Medicine, Los Angeles, CA, USA
1
Introduction: Alzheimer disease and Minor cognitive impairment (MCI) patients have a defect in the phagocytosis of amyloid-beta 1-42 (Abeta1-42) and abnormally low or high activation of inflammation. Objectives: We are studying the effects of the omega-3 drink (Smartfish, Oslo, Norway) and the specialized pro-resolving
Introduction: Chronic human diseases and conditions are often caused or worsened by excessive activation of macrophages and their monocyte precursors. During immune adaptation to the local environment, activated macrophages of both M1 and M2 phenotypes express a nanomolar-affinity folate receptor (FR)-b. This
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S234 phenomenon has allowed for a receptor-targeted approach for sitespecific delivery of both imaging and therapeutic agents. In patients with rheumatoid arthritis and osteoarthritis, etarfolatide (99mTcEC20), a FR-specific radioimaging agent, was found to preferentially accumulate in the inflamed joints or ‘‘hot spots’’. In the therapeutic realm, we have previously shown that EC0746, a folic acid (FA)aminopterin (AMT) small molecule drug conjugate, can modulate macrophage functionality and is effective in rodent models of adjuvant arthritis, autoimmune uveitis and encephalomyelitis. In order to facilitate therapeutic translation, the current investigation focuses on species differences in linker metabolism and potential implications in extrapolating metabolism data from animal models to humans. Methods: A series of FA-AMT conjugates bearing subtle or significant structural variations next to or in the linker region were synthesized. In-vitro and in vivo similarities and differences in pharmacodynamics, pharmacokinetics, and metabolism among different species were investigated using multiple relevant biological systems. Results: Release of free AMT (and its active analog) directly or indirectly from intact conjugates was largely enzymatic. Using wholecell homogenates and liver S9 fractions from different species, it was found that the linker region influences the rate of free AMT release. In preclinical models, activity and tolerability of the folate-AMT conjugates correlated well with differences seen in linker metabolism and pharmacokinetics. By fine-tuning the enzymatic substrate specificity in the linker region we could control the rate of free AMT release in vitro and in vivo. Conclusion: The main challenge in preclinical translation has always been to circumvent species differences in drug metabolism. The promiscuity of metabolic enzymes in rodents complicates the interpretation and prediction of linker metabolism in dogs and man. We have identified specific metabolic enzymes that are responsible for AMT release from our folate-AMT series. We have been able to utilize this knowledge and incorporate species-dependent and -independent structural variations to control the rate of linker metabolism and optimize conjugate efficacy and tolerability. Consequently, a lead compound has emerged with superior translational properties for advancement into the clinic.
Inflamm. Res. electrochemiluminescence (ECL). The reported ECL-ELISA method, which has a 96 well format, offers high sensitivity [limit of quantitation 0.04 nM nitrated bovine serum albumin (BSA-Tyr-NO2) equivalents] and good linearity across four dilutions (R2 = 0.96 ± 0.04, n = 3). To test the ability of the assay to detect Tyr-NO2 changes, in an example of an acute inflammatory response, we analysed serum samples from patients undergoing a range of surgical procedures, where serum had been collected both before and after surgery. A statistically significant increase in nitration was seen post-surgery (n = 35, p \ 0.05, Wilcoxon Matched Pairs) compared to the levels before surgery; median (IQR): 0.59 (0–1.3) and 0.97 (0–1.7) Tyr-NO2 (fmol BSA-Tyr-NO2 equivalents/mg protein) for pre- and post-surgery respectively. These results confirm that the new assay is indeed able to detect an increase in serum nitration following an acute inflammatory response. In conclusion we have developed and validated a new, sensitive and robust ECL-ELISA for the measurement of Tyr-NO2 that offers high-throughput analysis of human samples. Our assay is able to detect an increase in serum levels of Tyr-NO2 following an inflammatory response induced by surgery. The present Tyr-NO2 assay may be applicable in providing a pharmacodynamic endpoint in pre-clinical and clinical studies in which therapeutic effects on oxidative stress are implicated. For example the assay may be useful in testing novel anti-inflammatory drugs that lower ROS production. Several such drugs are currently in commercial development, including selective NADPH oxidase inhibitors and myeloperoxidase inhibitors.
B330 MEASUREMENT OF SERUM NITRATE CONCENTRATION FOR THE DIAGNOSIS OF INFECTIVE GASTROENTERITIS Miranda J. Smallwood1, Kyle Stewart2, Thalia Groom1, Jessica Cross1, Nigel Benjamin2, Paul G. Winyard1 University of Exeter Medical School, Exeter, UK; 2Torbay Hospital, Torquay, UK 1
B329 NEW ELECTROCHEMILUMINESCENCE-BASED ELISA FOR HIGH-THROUGHPUT ANALYSIS OF SERUM OXIDATIVE DAMAGE ASSOCIATED WITH INFLAMMATION Annie Knight1, Emma Taylor1, Jane E. Carre´1, Yuktee Dogra1, Jo Tarr1, Roman Lukaszewski2, Paul G. Winyard1 University of Exeter Medical School, Exeter, UK; 2Dstl, Porton Down, Salisbury, UK
1
Activation of an inflammatory response leads to the generation of reactive oxygen species (ROS); these species cause oxidative damage such as nitration of tyrosine residues within proteins, thereby generating 3-nitrotyrosine (Tyr-NO2). Previous studies have reported elevated serum Tyr-NO2 in many human inflammatory diseases, e.g. sepsis and systemic lupus erythematosus, and it has been suggested that Tyr-NO2 is a potential marker of inflammatory activity. Measurement of oxidative stress markers in clinical studies has been hindered by methodological issues such as low sensitivity and low throughput. Therefore a high-throughput method, for Tyr-NO2 detection, has been developed based on the highly sensitive platform,
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An increased serum concentration of nitrate, a metabolic end product of nitric oxide (NO), has been reported in patients with infective gastroenteritis, and this increase in nitrate is much greater than is seen in patients with sepsis or other causes of the inflammatory response. The observed extreme increase of serum nitrate concentration in infective gastroenteritis is thought to be due to a host inflammatory ‘superinduction’ of inducible nitric oxide synthase activity, over and above that seen in many autoimmune inflammatory diseases. In serum samples collected from 149 patients upon their admission to hospital with suspected infective gastroenteritis, the nitrate concentration was measured by both the established method of ozonebased chemiluminescence and by a new, validated, modification of a rapid and cheap spectrophotometric plate-based method. As measured by chemiluminescence, the median serum nitrate concentration was significantly increased (p \ 0.0001) in patients in whom a causative organism was identified (75.7 lM; IQR, 37.3–150.0 lM; n = 51) compared with those in whom no organism was found (43.6, 21.8–66.0 lM, n = 98). Patients with a bacterial cause of gastroenteritis (Campylobacter or Salmonella) had particularly high concentrations of plasma nitrate (median: 115.3, IQR: 78.6–268.8, and median: 205.4, 65.0–2472.0 lM respectively), compared with serum nitrate concentrations from patients who did not have a bacterial cause of gastroenteritis. Patients diagnosed with
Inflamm. Res. norovirus had similar serum nitrate concentrations (51.5, 25.1–145.0 lM) to patients with non-infective gastroenteritis (43.6, 21.8–66.0). In patients in whom a causative organism was identified, the serum nitrate concentration correlated with the severity of disease as measured by the amount of intravenous fluids required (R2 = 0.58; p \ 0.02, n = 18). However serum nitrate concentration was not correlated with CRP values (R2 = 0.02, n = 43). When measured by the spectrophotometric 96-well plate-based assay, serum nitrate concentrations correlated well with nitrate measured by chemiluminescence (R2 = 0.97), suggesting that the spectrophotometric method could serve as a high throughput assay. The present study provides evidence that a serum nitrate concentration of greater than 70 lM indicates bacterial gastroenteritis, particularly Campylobacter and Salmonella. We have also demonstrated the feasibility of screening hospital patients for serum nitrate, using a convenient high-throughput nitrate assay. We therefore suggest that serum nitrate measurement may be a useful diagnostic test in gastroenteritis patients.
B331 MECHANISMS OF DRUG-INDUCED SKIN RASH USING BIOMAPÒ HUMAN PRIMARY CELLBASED SYSTEMS Jason Ptacek, Karen Matta, Sylvie Privat, Dat Nguyen, Ellen Berg, Alison O’Mahony BioSeek, a division of DiscoveRx Corp, South San Francisco, CA, USA The relationship between drug efficacy and adverse events (AE) is dependent on (1) target selectivity, (2) primary and secondary effects of target modulation and (3) the concentration at which these interactions occur. BioMAP technology uses a systems biology approach to capture the complexity of physiologically relevant tissue microenvironments and provide information about how compounds behave in these settings with respect to efficacy and safety. BioMAP systems consist of cultures of human primary cells stimulated to model disease biology and protein-based biomarker endpoints to detect phenotypic changes resulting from compound exposure. Here, we use BioMAP technology to gain a phenotypic and mechanistic understanding of the differentiating properties between classes of MAPK inhibitors. We show these inhibitors have expected anti-inflammatory and anti-proliferative activities but differ in the induction of a pro-inflammatory response associated with skin rash, an AE reported clinically for the MEK and p38 inhibitor classes. We performed a phenotypic screen of three target-selective MAPK inhibitors: PD-184352 (MEKi), BIRB-796 (p38i), and JNK IX (JNKi). In BioMAP systems modeling inflammation, these compounds across a range of concentrations decreased several inflammatory biomarkers (CD69, IL-1a, TNFa). Interestingly, at the same exposure levels, BIRB-796 and PD-184352 showed pro-inflammatory activities (increased VCAM-1, IP-10, MIG) in a BioMAP system modeling wound healing. Multiple highly selective p38 inhibitors had similar activities, indicating that this effect is target-class, not compound specific. Conversely, JNK IX had the opposite profile in the wound healing model with anti-inflammatory activities over several concentrations. To further elucidate the differences between the MAPK inhibitors in the wound healing system, we used phosphorylation profiling of key signaling proteins to compare underlying network interactions at early and late time points. Specifically, PD-184352, BIRB-796 and JNK IX decreased the levels of p-ERK, p-p38, and p-c-Jun, respectively, at early time points. A broader, network impact is manifested
S235 at later times suggesting crosstalk between pathways; BIRB-796 and PD-184352 both decrease p-p38 at 24 h. Whereas PD-184352 and JNK IX decrease p-c-Jun, BIRB-796 unexpectedly increases p-c-Jun. Phosphorylation of HSP27, a cytoprotective protein implicated to have a role in skin injury, is decreased by p38i and JNKi but increased by MEKi. Together, this systems biology approach reveals that MAPK inhibitors do not act in a target-isolated manner but rather impact a network of inflammatory, proliferative and stress response pathways. In conclusion, phenotypic screening and phosphorylation profiling enables the identification of sentinel biomarkers that can be associated with efficacy and the potential for drug-related skin rash AE.
