Inflamm. Res. (2013) 62:981–990 DOI 10.1007/s00011-013-0656-6

Inflammation Research

ORIGINAL RESEARCH PAPER

Myeloperoxidase deficiency in mice exacerbates lung inflammation induced by nonviable Candida albicans Mizuki Homme • Nao Tateno • Noriko Miura Naohito Ohno • Yasuaki Aratani

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Received: 20 May 2013 / Accepted: 6 August 2013 / Published online: 18 August 2013 Ó Springer Basel 2013

Abstract Objective This study aimed to evaluate the effect of myeloperoxidase (MPO) deficiency on lung inflammation induced by nonviable Candida albicans (nCA). Methods Mice were inoculated intranasally with nCA, and accumulation of neutrophils and macrophages in the bronchoalveolar lavage fluid was analyzed by flow cytometry. The levels of macrophage inflammatory protein 2 (MIP-2), keratinocyte-derived chemokine (KC), tumor necrosis factor (TNF)-a, and interleukin (IL)-1b in the lung were measured by ELISA. Production of MIP-2 and KC from neutrophils and macrophages was quantified in vitro. MIP-2 mRNA expression in the neutrophils was analyzed by real-time reverse transcription-PCR, and the extent of phosphorylation of ERK1/2 and Syk in the neutrophils was analyzed by Western blotting. Results The MPO-/- mice that received nCA showed more severe pneumonia than wild-type mice. Within 12 h of nCA administration, MPO-/- mice had significantly higher numbers of alveolar neutrophils and increased production of MIP-2 and KC relative to the responses seen in wild-type mice. Neutralization of MIP-2 and KC in vivo significantly reduced neutrophil infiltration. In vitro, production of MIP-2, but not that of KC, was enhanced in the

Responsible Editor: Mauro Teixeira. M. Homme  N. Tateno  Y. Aratani (&) Graduate School of Nanobioscience, Yokohama City University, Seto 22-2, Kanazawa, Yokohama 236-0027, Japan e-mail: [email protected] N. Miura  N. Ohno Laboratory for Immunopharmacology of Microbial Products, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan

nCA-stimulated neutrophils from MPO-/- mice, concomitant with up-regulation of Syk and ERK1/2. At 1 and 3 days after nCA administration, MPO-/- mice had significantly higher lung concentrations of TNF-a and IL-1b than wild-type mice. Conclusion Pulmonary administration of nCA produced an altered inflammatory response in MPO-/- mice relative to wild-type mice. Enhanced MIP-2 production by MPO-/- neutrophils may at least partly contribute to exacerbated inflammation in mutant mice. Keywords Inflammation  Myeloperoxidase  Neutrophil  Candida

Introduction The increase in fungal infections over the last few decades, particularly with normally commensal or nonpathogenic fungi, has prompted renewed interest in elucidating the mechanisms of protective host anti-fungal immunity. Candida albicans is the most common opportunistic fungal pathogen in humans. The identification of innate recognition systems has been a focus in many laboratories. Several lines of evidence suggest that reactive oxygen species (ROS) derived from neutrophils are the main cellular component of the immune system responsible for defense against fungal infection [1]. Myeloperoxidase (MPO) [2, 3] is found mainly in neutrophils and to a lesser degree in monocytes; MPO produces a strong oxidant, hypochlorous acid (HOCl), from hydrogen peroxide (H2O2) and chloride ion (Cl-). We previously reported that this enzyme is essential for killing invading microorganisms in mice and that intranasal infection with C. albicans and other fungi results in a more

