Dig Dis Sci DOI 10.1007/s10620-015-3807-5

ORIGINAL ARTICLE

Deficiency of Group VIA Phospholipase A2 (iPLA2b) Renders Susceptibility for Chemical-Induced Colitis Li Jiao1,2 • Johannes Inhoffen2 • Hongying Gan-Schreier2 • Sabine Tuma-Kellner2 Wolfgang Stremmel2 • Zhiwei Sun1 • Walee Chamulitrat2

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Received: 12 February 2015 / Accepted: 9 July 2015 Ó Springer Science+Business Media New York 2015

Abstract Background Inflammatory bowel disease results from a combination of dysfunction of intestinal epithelial barrier and dysregulation of mucosal immune system. iPLA2b has multiple homeostatic functions and shown to play a role in membrane remodeling, cell proliferation, monocyte chemotaxis, and apoptosis. The latter may render chronic inflammation and susceptibility for acute injury. Aims We aim to evaluate whether an inactivation of iPLA2b would enhance the pathogenesis of experimental colitis induced by dextran sodium sulfate. Methods iPLA2b-null male mice were administered dextran sodium sulfate in drinking water for 7 days followed by normal water for 3 days. At day 10, mice were killed, and harvested colon and ileum were subjected for evaluation by histology, immunohistochemistry, and quantitative RT-PCR. Results Dextran sodium sulfate administration caused a significant increase in histological scores and cleaved caspase 3 (?) apoptosis concomitant with a decrease in colon length and crypt cell Ki67 (?) proliferation in iPLA2b-null mice in a greater extent than in control Electronic supplementary material The online version of this article (doi:10.1007/s10620-015-3807-5) contains supplementary material, which is available to authorized users. & Zhiwei Sun [email protected] & Walee Chamulitrat [email protected] 1

Department of Toxicology, School of Public Health, Jilin University, Changchun, China

2

Department of Internal Medicine IV, University of Heidelberg Hospital, Heidelberg, Germany

littermates. This sensitization by iPLA2b deficiency was associated with an increase in accumulation of F4/80 (?) macrophages, and expression of proinflammatory cytokines and chemokines, while the number of mucin-containing goblet cells and mucus layer thickness was decreased. Some of these abnormalities were also observed in the ileum. Conclusions An inactivation of iPLA2b exacerbated pathogenesis of experimental colitis by promoting intestinal epithelial cell apoptosis, inhibiting crypt cell regeneration, and causing damage to mucus barrier allowing an activation of innate immune response. Thus, iPLA2b may represent a susceptible gene for the development of inflammatory bowel disease. Keywords iPLA2b  Inflammatory bowel disease  Dextran sodium sulfate  Colitis  Apoptosis  Regeneration Abbreviations AB–FR Alcian blue–fast red Bcl-2 B cell lymphoma 2 Bcl-x Bcl-2-like protein 1 CD Crohn’s disease DSS Dextran sodium sulfate ER Endoplasmic reticulum H&E Hematoxylin–eosin IBD Inflammatory bowel disease IEC Intestinal epithelial cell IHC Immunohistochemistry iPLA2b Group VIA calcium-independent iPLA2 KO Knockout LC/MS– Liquid chromatography mass spectrometry MS mAb Monoclonal antibody MIP-1a Macrophage inflammatory protein-1a

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MIP-1b MCP1 LPA LPC PPARc qRT-PCR TNF-a UC WT

Macrophage inflammatory protein-1b Monocyte chemotactic protein1 Lysophosphatidic acid Lysophosphatidylcholine Peroxisome proliferator-activated receptor c Quantitative real-time polymerase chain reaction Tumor necrosis factor-a Ulcerative colitis Wild-type

Introduction Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn’s disease (CD), is a relapsing inflammatory disorder that results from abnormal crosstalk between commensal microbes and the local immune system [1]. Although the etiology of IBD remains unclear, it results from a combination of dysfunction of intestinal epithelial barrier and dysregulation of the mucosal immune system [2]. A number of studies in animal colitis models and IBD patients have shown that increased intestinal epithelial cell (IEC) apoptosis [3], impaired crypt regeneration [4, 5], and the loss of goblet cells [6, 7] contribute to the breakdown of epithelial barrier integrity. The dysfunction of epithelial barrier often occurs at early stages of this disease, leading to bacterial invasion to the mucosa where they could induce inflammation [8]. However, the molecular mechanisms underlying IEC apoptosis, crypt regeneration impairment, and goblet cell loss during the development of IBD remain poorly understood, particularly, regarding susceptible genes. iPLA2b is one of the phospholipase A2 (PLA2) family members which hydrolyze the sn-2 substituent of membrane phospholipids to release a free fatty acid and a lysophospholipid [9]. It has been shown that iPLA2b is an essential regulator of a variety of cellular activities including cell proliferation [10], eicosanoid metabolism [11], chemotaxis [12], and insulin secretion [13]. Despite the anticipated dysfunction caused by iPLA2b deficiency, iPLA2b-null mice with the deletion of the lipase-containing exon 9 live normally. However, they have defects in glucose metabolism [14], and they gain less body weights [15] and develop neuroaxonal dystrophy [16] as they age 1–2 years. Interestingly, a study by Lauber et al. [17] has shown that iPLA2b is involved in apoptotic cell clearance by generating lysophosphatidylcholine (LPC) which acts as a ‘‘find-me’’ signal for attracting monocytes. Moreover, iPLA2b-derived lysophosphatidic acid (LPA) has been found to play an essential role in the migration of