B332 CRP AND 14-3-3 ETA ARE EACH ASSOCIATED WITH JOINT DAMAGE PROGRESSION, THEIR TITRES DO NOT CORRELATE AND ARE BETTER PREDICTORS OF PROGRESSION TOGETHER THAN ALONE Anthony Marotta1, Nathalie Carrier4, Artur J. Fernandes2, Patrick Liang2, Ariel Masetto2, Yuan Gui1, Jane Savill1, Sara Michienzi1, Henri A. Menard6,3, Walter P. Maksymowych5, Gilles Boire2 1
Augurex Life Sciences Corp., British Columbia, Canada; 2University of Sherbrooke, Sherbrooke, Que´bec, Canada; 3McGill University Health Center Research Institute, Montreal, Quebec; 4Centre hospitalier universitaire de Sherbrooke, Sherbrooke, Que´bec, Canada; 5University of Alberta, Edmonton, Alberta, Canada; 6MSK Research Axis, Canada Background: As a marker of inflammation and an acute phase reactant, C-reactive protein (CRP) is routinely used in clinical practice and in RA classification criteria. Higher or sustained levels of CRP are associated with a worse prognosis. 14-3-3g is a joint derived mechanistic marker that up-regulates factors that perpetuate disease. Similar to CRP, higher or persistent levels of 14-3-3g are associated with a worse prognosis, and a corresponding decrease in circulating levels is associated with better clinical outcomes. Inflammation and joint damage are now understood to be processes that uncouple along the course of disease and treatment strategies have been tightened to achieve both clinical and joint damage remission. Objectives: The aim of this study was to examine both the independent and combined effects of CRP and 14-3-3g on radiographic progression. Methods: Baseline (BL) serum 14-3-3g titres were assessed in 331 recent onset polyarthritis patients from the Sherbrooke EUPA Cohort with 5 years of radiographic follow-up data. Patients were DMARD naı¨ve at BL, median age was 60 years, and 62 % were female. CRP and 14-3-3g positivity were defined as [8 mg/L and C0.19 ng/mL, respectively. Spearman correlation was performed to assess the relationship between 14-3-3g and CRP titres. Radiographic changes (change in total Sharp/van der Heijde (DSHS) score over 30 months were assessed in relation to CRP and 14-3-3g co-expression using ANOVA analysis. Chi square was used to assess the relationship between CRP positivity with radiographic changes at 30 months based on DSHS C1, 3, and 5 unit cut-offs. Results: Of 331 patients, 207 (63 %) and 153 (46 %) were CRP and 14-3-3g positive at BL, respectively. Spearman correlation revealed that titres of CRP and 14-3-3g did not correlate, r = -0.00025 p = 0.996. As noted in Table, Chi square analysis returned both CRP and 14-3-3g as significantly associated with radiographic changes.
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. Variable
DSHS BL to DSHS BL to DSHS BL to year 3 C 1 year 3 C 3 year 3 C 5 OR
pvalue
OR
pvalue
OR
pvalue
CRP positivity
1.73 0.034
2.47 \0.001 2.22 0.001
14-3-3g positivity
1.69 0.046
1.74 0.016
CRP and 14-3-3g positivity
2.82 0.002
3.68 \0.001 3.16 \0.001
1.70 0.023
The cumulative probability plot illustrates that patients who were positive for both CRP and 14-3-3g had the significantly greatest increase in radiographic progression (p \ 0.001) with over 50 % of patients having DSHS C5 over the 30 month period.
Conclusions: CRP and 14-3-3g are both associated with joint damage progression at 3 years and titres do not correlate consistent with their distinct roles in RA disease processes. Interaction analysis further reveals that the combination of these two markers is a better predictor of future radiographic damage than either marker alone. Concomitant serial testing of both these modifiable markers may assist with tight control RA treatment strategies.
B333 SUPAR: A NOVEL INFLAMMATORY BIOMARKER IN CLINICAL ROUTINE ASSOCIATED WITH READMISSION AND MORTALITY Line JH Rasmussen, Steen Ladelund, Thomas H. Haupt, Jørgen H. Poulsen, Gertrude Ellekilde, Ove Andersen Copenhagen University Hospital, Hvidovre, Denmark Background: Soluble urokinase plasminogen activator receptor (suPAR) is the circulating form of uPAR, which is expressed on immune and endothelial cells. suPAR levels increase with inflammation and immune activation. suPAR is an unspecific marker of chronic low-grade inflammation associated with prognosis in both infectious and chronic diseases. suPAR was recently introduced in clinical routine as one of the standard blood tests measured on all patients admitted to the Acute Medical Unit, Hvidovre Hospital, Denmark. Objectives: We aimed to investigate the association between the new inflammatory biomarker suPAR, readmission, and mortality as well as
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relation to the established inflammatory marker C-reactive protein (CRP). Methods: Patients admitted to the Acute Medical Unit, Hvidovre Hospital, between 11/18/2013 and 03/31/2014 were included. The study population comprised 4,343 patients. suPAR was measured with suPARnostic AUTO Flex ELISA. CRP was measured with COBAS 6000 analyzer. Data on admissions and vital status were extracted from Danish patient administrative registries using the patients’ unique Danish civil registry number. Duration of follow-up was 30 days. Results: Median suPAR was 3.2 ng/mL (95 % RI: 1.3–11.3 ng/mL), and suPAR levels increased with admission time (p \ 0.0001). CRP and suPAR were significantly correlated (Spearman’s rank correlation coefficient: 0.43, p \ 0.0001), however, 20.6 % of the patients had low CRP (\10 mg/l) but high suPAR levels ([4.5 ng/mL). The median suPAR level was higher in patients who were readmitted during follow-up (3.9 ng/mL, IQR 2.7–5.6, n = 854) compared to patients who were not readmitted (3.0 ng/mL, IQR 2.2–4.5, p \ 0.0001). Similarly, the median suPAR level was higher in patients who died during follow-up (6.8 ng/mL, IQR 4.7–9.9, n = 224) compared to survivors (3.1 ng/mL, IQR 2.2–4.5, p \ 0.0001). Both suPAR and CRP predicted 30 days mortality, and the ROC AUCs for suPAR and CRP were 0.84 (95 % CI 0.81–0.86) and 0.77 (95 % CI 0.74–0.80), respectively. In Cox regression analysis of log2-transformed suPAR values, suPAR was a strong independent predictor of readmission and mortality with a HR for 30 days mortality of 2.56 (95 % CI 2.13–3.08, p \ 0.0001) when adjusted for age, sex, Charlson score, and CRP. In patients with low CRP (\10 mg/l), suPAR remained an independent predictor of readmission and mortality. Conclusions: The new inflammatory biomarker suPAR is associated with readmission and mortality in acute medical patients. suPAR adds information on high risk patients to the established inflammation marker CRP.
B334 SERUM INTERLEUKIN-18 AND S100A8/A9 AS PREDICTION MARKERS FOR DISEASE ACTIVITY IN PATIENTS ADULT ONSET STILL’S DISEASE Dae Hyun Yoo, Jinju Kim, Seung Taek Song Department of Rheumatology, Hanyang University Hospital for Rheumatic Diseases, Hanyang, Japan Background: Adult-onset Still’s disease (AOSD) is a rare systemic inflammatory disease. Although the pathogenesis of AOSD is still unknown, proinflammatory cytokines including interleukin (IL)-1, IL6, IL-18 and TNF-a contribute to clinical manifestations and laboratory abnormalities. Determination of disease activity is difficult because there are lack of disease specific clinical findings and serologic markers. Objectives: To investigate the significance of serum IL-18 and S100A8/A9 protein in the assessment of disease activity among AOSD patients. Methods: Forty patients satisfying Yamaguchi’s criteria for AOSD, 26 healthy controls and 23 patients with rheumatoid arthritis (RA) were enrolled. We collected clinical data including demographic findings and laboratory findings in active state and inactive state. Serum levels of IL-18 and S100A8/A9 proteins were measured by enzyme-linked immunosorbent assay (ELISA). Activity state was divided by modified Pouchot’s (mPouchot’s) score. Inactive state was
Inflamm. Res. defined mPouchot’s score less than 4 at least consecutive 2 months, after 6.2 ± 1.4 months of active state. Results: There were significantly higher levels of serum IL-18 (107951.1 ± 94440.9 pg/mL) in active state than inactive state of AOSD (14772.2 ± 22126.7 pg/mL), RA patients (333.8 ± 309.0 pg/ mL) and healthy controls (350.7 ± 160.4 pg/mL). Although, serum S100A8/A9 protein were significantly higher in active state of AOSD (33340.3 ± 22029.3 ng/mL) than inactive state (11826.5 ± 15368.4 ng/mL), healthy controls (1812.7 ± 2506.1 ng/ mL) and RA (13577.5 ± 12893.6 ng/mL), S100A8/A9 in inactive state was not showed different compared to RA patients. The levels of serum IL-18 and S100A8/A9 protein showed mild to moderate correlations with other serologic markers such as WBC, ESR, CRP and ferritin. And they also showed moderate correlations with mPouchot’s score. The cutoff points of differentiating activity in AOSD were determined by receiver operator characteristic (ROC) curve. When using a 24399.85 pg/mL of IL-18 as an AOSD activity marker, sensitivity was 82.5 % and specificity was 82.5 %. And using a 12101.55 ng/mL of S100A8/A9 protein level showed lower sensitivity (72.5 %) and specificity (72.5 %) than IL-18. The 25 % of AOSD patients with clinically inactive state became active state and/ or elevated ESR and CRP within 2 months. AOSD patients showing higher IL-18 and S100A8/A9 protein than activity cutoff points in inactive state, showed disease flare within 2 months [OR = 14.0 (2.1–93.2), OR = 9.8 (1.9–50.6), respectively]. Conclusions: Serum IL-18 and S100A8/A9 protein were significantly increased in active AOSD patients compared with inactive AOSD, RA and healthy controls. However, IL-18 was more useful cytokine to distinguish from RA in inactive AOSD compared to S100A8/A9 protein. Serum level of IL-18 and S100A8/A9 protein may be useful predictors of disease activity within several months in inactive state of AOSD patients.