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severe pneumonia in MPO-deficient (MPO-/-) mice than in wild-type controls [4–8]. Impaired ROS production by neutrophils has previously been shown to cause an abnormal inflammatory response. Mice deficient in phagocyte NADPH-oxidase, which lack superoxide (O2-) production by phagocytes, developed exaggerated lung inflammation when intratracheally challenged with either sterile Aspergillus fumigatus [9] or zymosan, a stimulatory cell wall extract of Saccharomyces cerevisiae that is composed mainly of b-glucan [10]. Enhanced cutaneous inflammatory reactions to A. fumigatus [11] and ultraviolet exposure [12] have been observed in mutant mice. These studies suggest that impairment in O2production results in neutrophil dysfunction and causes an abnormal inflammatory response. We have previously reported that intranasal administration of zymosan to MPO-/- mice causes a more severe neutrophilic lung inflammation than in wild-type mice, and that MPO-/- neutrophils produce a greater amount of macrophage inflammatory protein (MIP)-2 in vitro than do wild type, which may modulate neutrophil accumulation during lung inflammation [13, 14]. These studies suggest that not only O2- but also HOCl derived from neutrophils is a factor that modulates inflammatory responses. Zymosan has been widely used to study immune function both in vitro and in vivo and is often used as a representative fungal particle, although it does not reflect the true complexity of intact fungal cell walls. In this study, we investigated the response of MPO-/mice to nonviable C. albicans (nCA). Intranasal administration of nCA produced a more severe pulmonary inflammation with a distinctive neutrophilic infiltrate in MPO-/- mice compared to wild-type mice. The severe lung inflammation in the mutant mice was associated with a significantly higher number of alveolar neutrophils and increased levels of MIP-2, keratinocyte-derived chemokine (KC), tumor necrosis factor (TNF)-a, and interleukin (IL)1b relative to wild-type mice. These data suggest that the absence of MPO can result in altered progression of lung inflammation even with nonviable Candida.

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Preparation of nCA Candida albicans NBRC 1385 purchased from the National Institute of Technology and Evaluation Biological Resource Center (Chiba, Japan) was maintained on Sabouraud agar medium (Difco) at 27 °C and transferred once every 3 months. A C-limiting medium originally described by Shepherd and Sullivan [15] was used to grow C. albicans. Five liters of medium was placed in the glass jar of a microferm fermentor (Sakura Seiki, Japan) and C. albicans was cultured at 27 °C with aeration as previously described [16]. Cells were collected by centrifugation and were killed with ethanol, and then dried with acetone. The resulting nCA was suspended in phosphate-buffered saline (PBS). Induction of lung inflammation by nCA administration Mice were anesthetized by intraperitoneal injection of 200 mg/kg of 2,2,2-tribromoethanol (Sigma-Aldrich). Wild-type and MPO-/- mice were challenged intranasally with 1 9 107 cells of nCA in a volume of 40 ll of PBS. Lung histology Mice were killed by cervical dislocation, and the lungs were removed and fixed in 10 % buffered formalin. For light microscopy, tissues were fixed overnight, dehydrated in graded ethanol solutions, embedded in paraffin, sectioned at 2 lm, and stained with hematoxylin and eosin (H&E) using standard protocols. For quantification of inflammation, two independent investigators microscopically examined five well-separated cross-sections per mouse, recording results for each of the following criteria: (1) alveolar and interstitial edema, (2) alveolar hemorrhage, and (3) infiltration of leukocytes. Each criterion was scored on a scale of 0–3 as described previously [17], where 0 = normal, 1 = mild change, 2 = moderate change, and 3 = severe change. The scores for criteria were summed to represent the inflammation score. Bronchoalveolar lavage fluid preparation and immunocytochemistry

Materials and Methods Animals Experiments on 8- to 12-week-old C57BL/6 mice were performed in accordance with the guidelines of Yokohama City University, Japan. C57BL/6 mice were purchased from Japan SLC (Hamamatsu, Japan). MPO-/- mice were generated as previously described [4]. All animals were housed under specific pathogen-free conditions.

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Lungs were lavaged in situ with ten instillations of 1 ml of PBS containing 1 mM EDTA. The bronchoalveolar lavage (BAL) fluid suspensions were pooled and kept on ice until centrifugation at 2609g for 5 min at 4 °C, and the resulting cell pellets were resuspended in PBS. Total cell counts were determined using a hemocytometer. FITC-conjugated antiGR1 (Ly-6G) was used to characterize pulmonary neutrophils. FITC-conjugated anti-CD11c (HL3) was used to identify mouse macrophages. All antibodies were obtained

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from BD Pharmingen. The cells were incubated with monoclonal antibodies for 15 min at 4 °C, then analyzed using a JSAN cell sorter (Bay Bioscience, Japan).

