The intestinal immune system is constantly challenged by commensal bacteria; therefore, it must maintain quiescence via several regulatory mechanisms. Although intestinal macrophages (Mϕs) 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 Mφs 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 Mφs as the main producers of IL-10 via αvβ1 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 Mφs during interactions between IECs and Mφs. Furthermore, we determined that the administration of recombinant Sema7A proteins 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 αvβ1 integrin, but also provide a novel mode of IL-10 induction via interactions between IECs and Mφs.

In the intestine, intimate contact continuously occurs between the immune system and exogenous Ags such as diverse microflora and food. Thus, immune responses in the intestine need to be tightly regulated to prevent excessive inflammation in response to these innocuous Ags. The breakdown of these regulatory mechanisms leads to development of inflammatory bowel disease (IBD), including Crohn’s disease and ulcerative colitis (1). In the intestine, several immunosuppressive factors, such as thymic stromal lymphopoietin, retinoic acid, TGF-β, and IL-10, shape the local microenvironment to confer anti-inflammatory properties (2, 3). Among these, one of the most important and best-characterized molecules is the immune regulatory cytokine IL-10. Either IL-10– or IL-10R–deficient mice spontaneously develop colitis in the presence of commensal bacteria (4, 5), and IL-10 is shown to be a pivotal factor in IBD patients (6). Indeed, reduced expression of IL-10 has been demonstrated in the inflamed regions and granulomas of patients with Crohn’s disease (7). Although various innate and adaptive immune cells can produce IL-10, increasing evidence suggests that intestinal macrophages (Mφs) are crucially involved in IL-10–mediated immunological homeostasis (810). IL-10–producing intestinal Mφs are characterized by lower expression levels of costimulatory molecules (CD40, CD80, and CD86) and lower production of proinflammatory cytokines (11) and produce IL-10 by recognition of exogenous Ags with pattern recognition receptors, such as TLRs and nucleotide-binding oligomerization domains (8, 12). However, it still remains unclear how intestinal intrinsic factors regulate IL-10 production in Mφs.

Semaphorins were originally identified as axon guidance cues in the nervous system, which are characterized by a conserved N-terminal Sema domain in their extracellular regions. The semaphorin family has been further divided into eight subclasses based on additional structural features. Two groups of protein families, plexins and neurophilins, have been shown to function as the main receptors for semaphorins. Among them, semaphorin 7A (Sema7A; also known as CD108) (13), a membrane-associated GPI-linked semaphorin, is unique because it has an integrin-binding motif, arginine–glycine–aspartate (RGD), in its Sema domain. Indeed, Sema7A uses β1 integrin as receptor to promote axon outgrowth and contributes to the formation of lateral olfactory tracts (14). In the immune system, Sema7A, which interacts with α1β1 integrin, stimulates inflammatory Mφs to produce proinflammatory cytokines (15). In addition, a vaccinia virus semaphorin A39R, a homolog of vertebrate Sema7A, binds to plexin-C1 and induces the activation of human monocytes in the context of aggregation and production of cytokines such as IL-6 and TNF-α (16). Accordingly, Sema7A-deficient mice are defective in T cell-mediated inflammatory responses in contact hypersensitivity (CHS), experimental autoimmune encephalomyelitis (EAE), and pulmonary fibrosis models, indicating that Sema7A exerts an important role in evoking inflammatory immune reactions (14, 15, 17). However, the role of Sema7A in the intestinal immune system still remains unclear.

In this study, we showed that Sema7A ameliorates intestinal inflammation by inducing IL-10 production from intestinal Mφs. Sema7A-deficient mice displayed more susceptibility to dextran sodium sulfate (DSS)-induced colitis compared with wild-type (WT) mice, primarily due to impaired IL-10 production. We also identified that αvβ1 integrin, but not α1β1 integrin or plexin-C1 on intestinal Mφs, functions as a Sema7A receptor to suppress intestinal inflammation. Moreover, we identified that Sema7A expressed in intestinal epithelial cells (IECs) is responsible for IL-10 production by intestinal Mφs, which is mediated through interactions between intestinal Mφs and IECs. Furthermore, we demonstrated that recombinant Sema7A (rSema7A) proteins exert therapeutic effects on the severities of colitis via IL-10 production.

Sema7A- (14), integrin α (Itga) 1- (18), and plexin-C1–deficient (14) and Ly5.1 mice were generated in the C57BL/6 background. Eight- to 10-wk-old mice were used for experiments. All mice used in this study were housed in specific pathogen-free conditions. All experimental procedures were performed following our institutional guidelines.