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monocytes [18]. These results indicate that iPLA2b expression has a critical role in the clearance of apoptotic cells and immune functions, thus contributing to homeostatic host defense in the body. iPLA2b functions have been reported in pancreatic islets [14], bone [15], and brain [16]. We have recently reported that iPLA2b-null mice were more susceptible for concanavalin A-induced autoimmune hepatitis than control littermates, and this was accompanied with an increased apoptosis in the intestine [19]. However, the direct role of iPLA2b in the gastrointestinal tract has not been studied. In view of homeostatic role of iPLA2b in apoptosis and inflammation, we hypothesize that iPLA2b might be involved in the pathogenesis of IBD. In this study, we reported that iPLA2b deficiency promoted dextran sodium sulfate (DSS)-induced colitis, and this was associated with an induction of IEC apoptosis, impairment of epithelial regeneration, and goblet cell loss. These events led to a breakdown of the intestinal epithelial barrier and secondary inflammatory response which enhanced the susceptibility to colitis. Our data demonstrate the protective role of iPLA2b in mouse intestine.

Materials and Methods Animals iPLA2b-null (iPLA2b-/-) mice with C57BL/6 background were kind gifts from Dr. John Turk (Washington University School of Medicine, St. Louis, MO, USA), and further breeding with C57BL/6 mice was performed to 20 generations prior to use. Genotyping was performed according to the published work [14]. Male iPLA2b-/- and wild-type (WT with iPLA2b?/? phenotype) mice at an age of 8–14 weeks were used in our study. All mice were bred and housed at the animal facility of the University Heidelberg. In some experiments, C57BL/6 male mice as WT controls were obtained from Charles Rivers (Sulzfeld, Germany). Chemical-Induced Colitis Model For an acute colitis induction, mice received 2.5 or 3 % DSS (40 kilodaltons, cat#42867, Sigma, Taufkirchen, Germany) added to drinking water for 7 days, followed by normal tap water for 3 days. Mice were weighed daily throughout the course of experiment. At the 10th day, mice were euthanized, and intestine and colon tissues were harvested. All animal experiments were approved by the Animal Care and Use Committee of the University of Heidelberg.

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iPLA2 Activity Assay

Histology and Immunohistochemistry (IHC)

Intestine samples were homogenized in PBS, and homogenates were obtained following centrifugation at 5000g for 15 min. The substrate arachidonyl phosphatidylcholine used at 125 lg was dissolved in 95 ll H2O and 95 ll iPLA2 assay buffer (300 mM NaCl, 0.5 % Triton X-100, 60 % glycerol, 4 mM EGTA, 10 mM HEPES, and 2 mg/ml BSA). This 190 ll substrate mixture was added with 10 ll intestinal homogenates and incubated at room temperature for 1 h. To stop the enzymatic reaction, 10 ll of 25 mM 5,50 dithiobis-2-nitrobenzoic acid containing 4.75 mM EGTA was added. Following a 5-min incubation at room temperature for color development, an absorbance at 405 was measured with a Multiscan Ascent microplate reader (Thermo Fischer Scientific, Langenselbold, Germany). An absorbance change was calculated according to A415/min = A415(sample) - A415(blank)/60 min. iPLA2 activity in U (lmol/min/ml) was calculated according to A415/ min 9 (0.2 ml/0.01 ml)/(10.66 mM-1) 9 sample dilution, which was normalized to U/g protein.