KC levels were measured using ELISA kits as described above. When used, piceatannol (Calbiochem), a Syk inhibitor, was added 1 h before nCA.

Measurement of cytokine and chemokine levels

Real-time reverse-transcription-PCR

At various time points after the nCA challenge, lungs were harvested and homogenized in PBS in the presence of protease inhibitors (Protease Inhibitor Cocktail, SigmaAldrich). The homogenates were centrifuged at 19,0009g for 10 min to remove cell debris. The supernatants were frozen at -80 °C until assayed. MIP-2, KC, TNF-a, and IL-1b quantifications were performed using murine ELISA kits (R&D Systems) according to the manufacturer’s instructions.

Total RNA was isolated from isolated neutrophils using TRIzol (Gibco BRL Co.) as recommended by the manufacturer. cDNA was synthesized from 0.8 lg of total RNA using a PrimeScript First Strand cDNA Synthesis Kit (TaKaRa), and was then analyzed by reverse transcription-PCR (RT-PCR) using Fast SYBR Green Master Mix (Applied Biosystems) according to the manufacturer’s instructions. Amplification was performed in a StepOnePlus real-time PCR system (Applied Biosystems) with the following thermocycling conditions: 20 s at 95 °C, followed by 40 cycles of 3 s at 95 °C, 30 s at 62.7 °C, and 15 s at 95 °C. Relative gene expression was normalized to b-actin using the change in threshold cycle (Ct) method, CtMIP-2–Ctb-actin. Specific primer sets used for the murine MIP-2 and the b-actin housekeeping gene are as follows: MIP-2 sense, 50 -CACTCT CAAGGGCGGTCAA-30 ; MIP-2 antisense, 50 -AGGCACAT CAGGTACGATCCA-30 ; b-actin sense, 50 -CATCCGTAAA GACCTCTATGCCAAC-30 ; b-actin antisense, 50 -ATGGAG CCACCGATCCACA-30 .

Effect of MIP-2 and KC antibodies on neutrophil migration induced by nCA Mice were injected intranasally with a monoclonal IgG against MIP-2 (50 lg per lung; R&D Systems) and KC (50 lg per lung; R&D Systems) at the same time as nCA challenge. Neutrophil migration into the alveoli was measured 12 h after nCA administration. In vitro stimulation of macrophages with nCA

Western blot analysis Approximately 10 ml of BAL fluid was harvested per mouse, resulting in the isolation of 2 to 3 9 105 macrophages per animal. The alveolar cells were washed with RPMI 1640 medium (Nissui Pharmaceutical, Tokyo, Japan), followed by cell counting and differential cell analysis. In both wild-type and MPO-/- mice, more than 90 % of the cells present in BAL fluid were macrophages. Macrophages were cultured at a concentration of 1 9 106 cells/ml in RPMI 1640 medium supplemented with 10 % fetal bovine serum (FBS) for 3 h in a 96-well culture plate (Nalge Nunc International), in the presence or absence of 5 9 106 cells of nCA. Isolation of bone marrow neutrophils from the femora of donor mice Neutrophils were purified using a 3-layer Percoll gradient (72, 62, and 52 %; Sigma-Aldrich) and washed twice with PBS. Approximately 85 % of the isolated cells were morphologically identified as neutrophils by Diff-Quick (Sysmex) in both wild-type and MPO-/- mice. The isolated neutrophils were then cultured at a concentration of 3 9 106 cells/ml in RPMI 1640 medium supplemented with 10 % FBS for 3 h in a 96-well culture plate in the presence or absence of 5 9 106 cells of nCA. MIP-2 and

Whole-cell lysates were prepared by harvesting the cells after culturing them in the presence or absence of nCA for 30 min. The cells were solubilized in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer with 2 % dithiothreitol. Samples were separated by 13 % SDS-PAGE, and gels were transferred to PVDF Transfer Membrane (Millipore). The membrane was blocked with PVDF Blocking Reagent for Can Get Signal (Toyobo, Japan), incubated overnight with primary antibody, and then incubated for 1 h with a 1:20,000 diluted secondary antibody conjugated to horseradish peroxidase. The reaction products were visualized using an enhanced chemiluminescence Western blotting detection system (Thermo Scientific). The primary antibodies included antiextracellular signal-regulated kinase 1/2 (ERK1/2), antiphospho-ERK1/2, anti-spleen tyrosine kinase (Syk), and anti-phospho-Syk (Cell Signaling Technology). Statistical analysis Data are presented as mean ± SD. Statistical comparison between two groups was performed with a 2-tailed Student’s unpaired t test. P \ 0.05 was considered statistically significant.

