Intestinal Epithelial Cell-Derived Semaphorin 7A Negatively Regulates Development of Colitis via αvβ1

This information is current as Sujin Kang, Tatsusada Okuno, Noriko Takegahara, Hyota of September 23, 2021. Takamatsu, Satoshi Nojima, Tetsuya Kimura, Yuji Yoshida, Daisuke Ito, Saori Ohmae, Dong-Ju You, Toshihiko Toyofuku, Myoung Ho Jang and Atsushi Kumanogoh J Immunol published online 23 December 2011

http://www.jimmunol.org/content/early/2011/12/23/jimmun Downloaded from ol.1102084

Supplementary http://www.jimmunol.org/content/suppl/2011/12/23/jimmunol.110208 Material 4.DC1 http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists by guest on September 23, 2021 • Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published December 23, 2011, doi:10.4049/jimmunol.1102084 The Journal of Immunology

Intestinal Epithelial Cell-Derived Semaphorin 7A Negatively Regulates Development of Colitis via avb1 Integrin

Sujin Kang,*,† Tatsusada Okuno,*,‡ Noriko Takegahara,* Hyota Takamatsu,*,† Satoshi Nojima,* Tetsuya Kimura,* Yuji Yoshida,† Daisuke Ito,*,† Saori Ohmae,*,† Dong-Ju You,x Toshihiko Toyofuku,*,† Myoung Ho Jang,x,{ and Atsushi Kumanogoh*,†

The intestinal immune system is constantly challenged by commensal bacteria; therefore, it must maintain quiescence via several regulatory mechanisms. Although intestinal macrophages (Mws) have been implicated in repression of excessive inflammation, it remains unclear how their functions are regulated during inflammation. In this study, we report that semaphorin 7A (Sema7A), a GPI-anchored semaphorin expressed in intestinal epithelial cells (IECs), induces IL-10 production by intestinal Mws to regulate intestinal inflammation. Sema7A-deficient mice showed severe signs of dextran sodium sulfate-induced colitis due to reduced intestinal IL-10 levels. We further identified CX3CR1+MHC class IIintF4/80hiCD11bhi Mws as the main producers of IL-10 via Downloaded from avb1 integrin in response to Sema7A. Notably, Sema7A was predominantly expressed on the basolateral side of IECs, and its expression pattern was responsible for protective effects against dextran sodium sulfate-induced colitis and IL-10 production by Mws during interactions between IECs and Mws. Furthermore, we determined that the administration of recombinant Sema7A ameliorated the severity of colitis, and these effects were diminished by IL-10–blocking Abs. Therefore, our findings not only indicate that Sema7A plays crucial roles in suppressing intestinal inflammation through avb1 integrin, but also provide a novel mode of IL-10 induction via interactions between IECs and Mws. The Journal of Immunology, 2012, 188: 000–000. http://www.jimmunol.org/

n the intestine, intimate contact continuously occurs between need to be tightly regulated to prevent excessive inflammation in the immune system and exogenous Ags such as diverse mi- response to these innocuous Ags. The breakdown of these regu- I croflora and food. Thus, immune responses in the intestine latory mechanisms leads to development of inflammatory bowel disease (IBD), including Crohn’s disease and ulcerative colitis (1). *Department of Immunopathology, World Premier International Research Center, In the intestine, several immunosuppressive factors, such as thy- Immunology Frontier Research Center, Osaka University, Suita City, Osaka 565- mic stromal lymphopoietin, retinoic acid, TGF-b, and IL-10, 0871, Japan; †Department of Respiratory Medicine, Allergy and Rheumatic Disease, Graduate School of Medicine, Osaka University, Suita City, Osaka 565-0871, Japan; shape the local microenvironment to confer anti-inflammatory by guest on September 23, 2021 ‡Department of Neurology, Graduate School of Medicine, Osaka University, Suita properties (2, 3). Among these, one of the most important and x City, Osaka 565-0871, Japan; Department of Gastrointestinal Immunology, World best-characterized molecules is the immune regulatory cytokine Premier International Research Center, Immunology Frontier Research Center, Osaka University, Suita City, Osaka 565-0871, Japan; and {Division of Integrative Bio- IL-10. Either IL-10– or IL-10R–deficient mice spontaneously sciences and Biotechnology, Pohang University of Science and Technology, Pohang develop colitis in the presence of commensal bacteria (4, 5), and City, Kyoungbuk 790-784, Korea IL-10 is shown to be a pivotal factor in IBD patients (6). Indeed, Received for publication July 21, 2011. Accepted for publication November 20, reduced expression of IL-10 has been demonstrated in the 2011. inflamed regions and granulomas of patients with Crohn’s disease This work was supported by research grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to A.K. and S.K., Research Fellowships for (7). Although various innate and adaptive immune cells can pro- Young Scientists), grants-in-aid from the Ministry of Health, Labor and Welfare, the pro- duce IL-10, increasing evidence suggests that intestinal macro- gram for Promotion of Fundamental Studies in Health Sciences of the National Institute w of Biomedical Innovation (to A.K.), the Target Research Program of the Japan phages (M s) are crucially involved in IL-10–mediated immu- Science and Technology Agency (to T.T. and A.K.), Funding Program for Next Generation nological homeostasis (8–10). IL-10–producing intestinal Mws are World-Leading Researchers (NEXT Program) and Special Coordination Funds for Promot- characterized by lower expression levels of costimulatory mole- ing Science and Technology (to A.K.), and the World Class University program, National Research Foundation, and Ministry of Education, Science, and Technology, Korea. cules (CD40, CD80, and CD86) and lower production of proin- Address correspondence and reprint requests to Dr. Tatsusada Okuno, Dr. Myoung flammatory cytokines (11) and produce IL-10 by recognition of Ho Jang, or Prof. Atsushi Kumanogoh, Department of Neurology, Graduate School of exogenous Ags with pattern recognition receptors, such as TLRs Medicine, Osaka University, 2-2 Yamada-oka, Suita City, Osaka 565-0871, Japan and nucleotide-binding oligomerization domains (8, 12). How- (T.O.), Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang City, Kyoungbuk 790-784, Korea (M.H.J.), or De- ever, it still remains unclear how intestinal intrinsic factors regu- partment of Respiratory Medicine, Allergy and Rheumatic Disease, Graduate School late IL-10 production in Mws. of Medicine, Osaka University, 2-2 Yamada-oka, Suita City, Osaka 565-0871, Japan (A.K.). E-mail addresses: [email protected] (T.O.), jang@ifrec. Semaphorins were originally identified as axon guidance cues osaka-u.ac.jp (M.H.J.), and [email protected] (A.K.) in the nervous system, which are characterized by a conserved The online version of this article contains supplemental material. N-terminal in their extracellular regions. The sem- Abbreviations used in this article: Aldh1a2, retinal dehydrogenase 2; BM, bone aphorin family has been further divided into eight subclasses based marrow; CHS, contact hypersensitivity; DC, dendritic cell; DSS, dextran sodium on additional structural features. Two groups of protein families, sulfate; EAE, experimental autoimmune encephalomyelitis; FAK, focal adhesion kinase; IBD, inflammatory bowel disease; IEC, intestinal epithelial cell; Itga, integrin plexins and neurophilins, have been shown to function as the main a; LP, lamina propria; Mw, macrophage; MHC II, MHC class II; RGD, arginine– receptors for semaphorins. Among them, semaphorin 7A (Sema7A; glycine–aspartate; rSema7A, recombinant semaphorin 7A; Sema7A, semaphorin 7A; also known as CD108) (13), a membrane-associated GPI-linked SP, spleen; Treg, regulatory T cell; WT, wild-type. semaphorin, is unique because it has an integrin-binding motif, Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 arginine–glycine–aspartate (RGD), in its Sema domain. Indeed,