Human colon slides were provided by the tissue bank of the National Center for Tumor Disease (NCT, Heidelberg, Germany) in accordance with the regulations of the tissue bank and the approval of the ethics committee of the University of Heidelberg. Intestine and colon specimens from mice were fixed in 10 % formalin at room temperature for 18 h, embedded in paraffin, and cut into 5-lm sections. Sections were stained with hematoxylin and eosin (H&E) for histology. Histological changes were graded according to the previously described method [22]. The sections were graded in a blinded manner with a scoring system according to the level of inflammation (ranging from 0 to 3 from acute to chronic), the extent of inflammation (ranging from 0 to 3 for mucosa, submucosa to transmural), the extent of crypt damage (ranging from 0 to 4 for basal 1/3 damaged, basal 2/3 damaged, only surface epithelium intact and entire crypt and epithelium lost), regeneration (ranging from 4 to 0), and percent involvement (ranging from 1 to 4 for 1–25, 26–50, 51–75, and 76–100 %). Histological scores were the combined numbers of the grading scores from 1 to 18. For IHC procedure, sections were deparaffinized and rehydrated with ethanol and xylene, and were heated to 98 °C in 10 mM citrate buffer (pH 6.0) for 20 min. Endogenous peroxidase was blocked by treating sections with 3 % H2O2 for 10 min at room temperature prior to an exposure to a primary antibody overnight at 4 °C. Primary antibodies used for IHC were iPLA2b (1:100; cat# ab103258, Abcam), iPLA2b (1:1000; cat# sc-166616, Santa Cruz Biotechnology), cleaved caspase 3 (1:200; cat# 9664, Cell Signaling), Ki67 (1:100; cat# ab16667, Abcam), F4/80 (1:100; cat# ab11110, Abcam), CD3 (1:100; cat# ab5690, Abcam), and CD45R (1:100; cat# ab64100, Abcam). Mouse IgG (1:1000; cat# sc-2025) or rabbit IgG (1:100; cat# sc-2027) from Santa Cruz Biotechnology was used as a negative control. IHC staining of cleaved caspase 3 and F4/80 was carried out by using an Avidin–Biotin Complex kit (cat# ab64261, Abcam). For iPLA2b (Abcam), Ki67 and CD3 staining, sections were incubated with a goat anti-rabbit (cat# ab6721, Abcam), and for iPLA2b (Santa Cruz Biotechnology) staining, sections were incubated with goat anti-mouse (cat# sc2005, Santa Cruz Biotechnology), and for CD45R staining, sections were incubated with goat anti-rat secondary antibody (cat# ab7010, Abcam) for 1 h at room temperature. Positive staining was detected by diaminobenzidine. Slides were counterstained with hematoxylin prior to mounting. Light microscopy was used to visualize stained cells with an Olympus AX 70 microscope. For computerized image analysis, the number of stained cells on a surface

LPC Analysis by LC/MS–MS Phospholipid standards LPC 18:1, LPC 18:0, LPC 16:0, and PC 28:0 were purchased from Avanti Polar Lipids (Alabaster, AL). Methanol and chloroform were of HPLC grade from Merck (Darmstadt, Germany), and ammonium acetate was of the highest analytical grade available from Fluka (Buchs, Switzerland). Lipid extraction was performed in 12-ml glass tubes according to Bligh and Dyer procedure [20] in the presence of 20 ng PC 28:0 as an internal standard. The chloroform phase was dried and dissolved in methanol for quantitative lipid analysis. Mass spectrometric analysis was performed on a Quattro microTM API mass spectrometer (Waters, UK) equipped with a Waters 2695 Separation Module. Data were acquired by using MassLynx V4.1 software. Typical values for capillary and cone voltages were 4.3 kV and 27 V, respectively. The injection volume was 10 ll. The mobile phase consisted of methanol and chloroform (3:1) containing 7 mM ammonium acetate. The flow rate was set to 50 ll/min for 1.6 min, a linear decrease to 20 ll/min for 1.4 min, a linear increase to 50 ll/min for 1.6 min, and a linear increase to 200 ll/min for 1.4 min, and finally at reequilibration with 50 ll/min for the final 1 min. LPC species were analyzed in the positive ion mode using collision energies ranging from 20 to 40 V, respectively [21]. The total LPC levels were the sum of LPC 18:1, LPC 18:0, and LPC 16:0 in ng/mg protein.

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area of epithelial cells was determined by using the Olympus Cell^F software. An evaluation of IHC sections was performed in a blinded manner. Three sections were used for each analysis, and on each section 20 random locations were used for the analysis.

Mucus Thickness Measurements and Goblet Cell Staining For measurement of mucus thickness, chloroform-based Carnoy’s fixative was used to better preserve mucus integrity because formaldehyde fixation has been reported to dehydrate the mucus [23]. A segment of mouse ileum or colon containing fecal materials was dissected and placed into Carnoy’s solution containing 60 % ethanol, 30 % chloroform, and 10 % glacial acetic acid for 24 h at room temperature. Tissues were transferred to 100 % methanol for two 30-min washes and followed by two 20-min washes in 100 % ethanol. The fixed tissues were then incubated twice in xylene for 15 min and subsequently embedded in paraffin. Paraffin sections were cut into 5 lm thickness and stained with Alcian blue–fast red (AB–FR) reagents (Sigma, Taufkirchen, Germany) according to instructions. Images of the stained slides were acquired on an Olympus AX 70 microscope and analyzed using Image J software. Three sections were used for each analysis, and, on each section, 20 random locations were used for mucus layer thickness analysis.

Gene Expression Analyses Total RNA was isolated by using the QIAGEN RNeasy Mini kit (Qiagen, Hilden, Germany). cDNA was synthesized from 2 lg RNA by using a Maxima First Strand cDNA synthesis kit (Fermentas, St. Leon-Rot, Germany). Gene expression was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) using Applied Biosystems TaqManÒ gene expression assays (including those of custom synthesized Bcl-x (L) and Bcl-x (S)) with TaqManÒ Universal PCR Master Mix, and run on an Applied Biosystem 7500. Mouse Bcl-x (L) primer sequences were: F: 50 GCG TAG ACA AGG AGA TGC AG 30 ; R: 50 TGT TCC CGT AGA GAT CCA CA 30 ; P: 50 AAG TGT CCC AGC CGC CGT TC30 . Mouse Bcl-x (S) primer sequences were: F: 50 CAG CAG TGA AGC AAG CGC TGA 30 ; R: 50 AAC CAG CGG TTG AAG CGC TC 30 ; P: 50 TGA ACA GGA CAC TTT TGT GGA TCT CTA CGG G 30 . The expression level of a target gene in quadruplets was calculated using the D–Ct transformation method and determined as a ratio of the target normalized to the house-keeping gene GAPDH.