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Results MPO deficiency enhances lung inflammation after intranasal nCA administration Untreated lungs showed negligible or minimal signs of inflammation in both wild-type (Fig. 1a, panel a) and MPO-/- mice (panel c). The lungs of wild-type mice at 6 d after intranasal administration of nCA showed only localized areas of inflammation (panel b). In contrast, there was prominent airway accumulation of inflammatory cells in the lungs of MPO-/- mice (panel d and e). Compared with the wild-type mice, the lung inflammation scores in the

Fig. 1 a Lung pathology observed in mice 6 days after intranasal administration of nCA. Lung tissues were obtained from wild-type (a and b) and MPO-/- (c–e) mice exposed to 1 9 107 nCA for 6 days (b and d, 910). Lung sections not exposed to nCA (a and c, 910) were also stained with H&E as a control. Higher magnification (9100) of the same section as panel d is shown in panel e. b Inflammation scores of histology sections. The severity of inflammation in the wild-type (black circles) and the MPO-/mice (white circles) was determined as described in ‘‘Materials and methods’’. Four mice were used for each group. c Number of BAL fluid cells recovered from the lung after intranasal inoculation with nCA. Wild-type (black bars) and MPO-/- mice (white bars) were inoculated intranasally with nCA and analyzed 12 and 24 h later. Four wild-type and six MPO-/- mice were used. In b and c, results are mean ± SD. *P \ 0.05, **P \ 0.001 compared to wild-type exposed mice

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MPO-/- mice were significantly and time-dependently elevated up to 6 d after nCA administration (Fig. 1b). The total number of BAL fluid cells showed obvious differences between wild-type and MPO-/- mice as early as 12 and 24 h after nCA exposure, and approximately 16-fold greater numbers of cells had already accumulated by 24 h in MPO-/- mice compared with wild type (Fig. 1c). Treatment of both wild-type and MPO-/- mice with the PBS control had no effect on cell number (data not shown). The Gr-1high? neutrophils were the predominant cells in both wild-type and MPO-/- alveoli (Fig. 1c), indicating that intranasal administration of nCA causes neutrophil-mediated lung inflammation, and that the lack of MPO enhances neutrophil accumulation.

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Fig. 3 Effect of MIP-2 and KC antibodies on the development of lung inflammation. MPO-/- mice were injected intranasally with MIP-2 antibody, KC antibody, or both at the same time as nCA challenge. BAL fluid cells were recovered from the mice 12 h after nCA administration, and the number of Gr-1high? neutrophils was determined using flow cytometry. Data are mean ± SD. *P \ 0.05 compared to control without antibody injection

Fig. 2 Production of MIP-2 and KC in the lung of wild-type and MPO-/- mice after nCA exposure. Lung tissues were collected from wild-type (black circles) and MPO-/- (white circles) mice at 6 h, 12 h, and 1, 3, and 6 days after nCA exposure, and then homogenized. MIP-2 (a) and KC (b) levels were measured using specific ELISA kits. At least four mice were used for each group. Results are mean ± SD. *P \ 0.05, **P \ 0.001 compared to wild-type mice

MIP-2 and KC contribute to nCA-induced neutrophil migration in the early phase of inflammation To elucidate the mechanisms by which MPO-/- mice developed a more severe lung inflammation in response to nCA, we determined the protein levels of MIP-2 (Fig. 2a) and KC (Fig. 2b) in lung homogenates 6 h, 12 h, and 1, 3, and 6 days after injection. A strong and significant increase in the amount of both chemokines was observed by 12 h, followed by a decrease. Strikingly, MIP-2 and KC levels were approximately 3.6- and 4.5-fold higher, respectively, in the lungs of MPO-/- mice compared to those of wildtype mice at 12 h after nCA treatment.