B335 CLINICAL AND BIOLOGIC CHARACTERIZATION OF CYTOKINE RELEASE SYNDROME AFTER CHIMERIC ANTIGEN RECEPTOR (CAR) T CELL THERAPY FOR ACUTE LYMPHOBLASTIC LEUKEMIA (ALL) David T. Teachey1,2, Simon Lacey2, Pamela A. Shaw2, Jos Melenhorst2, Noelle Frey2, David Barrett1,2, Fang Chen2, Julie Fitzgerald1,2, Vanessa Gonzalez2, Shannon Maude1,2, Edward Pequignot2, Scott Weiss1,2, Carl June2, David Porter2, Stephan A. Grupp1,2
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Children’s Hospital of Philadelphia, Philadelphia, USA; University of Pennsylvania, Philadelphia, USA
2
CAR T cells with anti-CD19 specificity have demonstrated considerable promise against highly refractory hematologic malignancies. Dramatic clinical responses with complete remission rates as high as 90 % have been reported in children with relapsed/refractory ALL treated with CTL019 (Maude et al. NEJM 2014). Marked in vivo CAR T cell proliferation (100 to 100,0009) leads to improved efficacy but can be associated with adverse advents, including cytokine release syndrome (CRS). To better understand the manifestations of CRS, we studied 39 children (median age 11; range 9–16) treated with CTL019 and present clinical, laboratory, biomarker data. T cells were lentivirally transduced with a CAR composed of antiCD19 scFv/4-1BB/CD3 as previously described (Porter, NEJM 2011). 44 cytokines were measured, using Luminex bead array. Other biomarkers were tested in a CLIA/CAP certified clinical lab. 36/39 patients developed grade 1-4 CRS (CRS1-4) at peak T cell expansion. 11 pts developed grade 4 CRS (CRS4), defined as requiring high dose vasoactive medications and/or mechanical ventilation. These 11 pts and 2 of 7 pts with grade 3 CRS (CRS3, defined by less severe critical illness) were treated with the IL-6 inhibitor tocilizumab, and all had rapid marked improvement in CRS. We hypothesized and demonstrated patients with CRS4 develop clinical and laboratory manifestations similar to macrophage activation syndrome (MAS)/hematophagocytic syndrome (HLH). 5 of 10 CRS4 pts developed splenomegaly (1 subject too obese to evaluate), and 7/10 developed hepatomegaly, whereas no CRS0-3 patients developed organomegaly. Fibrinogen (not sent on all subjects) was decreased (\150 mg/dl) in 9/10 with CRS4 vs 2/15 with CRS0-3. Ferritin and LDH were also markedly clinically and statistically significantly elevated in all pts with CRS4 compared to CRS1-3. Of the 44 tested cytokines, 16 have been previously studied in children with primary HLH. We found an identical pattern of cytokines differentially elevated in HLH also in patients with CRS4 compared with CRS0-3. There was a clinically, statistically, and biologically significant difference in IFN-g, IL10, sIL2Ra, IL6, IL8, MCP1, and MIP1B in CRS4 vs CRS0-3 and no clinically or biologically significant difference in IL1B, IL2, IL4, IL5, IL7, IL12, IL13, IL17, and TNF alpha. Both IL6 and IL6R were markedly elevated in patients with CRS4; this IL6 cytokine pattern, along with the pronounced response to tocilizumab, establish that IL6 trans-signaling is clinically relevant. These data represent the largest and most comprehensive profiling of the clinical and laboratory manifestations of CAR T cell related CRS and provide novel insights into the biology of CRS. They also provide direct therapeutic relevance and may guide future cytokine directed therapy.
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Author Index A Abboud, D. B061 Abboud, N. B277 Abdillahi, S.M. B048 Abels, C. B011 Aboelnazar, S. B283 About, I. B321 Abreu, L.C. B147 Abulrob, A. B259 Achacoso, P. B080 Adcock, I.M. B294, B295, B300 Ademowo, O.G. B155 Adenir, P. B299 Aderem, A. B094 Adler, D. B322 Afonso, L. B204 Africander, D.J. B263 Agapova, O.Y. 107 Agra, L.C. B205 Ahemad, N. B222 Ahuja, S. B151 Aitchison, R. 118 Ajayi, A.M. B155 Akama, T. B256 Akbari, O. 131 Akram, M. B043 Aksentijevich, I. B018 Alabi, A. B038 Albaladejo, B.T. B290 Al-Bustan, S.A. B124 Alcalay, R. B289 Alcorn, D. 103 Alenina, N. B148 Alexandre, E.C. B286 Aliev, L. B122 Alleyne, J. B199 Almeida, C.P. B317 Almeida, F.M. B296 Almeida, T.F.A. B208 Almeida-da-Silva, C.L.C. B085 Almolda, B. B206 Alpizar, Y.A. 059 Alrashid, M.H. B124 Alric, L. B267 Altavilla, D. B037 Altemeier, B. B094 Altier, C. 061 Alvarenga, P.B. B046 Alvarez, A.R.P. B292 Alvarez, J.M.M. B087 Alves, C.R. B309 Alves-Filho, J.C. B033, B064, B292 Amano, H. B107 Amaral, E.P. B087, B217 Amaral, F.A. B064 Amarante-Mendes, G.P. B217 Amaravadi, L.S. 102 Amison, R. B288 Amorim, N.R. B205 Ampuero, M.R. B204 An, D. 007, 089
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An, H. B284 Andersen, O. B333 Anderson, A.C. 068 Anderson, L. 118 Anderson, R. B262 Anderson, S. B220 Andrade, M.M. B112 Andrade, M.F. B249 Andrade-Silva, M. B312 Andre, D.M. B286 Andreotta, P.W. B298 Anhe, G.F. B286 Anisimova, L. B182 Annoni, R. B314 Ansari, M.A. B010 Ansari, T. B319 Antunes, A.M.S. B045 Antunes, E. B286 Antunes, F. B034 Anu Geo, J. B124 Anuforo, O. B278 AR, G. B313 Arantes, A.C.S. B304, B305 Arantes-Costa, F.M. B071, B291, B320 Ardekani, S. B053, B106 Ardizzone, S. 047 Arhatari, B.D. B235 Ariel, A. B276, B277 Ariga, S.K. B089 Arijs, I. B253 Arita, M. B221 Armstrong Junior, R. B187 Armuzzi, A. 047 Arnardottir, H.H. B275 Arnoletti, J.P. 039 Arriaga, A.M.C. B078, B158 Arron, J.R. 075, 110 Assis, E.L.D. B157, B258 Atreya, R. 047 Attia, S.M. B010 Ayaz, W. B321 Azevedo, R. B176 Aziz, Q. B113 Azzolini, A.E.C.S. B019, B249 B Baba, T. B062 Babaeijandaghi, F. 067 BabuRao, K. B313 Bacal, F. B165 Bachert, C. B041 Bader, M. B148, B270 Bae, O.-N. B043 Baillif, V. B149, B280 Bakheet, S.A. B010 Balasubramanian, W.R. B313 Balog, A. B012 Baltazar, G. B256 Banda, N. 083 Bandeira-Melo, C. B200, B205 Bandyopadhaya, S. B316
Banerjee, S. 046 Baranek, T. B282 Barata, J.T. B029 Barbeiro, D.F. B089, B112 Barbeiro, H. B112 Barbim, P. B033 Barbosa, A.S. B317 Barbosa-Lima, G. B143 Barcelos, L.S. B317 Baribaud, F. 076, B294 Barioni, E.D. B042 Barioni, E. B176 Barja-Fidalgo, C. B126 Barr, F. B216 Barral, A. B204 Barresi, S. B210 Barreto, A.K.S. B081 Barrett, D. B335 Barrett, P. B034 Barrett, T.J. B052 Barron, L. 054, B082 Barthelmes, J. B017 Bassuk, A.G. B116 Bastos, B.M. B078 Bastos, J.K. B249 Bastos, J.M. B076 Basyal, R.K. B316 Batista, A.C. B060 Batista, P.R. B309 Bauer, M.E. B324 Baumgarten, M. B048 Bautzova, T. B268 Baxter, A.G. B102 Beal, D.R. B028, B298 Becerra, A. B079 Beckett, E. B303 Bedard, A. B196 Belizowski, S.M. B032 Bell, S. 118 Bellas, I. B205 Bellio, M. B119 Belmonte, C. 059 Beloglazov, V. B131 Beltrame, M.H. B072 Beltran, J.S. B140 Benabdoune, H. B050 Ben-Azu, B. B155 Benderdour, M. B050 Benhar, M. B146 Benjamim, C.F. B046 Benjamin, N. B330 Bennett, A. B199 Benoist, C. 038 Bereshchenko, O. 035, 036 Berg, E. B331 Bernhagen, J. B163 Bernhart, E. B100 Berta, T. B325 Bertheau Mailhot, G. B055, B077 Bessi, V.L. B170 Bettano, K. B022