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102084 2 EPITHELIAL CELL-DERIVED Sema7A NEGATIVELY REGULATES COLITIS

Sema7A uses b1 integrin as receptor to promote axon outgrowth level of inflammation with evidence of wall thickening by inflammation; and contributes to the formation of lateral olfactory tracts (14). In and 4, maximal severity of inflammation with transmural leukocyte infil- the immune system, Sema7A, which interacts with a1b1integrin, tration and/or architectural distortion. stimulates inflammatory Mws to produce proinflammatory cyto- Colon organ culture kines (15). In addition, a vaccinia virus semaphorin A39R, a ho- 3 molog of vertebrate Sema7A, binds to plexin-C1 and induces the The 1 1 cm standardized segments of the colon were washed in cold PBS supplemented with penicillin and streptomycin. Epithelial integrity activation of human monocytes in the context of aggregation and was disrupted by treatment with 1 mM EDTA for 30 min at 37˚C on production of cytokines such as IL-6 and TNF-a (16). Accordingly, a shaker, followed by vortexing for 2 min. These segments were cultured Sema7A-deficient mice are defective in T cell-mediated inflam- in 24-well flat-bottom culture plates in serum-free RPMI 1640 medium matory responses in contact hypersensitivity (CHS), experimental (Invitrogen) supplemented with penicillin, streptomycin, and gentamicin. autoimmune encephalomyelitis (EAE), and pulmonary fibrosis mod- After 24 h of cultivation, supernatant fluid was collected and used for estimation of IL-10 and TGF-b secretion. els, indicating that Sema7A exerts an important role in evoking inflammatory immune reactions (14, 15, 17). However, the role of Isolation of lymphocytes and Mws from mouse colon and Sema7A in the intestinal immune system still remains unclear. spleen In this study, we showed that Sema7A ameliorates intestinal LP-lymphocytes and Mws were isolated as described (20), with some inflammation by inducing IL-10 production from intestinal Mws. modifications. Mice were sacrificed, and colons and spleens were removed Sema7A-deficient mice displayed more susceptibility to dextran and placed in PBS. The colon was opened longitudinally, washed in PBS, sodium sulfate (DSS)-induced colitis compared with wild-type and cut into 1-cm pieces. The pieces were treated for 30 min at 37˚C with

(WT) mice, primarily due to impaired IL-10 production. We PBS containing 5% (v/v) FCS, HEPES (20 nM) (pH 7.4), penicillin (100 U/ Downloaded from also identified that avb1 integrin, but not a1b1 integrin or plexin- ml), streptomycin (100 U/ml), sodium pyruvate (1 mM), EDTA (10 mM), and polymyxin B (10 mg/ml; Calbiochem) to remove epithelial cells and C1 on intestinal Mws, functions as a Sema7A receptor to suppress then washed extensively with PBS. Segments of the colon and SP were intestinal inflammation. Moreover, we identified that Sema7A digested for 45 min with continuous stirring at 37˚C, with collagenase D expressed in intestinal epithelial cells (IECs) is responsible for (800 Mandl units/ml; Roche), DNase I (10 mg/ml; Roche), and dispase I (10 IL-10 production by intestinal Mws, which is mediated through mg/ml; Invitrogen) in RPMI 1640 medium with 5% (v/v) FCS. EDTA was added (final concentration, 10 mM), and cell suspensions were incubated for interactions between intestinal Mws and IECs. Furthermore, we an additional 5 min at 37˚C. Cells were spun through a 17.5% (w/v) solution http://www.jimmunol.org/ demonstrated that recombinant Sema7A (rSema7A) proteins exert of Accudentz (Accurate Chemical & Scientific), and collected whole cells therapeutic effects on the severities of colitis via IL-10 production. were used in assays. LP-Mw were sorted on the basis of their expression of F4/80, CD11b, and I-A/I-E with an FACSAria (BD Biosciences). In addi- tion, inflammatory Mws from colon of DSS-fed WT mice were sorted on the Materials and Methods basis of CD14 and CD11b expression with FACSAria (BD Biosciences). Mice The purity of the sorted Mws was routinely .98%.Foranalysisoflym- phocytes, cells were subjected to density-gradient centrifugation in 40–75% Sema7A- (14), integrin a (Itga) 1- (18), and plexin-C1–deficient (14) and (v/v) Percoll (approximate density 1.058 g/ml and 1.093 g/ml, respectively) Ly5.1 mice were generated in the C57BL/6 background. Eight- to 10-wk- after enzyme treatment. Cells collected from the interface were washed and old mice were used for experiments. All mice used in this study were + + used as LP-lymphocytes; CD4 and B220 cells were purified by magnetic by guest on September 23, 2021 housed in specific pathogen-free conditions. All experimental procedures sorting with mouse anti-CD4 beads and mouse anti-B220 beads, respec- were performed following our institutional guidelines. tively. The purity of the sorted cells was routinely .90%.