The involvement of these chemokines in nCA-induced neutrophil migration into the lung was investigated using specific antibodies. MPO-/- mice were intranasally injected with MIP-2 antibody, KC antibody, or both at the same time as nCA challenge, and the number of neutrophils infiltrating into the alveoli was determined 12 h later. Although treatment with each of these antibodies had no significant effect, treatment with both antibodies resulted in a significant reduction in the number of BAL fluid neutrophils (Fig. 3), strongly suggesting that both chemokines produced by the lung at least partly contribute to neutrophil infiltration. These results suggest that the neutrophil-mediated inflammation observed after nCA administration is at least partly dependent on MIP-2 and KC, and that a higher production of these chemokines in the lung of MPO-/mice contributes to severe inflammation. MPO deficiency enhances nCA-stimulated production of MIP-2, but not KC, by neutrophils There is accumulated evidence that lung inflammation is due to activation of lung phagocytic cells, which produce numerous chemokines [18, 19]. We determined the production of MIP-2 in vitro in isolated macrophages and neutrophils

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Fig. 4 Production of MIP-2 and KC by neutrophils and macrophages in vitro. Bone marrow neutrophils and alveolar macrophages prepared from wild-type (black bars) and MPO-/- mice (white bars) were cultured with (plus) or without (minus) nCA for 3 h. The culture medium was collected and the levels of MIP-2 (a, n = 5) and KC (b, n = 4) were determined by ELISA. ND not detected, *P \ 0.05

(Fig. 4a). Analysis of supernatants of the cultured macrophages demonstrated that the production of MIP-2 was induced by nCA treatment, indicating that the alveolar macrophages are a source of MIP-2 in the lung exposed to nCA. Strikingly, stimulation of MPO-/- neutrophils with nCA in vitro for 3 h led to a 13-fold greater production of MIP-2 compared to wild-type neutrophils. To confirm that the increase in MIP-2 protein release from nCA-stimulated neutrophils was due to an increase in mRNA expression, we stimulated the neutrophils with nCA for 3 h to evaluate mRNA expression by real-time RT-PCR. Stimulation of MPO-/- neutrophils with nCA led to a significantly greater expression of MIP-2 than in the stimulated wild-type neutrophils (Fig. 5), indicating that the up-regulation of MIP-2 is regulated at the transcriptional level. In contrast, production of KC from both macrophages and neutrophils was extremely low (Fig. 4b). Moreover, in contrast to the production of MIP-2, there was no significant difference in the production of KC between the neutrophils with different genotypes. These data suggest that neither macrophages nor neutrophils were the major source of KC. nCA-stimulated MIP-2 production is dependent on the Syk/ERK pathway Pharmacologic Syk inhibition resulted in significantly reduced MIP-2 production (Fig. 6), suggesting that the Syk

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Fig. 5 MPO deficiency enhances MIP-2 gene expression in neutrophils stimulated with nCA in vitro. Bone marrow neutrophils prepared from wild-type (black bars) and MPO-/- mice (white bars) were cultured with (plus) or without (minus) nCA for 3 h. Total RNA was isolated from neutrophils and then analyzed by real-time RT-PCR as described in ‘‘Materials and methods’’. Data are mean ± SD of four independent experiments. *P \ 0.05

signaling pathway is required for MIP-2 production by mouse neutrophils stimulated with nCA. Intriguingly, we found that nCA-stimulated phosphorylation of Syk and ERK1/2 in MPO-/- neutrophils was higher than that observed in the wild type (Fig. 7a), suggesting that MPO deficiency up-regulates the activation of these kinases. The inhibition of Syk by piceatannol was accompanied by the suppression of ERK1/2 phosphorylation (Fig. 7b), suggesting that Syk acts upstream of ERK1/2 after nCA stimulation. Collectively, these results suggest that up-regulation of the Syk/ERK pathway in MPO-/- neutrophils leads to an enhanced production of MIP2, which may at least partly contribute to the severe lung inflammation observed in the MPO-/- mice exposed to nCA. Enhanced lung inflammation in MPO-/- mice is associated with higher lung concentrations of TNF-a and IL-1b To determine whether the differences in the inflammatory response to nCA between wild-type and MPO-/- mice observed by histologic examination were associated with differences in production of other cytokines, we determined the protein levels of TNF-a and IL-1b in the lung homogenates. As shown in Fig. 8, both cytokines showed very low levels in untreated mice, with no significant