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Statistics Results were expressed as mean ± SEM. Significance was accepted at p \ 0.05. For a multiple comparison, overall difference was determined by ANOVA. If the result from one-way ANOVA was significant, the difference between individual groups was analyzed by Turkey’s post test. For two group comparison, statistics analysis was determined by Mann–Whitney U test. All analyses were performed by using GraphPad Prism 5.

Results iPLA2b Deficiency Decreases Colonic LPC Levels and Worsens the DSS Effects To demonstrate the significance of homeostatic role of iPLA2b, we performed iPLA2b IHC staining on normal human colon by using two different monoclonal antibodies (mAb) from Abcam (mAb1) and Santa Cruz Biotechnology (mAb2); whereby the former detects a peptide sequence at 558–588 and the latter at 1–120 of human iPLA2b. Cytoplasmic localization of iPLA2b was detected by both antibodies (Fig. 1a). mAb1 detected iPLA2b nearby the nuclei of colonocytes which were on the apical side of the villous. With stronger brown staining, mAb2 detected iPLA2b in the cytoplasm of both colonocytes (indicated by a black arrow) and immune cells (indicated by a red arrow) (Fig. 1a). Appropriate IgG was used as a negative control for each mAb (Fig. 1a). The presence of iPLA2b in IECs and intestinal immune cells in human colon was suggestive of its physiological role in these two cell types. In our mouse model, the genetic deletion of exon 9 in the iPLA2b gene led to a complete absence of iPLA2b mRNA expression (Fig. 1b) and a significantly decreased iPLA2 activity (Fig. 1c) in the colon of iPLA2b-/- (KO) compared with WT mice. Consistent with decreased iPLA2 activity, a decrease in LPC levels was found in the colon of KO mice, and treatment with 2.5 % DSS further decreased LPC levels in a greater extent in KO than in WT mice (Fig. 1d). Following treatment of WT and KO mice with 2.5 or 3 % DSS for 7 days, both groups lost their body weight from day 5 onwards (Fig. 1e). All mice developed diarrhea and gross blood in their stool. At day 7, both WT and KO mice treated with 2.5 or 3 % DSS had severe colitis characterized by diarrhea and bloody stool associated with a body weight loss of 16 and 20 %, respectively. Upon DSS removal and feeding mice with normal water for further 3 days, the colon of KO mice at day 10 displayed a significant shrinkage compared with that of WT mice (Fig. 1f). At day 10, WT mice regained their body weights

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Fig. 1 iPLA2b deficiency decreases colonic LPC levels and worsens the effects of DSS by causing colonic shrinkage and increasing mortality. Wild-type and iPLA2b-/- mice were treated with 2.5 or 3 % DSS in drinking water for 7 days followed by regular drinking water for 3 days prior to killing at day 10. No DSS in drinking water was the control (CON) group. a iPLA2b IHC of human colon was analyzed by using a monoclonal antibody from Abcam (mAb1) and from Santa Cruz (mAb2). Positive DAB-brown staining was observed in colonocytes (indicated by a black arrow) and immune cells (indicated by a red arrow). These IHC was a representative picture from three samples of normal human colon. b qRT-PCR analysis of iPLA2b expression in the colon of WT and iPLA2b-/- (KO) mice (N = 6–7 per group). c iPLA2 activity (U/g protein) of the colon of

WT and KO mice (N = 14 per group). d Colonic LPC levels (ng/mg protein) of CON or 2.5 % DSS-treated WT and KO mice (N = 6–8 per group). e The body weights of WT and KO treated with either 2.5 % DSS (left panel) or 3 % DSS (right panel) were monitored over 10 days. The percentage (%) was calculated based on the original body weight. f Colon length in cm of CON, 2.5 % DSS-, or 3 % DSStreated WT and KO mice. g Mortality rates were monitored as % survival in WT and KO mice treated with 2.5 % DSS (left panel) and 3 % DSS (right panel). Data are mean ± SEM (in e–g, N = 6 for CON group, N = 6–7 for 2.5 % DSS group, and N = 4 for 3 % DSS group). #p \ 0.05 or ###p \ 0.001 versus WT; *p \ 0.05 or **p \ 0.005 versus DSS-treated WT. Scale bar 100 lm

while the mutant mice continued to lose weights with 23 and 33 % weight loss for WT and KO mice, respectively (Fig. 1e). Furthermore, iPLA2b-/- mice were more severely affected by DSS, leading to a 50 % survival rate compared with 86 % for WT mice in the 2.5 % DSS treatment group (Fig. 1g). Similar pattern was observed in the 3 % DSS treatment group with a 50 and 75 % survival rate for KO and WT mice, respectively. Taken together, these results provided a compelling evidence that

iPLA2b-/- mice were significantly more susceptible to DSS-induced colitis than control littermates. iPLA2b Deficiency Increases Colonic Damage, Apoptosis, and Defective Regeneration The protective role of iPLA2b in mouse colon was further confirmed by histological analysis. DSS administration of KO mice resulted in an increase in colonic ulceration