Inflamm. Res. Beurel, E. 040 Bezerra, M.M. B051, B076, B258 Bhatt, L. B151 Bhongir, R.K.V. B285 Biagioli, M. 035, 036 Biancone, L. 047 Bica, M.S.R. B231 Biglarnia, A. 082 Billin, A.N. 066 Bilton, D. B261 Bing, X. B116 Binge, L. B209 Biragyn, A. B047 Bishop-Bailey, D. B273 Bitto, A. B037 Blank, S. B109 Blanque´, R. B253 Blumberg, R.S. 048, 089 Bocchi, E. B165, B166 Bodin, A. B097, B114 Bodogai, M. B047 Boehm, M. B018 Bogo, M.R. B229 Boichot, E. B097, B114 Boire, G. B332 Boivin-Jahns, V. B169 Bokil, N.J. B323 Bomfim, C.C. B087 Bonifacio, J.P.P. B135, B141 Bonnart, C. B267 Bonnel, D. B092 Boraschi, D. B237 Borelli, P. B140 Borges, P.V. B309 Borges, V.M. B204 Borges, V.F. B064 Borilova Linhartova, P. B075 Borodovsky, A. 083 Borthwick, L.A. 054, B082, B095 Bortoluci, K.R. B217 Boshuizen, M.C.S. B164 Bosiljcic, M. B262 Bossa, F. 047 Botega, A. B112 Botham, K.M. B273 Bouchard, G. B191, B228 Bouche, G. B262 Bouchet, A. B002 Bouillet, P. B235 Bourassa, J. 099 Bourcier, N. B055 Bouttier, M. B218 Boylan, F. B251 Boyle, C. B034, B049 Bozza, F.A. B143 Bozza, P.T. B142, B143, B200, B214 Braga, Y.A.V. B231 Brandt, S. B007, B109 Braselmann, S. B311 Bra¨uer, E. B002 Brayner, M.M.B. B157, B158 Brazzell, K. 099 Brea, D. B282 Breit, S.N. B175, B178 Breithaupt Faloppa, A.C. B184, B185, B187 Brenchley, J. 006
S239 Brenck, L.F. B161 Brito, Gerly A.D.C. B051, B076, B157 Brito, N.M. B126 Brock, C. B113 Brodermann, M.H. B052 Brodmerkel, C. B030 Brodskyn, C. B204 Brogliato, A.R. B046 Broide, D. 130 Bro¨nnimann, D. B002 Brown, A.C. B230, B303 Brown, D.A. B175, B178 Bru¨ggemann, T.R. B071, B291, B320 Bruhns, P. 028 Brumbaugh, K. B004, B220 Brunelle, A.R. B106 Brunger, J.M. 009 Bruni, F.M. B291, B320 Brunialti, M.K.C. B306 Bruschetta, G. B188 Bruscoli, S. 035, 036 Bryant, S. B016 Brys, R. B252, B253 Buatois, V. 115 Budelsky, A.L. 054, B082 Burelle, Y. B170 Buret, A.G. B225 Burgel, P.-R. 029 Burgess, L. 118 Burkly, L.C. B098 Buryachkovskaya, L.I. 109, B162, B171 Bush, J. 083 Byford, A. B022 C Cabrera, A. B053 Cadwell, K. 016 Caffarena, E. B309 Cai, B. B213 Cain, D.W. B264 Calabro, S. B054 Calixto, M.C. B286 Campa, A. B042 Campbell, I.L. B206 Campos, M.M. B229 Can, G. B025 Can, S. B025 Cande´a, A. B312 Canesso, M.C.C. B317 Canetti, C. B119 Cantu, D.A. B095 Caperuto, L.C. B299 Caprioli, F. 047 Cardell, L.-O. B041 Caria, C.R.P. B130, B134 Carmona, A.K. B217, B270 Carneiro, A.B. B142 Carpentier, A.C. B170 Carrasco, S. B109 Carre´, J.E. B329 Carrier, N. B332 Carvalho, G.H. B142 Carvalho, H.N.S. B142 Carvalho, L.A.C. B135, B141 Caspi, R.R. 030 Cassali, G.D. B161, B292
Castanheira, F.V.S. B064 Castiglione, F. 047 Cavagnaro, J. 125 Cavalher-Machado, S.C. B312 Ceccotti, M.-C. B252, B253 Cehelsky, J. 083 Cenac, N. B268 Cenedeze, M.A. B144 Cervelli, J. B293 Cervilha, D.A.B. B269 Cha, J.J. B094 Chaabo, K. 120 Chagas, A.S. B108 Chagas, J.R. B270 Chai, J.C. B090 Chai, Y.G. B090 Chakravarthy, K. B242 Chalmin, F. B035 Chan, M. B311 Chang, C.-K. 067 Channon, K.M. B052 Chantry, D. 118 Chao, W. B186, B227 Charlotte, G. B280 Chaturvedi, P. 083 Chaudhuri, J. B316 Chaves, H.V. B051, B076, B078, B157, B158, B258 Chaves, M.M. B119 Chazaud, B. 065 Chazova, I.E. 107 Cheever, A.W. 054, B082 Chelur, S. B313 Chen, B. B110 Chen, F. B335 Chen, G. 082 Chen, G. B018 Chen, H. B227 Chen, H. 099 Chen, J. 030 Chen, M. B201 Chen, P. B054 Chen, Y. B311 Cheˆne, G. B149, B280 Cheng, Q. 023 Cheng, X. B284 Cherkas, Y. B030 Chiang, N. 014, 062, B271 Chiu, I.M. 058 Chmilewsky, F. B321 Cho, H. B091 Chollet-Martin, S. 027 Chong, W.P. 030 Chouinard, L. B196 Chowdhury, K. B316 Choy, E.H. 113, 115 Christensen, A.D. B027 Chumachenko, P.V. 109, B162 Chung, K.F.B295 Chung, S.H. B321 Ciambarella, B.T. B304, B305 Cicmil, M. B022, B242 Cidlowski, J.A. B264 Cimino, M. 035, 036 Clare-Salzler, M.J. 039 Clarke, J.M. B102
123
S240 Clarke, S. B310 Clatworthy, A. B240 Claudio Fantini, M. 047 Clausen, B.E. B198 Clottu, A. B035 Colas, R.A. 062, B125, B271 Cole, J 118 Coler, R B084 Colletti, N. B111 Collins, J.E. B216 Collison, A.M. B301 Colon, D.F. B180 Comeau, M.R. 054, B082 Cominelli, F. B032 Cook, A.D 019, B027, B153 Cooper, D. B052 Cooper, M.A. B099 Cooray, S.N. B274 Corazza, G.R. 047 Cordaro, M. B188 Cordeiro, N.M. B248, B250, B260 Corluka, S. B259 Cornicelli, J. B092, B132, B196 Correa da Rosa, J. 074 Correa, J.R. B129 Correa, L.B. B312 Correa, R. B129 Corrigall, V. 120 Costa, E.A. B156, B223, B245 Costa, H.C. B126 Costa, R.S. B180 Costa, S.K. B159, B172 Cotton, J.A. B238 Couillin, I. B114 Courad, P.O. B008 Courty, Y. B282 Cousens, L. B049 Coutinho, D.S. B305 Coutinho, I.C. B081 Coutinho-Silva, R. B046, B085, B119, B208 Couto, G.K. B172 Cox, A.J. B116 Croda, J. B154 Croft, M. 069, B066 Cross, J. B330 Cross, V. B328 Crupi, R. B188 Cuff, C. 119 Cummings, R.D. B105 Cunha, F.Q. 090, B033, B064, B180 Cunha, L.D. B118 Cunha, L.C. B156 Cunha, T.M. B033, B064 Curran, M. B030 Cuzzocrea, S. B188 Czepielewski, R.S. B233 D Daino-Laizure, H. B080 Dalli, J. 014, 062, B271, B275 Dalloneau, E. B282 Damasceno, K.A. B292 da Matta, M.C. B081 Darbro, B.W. B116 d´Areˆde, S. B204 Das, A. B090
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Inflamm. Res. Das, R. 088 Das, S. B316 Dasgupta, B. B030 Dasgupta, S. B215 David, B.A. B307 David, C. B252 ´ vila, H. B142, B214 D’A deGraaf, K. 115 De Groot, A.S. B034, B049 DeGroot, J. B092, B132, B243 Dejani, N.N. B007 de Jesus, F.N. B172 Delachaume, C. B252 Delafield, N. B109 de Lima, K.A. B033 de Lima, T.M. B089 Della-Casa, M. B144 Dellatorre-Teixeira, L. B200, B205 De Maeyer, R.P.H. B121, B279 de Min, C.115 Demir, A.M. B024 Demirtas, S. B024, B025 de Morais, I.C.O. B005 Denadai-Souza, A. B267, B268 Denlinger, B. 059 de Paula, M.A. B159 DePierri, K. B128 Deraison, C. B267, B268 De Vos, M. B252, B253 De Vos, S. B252, B253 De Vriendt, V. B252, B253 de Winther, M.P.J. B164 Dias Fernandes, P. B251 Dias, D.F. B305 Dias, N.H. B290 Dietrich, D. B139 Dill, V. B048 Dimasi, N. B110 Dimitrov, V. B218 D’Imperio-Lima, M.R. B087, B217 Diot, P. B282 Di Sabatino, A. 047 Di Stasi, L.C. B108 Djonov, V. B002 Djukanovic, R. B294 Do, A.T. B013 do Amaral Neto, E.S. B172 Doan, A. B080 Dodheri, S.S. B313 Dogra, Y. B329 Donadi, E.A. B019 Donahue, D. B095 Dong, C. B256 Donovan, K.A. B123 Dorwood, D. B095 dos Reis, G. B204 dos Santos, T.W. B130 Dotan, S. B146 Dotimas, H. B080 Dowds, M. 089 Drewes, A.M. B113 Du, X.-J. B235 DuBois, R.N. 011 Dubourdeau, M. B149, B280 Duchene, J. B148 Duffield, J.S. 052, B094, B098, B219
Dumas, A. B055 Dupont, S. B252, B253 Duran, A. B269 Durum, S.K. B029 E Eastman, R. B083 Ebel, P. B017 Eberhardt, C. 118 Eberle, M. B017 Eckman, J. B022 Edir, A. B267 Ediriweera, H. 044 Efremov, E. B162 Egesten, A. B285 Eisenbarth, S.C. B054 Eklund, K.K. B117 Elborn, S. 025, B261 El Bounkari, O. B163 Eliria, E. B004 Elkins, J.S. 102 Ellekilde, G. B333 Elmore, S. B310 Elson, G. 115 Emery, F.S. B249 Engelman, R. B146 Engstrom, L. B241 Enlow, E.M. 099 Errico, J.P. B113 Esmaeili, A. B168 Esposito, E. B188 Essilfie, A.T. B300, B303 Estelle, W. B280 Estes, B.T. 009 Eum, S. B106 Evans, E. 088 F Fahmi, H. B050 Fahoum, L. B136 Fairlie, D.P. B013 Fajardo, O. 059 Falach, R. B289 Faloppa, A.C.B. B296 Faltus, R. B022, B242 Famenini, S. B327 Fang, W. B201 Fantozzi, E.T. B184, B185 Farache, J. 038 Farber, J.M. B198 Farmer, A.D. B113 Farokhzad, O.C. 055, 123 Farsky, S.P. B021, B042, B176 Fassmann, A. B075 Faulkner, A.R. B273 Fautrel, A. B097 Fayaz Ahmad, S. B010 Febbraio, M. B170 Fedosov, M. B182 Feinberg, M.W. 045 Feldbru¨gge, L. B208 Felizardo, R.J.F. B144 Feng, Y. B186, B227 Ferguson, P.J. B116 Ferlin, W. 115 Fernandes, A.J. B305