Abs and reagents Cell culture and ELISA analysis For flow cytometry, cells from lamina propria (LP) and spleen (SP) were LP- and SP-derived whole cells or sorted LP-Mws(23 105 per well) were stained with the following Abs; anti–I-A/I-E (M5/114.15.2) conjugated added to flat-bottom 96-well plates coated with the indicated concentrations with eFluor 450, anti-F4/80 (BM8) conjugated with allophycocyanin, anti- of Fc–control (Millipore), Sema7A–Fc, or KGE–Fc and cultured for 24 h in CD11b (MJ1/70) conjugated with PE, anti-CD103 (2E7)-PE, anti-CD11c RPMI 1640 medium containing 5% FCS and gentamicin. The supernatant (HL3)-FITC, and anti-CD14 (Sa2-8)-allophycocyanin. For analysis of fluid was analyzed by ELISA (IL-10, IFN-g, TNF-a, and IL-12p70: R&D protein expression on LP-Mws, biotin-conjugated anti-Sema7A (4D9, Systems; TGF-b: eBioscience). For function-blocking assays, LP-Mws mouse IgG1), anti-a1 (HM alpha1), -av (10D5), or -b1 integrin (HM b (1 3 105 cells/well) were pretreated with anti-integrin mAbs or anti-mouse 1.1), anti–plexin-C1 mAbs (R&D Systems), and CX3CR1 (catalog number plexin-C1 mAb (R&D Systems) for 30 min on ice. Then, these cells were 2093; ProSci) (19) polyclonal Abs were used. FITC-conjugated strepta- added to flat-bottom 96-well plates coated with the indicated concentrations vidin was used for secondary staining. An anti-mouse Sema7A Ab was of Fc–control (Millipore) and Sema7A–Fc and cultured for 24 h. obtained by immunizing Sema7A-deficient mice with Sema7A–Fc as previously described (15) and conjugated with biotin using an Ab bio- Isolation of RNA and quantitative real-time or RT-PCR tinylation (American Qualex). For function-blocking assays, we used the following Abs against mouse: anti-a1 (HM alpha1), anti-av (10D5), Total RNAs were prepared from the colon tissue of DSS-fed WT and anti-a5 (5H10-27 [MFR5]), anti-a8 (aa38-1007), anti-b1 (HM b 1.1), Sema7A-deficient mice using an RNeasy Mini kit (Qiagen) and treated with anti-b3, -b5, -b8 (H-160), and anti–plexin-C1 (AF5375). MEK1/2 inhib- DNase I (Invitrogen) to eliminate genomic DNA. cDNA was synthesized itor, U0126, and U0124 were purchased from Calbiochem. using a SuperScript II cDNA synthesis kit (Invitrogen). A 7700 Sequence Detector (Applied Biosystems) was used for quantitative PCR of cDNA Induction of DSS-induced colitis amplified with 23 PCR Master Mix (Applied Biosystems) and primers Eight- to 10-wk-old Sema7A-, Itga1- and plexin-C1–deficient mice and specific for IL-10, TGF-b, retinal dehydrogenase 2 (Aldh1a2), and Sema7A their littermate WT mice as controls were used for DSS-induced colitis (Applied Biosystems) in a final volume of 20 ml. After incubation at 95˚C experiments. Acute colitis was induced by administration of 2% (w/v) DSS for 10 min, products were amplified by 35 cycles of 95˚C for 15 s, 60˚C for (36–50 kDa; MP Biomedicals) to the drinking water for 7 d. To test sur- 60 s, and 50˚C for 120 s. RT-PCR was performed with 35 cycles of 94˚C vival rate, 4% (w/v) DSS was used until day 4 followed by normal drinking for 30 s, 60˚C for 30 s, and 72˚C for 30 s using the -specific primers water until the end of the experiment (day 14). (mouse a1 integrin: forward, 59-AATGAGCCTGGAGCCTATCA-39 and reverse, 59-TATACACGGCTCCTCCGTGA-39;mouseav integrin: for- Histology and scoring ward, 59-CAAGCTCACTCCCATCAC-39 and reverse, 59-GGGTGTCTT- GATTCTCAAAGGG-39). The specimens were embedded in O.C.T. compound and fixed with methanol. Tissue sections were stained with H&E. Histological grading was Inflammatory Mw polarization based on observed inflammation, defined as follows: 0, no inflammation; 1, low level of inflammation with mildly increased inflammatory cells in the Bone marrow (BM) cells (2 3 106) were cultured in 24-well plates in 1 ml LP; 2, moderately increased inflammation in the LP (multiple foci); 3, high RPMI 1640 medium containing 10% FCS and 40 ng/ml murine GM-CSF The Journal of Immunology 3

(PeproTech). LPS (1 mg/ml; Sigma-Aldrich) was added to the culture 12 h transcripts were abundantly expressed in the intestine (data not before the end of the culture (21). shown), we decided to investigate the role of Sema7A in the in- Western blot testinal immune system. We first performed an experimental colitis model using WT and Sema7A-deficient mice treated by oral ad- LP-Mws were serum-starved for 4 h in RPMI 1640 medium supplemented with 0.1% BSA. Cells were resuspended in serum-free RPMI 1640 me- ministration of 4% DSS, which is toxic to colonic epithelial cells dium, and 1 3 106 cells were seeded into 96-well plates coated with Fc– and elicits acute inflammatory responses by disrupting the com- control or Sema7A–Fc (20 nM). Cell extracts were prepared by lysing the partmentalization of commensal bacteria. Only 10% of WT mice cells with a buffer (50 mM Tris-Cl [pH 7.5], 150 mM NaCl, and 2 mM died during the DSS administration period, but, unexpectedly, a EDTA, plus cocktails of protease inhibitors [Roche] and phosphatase . inhibitors [Sigma-Aldrich]) containing 2% Nonidet P-40, subjected to a 4– mortality rate 90% was noted for Sema7A-deficient mice (Fig. 12% gradient SDS gel (Invitrogen) in MOPS buffer, and transferred to 1A). The experiment was repeated with a lower DSS concentration a polyvinylidene fluoride membrane (Millipore). Membranes were probed (2%) to study the phenotype of Sema7A-deficient mice under with anti–phospho-focal adhesion kinase (FAK; Tyr397) and anti–phospho- 202 204 milder conditions. Sema7A-deficient mice exhibited loss of body ERK1/2 (Thr /Tyr ) (all from Cell Signaling Technology). The mem- weight and more severe colitis than WT mice (Fig. 1B)dueto brane was stripped with the Restore Western blot Stripping Buffer (Pierce) for 10 min at room temperature and reprobed with Abs to total FAK and severe diarrhea and rectal bleeding. In addition, the mutant mice ERK (Cell Signaling Technology). showed shorter colon and severe transmural inflammation with Preparation of IECs extensive crypt destruction, edema, and infiltration of immune cells, leading to severe histological scores (Fig. 1C–E). These IECs from colon of WT mice were isolated as described reference (22). results suggest that Sema7A plays a suppressive role in the intes- Colons were isolated, opened longitudinally, and rinsed with PBS. The epithelial integrity was disrupted by treatment with 1 mM DTT for 30 min tinal inflammatory responses. Downloaded from at 37˚C on a shaker, followed by vortexing for 1 min. The liberated IECs were collected, resuspended in 5 ml 20% Percoll, and overlaid on 2.5 ml Intestinal Mws are responsible for IL-10 production in 40% Percoll in a 15-ml Falcon tube. Percoll gradient separation was per- response to Sema7A formed by centrifugation at 2000 rpm for 20 min at 25˚C. The interface cells were collected and used as colonic IECs (purity .90%, survival rate 95%). To determine the responsible regulatory mechanisms, we measured the expression of several immunosuppressive factors such as IL-10, Cocultivation of Caco-2 cells and LP-Mws TGF-b, and Aldh1a2, which encodes Aldh1a2 in the colon of DSS- http://www.jimmunol.org/ Caco-2 cells were cultured for 7 d in the upper chambers of Transwell filters treated mice. Although the expression levels of TGF-b and Aldh1a2 (3 mm in pore diameter; Costar). LP-Mws were incubated for 24 h with me- were not affected, IL-10 expression levels were considerably de- dium alone or with Caco-2 cells. In the direct contact experiment, filters were turned upside down, and Caco-2 cells were seeded on the membrane of fil- creased by the absence of Sema7A (Fig. 2A). Also, in the DSS- ters. After 7 d, filters bearing Caco-2 cells were turned upside down, and LP- treated colon explant culture, IL-10 but not TGF-b concentrations Mws(4.53 105 cells/well) were incubated on the filter facing the basolateral in the supernatant from colon of DSS-fed Sema7A-deficient mice side of Caco-2 cells for 24 h. Supernatants were used for ELISA analysis. were significantly reduced (Fig. 2B, Supplemental Fig. 1A). BM chimeras Next, to examine whether rSema7A protein promotes cytokine production in the intestine, we cultured whole LP cells and SP