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Fig. 6 Inhibition of Syk prevents nCA-induced MIP-2 production. Bone marrow neutrophils prepared from wild-type (black bars) and MPO-/- mice (white bars) were pretreated with piceatannol, a Syk inhibitor, for 1 h followed by exposure to nCA for 3 h. MIP-2 levels of four mice with each genotype were determined by ELISA. Results are expressed as mean ± SD. *P \ 0.05 compared to the piceatannol-untreated sample in each genotype

differences observed between wild-type and MPO-/- mice. Although obviously increased in both wild-type and MPO-/- mice at 1 day after instillation, the production of TNF-a and IL-1b in MPO-/- mice was nearly 3.3- and 7.1fold, respectively, higher than levels seen in wild-type animals. By 3 days, the levels of both cytokines had dropped nearly to baseline levels in wild-type mice, while those in MPO-/- mice were even higher than at 3 days. These data suggest that higher concentrations of these proinflammatory cytokines in the lungs of MPO-/- mice may have been involved in the progression of lung inflammation observed in the mutant mice.

Discussion Previous work in our laboratory has demonstrated that MPO-/- mice develop an invasive bronchopneumonia after respiratory challenge with viable C. albicans [4]. In this study, we have shown that administration of nCA produced a severe lung inflammation in MPO-/- mice, indicating that viable organisms are not always a prerequisite for the development of inflammation in the mutant mice. We have shown that both MIP-2 and KC levels were 3.6- and 4.5-fold higher, respectively, in the lungs of MPO-/- mice compared to those of wild-type mice at an early stage (12 h) of nCA challenge (Fig. 2), and that neutralization of both chemokines by their specific antibodies led to significantly reduced lung inflammation (Fig. 3). These results strongly suggest that the underlying mechanisms responsible for the pathologic response to nCA in MPO-/- mice include aberrant production of MIP-2 and KC. Because MIP-2 and KC are 63 % identical

Fig. 7 Inhibition of Syk prevents nCA-induced phosphorylation of ERK1/2. a Bone marrow neutrophils prepared from wild-type and MPO-/- mice were stimulated with (plus) or without (minus) nCA for 30 min. Western blotting was carried out with antibodies against phospho-Syk (p-Syk), total Syk (Syk), phospho-ERK1/2 (p-ERK1/2), and total ERK1/2 (ERK1/2). b Wild-type (black bars) and MPO-/neutrophils (white bars) were pretreated with the indicated concentrations of piceatannol for 1 h and subsequently stimulated with (plus) or without (minus) nCA for 30 min. Cellular levels of p-ERK and were then determined by Western blotting. Representative blots and the ratios of the band intensities of p-ERK1/2 to those of ERK1/2 are shown. Results are expressed as mean ± SD of four independent experiments. *P \ 0.05 compared to the piceatannol-untreated sample in the presence of nCA in each genotype

in terms of amino acid sequence [20] and both bind to the same chemokine receptor, CXCR2 [21], their functional effects are similar and additive. Therefore, it is plausible that neutralization of both chemokines is required to prevent neutrophil recruitment. Our previous studies have clearly demonstrated that both alveolar macrophages and infiltrating neutrophils are sources of MIP-2 in both wild-type and MPO-/- mice at an early stage after intranasal administration of zymosan, and

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Fig. 8 Production of TNF-a and IL-1b in the lung of wild-type and MPO-/- mice after nCA exposure. Lung tissues were collected from wild-type (black circles) and MPO-/- (white circles) mice at 1, 3, and 6 days after nCA exposure, and then homogenized. TNF-a (a) and IL1b (b) levels were measured using specific ELISA kits. Four mice were used for each group. Results are mean ± SD. *P \ 0.05, **P \ 0.001 compared to wild-type mice

that, intriguingly, MPO-/- neutrophils produced larger amounts of MIP-2 than did wild-type neutrophils, in vitro, in response to zymosan [13, 14]. Similarly, in the present study, we showed that MPO-/- neutrophils produced larger amounts of MIP-2 than did wild-type neutrophils, in vitro, in response to nCA (Figs. 4a, 5). Together, these data indicate that MPO deficiency up-regulates the production of MIP-2 by neutrophils irrespective of fungal species. Because an early initial secretion of MIP-2 can promote the recruitment of additional neutrophils, the greater production of this chemokine by mutant neutrophils provides a possible explanation for the rapid accumulation of neutrophils in the lungs of mutant mice. Inhibition of Syk activity significantly diminished MIP-2 production by neutrophils (Fig. 6) and ERK activity (Fig. 7b).