Inflamm. Res. Fernandes, A.J. B332 Fernandes, C.M. B021 Fernandes, J. B050 Fernandes, L. B270 Fernandes, P.D. B248, B250, B257, B260 Fernandes, P. B071, B291, B320 Ferna´ndez-Pen˜a, C. 059 Ferreira, H.H.A. B290 Ferreira, L.C.S. B217 Ferreira, R.G. B292 Ferreira, S.G. B187 Ferreira, T.P.T. B305 Fiala, M. B327 Fichera, M. 047 Fiebich, B.L. B244 Fierro, I.M. B045, B126 Figliuolo, V.R. B208 Figueiredo, R.T. B231 Figueiredo-Rinhel, A.S.G. B249 Filgueiras, L.R. B101, B207 Filho, G.C. B078, B157, B258 Filho, S.M.P. B258 Finkel, D. B004 Fiore, D. 067 Fiorin, V. B147 Fischer, D.F. B092, B132, B243 Fitch, N. B111 Fitzgerald, J. B335 Florence, A.C. B267 Florence, G. B114 Florentino, I.F. B156, B223, B245 Flower, R. B195 Flynn, S.M. B297 Fock, R.A. B140 Fomochkina, I. B182 Fonseca, F.C.S. B272 Fontenot, J.D. 101, 102 Foster, P.S. B300, B303 Frade, T.I.C. B161 Fraga, C.A.M. B248, B250, B260 Frammartino, T. 035, 036 Franc¸a, A.L. B051 Franc¸a, E.L. B147 Franca, R.F.O. B180 Frances, R. B311 Francischi, J.N. B161, B272 Frates, C. B256 Fredman, G. B213 Fredman, G. 123 Freire, J.M.D.O. B076, B078 Freitas, A.R.D. B076, B158 Freitas, H.C. B157 Freitas, R.S. B051, B078, B157, B158, B258 Freitas, R.H.C.N. B248, B250, B260 Freitas-Limas, L.C. B317 Frey, N. B335 Freysdottir, J. B278 Fu, W. 038 Fu, X.-N. B073 Fuerstenberg, R.K. B220 Fujimori, M. B147 Fukuda, D. B173 Fukunaga, Y. B246 Fullerton, J. B279 Funchal, G.A. B233 Furuichi, K. B138
S241 Futerman, A. B017 G Gabay, C. 115, B139 Gadelha, C.A.D.A. B157, B258 Gal, Y. B289 Galien, R. B252, B253 Gallman, A. B054 Gambero, A. B130, B134 Gamble, J.R. 022 Ganiyu, O. B038 Gao, C. B110 Gapon, L.I. B133, B167 Garcia, C.C. 093, B307 Garg, P. 083 Garrood, T. 120 Gavin, M.A. 103 Gavins, F.N.E. B059, B190 Geisslinger, G. B017 Georgii, J.L. B046 Germain, R.N. 030 Gerner, E. B048 Gersbach, C.A. 009 Ghosh, K. B053, B106 Ghosh, P. B316 Giambelluca, M.S. B077 Gicquel, T. B097, B114 Giera, M. B278 Gillie´ron, C. B139 Gilligan, M.M. B125 Gilroy, D.W. 064, B121, B279 Giorno, T.B.S. B257 Gira˜o, V.C.C. B078 Glass, K.A. 009 Glosson-Byers, N.L. B007, B183 Gobbetti, T. B273, B274 Gochuico, B. B096 Goedken, M. B293 Goerge, T. B211 Goes, P. B051, B076, B078 Goess, C. B310 Goh, H.Y. B102 Gold, L.S. B036 Goldman, N. B128 Golitsyn, S. B162 Gomes, T.V. B081 Gomez, I.G. B094, B098, B219 Gon, Y. B014 Gonc¸alves, L.O. B223 Gonza´lez, B. B206 Gonzalez, V. B335 Gordienko, A. B131 Gorski, K. 103 Gosset, P. B282 Gotardo, E.M.F. B130, B134 Graber, W. B002 Gracias, DT. B066 Graff, C. B310 Graham, G.J. B064 Graham, N. B041 Graham, S.M. B028 Granger, D.N. B059, B190 Granucci, F. B210 Grassi-Oliveira, R. B324 Greaves, D.R. B052, B056 Grebe, K. B310
Greco, K.V. B319 Gretzer, C. B048 Gridley, S.B080 Gronert, K. 012 Groom, T. B330 Gros, A. B211 Gro¨sch, S. B017 Grosswald, R. B151, B261 Grupp, S.A. B335 Gu, J.T. B326 Guan, P. 088 Guerau-de-Arellano, M. 100 Gueugnon, F. B282 Gui, Y. B259, B332 Guigne´, C. B149 Guilak, F. 009 Guillen, E. B034 Guillon, A. B282 Guimara˜es, P.B. B270 Gumperz, J.E. 086 Gundra, U.M. 044 Gunn, H. B262 Gunthuri, S. B080 Gupalo, E.M. 109, B162 Gupta, P. B013 Gupta, S. B106 Guttman-Yassky, E. 072 Guyre, P.M. B216 H Haase, C. B027 Hafezi-Moghadam, A. 032 Haibara, A.S. B161 Hajishengallis, G. 082 Halai, R. B099 Halder, R. B327 Hall, C. 120 Hall, W.L. B273 Halsall, D. B195 Hamilton, J.D. 073, B041 Hamilton, J. B027, B153 Hammer, A. B100 Han, M.-S. B065 Han, Q. B325 Han, S. B065 Hanish, I. B301 Hannen, R.F. B195 Hansbro, P.M. B230, B300, B301, B302, B303 Hansen, F. B193 Hansen, R. B036 Hapgood, J.P. B263 Hardardottir, I. B278 Harrison, L.C. B102 Hart, K.M. 054, B082, B096 Hartigan, A. B022 Hartman, D. B310 Hartmann do Prado, C. B324 Harvey, R. B034 Hasegawa, Y. B189 Hashimoto, N. B189 Hassanpour Golakani, M. B175, B178 Hatterer, E. 115 Hattori, H. B254 Haupt, T.H. B333 Haw, T. B300, B301, B302
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S242 Hayden, K. B030 He, S. B088 Headland, S.E. B255 Heckrodt, T. B311 Hedrick, M.N. B198 Helluy, X. B169 Heluany, C.S. B021 Henriques, M. B309, B312 Henry, C. B282 Herbst, R. B110 Herrmann, M. B012 Hertzog, P.J. B230 Herwald, H. B145 Hibi, Y. B254 Hickey, M.J. 023 Hickey, P. B235 Hicks, A. B241 Higashikuni, Y. B173 Higuchi, M. B165, B166 Higuchi, T. B169 Hiller, K.-H. B169 Hirata, M. B087 Hixon, J.A. B029 Hobbie, S.N.K. B281 Hoda, U. B294 Hoeksema, M.A. B164 Hoff, C.C. B270 Holers, V.M. 083 Hollander, C.M. B047 Holloway, P.M. B059 Holtappels, G. B041 Honda, Y. B247 Hono´rio-Franc¸a, A.C. B147 Hopkins, R. 118 Horan, G. 047 Horner, M.J. 127 Horsley, A. B261 Horvat, J.C. B230, B300, B301, B303 Hoshino, T. B068 Ho-Tin-Noe´, B. B211 Hottz, E.D. B143 Hsieh, C.-M. 119 Huang, S. B125, B127 Huang, Y. B018 Huard, J. 084 Huber-Lang, M. 082 Hugunin, M. 119 Huh, J. B179 Huss, D.J. 102 Huynh, D.N. B170 I Iadonato, S.P. 033 Ianni, B. B166 Icimoto, M.Y. B217 Ikegami, R.N. B165, B166 Imai, R. B003 Imajo, K. B247 Inagaki, Y. B093 Inal, A. B001, B026 Inman, M.D. B301 Iqbal, A.J. B052, B056 Irimia, D. B239 Isaacs, J.D. 116 Isaias, P.H. B051 Ishida, K. 103
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Inflamm. Res. Ishizuka, E.K. B101, B207 Italiani, P. B237 Ito, J.T. B269, B314 Iula, L. B234 Ivanov, A.V. 119, B016 Ivashkiv, L. 004 Iwabuchi, K. B107 Iwata, Y. B138 Iyer, A. B013 Izakovicova Holla, L. B075 Izuhara, K. 118 Izumi, K. B070 J Jack, R. B219 Jaeger, N. B233 Jahns, R. B169 Jakob, P. B169 James, A. B004 Jancar, S. B101, B207 Jancic, C.C. B234 Jandrot-Perrus, M. B211 Jang, J. B065 Jankovic, D. B083, B150 Jarnagin, K. B256 Jarolimek, W. 111 Jelinsky, S. B088 Jenne, C.N. B232 Jensen, N.M. B216 Jeon, S.G. B152 Jeong, Y.-J. B152 Ji, R.-R. B325 Jian, W. B186 Jiang, J. B284 Jijon, H. B240 Jittayasothorn, Y. (Ed), 030 Jiwrajka, N. B096 Johnson, B. B094 Jonasdottir, H.S. B278 Jonassen, T.E.N. B274 Jones, B. B300 Jones, H.E. 001, 043, 079 Jones, H.R. B103 Jones, S. 115 Jonesiatauro, S. B256 Jorge, R.J. B006 Jouan, Y. B282 Juban, G. 065 June, C. B335 Jung, K.H. B090 Jung, Y.-K. B065 K Kagawa, L. B107 Kaieda, S. B068 Kaiser, F. B169 Kaliannan, K. B224 Kalyan, S. B262 Kamaly, N. 123 Kamboj, D. B315 Kamimura, Y. B137 Kaminski, H. 083 Kamp, M. B209 Kanashiro, A. B064 Kaneko, Y. B070 Kaner, Z. B146
Kaneva, M.K. B255 Kang, J.X. B224 Kang, M.-J. B152 Kanga, U. B316 Kanjilal, M. B316 Kankova, K. B075 Kannan, G. 103 Kano, F. B318 Karaca, T. B024, B025 Karnell III, F. B110 Karow, M. 103 Karp, J.M. 057 Kasaian, M.T. B096 Kaser, A. 089 Kasetty, G. B145, B285 Kasper, D.L. 089, B215 Kassuya, C.A.L. B154, B177 Kawahata, N. 083 Kawai, S. 078 Kawakami, J.T. B165, B166 KB, Charamanna, B313 Keely, S. B300 Kehoe, P.G. B120 Keitelman, I. B234 Kelley, R. B311 Kelly Palma, R. B308 Kelsall, B.L. B198 Kendall, A. B273 Kennedy, T.P. B039, B040 Kerwin, E. 118 Keskin, D. B026 Keskin, G. B001, B026 Kessoku, T. B247 Khanna, K.M. B104 Kharchenko, V. B182 Khimich, N. B131 Kichko, T. 059 Kilty, I. 041 Kim, B.Y. B174 Kim, G.-W. B065 Kim, H.-R. B067 Kim, J. B194, B334 Kim, J.-A. B265 Kim, K. B174 Kim, K.W. B067 Kim, R.Y. B230, B300, B301 Kim, Y.H., B325 Kindrachuk, K.N. 054, B082, B095 Kirchner, J. 103 Kiriazis, H. B235 Kirkham, B. 120 Kita, H. 129 Kitasato, H. B003, B107 Klase, Z. 006 Klasen, C. B163 Klaunberg, B. B095 Klaus, E.J. B281 Kleef, R. B262 Klein, A. B292 Klein, P. B328 Kleindl, P. B328 Klingel, K. B169 Knight, A. B120, B329 Knight, D.A. B301 Ko, H.-K. B287 Ko, K.-M. B044