BM transfer experiment was used to generate Sema7A-deficient chimera by guest on September 23, 2021 mice wherein the genetic deficiency of Sema7A was limited to either cells in the presence of rSema7A. As shown in Fig. 2C, rSema7A circulating cells or nonhematopoietic cells. In brief, BM samples were dose-dependently increased IL-10 levels from LP cells, whereas collected from femur and tibia of congenic WT (expressing CD45.1 leu- rSema7A did not affect the production of other proinflammatory kocyte Ag) or Sema7A-deficient and littermate WT (expressing CD45.2 cytokines such as IFN-g, IL-12p70, and TNF-a (Supplemental leukocyte Ag) donor mice by flushing with HBSS. After several washing steps, cells were resuspended in PBS at a concentration of 3 3 107 cells/ml. Fig. 1B). In addition, Sema7A had less of an effect on IL-10 A total of 100 ml of this cell suspension was injected in irradiated recipient production by SP cells (Fig. 2C). Thus, Sema7A preferentially mice. Four chimera groups were generated: WT . WT (WT cells induces IL-10 production by LP cells. expressing CD45.2 into WT expressing CD45.1); WT . 7A2/2 (WT cells 2/2 2/2 2/2 Several studies have identified that various immune cells, such expressing CD45.1 into Sema7A expressing CD45.2); 7A . 7A + 2 2 2 2 as regulatory T cells (Treg; Tr1 and Foxp3 ), regulatory B cells, (Sema7A / cells expressing CD45.2 into Sema7A / expressing CD45.1); and 7A2/2 . WT (Sema7A2/2 cells expressing CD45.2 into dendritic cells (DCs), and resident Mws, can produce IL-10 in the WT expressing CD45.1). The use of CD45.1-expressing congenic mice intestine (8, 23, 24). So, we examined which cells are responsible facilitated verification of proper reconstitution in the chimeric mice. BM for IL-10 production in response to Sema7A. Neither T cells, reconstitution was verified after 7 wk by staining for CD45.1 and CD45.2 B cells, DCs, nor inflammatory Mws produced IL-10 in response in cells with PE-conjugated anti-CD45.1 and FITC-conjugated anti- int CD45.2. Eight weeks after BM transfer, mice were treated with 2% DSS to rSema7A (Fig. 2D). In contrast, MHC class II (MHC II) F4/ hi hi for 7 d. Body weight change was monitored daily. At day 7, mice were 80 CD11b resident Mws (LP-Mws) produced IL-10 in response sacrificed to collect colon tissue for H&E staining. to rSema7A in a dose-dependent manner (Fig. 2E). Moreover, we Immunohistochemistry tested whether IL-10 production is impaired in LP-Mws from DSS-fed Sema7A-deficient mice. We isolated LP-Mws from the The 4% paraformaldehyde in PBS (paraformaldehyde)-fixed colon tissue colon of WT or Sema7A-deficient mice before and 6 d after DSS slides were stained for Sema7A-positive cells via the immunoperoxidase method with anti-mouse Sema7A Abs and then counterstaining with administration. Consistent with the data in Fig. 2B, LP-Mws from hematoxylin. The Sema7A-positive cells were observed by microscopy DSS-fed Sema7A-deficient mice displayed significantly reduced (Nikon ELIPSE E600; Nikon). IL-10 production compared with those from WT mice (Fig. 2F). Statistical analysis Additionally, these LP-Mws are characterized by their potent IL-10 production and expression of the fractalkine receptor To analyze statistical significance, we used an unpaired, two-tailed Student t test. We considered p values ,0.05 to be significant. CX3CR1 on their surface (Supplemental Fig. 2A) (25–27). There- fore, these results indicate that LP-Mws are compatible to CX3CR1+ Results Mws and responsible for Sema7A-induced IL-10 production. Sema7A-deficient mice showed severe colonic inflammation avb1 integrin is a functional receptor for Sema7A in LP-Mws A previous study showed that Sema7A promotes T cell-mediated Sema7A has been reported to use a1b1 integrin in inflammatory inflammatory responses in the skin and CNS (15). Because Sema7A Mws and plexin-C1 in human monocyte (15, 28). We then ex- 4 EPITHELIAL CELL-DERIVED Sema7A NEGATIVELY REGULATES COLITIS Downloaded from

FIGURE 1. Sema7A-deficient mice are hypersusceptible to DSS-induced colitis. A, Survival rate. WT (n = 10) or Sema7A-deficient (n = 10) mice were orally administrated 4% DSS solution in drinking water for 4 d. Survival was monitored until day 14 after the start of DSS treatment. B, Percentage of weight changes after 2% DSS administration. Initial weight of each mouse was defined as 100%. C, Colon length of mice. The average of each group is indicated with a bold red line. D, Histopathological changes in colon tissue from WT and Sema7A-deficient mice were analyzed by H&E staining at day 8 of DSS administration. Scale bars, 100 mm. E, Semiquantitative scoring of histopathology was performed as described in the Materials and Methods. B–E, http://www.jimmunol.org/ WT or Sema7A-deficient mice were administered 2% DSS in drinking water for 8 d. Data are from six mice; error bars indicate SD. Data are representative of three independent experiments. *p , 0.05, **p , 0.01, ****p , 0.0001. amined whether Sema7A-induced IL-10 production by LP-Mwsis posed to a plate coated with rSema7A. Sema7A-induced IL-10 mediated by or plexin-C1. LP-Mws were pretreated with production from these Mws was significantly inhibited by Abs Abs against a1 integrin, b1 integrin, or plexin-C1 and then ex- against anti-b1 integrin but not by anti–plexin-C1 Ab. Unexpect-