For flow cytometry, cells from lamina propria (LP) and spleen (SP) were stained with the following Abs; anti–I-A/I-E (M5/114.15.2) conjugated with eFluor 450, anti-F4/80 (BM8) conjugated with allophycocyanin, anti-CD11b (MJ1/70) conjugated with PE, anti-CD103 (2E7)-PE, anti-CD11c (HL3)-FITC, and anti-CD14 (Sa2-8)-allophycocyanin. For analysis of protein expression on LP-Mφs, biotin-conjugated anti-Sema7A (4D9, mouse IgG1), anti-α1 (HM alpha1), -αv (10D5), or -β1 integrin (HM β 1.1), anti–plexin-C1 mAbs (R&D Systems), and CX3CR1 (catalog number 2093; ProSci) (19) polyclonal Abs were used. FITC-conjugated streptavidin was used for secondary staining. An anti-mouse Sema7A Ab was obtained by immunizing Sema7A-deficient mice with Sema7A–Fc as previously described (15) and conjugated with biotin using an Ab biotinylation kit (American Qualex). For function-blocking assays, we used the following Abs against mouse: anti-α1 (HM alpha1), anti-αv (10D5), anti-α5 (5H10-27 [MFR5]), anti-α8 (aa38-1007), anti-β1 (HM β 1.1), anti-β3, -β5, -β8 (H-160), and anti–plexin-C1 (AF5375). MEK1/2 inhibitor, U0126, and U0124 were purchased from Calbiochem.

Eight- to 10-wk-old Sema7A-, Itga1- and plexin-C1–deficient mice and their littermate WT mice as controls were used for DSS-induced colitis experiments. Acute colitis was induced by administration of 2% (w/v) DSS (36–50 kDa; MP Biomedicals) to the drinking water for 7 d. To test survival rate, 4% (w/v) DSS was used until day 4 followed by normal drinking water until the end of the experiment (day 14).

The specimens were embedded in O.C.T. compound and fixed with methanol. Tissue sections were stained with H&E. Histological grading was based on observed inflammation, defined as follows: 0, no inflammation; 1, low level of inflammation with mildly increased inflammatory cells in the LP; 2, moderately increased inflammation in the LP (multiple foci); 3, high level of inflammation with evidence of wall thickening by inflammation; and 4, maximal severity of inflammation with transmural leukocyte infiltration and/or architectural distortion.

The 1 × 1 cm standardized segments of the colon were washed in cold PBS supplemented with penicillin and streptomycin. Epithelial integrity was disrupted by treatment with 1 mM EDTA for 30 min at 37°C on a shaker, followed by vortexing for 2 min. These segments were cultured in 24-well flat-bottom culture plates in serum-free RPMI 1640 medium (Invitrogen) supplemented with penicillin, streptomycin, and gentamicin. After 24 h of cultivation, supernatant fluid was collected and used for estimation of IL-10 and TGF-β secretion.

LP-lymphocytes and Mφs were isolated as described (20), with some modifications. Mice were sacrificed, and colons and spleens were removed and placed in PBS. The colon was opened longitudinally, washed in PBS, and cut into 1-cm pieces. The pieces were treated for 30 min at 37°C with PBS containing 5% (v/v) FCS, HEPES (20 nM) (pH 7.4), penicillin (100 U/ml), streptomycin (100 U/ml), sodium pyruvate (1 mM), EDTA (10 mM), and polymyxin B (10 μg/ml; Calbiochem) to remove epithelial cells and then washed extensively with PBS. Segments of the colon and SP were digested for 45 min with continuous stirring at 37°C, with collagenase D (800 Mandl units/ml; Roche), DNase I (10 μg/ml; Roche), and dispase I (10 μg/ml; Invitrogen) in RPMI 1640 medium with 5% (v/v) FCS. EDTA was added (final concentration, 10 mM), and cell suspensions were incubated for an additional 5 min at 37°C. Cells were spun through a 17.5% (w/v) solution of Accudentz (Accurate Chemical & Scientific), and collected whole cells were used in assays. LP-Mφ were sorted on the basis of their expression of F4/80, CD11b, and I-A/I-E with an FACSAria (BD Biosciences). In addition, inflammatory Mφs from colon of DSS-fed WT mice were sorted on the basis of CD14 and CD11b expression with FACSAria (BD Biosciences). The purity of the sorted Mφs was routinely >98%. For analysis of lymphocytes, cells were subjected to density-gradient centrifugation in 40–75% (v/v) Percoll (approximate density 1.058 g/ml and 1.093 g/ml, respectively) after enzyme treatment. Cells collected from the interface were washed and used as LP-lymphocytes; CD4+ and B220+ cells were purified by magnetic sorting with mouse anti-CD4 beads and mouse anti-B220 beads, respectively. The purity of the sorted cells was routinely >90%.

LP- and SP-derived whole cells or sorted LP-Mφs (2 × 105 per well) were added to flat-bottom 96-well plates coated with the indicated concentrations of Fc–control (Millipore), Sema7A–Fc, or KGE–Fc and cultured for 24 h in RPMI 1640 medium containing 5% FCS and gentamicin. The supernatant fluid was analyzed by ELISA (IL-10, IFN-γ, TNF-α, and IL-12p70: R&D Systems; TGF-β: eBioscience). For function-blocking assays, LP-Mφs (1 × 105 cells/well) were pretreated with anti-integrin mAbs or anti-mouse plexin-C1 mAb (R&D Systems) for 30 min on ice. Then, these cells were added to flat-bottom 96-well plates coated with the indicated concentrations of Fc–control (Millipore) and Sema7A–Fc and cultured for 24 h.