Inflamm. Res. Koga, M.M. B101 Kogelnik, N. B008 Kojima, F. B003, B107 Kolaczkowska, E. B232 Komuro, I. B173 Konkiewitz, E.C. B154, B177 Korenchan, D. B326 Kosco-Vilbois, M. 115 Kou, Y.R., B287 Koutsogiannaki, S., 082 Kovalenko, E., B122 Kovanen, P.T., B117 Kra¨henbu¨hl, S. B058 Kranich, J. B102 Kraus, V. B010 Krishna Babu, R.D. B313 Krishnaswamy, J.K. B054 Kronman, C. B289 Krueger, J. 071 Kubes, P. 087, B232 Kubyshkin, A.V. B122, B131, B182 Kubyshkin, V. B122 Kudo, G.K. B187 Kumar, A. B244 Kumar, P. B316 Kumar, U. B316 Kumari, S. B197 Kuo, S. B294 Kupa, L.K. B021 Kurhanewicz, J. B326 Kusner, L. 083 Kuznetsov, V.A. B133, B167 Kyeong-A, K. B043 L Labe´gue`re, F. B253 Lacerda, J.T.J.G.D. B157, B258 Lacerda-Queiroz, N. B083 Lacey, D.C. B153, B235 Lacey, S. B335 Lacroix, S. B055 Ladelund, S. B333 Laflamme, C. B055, B077 Lage, S.L. B214, B217 Lagente, V. B097, B114 Laissue, J.A. B002 Lakshminarasimhan, A. B313 Lam, F.F.Y. B192 Lamagna, C. B311 Lamana, S.M.S B158 Lamberth, S. B030 Lambris, J.D. 031, 082 Lamerdin, J. B080 Lamrani, L. B211 Lanes, V.R. B087 Langer, M.N. B048 Lapeyre, G. 115 Laskin, D.L. B293, B322 Laskin, J.D. B293, B322 Lassounskaia, E. B087 Latham, S.H. B273 Laukens, D. B252, B253 Le, W. B212 Leaf, I. B094 Leamon, C.P. B328 Lee Chang, C. B047
S243 Lee, E.-J. B065 Lee, J.-Y. B152 Lee, N. B004 Lee, S.-H. B015, B067 Lee, T.-S. B287 Lee, W.Y. B039, B040 Lee, W.-Y. B232 Lee, Y.S. B090 Lehner, K.A. B127 Leita˜o, R.F.D.C. B157 Leite, C.E. B229 Lelias, S. B034 Lemos, D. 067 Leong, P.K. B044 Lesmes, U. B136 Leung, J. B327 Leverson, J. B310 Levy, B.D. 091 Lewis, E.C. B146 Li, C.-Y. B226 Li, D. B086 Li, H. B175, B178, B311 Li, J. 022 Li, L. 103 Li, L. 005 Li, S. B212 Li, W. B029 Liang, P. B332 Liao, W. B201 Libert, C. 034 Lichtnekert, J. B094 Lidani, K.C.F. B072 Lightman, S. B195 Lim, Y.M. B222 Lima, C.B. B217 Lima, C.K.F. B160 Lima, D.B. B006 Lima, L.D.C. B317 Lima, M.A.S. B076 Lima, O.C.O. B148 Lima, W.T. B296 Lima-Junior, D.S. B118 Lin, A.-H. B287 Lin, L.-L. B088 Linhares, A.G.F. B051 Linhares, E. B051 Lino, R.C. B223, B245 Lira, F.B. B159 Litherland, S.A. 039 Liu, D. B054 Liu, G. B301 Liu, J. B191, B228 Liu, M.-H. B287 Liu, T. B325 Llewellyn, D. B120 Lloyd, A. B328 Lobo, B.W. B160 Locati, M. B064, B305 Lockett, T.J. B102 Lohse, M. B169 Loiola, R.A. B042 Loke, P. 044 Lomakin, N.V. B171 Lombardi, M.S. B139 Lopes, F.D.T.Q.S. B269, B314 Lopes, F.M.L.D.S. B158
Lopes, J.F. B143 Lo´pez-Lo´pez, J.R. 059 Loser, K. B011 Lourenc¸o, J.D. B269 Louw-du Toit, R. B263 Louzada, P. B033 Lovett-Racke, A. 100 Low, M. 067 Low, P.S. B328 Loyau, S. B211 Loyer, P. B114 Loza, M. B294 Lu, A. B201 Lu, Y. B328 Lubell, W.D. B170 Lucisano-valim, Y.M. B249 Ludwig, N. B262 Luger, T.A. B011 Luis, E. 059 Lukaszewski, R. B329 Luna-Gomes, T. B200, B205 Luo, L. B323 Lupher, M. B219 Lust, J.A. B123 Lyng, G.D. B028, B298 M Macchione, M. B299 Macedo, C.G.D. B158 MacGregor, G. B261 Machado, F.R. B306 Mackay, C.C. B317 Mackay, C.R. B102, B209 MacKenzie, J.D. B326 MacKenzie, R. B259 Maddox, D.E. B297 Maehana, S. B003, B107 Mafezoli, J. B078 Mafra de Lima, F. B308 Magalha˜es, J.T. B051 Magalha˜es, K.G. B129 Mahalingam, N. B313 Mahalingam, S. 092 Maher, L. B104 Maher, T.M. 026 Maia, M.B.D.S. B158 Maia-Dantas, A. B172 Maier, P. B281 Mailhot, B. B055 Majima, M. B003, B107 Maksymowych, W.P. B259, B332 Malaviya, R. B293 Malchenko, O. B182 Malfait, A.-M. 008 Malkina, T. B162 Malle, E. B008 Malmstro¨m, E. B145 Malmstro¨m, J. B145 Mancuso, R.V. B058 Manenschijn, J.-A. 059 Mangan, N.E. B230 Mangini, S. B165, B166 Manko, A. B238 Mannent, L. B041 Mansikka, H. 119 Mantovani, A. 002
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S244 Maradana, M.R. B302 Marchi, L.F. B019 Marcy, B. B280 Marinho, A.D. B006 Marino, E. B102 Mario, R.N. B016 Marks, D. B279 Marleau, S. B170 Marotta, A. 095, B030, B259, B332 Martin, C. 029 Martin, D. 103 Martin, W. B034, B049 Martins, A.M.C. B005, B006 Martins, C.D.S. B051, B076 Martins, M.A. B304, B305 Martins, M.A. B071, B269, B291, B296, B299, B314, B320 Martins, P.R. B307 Masenko, V.P. 107 Masetto, A. B332 Mason, L.J. B209 Massis, L.M. B217 Masuda, E. B311 Mathis, D. 038 Matos, N.A. B292 Matsubara, K. B318 Matsushima, K. B093 Matsushita, Y. B189 Matta, K. B331 Mattes, J. B300, B301 Matteucci, K.C. B217 Mauad, T. B314 Maude, S. B335 Mauro, P. B274 Maus, M. 117 Mayall, J.R. B230, B300 Maya-Monteiro, C.M. B200, B214 Mayranpaa, M.I. B117 McElroy, M. B092 McInnes, I.B. 094, 115 McIntosh, F. B196 McKay, D. B267 McLeod, K.H. B102 McMurtrie, D. B311 McNeill, E. B052 Medeiros, A.I. B007 Medeiros, A.C.D. B078 Medeiros-de-Moraes, I. B143 Mehta, D.S. 102 Melenhorst, J. B335 Mello, P.A. B208 Melo, P.H. B064 Melo, R. B214 Melton, L. 083 Memari, B. B218 Menard, H.A. B332 Me´nard, L. B170 Mendes, J.A. B290 Mendonc¸a, K.M. B051 Menegatti, R. B156, B223, B245 Menezes, R.r.P.P.b. B006 Meng, G. B201 Meng, J. 046 Menon, M. B104 Meotti, F.C. B135, B141 Mercier, M. B259
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Inflamm. Res. Merciris, D. B252, B253 Mernak, M.M.B. B299 Meseguer, V. 059 Messias-Reason, I.J.T. B072 Metzler, S. B216 Meyaard, L. 050 Meyer, J.R. B046 Meyron, E.G. B136 Meyron-Holtz, E. B032 Michienzi, S. B259, B332 Mikchailichenko, V. B182 Milanesi, L. B237 Mille-Baker, B. B092, B132, B243 Mimura, K.K.O. B319 Ming, J.E. B041 Mingozzi, F. B210 Miranda, A.L.P. B160 Miriam, G.S. B055 Mironova, N.A. 109, B162 Misaki, K. B014 Mita, T. B254 Mitra, D.K. B084, B315, B316 Mittelsteadt, K. B094 Miyoshi, E. B177 Mizrachi Nebenzahl, Y. B146 Moaaz, M. B283 Mohammad, M.G. B175, B178 Mohan, A. B315 Mohideen, U. B106 Moller, T. 022 Monaghan, K. 020 Monk, P.N. 081 Monnet, E. 115 Monteiro, C.M. B309 Monteiro, H.S.A. B006 Monteiro, R.C. 051 Monteleone, G. 047 Montero-Melendez, T. B274 Montes, M.B.A. B101 Moore, A. B255 Moore, K.J. 044 Moraes Sernaglia, E. B203 Moraes, A.R. B319 Morais, I.C.O. B006 Morais, T.C. B147 Morand, E.F. 020, 023 Morandi, V. B126 Morandini, A.C.F. B085 Morato, P.N. B154, B177 Moreira, G.C.P. B290 Moreira, L.F.P. B187 Morello, E. B282 Moret, K.H. B309 Mo¨rgelin, M. B048, B285 Mori, P. B255 Moritoh, K. B047 Morris, D. B095 Mosca, E. B237 Motta, J.-P. B225, B238, B267, B268 Motwani, M. B121, B279 Mounier, R. 065 Moutos, F.T. 009 Mowen, K. B232 Mueller, A. B086 Mukaida, N. B062 Mukherjee, S. B313