FIGURE 2. Sema7A induces IL- 10 production by MHC IIintF4/80hi by guest on September 23, 2021 CD11bhi Mws. A, Relative tran- script levels of IL-10, TGF-b, and Aldh1a2 normalized to GAPDH in colon of DSS-fed WT and Sema7A- deficient mice (n = 3). B, IL-10 production in the supernatants of colon explant cultures at days 0 and 6 from DSS administration. C, IL-10 production by whole cells from SP and LP. Cells were stimulated with the indicated rSema7A and Fc–con- trol in the presence or absence of LPS (1 mg/ml) for 24 h. D, IL-10 production by MHC IIintF4/80hi CD11bhi, CD103+CD11c+, CD14+ CD11b+, CD4+, and B220+ cells from LP. Cells were stimulated with the 40 nM rSema7A and Fc–control. E, IL-10 production of LP-Mws. Cells were stimulated with various concentrations of rSema7A or Fc– control in the presence or absence of LPS (1 mg/ml). F, IL-10 production by LP-Mws. LP-Mws from WT and Sema7A-deficient mice before and 6 d after DSS administration were cultured for 12 h. B–F, Cytokine production was measured by ELISA. Data are representative of three in- dependent experiments (error bars, SD of averages from triplicate cul- tures). *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. The Journal of Immunology 5 edly, anti-a1 integrin Ab could not inhibit Sema7A-induced IL-10 Notably, pretreatment of LP-Mws with Ab against av integrin production in these Mws(Fig.3A). Consistent with this, a1 significantly abrogated IL-10 production by Sema7A stimulation, integrin and plexin-C1 were not expressed in LP-Mws (Supple- whereas treatment with anti-a5or-a8 integrin Abs did not (Fig. mental Fig. 2B,2C). Also, it was confirmed that a1 integrin- 3C). The av integrin is the most promiscuous a subunit, assem- deficient or plexin-C1–deficient mice displayed rather mild coli- bling with four different b subunits, including b1, b3, b5, and b8 tis compared with WT mice in terms of loss of body weight and integrin, which are involved in the intestinal homeostasis through shortening of the colon in a DSS-induced colitis model (Supple- TGF-b production (30, 31). However, we found that b subunits mental Fig. 2D,2E). other than b1 had no effects on IL-10 production by LP-Mws (Fig. Because Sema7A can bind to integrin through the RGD motif, 3D). In addition, pretreatment of LP-Mws with anti-integrin Abs we further examined whether the RGD motif is important for did not show any influences on TGF-b production (data not Sema7A-induced IL-10 production. The mutated Sema7A proteins shown). Collectively, these data imply that Sema7A uses avb1 (KGE–Fc) that lack integrin-binding activity failed to increase IL- integrin as a receptor to promote IL-10 production by LP-Mws. 10 production (Fig. 3B), suggesting Sema7A-induced IL-10 pro- Previous studies demonstrated that Sema7A exerts its biological duction in LP-Mws is dependent on integrin signaling. b1 integrin functions through activation of FAK and subsequently ERK sig- can be noncovalently associated with several different a integrin naling pathways, downstream of integrins in both immune and subunits, such as a5, av, and a8, and act as RGD receptor (29). neuronal cells (14, 15). In addition, IL-10 production by human Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 3. Sema7A increases IL-10 from LP-Mws through avb1 integrin signaling. A–D, IL-10 levels in the culture supernatant were measured. A, LP-

Mws were treated with anti-a1orb1 integrin or anti–plexin-C1 mAbs (25 mg/ml) and cultured with 40 nM rSema7A and Fc–control. Hamster anti-mouse IgG [IgG (H)] and sheep anti-mouse IgG [IgG (S)] were used as control. B, LP-Mws were cultured with plate-coated 40 nM rSema7A or a mutant protein containing the altered RGD motif (KGE–Fc) or Fc–control as negative control. C, LP-Mws were treated with anti-a5, -av, or -a8 integrin mAbs (25 mg/ml) and stimulated with plate-coated 40 nM rSema7A and Fc–control. D, LP-Mws were treated with anti-b3,-b5,or-b8 integrin mAbs (25 mg/ml) and stimulated with plate-coated 40 nM rSema7A and Fc–control. E, Western blot analysis of FAK and ERK1/2 phosphorylation in LP-Mws following rSe- ma7A and Fc–control (Cont.) stimulation (left panel) and in the pretreatment with anti-av integrin Ab or isotype Ab (right panel). F, LP-Mws were pretreated with vehicle (0.001% DMSO) or various concentrations of U0126 or U0124 (a negative control) and then stimulated with plate-coated 40 nM rSema7A. Concentrations of IL-10 in culture supernatants are presented as a percentage of control (vehicle) values. A–D and F, IL-10 concentration was determined by ELISA analysis. Data are representative of three independent experiments (error bars, SD of averages from triplicate cultures). **p , 0.01, ***p , 0.001, ****p , 0.0001. 6 EPITHELIAL CELL-DERIVED Sema7A NEGATIVELY REGULATES COLITIS monocytes is mediated by ERK signaling (32). Consistent with the When LP-Mws were faced to the basolateral side of Caco-2 cells, previous findings, Sema7A induced the phosphorylation of FAK they could produce IL-10 (Fig. 4C). By contrast, when LP-Mws and ERK1/2 in LP-Mws (Fig. 3E, left panel), and this effect was could not contact with Caco-2 cells or faced to the apical side of completely abolished by blocking Abs against av integrin (Fig. Caco-2 cells, they released less IL-10 (Fig. 4C). In addition, this 3E, right panel). Furthermore, Sema7A-induced IL-10 production effect was completely abolished by neutralizing Abs against av by LP-Mws was dose-dependently abolished by U0126, a specific integrins (Fig. 4D). Collectively, these findings suggest that inhibitor of ERK signaling (MEK-1 and MEK-2), but not U0124, interactions between IECs and LP-Mws are important for regu- an inactive analog compound (Fig. 3F). Thus, our findings suggest lating IL-10 production through Sema7A–avb1 integrin signaling. that IL-10 production by LP-Mws requires ERK activation through Nonhematopoietic cell-derived Sema7A is indispensable for Sema7A–avb1 integrin interactions. protection against DSS-induced colitis Sema7A expressed on IECs is critical for interactions with Several previous studies have reported that activated T cells are w LP-M s the main source of Sema7A during the development of CHS and Next, we tried to identify the type of cells that express Sema7A in EAE inflammation (22). However, as shown in Fig. 4A and Sup- the intestine by immunohistochemical analysis. When we stained plemental Fig. 3C, Sema7A was preferentially expressed in IECs. colon slice sections with anti-Sema7A Ab, Sema7A was highly Therefore, the question arose whether hematopoietic or non- expressed in the IEC and crypt enterocytes, rather than in LP hematopoietic cell-derived Sema7A is relevant for regulating in- immune cells (Fig. 4A). We also confirmed Sema7A expression testinal inflammation. To address this issue, we generated four

on IECs by flow cytometry (Supplemental Fig. 3A, left panel). In types of BM chimera mice by criss-cross transplantation of WT Downloaded from addition, quantitative analysis of the fluorescence intensity for or Sema7A-deficient BM cells to WT or Sema7A-deficient Sema7A indicated that Sema7A was predominantly localized on (7A2/2) recipient mice (WT . WT, 7A2/2 . WT, WT . 7A2/2, the basolateral but not the apical side of IECs (Supplemental Fig. or 7A2/2 . 7A2/2) and induced colitis in these animals by DSS 3B). feeding. Although mice that systemically lacked Sema7A ex- Recently, it has been reported that during inflammation, IECs pression (7A2/2 . 7A2/2) showed more severe loss of body

increased the expression of fractalkine and CX3CL1 and subse- weight, shortening of the colon, and extensive crypt destruction http://www.jimmunol.org/ quently induced the recruitment of CX3CR1+ cells to IECs (33). with infiltration of lymphocytes than WT . WT mice, mice that Therefore, we hypothesized that Sema7A expression on the lacked Sema7A expression in hematopoietic cells (7A2/2 . WT) basolateral side of IECs is crucial for IL-10 production by Mws exhibited colitis comparable to that observed in WT . WT mice that express CX3CR1 during interactions between IECs and Mws. (Fig. 5). Conversely, mice lacking nonhematopoietic Sema7A To demonstrate this, Caco-2 cells, which are human IEC lines and (WT . 7A2/2) displayed colitis as severe as that observed in expressed Sema7A (Supplemental Fig. 3A, right panel), were Sema7A-null mice (7A2/2 . 7A2/2). These results suggest that polarized on a transwell membrane and then added LP-Mws to the Sema7A expressed in IECs is indispensable for protection against apical or basolateral sides of epithelial cell monolayers (Fig. 4B). DSS-induced colitis. by guest on September 23, 2021