Total RNAs were prepared from the colon tissue of DSS-fed WT and Sema7A-deficient mice using an RNeasy Mini kit (Qiagen) and treated with DNase I (Invitrogen) to eliminate genomic DNA. cDNA was synthesized using a SuperScript II cDNA synthesis kit (Invitrogen). A 7700 Sequence Detector (Applied Biosystems) was used for quantitative PCR of cDNA amplified with 2× PCR Master Mix (Applied Biosystems) and primers specific for IL-10, TGF-β, retinal dehydrogenase 2 (Aldh1a2), and Sema7A (Applied Biosystems) in a final volume of 20 μl. After incubation at 95°C for 10 min, products were amplified by 35 cycles of 95°C for 15 s, 60°C for 60 s, and 50°C for 120 s. RT-PCR was performed with 35 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s using the gene-specific primers (mouse α1 integrin: forward, 5′-AATGAGCCTGGAGCCTATCA-3′ and reverse, 5′-TATACACGGCTCCTCCGTGA-3′; mouse αv integrin: forward, 5′-CAAGCTCACTCCCATCAC-3′ and reverse, 5′-GGGTGTCTTGATTCTCAAAGGG-3′).

Bone marrow (BM) cells (2 × 106) were cultured in 24-well plates in 1 ml RPMI 1640 medium containing 10% FCS and 40 ng/ml murine GM-CSF (PeproTech). LPS (1 μg/ml; Sigma-Aldrich) was added to the culture 12 h before the end of the culture (21).

LP-Mφs were serum-starved for 4 h in RPMI 1640 medium supplemented with 0.1% BSA. Cells were resuspended in serum-free RPMI 1640 medium, and 1 × 106 cells were seeded into 96-well plates coated with Fc–control or Sema7A–Fc (20 nM). Cell extracts were prepared by lysing the cells with a buffer (50 mM Tris-Cl [pH 7.5], 150 mM NaCl, and 2 mM EDTA, plus cocktails of protease inhibitors [Roche] and phosphatase inhibitors [Sigma-Aldrich]) containing 2% Nonidet P-40, subjected to a 4–12% gradient SDS gel (Invitrogen) in MOPS buffer, and transferred to a polyvinylidene fluoride membrane (Millipore). Membranes were probed with anti–phospho-focal adhesion kinase (FAK; Tyr397) and anti–phospho-ERK1/2 (Thr202/Tyr204) (all from Cell Signaling Technology). The membrane was stripped with the Restore Western blot Stripping Buffer (Pierce) for 10 min at room temperature and reprobed with Abs to total FAK and ERK (Cell Signaling Technology).

IECs from colon of WT mice were isolated as described reference (22). Colons were isolated, opened longitudinally, and rinsed with PBS. The epithelial integrity was disrupted by treatment with 1 mM DTT for 30 min 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 40% Percoll in a 15-ml Falcon tube. Percoll gradient separation was performed 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%).

Caco-2 cells were cultured for 7 d in the upper chambers of Transwell filters (3 μm in pore diameter; Costar). LP-Mφs were incubated for 24 h with medium 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 filters. After 7 d, filters bearing Caco-2 cells were turned upside down, and LP-Mφs (4.5 × 105 cells/well) were incubated on the filter facing the basolateral side of Caco-2 cells for 24 h. Supernatants were used for ELISA analysis.

BM transfer experiment was used to generate Sema7A-deficient chimera mice wherein the genetic deficiency of Sema7A was limited to either circulating cells or nonhematopoietic cells. In brief, BM samples were collected from femur and tibia of congenic WT (expressing CD45.1 leukocyte Ag) or Sema7A-deficient and littermate WT (expressing CD45.2 leukocyte Ag) donor mice by flushing with HBSS. After several washing steps, cells were resuspended in PBS at a concentration of 3 × 107 cells/ml. A total of 100 μl of this cell suspension was injected in irradiated recipient mice. Four chimera groups were generated: WT > WT (WT cells expressing CD45.2 into WT expressing CD45.1); WT > 7A−/− (WT cells expressing CD45.1 into Sema7A−/− expressing CD45.2); 7A−/− > 7A−/− (Sema7A−/− cells expressing CD45.2 into Sema7A−/− expressing CD45.1); and 7A−/− > WT (Sema7A−/− cells expressing CD45.2 into WT expressing CD45.1). The use of CD45.1-expressing congenic mice facilitated verification of proper reconstitution in the chimeric mice. BM reconstitution was verified after 7 wk by staining for CD45.1 and CD45.2 in blood cells with PE-conjugated anti-CD45.1 and FITC-conjugated anti-CD45.2. Eight weeks after BM transfer, mice were treated with 2% DSS for 7 d. Body weight change was monitored daily. At day 7, mice were sacrificed to collect colon tissue for H&E staining.