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Dectin-1 [22] and Dectin-2 [23] are the specific receptors for fungal b-glucans and a-mannans, respectively. These receptors in dendritic cells induce cytokines in a signaling pathway involving Syk kinase [23–25]. Microbe-mediated stimulation of Dectin-1 results in cytokine/chemokine production, including MIP-2, in cultured macrophages [26]. Complement receptor 3 (CR3) [27] and lactosylceramide [28] also recognize C. albicans, and signaling via CR3 results in the production of some inflammatory cytokines such as TNF-a in a Syk-dependent manner [29]. Because neutrophils express these receptors [30], it is plausible that nCA induces the production of MIP-2 by mouse neutrophils through one or some of these receptors. It remains to be elucidated how MPO deficiency up-regulates the Syk/ERK pathway. We observed that the KC level in the nCA-challenged MPO-/- mice was also significantly higher than in the wild type (Fig. 2b). However, production of this chemokine by neutrophils and macrophages was very low in vitro (Fig. 4b), and we found no significant difference between the neutrophils with different genotypes in the production of KC in response to nCA (Fig. 4b). These data suggest that, unlike the case of MIP-2, neither macrophages nor neutrophils were the major source of KC in mouse lung exposed to nCA. Because several other cell types including epithelial cells [31, 32] have been identified as sources of KC, such cell types in the lung may produce the chemokine abundantly in response to nCA. Further study is needed to identify the source of KC. The levels of MIP-2 and KC in the nCA-challenged MPO-/- mice peaked at 12 h followed by a decrease, and they reached nearly to baseline levels before 3 days (Fig. 2a, b). Nevertheless, the MPO-/- mice showed a continuous development of inflammation at least up to 6 days after nCA administration (Fig. 1a, b), suggesting that, in addition to MIP-2 and KC, additional cytokines or chemokines may contribute to the development of a late stage (after 12 h) of inflammation observed in the mutant mice. We found that the lung concentrations of TNF-a and IL-1b were significantly higher in the MPO-/- mice than in the wild-type mice at 1 and 3 days after nCA administration (Fig. 8). Since TNF-a and IL-1b are potent cytokines that play an important role in acute and chronic lung inflammatory diseases by inducing the production of various chemokines, growth factors, and adhesion molecules [33, 34], these proinflammatory cytokines may contribute to the late stage of inflammation observed in the MPO-/- mice. Ramos et al. [35] demonstrated that administration of MIP-2 to the peritoneum of mice induces TNF-a production. A similar mediator cascade may lead to a continuous neutrophil influx into the lung following nCA challenge of the MPO-/- mice. Several pathogens, particularly Candida, are sometimes problematic in MPO-deficient patients [3]. Indeed, we have previously reported that MPO-/- mice are sensitive to

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respiratory infection by opportunistic fungi such as C. albicans [4] and Cryptococcus neoformans [5], and that they cause a severe neutrophilic lung inflammation. Similarly, we demonstrated in this study that instillation of nCA into the lung resulted in a severe neutrophil-rich pneumonia. This is the first experimental evidence that the absence of MPO can be associated with the development of inflammatory lesions even without an inciting Candida infection. Our studies also suggest that the underlying mechanisms responsible for the pathologic response to nonviable Candida in MPO-/- mice include aberrant production of proinflammatory cytokines and chemokines. Thus, the mutant mice have significant abnormalities in both host defense and inflammation, confirming the importance of the neutrophil-derived HOCl in innate immunity. Acknowledgments We thank Minami Sugimura for technical support. This work was supported in part by JSPS KAKENHI Grant number 23580406, and a grant from the Japanese Ministry of Health, Labor and Welfare.

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