FIGURE 4. Sema7A, which is expressed on IECs, directly interacts with LP-Mws to induce IL-10 production. A, Representative photomi- crographs of colon from WT (left panel)and Sema7A-deficient (right panel) mice after 3,39- diaminobenzidine staining using anti-Sema7A Ab (arrowheads indicate positive cells) and hematox- ylin counter staining. Scale bars, 10 mm. B–D, Coculture system of Caco-2 and Mws. B, Brief scheme of experiment. C and D, IL-10 levels in culture supernatant were measured. C, Caco-2 cells (ECs) were grown on a transwell membrane. LP- Mws(Mw) were incubated facing to the basolateral (Baso-IEC) or apical (Api-IEC) side of the EC monolayer or lower chamber (noncontact [N.C.] between IECs and Mws) for 24 h. D, LP-Mws were pretreated with anti-av integrin mAbs (25 mg/ml) and incubated facing on the basolateral side of a Caco-2 monolayer for 24 h. C and D, IL-10 levels in supernatant were analyzed by ELISA. Data are representative of three independent experiments (error bars, SD of averages from triplicate cultures). *p , 0.05, ****p , 0.0001. The Journal of Immunology 7

The rSema7A has therapeutic effects on the development of cially involved in negative regulation of intestinal inflammation colitis via an interaction with avb1 integrin in intestinal Mws. Indeed, We finally examined the therapeutic effects of rSema7A against we found that inflammatory Mws expressed a1 integrin but not av intestinal inflammation and whether the effects of Sema7A depend integrin; vice versa, intestinal regulatory Mws expressed av on IL-10 in vivo. WT mice were given 2% DSS solution and treated integrin but not a1 integrin (Supplemental Fig. 2B). Regarding with rSema7A in the absence or presence of anti–IL-10–blocking suppressive functions of Sema7A, Czopik et al. (38) have previ- Abs. Mice receiving rSema7A were rescued from the loss of body ously reported that Sema7A expressed in T cells downregulates weight and shortening of the colon and improved histological T cell activation by functioning as a self-limiting molecule for scores for colitis compared with control mice. In addition, TCR signaling. In contrast, we confirmed in this study that IEC- blocking Abs against IL-10 eliminated the therapeutic effects of derived Sema7A induces IL-10 producing signaling in Mws, rSema7A (Fig. 6). Thus, Sema7A confers protection against DSS- showing the different negative machineries from peripheral induced colitis, depending on IL-10 production. Collectively, our T cells. Of note, DSS-induced colitis has been shown to develop findings demonstrated that Sema7A expressed on IECs is an im- even in the absence of T cells. It thus appears that Sema7A reg- portant element to induce IL-10 production in intestinal Mws and ulates immune responses, depending on different receptor use or prevent excessive intestinal inflammation (Supplemental Fig. 4). environmental contexts. av integrin plays pivotal roles in the maintenance of intestinal Discussion immune homeostasis by increasing TGF-b and subsequent Treg Semaphorins were originally identified as axon guidance molecules differentiation. Indeed, Itgav-deficient mice under the tie2 pro- Downloaded from in neural development; however, the accumulated evidence now moter (av-tie2 mice), in which av integrin is deleted in myeloid indicates that several semaphorins play crucial roles in physio- cells, developed spontaneous colitis due to failure of generation of logical and pathological immune responses (34–36). Sema7A has Treg cells and removed apoptotic cells (39). In addition, it has been shown to activate inflammatory Mws and monocytes (37). In been reported that the deficiency of avb5oravb8 integrin on DCs this study, we highlighted a negative regulatory role of Sema7A in also resulted in spontaneous colitis due to reduction of TGF-b in intestinal inflammation, in which Sema7A induces production of the intestine (31, 39). In this study, we found that avb1 integrin the anti-inflammatory cytokine IL-10 via use of a specific integrin on intestinal Mws is critically involved in IL-10 production by http://www.jimmunol.org/ receptor, avb1 integrin. We further demonstrated that Sema7A is Sema7A stimulation. However, we could not observe any differ- predominantly expressed on the basolateral side of IECs and that ences in the levels of TGF-b between DSS-fed Sema7A and WT Sema7A in IECs is involved in IL-10 production as a result of mice (Supplemental Fig. 1A), implying that TGF-b is not pri- interactions between IECs and intestinal Mws. marily involved in the protective role of Sema7A–avb1 integrin Previous studies have emphasized the critical role of Sema7A signaling. Indeed, deficiency of av integrin causes more severe in inflammation through interactions with either a1b1 integrin signs of colitis than those of DSS-fed Sema7A-deficient mice. or plexin-C1, resulting in the activation of inflammatory Mwsor Thus, it is possible that av integrin mediates both TGF-b and IL-10–producing signaling by coupling with the different integrin monocytes in the CHS, EAE, and pulmonary fibrosis models (14, by guest on September 23, 2021 15, 17). In contrast, we unexpectedly found that Sema7A is cru- b subunits in the intestinal immune system.

FIGURE 5. Sema7A expressed in nonhematopoietic cells but not he- matopoietic cells is critical for pro- tection against DSS-induced colitis. A, Percentage of weight changes of BM chimeric mice after DSS ad- ministration. Initial weight of each mouse was defined as 100%. B, Colon length of mice. The average of each group is indicated with a bold red line. C, Histopathological changes in colon tissues were ex- amined by H&E staining. Scale bars, 100 mm. D, Semiquantitative scor- ing of histopathology was per- formed. A–D,BMchimeramice were generated as described in Materials and Methods. Mice were treated with 2% DSS for 7 d. Data are from six mice in each group; error bars indicate SD. Data are representative of three independent experiments. **p , 0.01, ***p , 0.002, ****p , 0.0001. 8 EPITHELIAL CELL-DERIVED Sema7A NEGATIVELY REGULATES COLITIS

FIGURE 6. Sema7A ameliorates colitis by inducing IL-10 production. A, Percentage of weight change after DSS administration. Initial weight of each mouse was defined as 100%. B, Colon length of mice. The aver- age of each group is indicated with a bold red line. C, Histopathological changes in colon tissues were ex- amined by H&E staining. Scale bars, 100 mm. D, Semiquantitative scor- ing of histopathology was per- formed. A–D,WTmicewere administrated 2% DSS in drinking water for 7 d and injected with 50 Downloaded from mg of Fc–control (opened circle), rSema7A (red circle), anti–IL-10 Abs (black triangle), or rSema7A plus anti–IL-10 Abs (gray circle) every 2 d starting at day 0. Data are from four mice per each group; error bars indicate SD. Data are repre- http://www.jimmunol.org/ sentative of three independent ex- periments. *p , 0.05, **p , 0.01. by guest on September 23, 2021