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associated with larger areas of epithelial crypt loss, and inflammatory cell infiltration throughout the mucosa. The histological scores reflected a significant difference in DSS-induced pathological changes with iPLA2b deficiency (Fig. 2a). The rapid turnover of IECs because of vigorous proliferation of epithelial progenitors is thought to be an

Fig. 2 iPLA2b deficiency increases colonic mucosal damage associated with a defect in epithelial regeneration and an induction of apoptosis in response to DSS. DSS treatment protocol was the same as described in Fig. 1. a Representative H&E staining (left panel) and histological scores (right panel) of the colon of WT and KO mice treated with either 2.5 % DSS or 3 % DSS. b Representative Ki67 IHC staining (left panel) and Ki67 (?) quantification (right panel) of the colonic crypts of CON, 2.5 % DSS-, or 3 % DSStreated WT and KO mice. c Representative cleaved caspase 3 IHC staining (left panel) and its quantification (right panel) of the colon of CON, 2.5 % DSS-, or 3 % DSStreated WT and KO mice. Data are mean ± SEM (N = 6 for CON group, N = 6–7 for 2.5 % DSS group, and N = 4 for 3 % DSS group). *p \ 0.05 or ***p \ 0.001 versus DSStreated WT. Scale bar 100 lm

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important defensive mechanism by expelling colonized pathogens, confining bacterial spreading, and localizing inflammation [24, 25]. We used Ki67 staining to examine crypt cell proliferation in colonic tissue sections. With DSS treatment, Ki67 (?) proliferating cells were abundant at the bottom of virtually all colonic crypts of control mice,

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whereas Ki67 (?) cells were nearly absent in the colonic crypts of KO mice (Fig. 2b). The severe mucosal damage and markedly suppressed cell proliferation suggest that iPLA2b-deficient colonic epithelium was unable to renew itself and failed to recover from DSS insult. An abnormal intestinal barrier has been noted in patients with IBD and in experimental colitis [2, 26]. It has been suggested that IEC apoptosis is one of the major contributors to the disruption of intestinal epithelial integrity [27, 28]. Indeed, intestinal samples from patients with UC or CD showed a 25-fold increase in active caspase 3-positive IEC compared with those from control patients [29]. To determine whether iPLA2b deficiency disrupts the intestinal barrier after DSS treatment through apoptosis induction, we examined colonic apoptosis. We observed an increased number of cleaved caspase 3-stained colonocytes in DSS-treated iPLA2b-/- mice when compared to WT controls (Fig. 2c). However, the mRNA expression of antiapoptotic protein Bcl-2 remained similar among DSStreated WT and KO mice (Supplementary Fig. 1A); hence the modulation of Bcl-2 was not involved in the sensitization of colonic injury by iPLA2b deficiency. It has been suggested that abnormal endoplasmic reticulum (ER) stress is a critical contributor to IEC apoptosis and IBD development [30]. Administration of DSS upregulated the colonic mRNA expression of ER-stress markers GRP78 and PUMA, but no difference in expression level was observed among DSS-treated WT and KO mice (Supplementary Fig. 1B and C). Anti-apoptosis protein Bcl-x, which belongs to the Bcl-2 family, has been shown to be regulated by iPLA2b by inducing Bcl-x alternative splicing, leading to ER stress during thapsigargin-induced apoptosis in b-cells [31]. To further explore whether an alteration of Bcl-x splicing could contribute to the observed susceptibility, we examined colonic mRNA expression of Bcl-x (L) and Bcl-x (S). We observed no difference in the expression level of these genes as well as the Bcl-x (L)/Bcl-x (S) ratio in DSS-treated WT and KO colons (Supplementary Fig. 1D-F). These results excluded a possibility that ER stress and Bcl-x alternative splicing could contribute to observed susceptibility to DSS-induced colitis in iPLA2b-deficient mice. iPLA2b Protects Colonic Mucus Layer Integrity by Stabilizing the Retention of Goblet Cells Colonic epithelium is covered by an outer mucous layer inhabited by microbiota and an inner mucous layer adherent to IECs which is impervious to bacteria [32]. Mucus is continuously secreted into the lumen by goblet cells, highlighting the protective role of goblet cells and their secreted mucin. It has been noted that a decrease in number of goblet cells and thinner mucus layer has been