The 4% paraformaldehyde in PBS (paraformaldehyde)-fixed colon tissue slides were stained for Sema7A-positive cells via the immunoperoxidase method with anti-mouse Sema7A Abs and then counterstaining with hematoxylin. The Sema7A-positive cells were observed by microscopy (Nikon ELIPSE E600; Nikon).

To analyze statistical significance, we used an unpaired, two-tailed Student t test. We considered p values <0.05 to be significant.

A previous study showed that Sema7A promotes T cell-mediated inflammatory responses in the skin and CNS (15). Because Sema7A transcripts were abundantly expressed in the intestine (data not shown), we decided to investigate the role of Sema7A in the intestinal immune system. We first performed an experimental colitis model using WT and Sema7A-deficient mice treated by oral administration of 4% DSS, which is toxic to colonic epithelial cells and elicits acute inflammatory responses by disrupting the compartmentalization of commensal bacteria. Only 10% of WT mice died during the DSS administration period, but, unexpectedly, a mortality rate >90% was noted for Sema7A-deficient mice (Fig. 1A). The experiment was repeated with a lower DSS concentration (2%) to study the phenotype of Sema7A-deficient mice under milder conditions. Sema7A-deficient mice exhibited loss of body weight and more severe colitis than WT mice (Fig. 1B) due to severe diarrhea and rectal bleeding. In addition, the mutant mice showed shorter colon and severe transmural inflammation with extensive crypt destruction, edema, and infiltration of immune cells, leading to severe histological scores (Fig. 1C–E). These results suggest that Sema7A plays a suppressive role in the intestinal inflammatory responses.

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 μm. E, Semiquantitative scoring of histopathology was performed as described in the 1Materials and Methods. BE, 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.

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 μm. E, Semiquantitative scoring of histopathology was performed as described in the 1Materials and Methods. BE, 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.

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To determine the responsible regulatory mechanisms, we measured the expression of several immunosuppressive factors such as IL-10, TGF-β, and Aldh1a2, which encodes Aldh1a2 in the colon of DSS-treated mice. Although the expression levels of TGF-β and Aldh1a2 were not affected, IL-10 expression levels were considerably decreased by the absence of Sema7A (Fig. 2A). Also, in the DSS-treated colon explant culture, IL-10 but not TGF-β concentrations in the supernatant from colon of DSS-fed Sema7A-deficient mice were significantly reduced (Fig. 2B, Supplemental Fig. 1A).

FIGURE 2.

Sema7A induces IL-10 production by MHC IIintF4/80hiCD11bhi Mφs. A, Relative transcript levels of IL-10, TGF-β, 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–control in the presence or absence of LPS (1 μg/ml) for 24 h. D, IL-10 production by MHC IIintF4/80hiCD11bhi, 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-Mφs. Cells were stimulated with various concentrations of rSema7A or Fc–control in the presence or absence of LPS (1 μg/ml). F, IL-10 production by LP-Mφs. LP-Mφs from WT and Sema7A-deficient mice before and 6 d after DSS administration were cultured for 12 h. BF, Cytokine production was measured by ELISA. Data are representative of three independent experiments (error bars, SD of averages from triplicate cultures). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 2.

Sema7A induces IL-10 production by MHC IIintF4/80hiCD11bhi Mφs. A, Relative transcript levels of IL-10, TGF-β, 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–control in the presence or absence of LPS (1 μg/ml) for 24 h. D, IL-10 production by MHC IIintF4/80hiCD11bhi, 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-Mφs. Cells were stimulated with various concentrations of rSema7A or Fc–control in the presence or absence of LPS (1 μg/ml). F, IL-10 production by LP-Mφs. LP-Mφs from WT and Sema7A-deficient mice before and 6 d after DSS administration were cultured for 12 h. BF, Cytokine production was measured by ELISA. Data are representative of three independent experiments (error bars, SD of averages from triplicate cultures). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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Next, to examine whether rSema7A protein promotes cytokine production in the intestine, we cultured whole LP cells and SP cells in the presence of rSema7A. As shown in Fig. 2C, rSema7A dose-dependently increased IL-10 levels from LP cells, whereas rSema7A did not affect the production of other proinflammatory cytokines such as IFN-γ, IL-12p70, and TNF-α (Supplemental Fig. 1B). In addition, Sema7A had less of an effect on IL-10 production by SP cells (Fig. 2C). Thus, Sema7A preferentially induces IL-10 production by LP cells.