IECs are crucially involved in maintaining intestinal immune cell-derived Sema7A. Thus, IEC-derived Sema7A, of which ex- homeostasis by influencing the function of intestinal immune cells, pression levels were maintained during DSS-induced colitis including Mws, DCs, and lymphocytes (40–42); in particular, (Supplemental Fig. 3C), is crucial for intestinal immune homeo- interactions between intestinal Mws and IECs have been empha- stasis. It is possible that DSS exerts a cytolytic effect on IECs, sized. For instance, these interactions have been shown to direct resulting in the generation of soluble Sema7A. However, we did monocytes to differentiate into anti-inflammatory cells (26, 42) not detect the activities of soluble Sema7A on IL-10 production and play a cardinal role in initiating oral tolerance (25). Addi- (Fig. 4C). Therefore, our observations support the scenarios that in tionally, accumulated experimental findings also indicate that IL- the context of intestinal inflammation, CX3CL1 is upregulated by 10– producing intestinal Mws play important roles in repression of IECs to recruit CX3CR1+MHC IIintF4/80hiCD11bhi Mws, and, excessive intestinal inflammation, in that they actively counteract under these conditions, Mws adhere to a rigid scaffold composed the onset of inflammation. Recently, it has been suggested that of extracellular matrix and Sema7A on the basolateral side of intestinal Mws are located just below IECs and characterized by IECs and interact with avb1 integrin, resulting in IL-10 produc- CX3CR1+MHC IIintF4/80hiCD11bhi expression (26). Indeed, these tion (Supplemental Fig. 4). Mws are capable of producing a large amount of IL-10, which is In conclusion, we demonstrated in this study that the interaction regulated by intestinal intrinsic CX3CR1 signaling (25). Notably, between IECs and intestinal Mws plays a crucial role in controlling the CX3CR1 ligand CX3CL1 is expressed as a membrane-bound intestinal inflammation through Sema7A–avb1 integrin inter- form by IECs and upregulated in the inflamed intestine, recruiting actions. Defects in this mechanism resulted in impaired IL-10 the regulatory Mws to the basolateral side of IECs (33). These production, exacerbating intestinal inflammation. The results not dynamic interactions between IECs and Mws seem to contribute to only elucidate a pivotal role for Sema7A as an intrinsic suppressor maintain the intestinal homeostasis. In this context, it is worthy of of intestinal inflammation, but also provide a novel insight into note that Sema7A was expressed at the basolateral side of IECs, mechanisms for controlling the intestinal inflammation and clin- and its receptor avb1 integrin was expressed in the CX3CR1- ical applications in IBDs. positive intestinal Mws. Indeed, we found in this study that only CX3CR1+MHC IIintF4/80hiCD11bhi Mws produced IL-10 upon Acknowledgments Sema7A stimulation, whereas other cells, including CD103+ DCs, We thank T. Ishikawa for technical support. T cells, and B cells, did not. In addition, mice deficient for non- hematopoietic cell-derived Sema7A showed more severe signs of Disclosures colitis than either WT mice or mice deficient for hematopoietic The authors have no financial conflicts of interest. The Journal of Immunology 9

References 22. Greten, F. R., L. Eckmann, T. F. Greten, J. M. Park, Z. W. Li, L. J. Egan, M. F. Kagnoff, and M. Karin. 2004. IKKbeta links inflammation and tumori- N. Engl. J. Med. 1. Podolsky, D. K. 2002. Inflammatory bowel disease. 347: 417– genesis in a mouse model of colitis-associated cancer. Cell 118: 285–296. 429. 23. Barnes, M. J., and F. Powrie. 2009. Regulatory T cells reinforce intestinal ho- 2. Round, J. L., and S. K. Mazmanian. 2009. The gut microbiota shapes intestinal meostasis. Immunity 31: 401–411. immune responses during health and disease. Nat. Rev. Immunol. 9: 313–323. 24. Izcue, A., J. L. Coombes, and F. Powrie. 2009. Regulatory lymphocytes and 3. Schenk, M., and C. Mueller. 2007. Adaptations of intestinal macrophages to an intestinal inflammation. Annu. Rev. Immunol. 27: 313–338. antigen-rich environment. Semin. Immunol. 19: 84–93. 25. Hadis, U., B. Wahl, O. Schulz, M. Hardtke-Wolenski, A. Schippers, N. Wagner, 4. Kuhn, R., J. Lohler, D. Rennick, K. Rajewsky, and W. Muller. 1993. Interleukin- ¨ ¨ ¨ W. Mu¨ller, T. Sparwasser, R. Fo¨rster, and O. Pabst. 2011. Intestinal tolerance 10-deficient mice develop chronic enterocolitis. Cell 75: 263–274. requires gut homing and expansion of FoxP3+ regulatory T cells in the lamina 5. Spencer, S. D., F. Di Marco, J. Hooley, S. Pitts-Meek, M. Bauer, A. M. Ryan, propria. Immunity 34: 237–246. B. Sordat, V. C. Gibbs, and M. Aguet. 1998. The orphan receptor CRF2-4 is an 26. Schulz, O., E. Jaensson, E. K. Persson, X. Liu, T. Worbs, W. W. Agace, and essential subunit of the interleukin 10 receptor. J. Exp. Med. 187: 571–578. O. Pabst. 2009. Intestinal CD103+, but not CX3CR1+, antigen sampling cells 6. Franke, A., T. Balschun, T. H. Karlsen, J. Sventoraityte, S. Nikolaus, G. Mayr, migrate in lymph and serve classical dendritic cell functions. J. Exp. Med. 206: F. S. Domingues, M. Albrecht, M. Nothnagel, D. Ellinghaus, et al; IBSEN study 3101–3114. group. 2008. Sequence variants in IL10, ARPC2 and multiple other loci con- 27. Varol, C., E. Zigmond, and S. Jung. 2010. Securing the immune tightrope: tribute to ulcerative colitis susceptibility. Nat. Genet. 40: 1319–1323. mononuclear phagocytes in the intestinal lamina propria. Nat. Rev. Immunol. 10: 7. Schreiber, S., T. Heinig, H. G. Thiele, and A. Raedler. 1995. Immunoregulatory 415–426. role of interleukin 10 in patients with inflammatory bowel disease. Gastroen- 28. Tamagnone, L., S. Artigiani, H. Chen, Z. He, G. I. Ming, H. Song, A. Chedotal, terology 108: 1434–1444. M. L. Winberg, C. S. Goodman, M. Poo, et al. 1999. Plexins are a large family of 8. Denning, T. L., Y. C. Wang, S. R. Patel, I. R. Williams, and B. Pulendran. 2007. receptors for transmembrane, secreted, and GPI-anchored semaphorins in ver- Lamina propria macrophages and dendritic cells differentially induce regulatory tebrates. Cell 99: 71–80. and interleukin 17-producing T cell responses. Nat. Immunol. 8: 1086–1094. 29. Hynes, R. O. 2002. Integrins: bidirectional, allosteric signaling machines. Cell 9. Murai, M., O. Turovskaya, G. Kim, R. Madan, C. L. Karp, H. Cheroutre, and 110: 673–687. M. Kronenberg. 2009. Interleukin 10 acts on regulatory T cells to maintain 30. Albert, M. L., S. F. Pearce, L. M. Francisco, B. Sauter, P. Roy, R. L. Silverstein, expression of the transcription factor Foxp3 and suppressive function in mice and N. Bhardwaj. 1998. Immature dendritic cells phagocytose apoptotic cells via Downloaded from with colitis. Nat. Immunol. 10: 1178–1184. alphavbeta5 and CD36, and cross-present antigens to cytotoxic T lymphocytes. 10. Qualls, J. E., A. M. Kaplan, N. van Rooijen, and D. A. Cohen. 2006. Suppression J. Exp. Med. 188: 1359–1368. of experimental colitis by intestinal mononuclear phagocytes. J. Leukoc. Biol. 31. Travis, M. A., B. Reizis, A. C. Melton, E. Masteller, Q. Tang, J. M. Proctor, 80: 802–815. Y. Wang, X. Bernstein, X. Huang, L. F. Reichardt, et al. 2007. Loss of integrin 11. Smith, P. D., C. Ochsenbauer-Jambor, and L. E. Smythies. 2005. Intestinal alpha(v)beta8 on dendritic cells causes autoimmunity and colitis in mice. Nature macrophages: unique effector cells of the innate immune system. Immunol. Rev. 449: 361–365. 206: 149–159. 32. Lang, R., D. Patel, J. J. Morris, R. L. Rutschman, and P. J. Murray. 2002.