observed in patients with IBD [33] and in experimental colitis [7]. We further investigated whether the dysfunction of mucus layer and goblet cells could contribute to the observed susceptibility in iPLA2b-deficient mice. By ABFR staining, the colon of DSS-treated WT mice was filled with goblet cells as seen by blue staining of mucin, and an inner mucus layer could be observed (marked with red ‘‘I’’ in Fig. 3a, left panel). On the other hand, the colon of DSStreated mutant mice contained less number of mucin-containing goblet cells showing crypts devoid of blue-stained goblet cells (marked with red ‘‘*’’ in Fig. 3a, right panel). By way of quantification, the number of mucin-containing goblet cells was reduced in KO colons (Fig. 3b), and the thickness of a continuous inner mucus layer was also drastically decreased (Fig. 3c). Thus, iPLA2b ablation in response to DSS caused a dysfunctional mucus barrier by decreasing the numbers of goblet cells and forming thinner mucus layers. iPLA2b Deficiency Increases DSS-Induced Inflammatory Response in the Colon To determine whether iPLA2b affects the infiltration of inflammatory cells in DSS-induced colitis, we measured F4/80 a marker for macrophages, CD11b and CD11c markers for dendritic cells, CD45R a B cell marker, and CD3 a T cell marker in the colon. We found a marked increase in infiltrating F4/80 (?) macrophages in the lamina propria (indicated by arrows in Fig. 4a) and F4/80 mRNA expression (Fig. 4b) of DSS-treated KO mice. This was concomitant with increased CD11b and CD11c mRNA expression supporting the role of macrophages and dendritic cells in the DSS and iPLA2b-deficiency sensitization (Fig. 4b). On the contrary, an infiltration of CD45R (?) and CD3 (?) cells was found to be similar among WT and KO mice treated with 3 % DSS (Fig. 4c, d). This indicated that T and B cells did not play an essential role in the observed colitis aggravation by the deficiency. Thus, iPLA2b ablation caused severe colitis after DSS treatment driven by an innate rather than adaptive immune response. We further determined whether iPLA2b-deficiency dependent injury from activated immune cells by DSS was associated with increased expression of inflammatory cytokines and chemokines. In a greater extent in KO compared with WT mice, DSS treatment increased colonic expression of TNF-a, IL-1b, IL-6, macrophage inflammatory protein-1a (MIP-1a), macrophage inflammatory protein-1b (MIP-1b), and monocyte chemotactic protein1 (MCP1) (Fig. 5). It is known that iPLA2b catalyzes phosphatidic acid to LPA [18], which is an important lipid mediator in chemotaxis. In order to delineate possible role of iPLA2b-derived LPA during DSS and iPLA2b-deficiency sensitization, we analyzed the expression of an

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Dig Dis Sci Fig. 3 iPLA2b protects colonic mucus layer integrity by stabilizing the retention of goblet cells. DSS treatment protocol was the same as described in Fig. 1. a Representative AB/Fast Red staining of the colonic sections of CON or 3 % DSS-treated WT and KO mice. ‘‘I’’ in red indicates inner mucus layer. ‘‘*’’ in red indicates a decreased number of mucin-containing goblet cells. b Quantification of the number of AB/Fast Red (?) or mucin-containing goblet cells in the colon of CON, 2.5 % DSS-, or 3 % DSS-treated WT and KO mice. c Quantification of inner mucus layer (red ‘‘I’’ indicated in a) thickness in lm of CON, 2.5 % DSS- or 3 % DSS-treated WT and KO mice. Data are mean ± SEM (N = 6 for CON group, N = 6–7 for 2.5 % DSS group, and N = 4 for 3 % DSS group). ***p \ 0.001 versus DSStreated WT

intracellular LPA receptor peroxisome proliferator-activated receptor c (PPARc) [34]. DSS treatment caused a significant increase in PPARc mRNA expression in WT colon (Fig. 5c), and there was a delay in this increase in the colon of DSS-treated KO mice. DSS-Induced Colitis by iPLA2b Deficiency Applies to the Ileum As Well DSS treatment of WT mice caused no significant histological abnormalities in the distal ileum showing nearly normal appearing villi (Fig. 6a). However, this treatment

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caused a severe damage to the ileum of iPLA2b-/- mice with a disturbed mucosal architecture, denuded epithelium, exposure of lamina propria, and shorter villi (Fig. 6a). Consistently, the number of goblet cells was dramatically decreased in the ileum of DSS-treated mutants as seen by AB-FR staining (Fig. 6b). Without any effects in the WT ileum, DSS treatment of KO mice increased the staining of cleaved caspase 3 in the ileum found to be located in the crypts (Fig. 6c). Although iPLA2b deletion enhanced the sensitivity of IECs to DSS-induced apoptosis, the crypt cell proliferation remained to be the same in the ileum of WT and KO mice after DSS challenge (Supplementary Fig. 2).