Several studies have identified that various immune cells, such as regulatory T cells (Treg; Tr1 and Foxp3+), regulatory B cells, dendritic cells (DCs), and resident Mφs, can produce IL-10 in the intestine (8, 23, 24). So, we examined which cells are responsible for IL-10 production in response to Sema7A. Neither T cells, B cells, DCs, nor inflammatory Mφs produced IL-10 in response to rSema7A (Fig. 2D). In contrast, MHC class II (MHC II)intF4/80hiCD11bhi resident Mφs (LP-Mφs) produced IL-10 in response to rSema7A in a dose-dependent manner (Fig. 2E). Moreover, we tested whether IL-10 production is impaired in LP-Mφs from DSS-fed Sema7A-deficient mice. We isolated LP-Mφs from the colon of WT or Sema7A-deficient mice before and 6 d after DSS administration. Consistent with the data in Fig. 2B, LP-Mφs from DSS-fed Sema7A-deficient mice displayed significantly reduced IL-10 production compared with those from WT mice (Fig. 2F). Additionally, these LP-Mφs are characterized by their potent IL-10 production and expression of the fractalkine receptor CX3CR1 on their surface (Supplemental Fig. 2A) (2527). Therefore, these results indicate that LP-Mφs are compatible to CX3CR1+ Mφs and responsible for Sema7A-induced IL-10 production.

Sema7A has been reported to use α1β1 integrin in inflammatory Mφs and plexin-C1 in human monocyte (15, 28). We then examined whether Sema7A-induced IL-10 production by LP-Mφs is mediated by integrins or plexin-C1. LP-Mφs were pretreated with Abs against α1 integrin, β1 integrin, or plexin-C1 and then exposed to a plate coated with rSema7A. Sema7A-induced IL-10 production from these Mφs was significantly inhibited by Abs against anti-β1 integrin but not by anti–plexin-C1 Ab. Unexpectedly, anti-α1 integrin Ab could not inhibit Sema7A-induced IL-10 production in these Mφs (Fig. 3A). Consistent with this, α1 integrin and plexin-C1 were not expressed in LP-Mφs (Supplemental Fig. 2B, 2C). Also, it was confirmed that α1 integrin-deficient or plexin-C1–deficient mice displayed rather mild colitis compared with WT mice in terms of loss of body weight and shortening of the colon in a DSS-induced colitis model (Supplemental Fig. 2D, 2E).

FIGURE 3.

Sema7A increases IL-10 from LP-Mφs through αvβ1 integrin signaling. AD, IL-10 levels in the culture supernatant were measured. A, LP-Mφs were treated with anti-α1 or β1 integrin or anti–plexin-C1 mAbs (25 μg/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-Mφs 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-Mφs were treated with anti-α5, -αv, or -α8 integrin mAbs (25 μg/ml) and stimulated with plate-coated 40 nM rSema7A and Fc–control. D, LP-Mφs were treated with anti-β3, -β5, or -β8 integrin mAbs (25 μg/ml) and stimulated with plate-coated 40 nM rSema7A and Fc–control. E, Western blot analysis of FAK and ERK1/2 phosphorylation in LP-Mφs following rSema7A and Fc–control (Cont.) stimulation (left panel) and in the pretreatment with anti-αv integrin Ab or isotype Ab (right panel). F, LP-Mφs 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. AD 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.

FIGURE 3.

Sema7A increases IL-10 from LP-Mφs through αvβ1 integrin signaling. AD, IL-10 levels in the culture supernatant were measured. A, LP-Mφs were treated with anti-α1 or β1 integrin or anti–plexin-C1 mAbs (25 μg/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-Mφs 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-Mφs were treated with anti-α5, -αv, or -α8 integrin mAbs (25 μg/ml) and stimulated with plate-coated 40 nM rSema7A and Fc–control. D, LP-Mφs were treated with anti-β3, -β5, or -β8 integrin mAbs (25 μg/ml) and stimulated with plate-coated 40 nM rSema7A and Fc–control. E, Western blot analysis of FAK and ERK1/2 phosphorylation in LP-Mφs following rSema7A and Fc–control (Cont.) stimulation (left panel) and in the pretreatment with anti-αv integrin Ab or isotype Ab (right panel). F, LP-Mφs 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. AD 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.

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Because Sema7A can bind to integrin through the RGD motif, we further examined whether the RGD motif is important for Sema7A-induced IL-10 production. The mutated Sema7A proteins (KGE–Fc) that lack integrin-binding activity failed to increase IL-10 production (Fig. 3B), suggesting Sema7A-induced IL-10 production in LP-Mφs is dependent on integrin signaling. β1 integrin can be noncovalently associated with several different α integrin subunits, such as α5, αv, and α8, and act as RGD receptor (29). Notably, pretreatment of LP-Mφs with Ab against αv integrin significantly abrogated IL-10 production by Sema7A stimulation, whereas treatment with anti-α5 or -α8 integrin Abs did not (Fig. 3C). The αv integrin is the most promiscuous α subunit, assembling with four different β subunits, including β1, β3, β5, and β8 integrin, which are involved in the intestinal homeostasis through TGF-β production (30, 31). However, we found that β subunits other than β1 had no effects on IL-10 production by LP-Mφs (Fig. 3D). In addition, pretreatment of LP-Mφs with anti-integrin Abs did not show any influences on TGF-β production (data not shown). Collectively, these data imply that Sema7A uses αvβ1 integrin as a receptor to promote IL-10 production by LP-Mφs.