Mullins, D.W. B262 Muniz, V.S. B205, B231 Munoz, E.J. 033 Munoz, L. B012 Murakami, R. B003 Murakumo, Y. B107 Muraro, S.P. B233 Murtaza, A. B242 Muscara, M.N. B159, B172 Musikhina, N.A. B133, B167 N Nagamoto, T. B014 Nahrendorf, M. 121 Nair, P.M. B301, B302 Nakada, N. B107 Nakagawa, N. B098, B219 Nakagawa, S. B094 Nakajima, A. B247 Nakamura, M. B003, B107 Nakamura, Y. B014 Nakano, Y. B137 Namdeo, M. B084 Napimoga, J.T.C. B158 Nascimento, M.S.L. B054 Nascimento, T.S. B260 Natoli, G. 003 Navarro, R. B214 Navia, B. 059 Navrotska, V. B236 Nedospasov, S.A. 067, 108 Neele, A.E. B164 Negro, A. B018 Nellore, K. B313 Nery Porto, B. B324 Nery, L.R. B229 Neto, B.A.D. B129 Neurath, M.F. 047 Neves, J.F. 089 Neves, J.S. B046, B231 Newson, J.S. B121 Ng, E.S.K. B192 Ng, N.H. B192 Nguyen, D. B091, B331 Nguyen, D.H. B301 Nguyen, J. B326 Nguyen, L. B326 Nichols, K.E. 088 Nicolaou, A. B273 Nieswandt, B. B211 Niewoehner, J. B036 Nilsson, B. 082 Nilsson-Ekdahl, K. 082 Nishikawa, M. B189 Nisihara, R.M. B072 Noels, H. B163 Nordin, S. B048 Norling, L.V. B103 Norris, P. 062 Norton, K. 033 Nowak, P. 099 Nurmi, K. B117 Nusshold, C. B008 Nys, K. B253 Nyska, A. B032
Inflamm. Res. O O’Byrne, P.M. 024 O’Connor, L. B310 Ogawa, F. B003 Ogawa, Y. B247 Oh, S. 089 Ohayon, D. 029 Ojcius, D.M. B085 Okhale, S.E. B155 Olajide, O. B244 Oland, S.D. B096, B150 Oliani, S.M. B319 Oliveira Correa, R. B203 Oliveira Filho, R.M. B184, B185 Oliveira, A.C. B102, B209 Oliveira, C.C. B130 Oliveira, D.C. B140 Oliveira, E.M. B042 Oliveira, J.M. B071 Oliveira, M.C.V.C. B081 Oliveira, R.R. B019 Oliveira, S.S. B208 Oliveria, R.A. B045 Olivo, C.R. B269, B291 Ollivier, V. B211 Olsen Saraiva Camara, N. B308 Olson, L.M. B016, B310 Olszak, T. 089 Oluwole, O.G. B038 O’Mahony, A. B091, B331 Onali, S. 047 Ong, H. B170 Ong, W. 099 Ongenaert, M. B253 Ono, J. 118 Oon, S. 020 Oorni, K. B117 Opdenakker, G. B232 O’Reilly, L. B235 Orlandi, I. B210 Orlando, A. 047 Orr, S.K. B271, B275 Orza´ez, M. B005 Osipova, I.V. B133, B167 Ostanin, K. B086 O’Sullivan, B. B302 Ouimet, M. 044 Ouwehand, K. B092 Owen, D. B088 Oxenkrug, G. B236 Ozısık, L. B001 P Pacheco, P. B309 ´ . B144 Pacheo-Silva, A Padley, R. 119 Padua, L. 093 Page, C.P. B288 Paiva, L.A. B205 Pallio, G. B037 Pallone, F. 047 Pamuk, G.E. B023, B024, B025 Pamuk, O.N. B023, B024, B025 Panayi, G.S. 120 Pandit, C. B313 Panigrahy, D. B125, B127
S245 Panjwani, P.A. B169 Pantaleao, L.N. B176 Pa˜o, C.R.R. B205 Papanastasiou, M. 082 Papareddy, P. B145 Parada Kusz, M.M. B240 Parent, I. B253 Parente, A.T.D.P.P. B076 Park, C.-K., B325 Park, G. B311 Park, H.-R. B065 Park, J.-H. B152 Park, S.-H. B057 Paschoalato, A.P. B019 Paschoalin, T. B270 Pasparakis, M. B197 Pasricha, T. B020, B096 Paterniti, I. B188 Patricio, E.S. B135 Patton, D. B262 Pavlides, S. B294, B295 Paya´, E.P. B005 Payan, D. B311 Pearson, J. 103 Peckham, D.W. 033 Peer, D. 056 Penido, C. B309 Pequignot, E. B335 Pereira, G.J.S. B005 Pereira, J.J. B166 Pereira, J.A. B290 Pereira, K.M.A. B051, B076, B078, B157, B258 Pereira, T.C.B. B229 Peres, R.S. B180 Perez, P. B195 Pe´rez-Garcı´a, M.T. 059 Perini, A. B296 Perng, D.-W. B287 Perper, S. B310 Perret, L. B252 Perretti, M. 063, B103, B176, B255, B273 Pesquero, J.B. B270 Petasis, N.A. B271 Petelina, T.I. B133, B167 Peterfy, C. 097 Petersen, L. B324 Petrlova, J. B193 Petrof, B.J. 065 Petroni, R.C. B089 Pewzner-Jung, Y. B017 Philbin, M. B036 Philibert, R.A. 037 Philpot, E. B261 Philpott, M. B195 Pica, R. 047 Piccinelli, A.C. B154, B177 Pichavant, M. B282 Pimenta, A.T.A. B076 Pinheiro, N.M. B296, B299 Pinkerton, J.W. B300 Pinto, A.C. B257 Pinto, I.R. B258 Pinto, M.C.X. B148 Pinto, V.P.T. B078, B157, B258 Pioli, P.A. B216
Piquereau, J. B170 Pirozzi, G. B041 Pitchford, S. B288 Pizzino, G. B037 Plastira, J. B100 Pochapski, J.A. B177 Polo, J.M. B102 Pomerantzeff, P. B165 Porter, D. B335 Porto, B.N. B233 Poskerova, H. B075 Pot, C. B035 Pouliot, M. B055, B077 Poulsen, J.H. B333 Powell, C. 083 Prada, P.O. B101 Prado, C.M. B296, B299 Prado, M.A.M. B296, B299 Prado, R. B096 Prado, V.F. B296, B299 Prestwich, G.D. B039, B040 Prevost, T. 120 Primikyri, A. 082 Prinjha, R. 038 Privat, S. B091, B331 Proulx, C. B170 Ptacek, J. B331 Pugh, M. B328 Pulsipher, A. B039, B040 Pupo, M.T. B249 Purcell, R. B273 Q Qin, S. B050 Quaresma, P.G. B101 Queiroz-Junior, C.M, B307 R Rachid, M.A. B060, B307 Racke, M. 100 Rajamaki, K. B117 Ramachandra, M. B313 Ramadas, R.A. B241 Ramakrishnan, S. 046 Ramalingam, T. 054, B020, B082, B095, B096 Ramanathan, A. B313 Ramirez Vinolo, M.A. B203 Ramon, S. 062, B125, B127 Ramos-Ju´nior, E.S. B085 Ranger, P. B050 Rao, N.K. B313 Rao, N. B039 Rao, N. B295 Rao, S. B328 Raphael, S. B259 Rashid, H. B196 Rasmussen, L.J.H. B333 Rathinam, V. 017 Rathod, S.B. B313 Rayner, K.J. 044 Rebelatto, M.C. B110 Rechberger, G. B008 Reed, S.G. B084 Reeh, P. 059 Regan-Komito, D. B056
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S246 Rehal, S. B031 Reicher, H. B008 Reico, C. B052 Reis, M.M. B165, B166 Renauld, J.-C. B282 Riani, A. B269 Ribault, C. B114 Ribeiro de Carvalho, P. B251 Ribeiro, K.A. B258 Ribeiro, M.C. B290 Ribeiro, S.C.M.B087 Ricardo da Silva, F.Y. B184, B185 Riccardi, C. 035, 036 Richard, G. B049 Richards, J.L. B102 Ricklin, D. 082 Riester, K.A. 102 Rietdorf, J. B214 Rigato, O. B306 Riggs, J.E. B128 Rios, F.J. B101 Risitano, A.M. 082 Riteau, N.D. B083, B150 Rivard, J. B004 Robert, S. B097, B114 Robinson, G. B022 Robson, S.C. B208 Rocha, G.C. B085 Rocha, T. B290 Rodrigues Garbin, S. B184, B185 Rodrigues, F.E.A. B078 Rodrigues, M.M. B217 Rogai, F. 047 Rogers, S. B298 Rohrbeck, L., B235 Rojas, M. B079 Roland, C. B267 Rolland Fourcade, C. B268 Rollet-Labelle, E. B077 Romano-Silva, M.A. B317 Ropero, D. B251 Roque, N.R. B214 Rosa, D.V. B317 Rosado, M. B298 Rosas, E.C. B312 Ross, A.K. 009 Rossi, F. 067 Rossios, C. B294, B295 Rossmann, C. B008 Rossoni, L.V. B172 Rothlin, C. 015 Rowe, A. B294 Rowe, D.C. B110 Roy, S. 046 Rubattino, R. B327 Ruitenberg, M. B175, B178 Ruscher, R. B302 Russell, K. B295 Russo, R.C. B060, B064, B307 Rutgeerts, P. B253 Ruzek, M. 119 Ryan, M.E. B039 S Sa, Y.A.J. B304 Saad, M.S. B101
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Inflamm. Res. Saadi, J. B277 Sabbione, F. B234 Sabo, T. B289 Saclier, M. 065 Sacramento, E.K. B317 Sagong, B. B327 Saito, R. B014 Saito, S. B070 Sakai, N. B138 Salant, D. 083 Saldiva, P.H. B299 Saleh, R.A. B153 Sales, D.S. B269, B314 Salgado, R.M. B087 Salles, T.S. B142 Salomao, R. B306 Salvemini, D. 106 Samadfam, R. B196 Samajdar, S. B313 Sanchez Crespo, M. 013 Sa´nchez, A. 059 Sandri, T.L. B072 Sang, H.-W. B073 Sannomiya, P. B187 Santana, F.P.R. B296, B299 Santana, P.T. B208 Santi-Gadelha, T. B157, B258 Santos, A.R.D. B158 Santos, E.W. B140 Santos, E.P.A. B160 Santos, R.A.S. B317 Santos, R.A.S. B148 Santos, R.D.S. B158 Santus, W. B210 Sanz, M.J. B232 Sapoznikov, A. B289 Saraiva-Romanholo, B.M. B071 Sasaki, S.D. B269 Sasaki, S. B063 Sasso, E.H. 096 Sata, M. B173 Satoh, M. B107 Sattler, W. B008, B100 Satyanarayanan, S.K. B276, B277 Sauer, J.M. 126 Saunders, M. 118 Savage, J.R. B039, B040 Savarino, V. 047 Savill, J. B259, B332 Savio, L.E.B. B208 Savion, O. B032 Sawchenko, P.E. B175 Scharfstein, J. B085 Schaub, R. 088 Schepman, P. B036 Schett, G. B012 Schettini, J.A.C. B081 Scheuplein, F. 088 Schiding, J. B111 Schiffmann, S. B017 Schif-Zuck, S. B276, B277 Schindler, S. B244 Schmitz, C. B163 Schneider, C. B105 Schofield, Z.V. B099 Schopf, L.R. 099