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Fig. 4 iPLA2b ablation causes increased infiltration of innate immune cells. DSS treatment protocol was the same as described in Fig. 1. a Representative F4/80 IHC staining (indicated by arrows in left panel) and F4/80 (?) quantification (right panel) of the colonic sections of CON, 2.5 % DSS- or 3 % DSS-treated WT and KO mice. b qRT-PCR analysis of F4/80, CD11b, and CD11c mRNA expression in the colon of CON, 2.5 % DSS- or 3 % DSS-treated WT and KO

mice. c Representative CD45R IHC staining of CON or 3 % DSStreated WT and KO mice. d Representative CD3 IHC staining of the colon of CON or 3 % DSS-treated WT and KO mice. Data are mean ± SEM (N = 6 for CON group, N = 6–7 for 2.5 % DSS group, and N = 4 for 3 % DSS group). #p \ 0.05, ##p \ 0.01 or ### p \ 0.001 versus WT; *p \ 0.05, **p \ 0.01 or ***p \ 0.001 versus DSS-treated WT. Scale bar 100 lm

Discussion

[35]. The removal of DSS in drinking water stimulates colonic progenitors to proliferate to completely renew IECs within 3–4 days [36]. The highly proliferative nature of colonocytes enables an efficient repair and regeneration after DSS injury. Mice with a heterozygous deletion of Kru¨pple-like factor 5, a transcription factor responsible for the proliferation of crypt cells, have been shown to exhibit increased sensitivity to DSS due to a slower proliferation rate of epithelial cells in injured mucosal region [37]. Moreover, the rapid epithelial turnover ability is essential in host defense regarding an expelling of invasive intestinal pathogens [38]. Therefore, if these mechanisms are impaired during epithelial renewal, there is an increased risk of epithelial injury, microbial infection, and secondary inflammation. We observed a significant decrease in colonic proliferation in iPLA2b-null mice relative to WT controls after DSS administration. This decrease may be relevant to a greater degree of crypt damage and epithelial apoptosis in these mutant mice, which further leads to a decrease in epithelial progenitors during injury phase. This

In this study, we provided evidence that iPLA2b, which is localized in intestinal epithelium and immune cells, plays a critical role in intestinal homeostasis and that an inactivation of iPLA2b exacerbated clinical activity and colonic pathology of DSS-induced acute colitis. By way of mechanisms, we demonstrated that iPLA2b deficiency promoted the effects of DSS by inducing (1) IEC apoptosis and inflammation mediated by innate immune cells, (2) defect in epithelial regeneration, and (3) dysfunction of goblet cells leading to breakdown of the mucosal barrier. This sensitization was also observed in the ileum bolstering the protective role of iPLA2b in the distal gastrointestinal tract. We found that DSS treatment in drinking water caused nearly no damage to the liver with an exception of one iPLA2b-/- mouse showing hepatic necrosis (data not shown). Hence, DSS exhibits specificity in causing damage to the colon showing colonic shrinkage, the loss of epithelial integrity, and the breakdown of mucosal barrier

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Fig. 5 iPLA2b deficiency sensitizes DSS-induced inflammatory response in the colon. DSS treatment protocol was the same as described in Fig. 1. a qRT-PCR analysis of inflammatory cytokines TNF-a, IL-1b, and IL-6 in the colon of CON, 2.5 % DSS-, or 3 % DSS-treated WT and KO mice. b qRT-PCR analysis of inflammatory chemokines MIP-1a, MIP-1b, and MCP1 in the colon of CON, 2.5 %

DSS-, or 3 % DSS-treated WT and KO mice. c qRT-PCR analysis of PPARc expression in the colon of CON, 2.5 % DSS- or 3 % DSStreated WT and KO mice. Data are mean ± SEM (N = 6 for CON group, N = 6–7 for 2.5 % DSS group, and N = 4 for 3 % DSS group). ##p \ 0.01 versus WT; *p \ 0.05, **p \ 0.005 or ***p \ 0.001 versus DSS-treated WT

can be supported by the findings that DSS affects stem cells and progenitor cells in the base of the crypt [3]. Thus the reduction in colonic proliferation may be due to increased apoptosis of progenitor cells caused by DSS in an iPLA2bdeficient epithelium. Interestingly, it has been shown that decreased iPLA2 activity is necessary for an accumulation of phosphatidylcholine which is essential for a proper cellcycle function [10], and thus an increased proliferation would be expected in iPLA2b-deficient intestinal cells. We surmise that the observed decreased proliferation might be indirectly caused by iPLA2b deficiency through increased IEC apoptosis, likely by the inability of macrophages to remove apoptotic intestinal cells [9, 12, 17]. We found cytoplasmic localization of iPLA2b nearby the nuclei of colonocytes in normal human colon. Further experiments are still warranted to determine iPLA2b localization in the ER and/or mitochondria since both organelles are important in apoptosis. Concurrently, it has been shown that iPLA2b which is localized in both organelles of b cells can confer protection [39] and that iPLA2b deficiency in hepatocytes renders the susceptibility for acute liver injury [19, 40]. In line with IEC apoptosis being