Previous studies demonstrated that Sema7A exerts its biological functions through activation of FAK and subsequently ERK signaling pathways, downstream of integrins in both immune and neuronal cells (14, 15). In addition, IL-10 production by human monocytes is mediated by ERK signaling (32). Consistent with the previous findings, Sema7A induced the phosphorylation of FAK and ERK1/2 in LP-Mφs (Fig. 3E, left panel), and this effect was completely abolished by blocking Abs against αv integrin (Fig. 3E, right panel). Furthermore, Sema7A-induced IL-10 production by LP-Mφs was dose-dependently abolished by U0126, a specific inhibitor of ERK signaling (MEK-1 and MEK-2), but not U0124, an inactive analog compound (Fig. 3F). Thus, our findings suggest that IL-10 production by LP-Mφs requires ERK activation through Sema7A–αvβ1 integrin interactions.

Next, we tried to identify the type of cells that express Sema7A in the intestine by immunohistochemical analysis. When we stained colon slice sections with anti-Sema7A Ab, Sema7A was highly expressed in the IEC and crypt enterocytes, rather than in LP immune cells (Fig. 4A). We also confirmed Sema7A expression on IECs by flow cytometry (Supplemental Fig. 3A, left panel). In addition, quantitative analysis of the fluorescence intensity for Sema7A indicated that Sema7A was predominantly localized on the basolateral but not the apical side of IECs (Supplemental Fig. 3B).

FIGURE 4.

Sema7A, which is expressed on IECs, directly interacts with LP-Mφs to induce IL-10 production. A, Representative photomicrographs of colon from WT (left panel) and Sema7A-deficient (right panel) mice after 3,3′-diaminobenzidine staining using anti-Sema7A Ab (arrowheads indicate positive cells) and hematoxylin counter staining. Scale bars, 10 μm. BD, Coculture system of Caco-2 and Mφs. 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-Mφs (Mφ) 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 Mφs) for 24 h. D, LP-Mφs were pretreated with anti-αv integrin mAbs (25 μg/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.

FIGURE 4.

Sema7A, which is expressed on IECs, directly interacts with LP-Mφs to induce IL-10 production. A, Representative photomicrographs of colon from WT (left panel) and Sema7A-deficient (right panel) mice after 3,3′-diaminobenzidine staining using anti-Sema7A Ab (arrowheads indicate positive cells) and hematoxylin counter staining. Scale bars, 10 μm. BD, Coculture system of Caco-2 and Mφs. 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-Mφs (Mφ) 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 Mφs) for 24 h. D, LP-Mφs were pretreated with anti-αv integrin mAbs (25 μg/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.

Close modal

Recently, it has been reported that during inflammation, IECs increased the expression of fractalkine and CX3CL1 and subsequently induced the recruitment of CX3CR1+ cells to IECs (33). Therefore, we hypothesized that Sema7A expression on the basolateral side of IECs is crucial for IL-10 production by Mφs that express CX3CR1 during interactions between IECs and Mφs. To demonstrate this, Caco-2 cells, which are human IEC lines and expressed Sema7A (Supplemental Fig. 3A, right panel), were polarized on a transwell membrane and then added LP-Mφs to the apical or basolateral sides of epithelial cell monolayers (Fig. 4B). When LP-Mφs were faced to the basolateral side of Caco-2 cells, they could produce IL-10 (Fig. 4C). By contrast, when LP-Mφs could not contact with Caco-2 cells or faced to the apical side of Caco-2 cells, they released less IL-10 (Fig. 4C). In addition, this effect was completely abolished by neutralizing Abs against αv integrins (Fig. 4D). Collectively, these findings suggest that interactions between IECs and LP-Mφs are important for regulating IL-10 production through Sema7A–αvβ1 integrin signaling.

Several previous studies have reported that activated T cells are the main source of Sema7A during the development of CHS and EAE inflammation (22). However, as shown in Fig. 4A and Supplemental Fig. 3C, Sema7A was preferentially expressed in IECs. Therefore, the question arose whether hematopoietic or nonhematopoietic cell-derived Sema7A is relevant for regulating intestinal inflammation. To address this issue, we generated four types of BM chimera mice by criss-cross transplantation of WT or Sema7A-deficient BM cells to WT or Sema7A-deficient (7A−/−) recipient mice (WT > WT, 7A−/− > WT, WT > 7A−/−, or 7A−/− > 7A−/−) and induced colitis in these animals by DSS feeding. Although mice that systemically lacked Sema7A expression (7A−/− > 7A−/−) showed more severe loss of body weight, shortening of the colon, and extensive crypt destruction with infiltration of lymphocytes than WT > WT mice, mice that lacked Sema7A expression in hematopoietic cells (7A−/− > WT) exhibited colitis comparable to that observed in WT > WT mice (Fig. 5). Conversely, mice lacking nonhematopoietic Sema7A (WT > 7A−/−) displayed colitis as severe as that observed in Sema7A-null mice (7A−/− > 7A−/−). These results suggest that Sema7A expressed in IECs is indispensable for protection against DSS-induced colitis.