12. Noguchi, E., Y. Homma, X. Kang, M. G. Netea, and X. Ma. 2009. A Crohn’s Shaping in activated and resting primary macrophages by IL-10. http://www.jimmunol.org/ disease-associated NOD2 mutation suppresses transcription of human IL10 by J. Immunol. 169: 2253–2263. inhibiting activity of the nuclear ribonucleoprotein hnRNP-A1. Nat. Immunol. 33. Mizutani, N., T. Sakurai, T. Shibata, K. Uchida, J. Fujita, R. Kawashima, 10: 471–479. Y. I. Kawamura, N. Toyama-Sorimachi, T. Imai, and T. Dohi. 2007. Dose- 13. Xu, X., S. Ng, Z. L. Wu, D. Nguyen, S. Homburger, C. Seidel-Dugan, A. Ebens, dependent differential regulation of cytokine secretion from macrophages by and Y. Luo. 1998. Human semaphorin K1 is glycosylphosphatidylinositol-linked fractalkine. J. Immunol. 179: 7478–7487. and defines a new subfamily of viral-related semaphorins. J. Biol. Chem. 273: 34. Kumanogoh, A., S. Marukawa, K. Suzuki, N. Takegahara, C. Watanabe, 22428–22434. E. Ch’ng, I. Ishida, H. Fujimura, S. Sakoda, K. Yoshida, and H. Kikutani. 2002. 14. Pasterkamp, R. J., J. J. Peschon, M. K. Spriggs, and A. L. Kolodkin. 2003. Class IV semaphorin Sema4A enhances T-cell activation and interacts with Tim- Semaphorin 7A promotes axon outgrowth through integrins and MAPKs. Nature 2. Nature 419: 629–633. 424: 398–405. 35. Suzuki, K., A. Kumanogoh, and H. Kikutani. 2008. Semaphorins and their 15. Suzuki, K., T. Okuno, M. Yamamoto, R. J. Pasterkamp, N. Takegahara, receptors in immune cell interactions. Nat. Immunol. 9: 17–23. H. Takamatsu, T. Kitao, J. Takagi, P. D. Rennert, A. L. Kolodkin, et al. 2007. 36. Takamatsu, H., N. Takegahara, Y. Nakagawa, M. Tomura, M. Taniguchi, by guest on September 23, 2021 Semaphorin 7A initiates T-cell-mediated inflammatory responses through R. H. Friedel, H. Rayburn, M. Tessier-Lavigne, Y. Yoshida, T. Okuno, et al. alpha1beta1 integrin. Nature 446: 680–684. 2010. Semaphorins guide the entry of dendritic cells into the lymphatics by 16. Comeau, M. R., R. Johnson, R. F. DuBose, M. Petersen, P. Gearing, activating myosin II. Nat. Immunol. 11: 594–600. T. VandenBos, L. Park, T. Farrah, R. M. Buller, J. I. Cohen, et al. 1998. A 37. Holmes, S., A. M. Downs, A. Fosberry, P. D. Hayes, D. Michalovich, poxvirus-encoded semaphorin induces cytokine production from monocytes and P. Murdoch, K. Moores, J. Fox, K. Deen, G. Pettman, et al. 2002. Sema7A is binds to a novel cellular semaphorin receptor, VESPR. Immunity 8: 473–482. a potent monocyte stimulator. Scand. J. Immunol. 56: 270–275. 17. Kang, H. R., C. G. Lee, R. J. Homer, and J. A. Elias. 2007. Semaphorin 7A plays 38. Czopik, A. K., M. S. Bynoe, N. Palm, C. S. Raine, and R. Medzhitov. 2006. a critical role in TGF-beta1-induced pulmonary fibrosis. J. Exp. Med. 204: 1083– Semaphorin 7A is a negative regulator of T cell responses. Immunity 24: 591– 1093. 600. 18. Gardner, H., J. Kreidberg, V. Koteliansky, and R. Jaenisch. 1996. Deletion of 39. Lacy-Hulbert, A., A. M. Smith, H. Tissire, M. Barry, D. Crowley, R. T. Bronson, by homologous recombination permits normal murine develop- J. T. Roes, J. S. Savill, and R. O. Hynes. 2007. Ulcerative colitis and autoim- ment but gives rise to a specific deficit in cell adhesion. Dev. Biol. 175: 301–313. munity induced by loss of myeloid alphav integrins. Proc. Natl. Acad. Sci. USA 19. Combadiere, C., J. Gao, H. L. Tiffany, and P. M. Murphy. 1998. Gene cloning, 104: 15823–15828. RNA distribution, and functional expression of mCX3CR1, a mouse chemotactic 40. Artis, D. 2008. Epithelial-cell recognition of commensal bacteria and mainte- receptor for the CX3C chemokine fractalkine. Biochem. Biophys. Res. Commun. nance of immune homeostasis in the gut. Nat. Rev. Immunol. 8: 411–420. 253: 728–732. 41. Rimoldi, M., M. Chieppa, V. Salucci, F. Avogadri, A. Sonzogni, 20. Uematsu, S., K. Fujimoto, M. H. Jang, B. G. Yang, Y. J. Jung, M. Nishiyama, G. M. Sampietro, A. Nespoli, G. Viale, P. Allavena, and M. Rescigno. 2005. S. Sato, T. Tsujimura, M. Yamamoto, Y. Yokota, et al. 2008. Regulation of Intestinal immune homeostasis is regulated by the crosstalk between epithelial humoral and cellular gut immunity by lamina propria dendritic cells expressing cells and dendritic cells. Nat. Immunol. 6: 507–514. Toll-like receptor 5. Nat. Immunol. 9: 769–776. 42. Spo¨ttl, T., M. Hausmann, M. Kreutz, A. Peuker, D. Vogl, J. Scho¨lmerich, 21. Stout, R. D., C. Jiang, B. Matta, I. Tietzel, S. K. Watkins, and J. Suttles. 2005. W. Falk, R. Andreesen, T. Andus, H. Herfarth, and G. Rogler. 2001. Monocyte Macrophages sequentially change their functional phenotype in response to differentiation in intestine-like macrophage phenotype induced by epithelial changes in microenvironmental influences. J. Immunol. 175: 342–349. cells. J. Leukoc. Biol. 70: 241–251.