a major pathogenic mechanism of IBD and DSS-induced colitis [3, 4, 26, 27], the observed increased IEC apoptosis caused by iPLA2b deficiency might be the main factor contributing to aggravated colitis induced by DSS. An improper clearance of IEC apoptosis might also contribute to an impairment of intestinal repair during experimental colitis [41]. It has been shown that iPLA2b produces LPC which acts as a chemotactic ‘‘find-me’’ signal attracting phagocytes for the clearance of apoptotic cells [17]. Moreover, iPLA2 activation during apoptosis was shown to promote the exposure of membrane LPC, leading to the binding of IgM antibodies and complement activation [42]. Consistent with the role of phagocytes in an initiation or resolution of inflammation, a report in sepsis patients has shown an inverse association between LPC levels and mortality rates [43]. As we observed decreased LPC levels with iPLA2b deficiency, it is very likely that LPC regulation of IEC apoptosis may be one of the major mechanisms which contribute to the susceptibility toward DSS insult. Interestingly, DSS-treated KO mice further decreased colonic LPC levels, and this may reflect a protective role of LPC in WT colon rendering the ability to recover from

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Fig. 6 iPLA2b deficiency disrupts the intestinal barrier integrity associated with increased IEC apoptosis in the ileum. DSS treatment protocol was the same as described in Fig. 1. a. Representative H&E staining of the ileum of CON or 3 % DSS-treated WT and KO mice. b. Representative AB/PAS staining (left panel) and goblet cell quantification (right panel) of the ileal sections of CON, 2.5 % DSS-

or 3 % DSS-treated WT and KO mice. c. Representative cleaved caspase 3 IHC staining of the ileum of CON or 3 % DSS-treated WT and KO mice. Data are mean ± SEM (N = 6 for CON group, N = 6–7 for 2.5 % DSS group, and N = 4 for 3 % DSS group). ***p \ 0.001 versus DSS-treated WT. Scale bar 100 lm

DSS insult. Our observation was consistent with the previous report showing the decreased LPC levels in the colon of UC patients [44]. Furthermore, LPA produced by iPLA2b has been shown to play an important role in directionality and speed of macrophage migration [18]. Interestingly, an administration of LPA is shown to reduce the severity of DSS-induced colitis [45], and the deletion of the LPA target PPARc [34] worsens DSS-induced colitis [46]. Upon DSS treatment, we found that PPARc mRNA expression in KO colon was lower than that in WT colon, and this may reflect a decrease in LPA contents rendering the inability of KO colon to recover from DSS insult. Furthermore, the exacerbated apoptosis in iPLA2b-deficient colon following DSS treatment was not due to increased ER stress or a modulation of anti-apoptosis protein Bcl-2. The oral administration of DSS stimulates nonlymphoid cells such as IEC and phagocytes, causing the destruction of epithelial layer and inducing monocyte infiltration eventually resulting in the release of proinflammatory cytokines [47, 48]. It has been reported that DSS-induced colitis does not require T and B cells since no difference in DSS-induced colitis is observed in mice lacking T and B

cells [49, 50]. In line with this, we did not observe any difference in T and B cell infiltration in the colon of DSStreated KO mice. This indicates that other cell types involved in an innate immune response might be crucial for the increased colitis severity in iPLA2b-deficient colon. Consistently, we found increased numbers of macrophages and dendritic cells infiltrating into colonic lamina propria of DSS-treated mutants. We suggest that intestinal macrophages and dendritic cells may play an important role in triggering innate immunity under iPLA2b deficiency [51]. DSS-induced colitis is associated with an upregulation of proinflammatory cytokines [52, 53]. Likewise, mutant mice treated with DSS showed an increase in colonic mRNA expression of cytokines and chemokines by six- to eightfold being greater than two- to fourfold seen in DSStreated WT mice. These changes are most likely due to increased apoptosis rates together with the breakdown of epithelial barrier, leading to microflora infiltration into lamina propria which further aggravates inflammatory response. TNF-a is a common pathogenic factor in many models of colitis and is known to trigger apoptosis of epithelial cells [54]. Increased expression of TNF-a in iPLA2b-deficient colon correlates well with increased

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colonic apoptosis and may be one of the contributing factors to the observed increased mucosal cell death in the ileum. Synthesis of IL-1b can be induced by Toll-like receptors in response to bacteria and bacterial fragments [55]. The increased colonic expression of IL-1b in DSStreated mutant mice may be in response to the increased bacterial infiltration caused by a breakdown of intestinal epithelium barrier. In summary, our findings show that iPLA2b deficiency promotes IEC apoptosis, impairs the regeneration of epithelial cells, reduces the numbers of goblet cells, and destroys the integrity of intestinal barrier. This results in hypersensitivity to DSS seen by increased colitis severity, and ileal damage. The data support homeostatic role of iPLA2b in the distal gastrointestinal tract and that an inactivation of iPLA2b contributes to the pathogenesis of IBD. Hence, iPLA2b may be used as a target gene for development of a new strategy for IBD treatment. Acknowledgments We thank Dr. John Turk who provided us with iPLA2b-null mice and genotyping method used in our study. This study was supported by Deutsche Forschungsgemeinschaft Grants (CH 288/6-1 and STR 216/15-3). Conflict of interest

The authors have no conflict of interest.

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