FIGURE 5.

Sema7A expressed in nonhematopoietic cells but not hematopoietic cells is critical for protection against DSS-induced colitis. A, Percentage of weight changes of BM chimeric mice after DSS administration. 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 examined by H&E staining. Scale bars, 100 μm. D, Semiquantitative scoring of histopathology was performed. AD, BM chimera mice were generated as described in 1Materials 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.

FIGURE 5.

Sema7A expressed in nonhematopoietic cells but not hematopoietic cells is critical for protection against DSS-induced colitis. A, Percentage of weight changes of BM chimeric mice after DSS administration. 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 examined by H&E staining. Scale bars, 100 μm. D, Semiquantitative scoring of histopathology was performed. AD, BM chimera mice were generated as described in 1Materials 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.

Close modal

We finally examined the therapeutic effects of rSema7A against intestinal inflammation and whether the effects of Sema7A depend on IL-10 in vivo. WT mice were given 2% DSS solution and treated with rSema7A in the absence or presence of anti–IL-10–blocking Abs. Mice receiving rSema7A were rescued from the loss of body weight and shortening of the colon and improved histological scores for colitis compared with control mice. In addition, blocking Abs against IL-10 eliminated the therapeutic effects of rSema7A (Fig. 6). Thus, Sema7A confers protection against DSS-induced colitis, depending on IL-10 production. Collectively, our findings demonstrated that Sema7A expressed on IECs is an important element to induce IL-10 production in intestinal Mφs and prevent excessive intestinal inflammation (Supplemental Fig. 4).

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 average of each group is indicated with a bold red line. C, Histopathological changes in colon tissues were examined by H&E staining. Scale bars, 100 μm. D, Semiquantitative scoring of histopathology was performed. AD, WT mice were administrated 2% DSS in drinking water for 7 d and injected with 50 μg 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 representative of three independent experiments. *p < 0.05, **p < 0.01.

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 average of each group is indicated with a bold red line. C, Histopathological changes in colon tissues were examined by H&E staining. Scale bars, 100 μm. D, Semiquantitative scoring of histopathology was performed. AD, WT mice were administrated 2% DSS in drinking water for 7 d and injected with 50 μg 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 representative of three independent experiments. *p < 0.05, **p < 0.01.

Close modal

Semaphorins were originally identified as axon guidance molecules in neural development; however, the accumulated evidence now indicates that several semaphorins play crucial roles in physiological and pathological immune responses (3436). Sema7A has been shown to activate inflammatory Mφs and monocytes (37). In this study, we highlighted a negative regulatory role of Sema7A in intestinal inflammation, in which Sema7A induces production of the anti-inflammatory cytokine IL-10 via use of a specific integrin receptor, αvβ1 integrin. We further demonstrated that Sema7A is predominantly expressed on the basolateral side of IECs and that Sema7A in IECs is involved in IL-10 production as a result of interactions between IECs and intestinal Mφs.

Previous studies have emphasized the critical role of Sema7A in inflammation through interactions with either α1β1 integrin or plexin-C1, resulting in the activation of inflammatory Mφs or monocytes in the CHS, EAE, and pulmonary fibrosis models (14, 15, 17). In contrast, we unexpectedly found that Sema7A is crucially involved in negative regulation of intestinal inflammation via an interaction with αvβ1 integrin in intestinal Mφs. Indeed, we found that inflammatory Mφs expressed α1 integrin but not αv integrin; vice versa, intestinal regulatory Mφs expressed αv integrin but not α1 integrin (Supplemental Fig. 2B). Regarding suppressive functions of Sema7A, Czopik et al. (38) have previously reported that Sema7A expressed in T cells downregulates T cell activation by functioning as a self-limiting molecule for TCR signaling. In contrast, we confirmed in this study that IEC-derived Sema7A induces IL-10 producing signaling in Mφs, showing the different negative machineries from peripheral T cells. Of note, DSS-induced colitis has been shown to develop even in the absence of T cells. It thus appears that Sema7A regulates immune responses, depending on different receptor use or environmental contexts.