Schumacher, J. B322 Schwartz, A. B310 Sciurba, J. B096 Scott, H.A. B053, B106 Scribano, M.L. 047 Seliger, N. B289 Sen˜arı´s, R. 059 Seong, J. 082 Serduc, R. B002 Serezani, C.H. B007, B109, B183, B207 Serhan, C.A. B271 Serhan, C.N. 014, 062, B125, B127, B275 Serra, M.F. B305 Servant, M.J. B077 Severin, M. 100 Sevilla, L. B195 Sevilla, R. B022 Shah, S. B265 Shahaf, G. B146 Shang, L. 115 Sharf, T. B162 Sharma, A. 102 Sharma, N.K. B306 Shaw, M. B111 Shaw, P.A. B335 Sher, A. B083, B150 Sher, L. 118 Sher, N. B276 Sheridan, J.P. 102 Shichino, S. B093 Shim, R. B209 Shimojima, C. B254 Shinozaki, Y. B138 Shirinian, L. B110 Shramko, Y. B131 Silva Jr., C.D. B081 Silva, A.L.N. B118 Silva, A.A.R. B258 Silva, A.G.S. B305 Silva, B.V. B257 Silva, C.L.M. B208 Silva, D.P.B. B156 Silva, D.P.B. B245 Silva, E. B306 Silva, F.R.L.D. B078, B158 Silva, F.S. B309 Silva, J.M. B060 Silva, J.R. B180 Silva, J.S. B054 Silva, L.F.F. B129 Silva, M.S. B290 Silva, P.M.R. B304, B305 Silva, R.V. B160 Silva, R.C. B144 Silva, T.S. B245 Silva, T.A. B060 Silveira, J.A.M. B006 Silver, P.B. 030 Simo˜es, R.L. B126 Simon, B. B113 Simon, F. B079 Sims, G.P. B110 Sinflo´rio, D.A. B119 Singh, R. B311 Singh, T.P. B198 Siracusa, R. B188
Inflamm. Res. Si-Tahar, M. 060, B282 Sites, B.D. B216 Siu, S. B311 Skoblov, Y.S. 107 Sleep, D. B034 Smaili, S.S. B005 Smallwood, M.J. B120, B330 Smith, A. B279 Smith, D.F. B105 Smith, D.E. 054, B082 smith, H.K. B059, B190 Smith, R.J. 082 Soeberdt, M. B011 Soken-Nakazawa, J.S. B069 Sokolove, J. 115 Solito, E. B176 Sollon, C.S. B286 Somerville, J. B128 Sonego, F. B064 Song, S.T. B334 Sonis, S.T. B028, B298 Sorcini, D. 035, 036 Sorensen, B. 083 Soriano, F.G. B089, B112 Souers, A. B310 Sousa, F.C.F.D. B258 Sousa, L.P. B307 Sousa, L.T. B051 Souza, D.G. 093 Souza, G.F. B233 Souza, P.R.S. B103 Spector, S. 118 Spits, H. 128 Spolidorio, L. B172 Spreadbury, I. B268 Springman, E. B151, B261 Squadrito, F. B037 Sriram, R. B326 St. Hilaire, C. B018 St.Pierre, C.A. B241 Stanley, D. B102 Stappenbeck, T. 049 Starkey, M. R. B230, B300, B301, B302 Stauber, J. B092 Stein, R.T. B233 Steinberg, G. 044 Sterk, P.J. B294 Sternemann, C. B011 Stevenson, C.S. 042 Stewart, J. B261 Stewart, K. B330 Stow, J.L. B323 Straumann, A. B105 Stricker-Krongrad, A. B191, B228 Strijbosch, A. B132 Stukalova, O. B162 Sturniolo, G.C. 047 Su, X. B212 Su, X. B083 Suarez, M. B204 Suarez-Farinas, M. 074 Sulciner, M.L. B125, B127 Sumarokov, A.B. B171 Summergrad, P. B236 Sun, K.-T. B226 Sunil, V.R. B293
S247 Surewaard, B.G. B232 Sutcliffe, S. B262 Suzuki, K. B070 Suzuki, M. B137 Swanson, B.N. B041 Sweet, M.J. 021, B323 Syvannarath, V. B211 Szymkowski, D.E. B304 T Tabas, I. 123, B213 Tager, A.M. 112 Tainaga, Y. B137 Tajada, S. 059 Takatori, S. B137 Takenaka, S.-I. B068 Takeuchi, T. 077, B070 Talavera, A. 059 Talbot, J. B033 Tanaka, J. B246 Tanimoto, A. B108 Tanioka, T. B137 Tarr, J. B329 Tashima, A.K. B306 Tasmo-Amorim, N. B200 Tati, R. B048 Taubel, J. 083 Tavares de Lima, W. B184, B185, B187 Tavares, J.C. B148 Tavares, L.P. B307 Tavares, N.M. B204 Taylor, E. B120, B329 Taylor, L. B052 Taylor, V. B311 Teachey, D.T. B335 Teixeira, A.H. B076, B078 Teixeira, L.H. B076, B078 Teixeira, M.M. 093, B060, B064, B307, B317 Teixeira, S.A. B159, B172 Teixiera, M.V.S. B158 Thakral, D. B084 Thanabalasuriar, A. B232 Thannickal, V.J. 053 Thomas, R. B302 Thompson, R. 054, B082, B095 Thorburn, A. B303 Tian, N. B041 Tibau, F. B045 Tibe´rio, I.F.L.C. B296, B299 Ting, K. 022 Toldi, G. B012 Tonussi, C.R. B245 Topping, D.L. B102 Torres, G. B128 Torres, L.C. B081 Toyama, M.H. B005 Trevani, A.S. B234 Tritapoe, J. B029 Tsai, V.W. B175 Tsokos, G.C. B025 Tsukui, T. B093 Tuimala, J. B117 Turchetti-Maia, R.M.M. B161 U Uchitel, I.A. 109, B171
Udeh-Momoh, C. B195 Ueha, S. B093 Ujjinamatada, R.K. B313 Uno, K. B069 Urban, L.A. 124 Uthaman, G. B054 Uyanik, M.S. B023, B024 V Vadas, M.A. 022 Vago, J.P. B307 Vai, M. B210 Vaishnaw, A. 083 Val, D.R.D. B051, B076, B078, B157, B158, B258 Valencia, J.C. B074 Vale´rio, T.S. B250, B260 Valim, Y.L. B019 Vallance, B.A. B238 Vallejos, A. B079 Vallie`res, L. B055 Valova, S. B075 Van, T. B151 Van Dam, B. B028 Vandeghinste, N. B253 Van den Bossche, J. B164 van der Velden, S. B164 Van Goethem, E. B149, B280 van Griensven, M. 082 Vannella, K.M. 054, B082, B096 Vanners, S.J. B268 van Panhuys, N. 030 Vargaftig, B.B. B184, B185 Vasconcelos, A.S. B258 Vasconcelos, J. 120 Vasquez, Y.R. B288 Vayas, K.P. B293 Vayssie`re, B. B252 Vecchi, M. 047 Velagapudi, R. B244 Veldman, T.M. B016 Velichko, S. B091 Venosa, A. B322 Verdu, E. B267 Vergnolle, N. B267, B268 Verkaar, F. B132 Vermeire, S. B253 Viana, F. 059 Viana, M.N. B021 Victoni, T. B114 Vieira, A. B203 Vieira, A.T. B317 Vieira, L.V. B157 Vignali, D.A.A. 070 Villablanca, E.J. B240 Villacampa, N. B206 Vincent, A. 120 Viney, J. 098 Vinhal, D.C. B156 Viola, J.P. B142 Vital, S. B190 Vlahov, I.P. B328 Voets, T. 059 Vokurka, J. B075 von der Weid, P.-Y. B031 von Gunten, S. B105
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S248 Von Ranke, N.L. B045 Vorobyova, L. B236 Voss, J. 119 Voynov, V. B110 W Wada, K. B247 Wada, T. B138 Waegell, W.O. B016 Wagers, A.J. 085 Wagner, D.D. 122 Wakayama, H. B189 Wallace, J.L. 105, B225 Walts, A. B018 Wanecq, E. B149 Wang, H. 082 Wang, L.C. B310 Wang, R. 104 Wang, S. B007, B109, B183 Wang, Y. B041 Ward, E. B110 Ward, P.A. 080 Warner, G. 083 Wegner, G. B004 Wehrli, M. B105 Weinblatt, M.E 114 Weiss, S. B335 Weitz-Schmidt, G. B058 Wen, L. B115 Wenceslau, C.F. B172 Wenzel, S. 118 Werneck, M.B. B142 Wersto, R. B047 Westerbeck, A. B262 Westmorland, S. B310 Westrick, E. B328 Weyrich, A.S. B143 Wheeler II, L B328 Wheeler-Jones, C.P. B273 White, G.E. B052 White, J.H. B218 White, S. 054, B082 White, S. B095 Whiteman, M. B159 Wicks, I. 020 Wicks, I.P. 018 Wieck, A. B324 Willard, V.P. 009
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Inflamm. Res. Willecke, K. B017 Willis, D. B266 Wilson, N.J. 020 Wilson, S.M. B106 Winkler, J.W. B271 Winyard, P.G. B120, B329, B330 Witaicenis Fantinati, A. B108 Witko-Sarsat, V. 029 Wolfe, J.D. 118 Wong, C.H. B209 Wood, M. B159 Woodruff, T.M. B099 Woolf, C.J. 058 Wright, M. B195 Wu, Y. B208 Wynn, T.A. 054, B020, B082, B095, B096
Yi, S. B311 Yildiz, M. B001 ¨ . B085 Yilmaz, O Yokota, T. B014 Yoo, D.H.B334 Yoon, C.-H. B015 Yoshida, M. B266 Yoshizaki, K. B069 Yoshizaki, K. B299 You, F. B328 You, X. 102 Young, A. B092 Young, C. B311 Yu, Z. B018 Yucius, K. 083 Yurkow, E. J. B322
X Xie, R.D. B206 Xing, Y. B201 Xiong, P. B073 Xu, G. B186, B227 Xu, L. B054 Xu, Z.-Z. B325
Z Zamboni, D.S. B118, B119 Zamore, P. 044 Zanchetta, I.A. B314 Zanoni, I. B210 Zauderer, M. 088 Zeissig, S. 089 Zemska, O. B256 Zhang, D. B041 Zhang, H.H. B198 Zhang, R.-H. 067 Zhang, W. B073 Zhang, W. B022 Zhang, Y. B115 Zhang-Hoover, J. B022 Zhao, Y. 022 Zheng, T.S. B098 Zhong, M. B191, B228 Zhou, Q.B018 Zhu, M. B073 Zhu, W. B086 Zielstorff, M. B022 Ziff, E.B. B154, B177 Zimmerman, G.A. B143 Zoheir, K.M.A. B010 Zou, L. B186, B227 Zuckerman, A. B032 Zu¨rcher, A. B105 Zutshi, A. 009 Zykov, K.A. 107
Y Yagita, H. B066, B301, B303, B303 Yagnik, D. B202 Yamada, K. B254 Yamada, M. B137 Yamamoto, A. B189, B254, B318 Yamamoto, N. B189 Yamaoka, K. B070 Yan, D. B227 Yanai, H. B181 Yancopoulou, D. 082 Yang, C.-S. B090 Yang, D. B018 Yang, L. B047 Yang, L. 065 Yang, X. B053, B106 Yao, H. B115 Yap, W.H. B222 Yap, Y.A. B102 Yasuda, H. B138 Ye, Y.-X. B169 Yeager, M.P. B216 Yen, R. B311