αv integrin plays pivotal roles in the maintenance of intestinal immune homeostasis by increasing TGF-β and subsequent Treg differentiation. Indeed, Itgav-deficient mice under the tie2 promoter (αv-tie2 mice), in which αv integrin is deleted in myeloid cells, developed spontaneous colitis due to failure of generation of Treg cells and removed apoptotic cells (39). In addition, it has been reported that the deficiency of αvβ5 or αvβ8 integrin on DCs also resulted in spontaneous colitis due to reduction of TGF-β in the intestine (31, 39). In this study, we found that αvβ1 integrin on intestinal Mφs is critically involved in IL-10 production by Sema7A stimulation. However, we could not observe any differences in the levels of TGF-β between DSS-fed Sema7A and WT mice (Supplemental Fig. 1A), implying that TGF-β is not primarily involved in the protective role of Sema7A–αvβ1 integrin signaling. Indeed, deficiency of αv integrin causes more severe signs of colitis than those of DSS-fed Sema7A-deficient mice. Thus, it is possible that αv integrin mediates both TGF-β and IL-10–producing signaling by coupling with the different integrin β subunits in the intestinal immune system.

IECs are crucially involved in maintaining intestinal immune homeostasis by influencing the function of intestinal immune cells, including Mφs, DCs, and lymphocytes (4042); in particular, interactions between intestinal Mφs and IECs have been emphasized. For instance, these interactions have been shown to direct monocytes to differentiate into anti-inflammatory cells (26, 42) and play a cardinal role in initiating oral tolerance (25). Additionally, accumulated experimental findings also indicate that IL-10– producing intestinal Mφs play important roles in repression of excessive intestinal inflammation, in that they actively counteract the onset of inflammation. Recently, it has been suggested that intestinal Mφs are located just below IECs and characterized by CX3CR1+MHC IIintF4/80hiCD11bhi expression (26). Indeed, these Mφs are capable of producing a large amount of IL-10, which is regulated by intestinal intrinsic CX3CR1 signaling (25). Notably, the CX3CR1 ligand CX3CL1 is expressed as a membrane-bound form by IECs and upregulated in the inflamed intestine, recruiting the regulatory Mφs to the basolateral side of IECs (33). These dynamic interactions between IECs and Mφs seem to contribute to maintain the intestinal homeostasis. In this context, it is worthy of note that Sema7A was expressed at the basolateral side of IECs, and its receptor αvβ1 integrin was expressed in the CX3CR1-positive intestinal Mφs. Indeed, we found in this study that only CX3CR1+MHC IIintF4/80hiCD11bhi Mφs produced IL-10 upon Sema7A stimulation, whereas other cells, including CD103+ DCs, T cells, and B cells, did not. In addition, mice deficient for nonhematopoietic cell-derived Sema7A showed more severe signs of colitis than either WT mice or mice deficient for hematopoietic cell-derived Sema7A. Thus, IEC-derived Sema7A, of which expression levels were maintained during DSS-induced colitis (Supplemental Fig. 3C), is crucial for intestinal immune homeostasis. It is possible that DSS exerts a cytolytic effect on IECs, resulting in the generation of soluble Sema7A. However, we did not detect the activities of soluble Sema7A on IL-10 production (Fig. 4C). Therefore, our observations support the scenarios that in the context of intestinal inflammation, CX3CL1 is upregulated by IECs to recruit CX3CR1+MHC IIintF4/80hiCD11bhi Mφs, and, under these conditions, Mφs adhere to a rigid scaffold composed of extracellular matrix and Sema7A on the basolateral side of IECs and interact with αvβ1 integrin, resulting in IL-10 production (Supplemental Fig. 4).

In conclusion, we demonstrated in this study that the interaction between IECs and intestinal Mφs plays a crucial role in controlling intestinal inflammation through Sema7A–αvβ1 integrin interactions. Defects in this mechanism resulted in impaired IL-10 production, exacerbating intestinal inflammation. The results not only elucidate a pivotal role for Sema7A as an intrinsic suppressor of intestinal inflammation, but also provide a novel insight into mechanisms for controlling the intestinal inflammation and clinical applications in IBDs.

We thank T. Ishikawa for technical support.

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 Young Scientists), grants-in-aid from the Ministry of Health, Labor and Welfare, the program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (to A.K.), the Target Protein Research Program of the Japan Science and Technology Agency (to T.T. and A.K.), the Funding Program for Next Generation World-Leading Researchers (NEXT Program) and Special Coordination Funds for Promoting Science and Technology (to A.K.), and the World Class University program, National Research Foundation, and the Ministry of Education, Science, and Technology, Korea.

The online version of this article contains supplemental material.

Abbreviations used in this article:

Aldh1a2

retinal dehydrogenase 2

BM

bone marrow

CHS

contact hypersensitivity

DC

dendritic cell

DSS

dextran sodium sulfate

EAE

experimental autoimmune encephalomyelitis

FAK

focal adhesion kinase

IBD

inflammatory bowel disease

IEC

intestinal epithelial cell

Itga

integrin α

LP

lamina propria

macrophage

MHC II

MHC class II

RGD

arginine–glycine–aspartate

rSema7A

recombinant semaphorin 7A

Sema7A

semaphorin 7A

SP

spleen

Treg

regulatory T cell

WT

wild-type.

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The authors have no financial conflicts of interest.