International Immunology, Vol. 22, No. 12, pp. 953962 doi:10.

1093/intimm/dxq449 Advance Access publication 3 November 2010

The Japanese Society for Immunology. 2010. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Commensal microbiota induce LPS hyporesponsiveness in colonic macrophages via the production of IL-10
Yoshiyasu Ueda1,2,3,*, Hisako Kayama1,2,*, Seong Gyu Jeon1,2, Takashi Kusu1,2, Yoshitaka Isaka3, Hiromi Rakugi3, Masahiro Yamamoto1,2 and Kiyoshi Takeda1,2
1 Laboratory of Immune Regulation, Department of Microbiology and Immunology, Graduate School of Medicine, 2WPI Immunology Frontier Research Center and 3Department of Geriatric Medicine and Nephrology, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan

*These authors contributed equally to this study. Correspondence to: K. Takeda; E-mail: ktakeda@ongene.med.osaka-u.ac.jp Received 1 September 2010, accepted 1 October 2010

Abstract Several subsets of innate immune cells, all with unique properties, reside within the intestinal lamina propria. However, compared with intestinal dendritic cells (DCs), intestinal macrophages are less well characterized. In this study, we examined the properties of macrophages in the colonic lamina propria (LMf). Colonic DCs (LDC) showed LPS-induced production of IL-12p40. In contrast, LMf showed constitutive IL-10 production and unresponsiveness to LPS in terms of inammatory cytokine production. Comparison of the gene expression proles between LMf and LDC revealed that LMf preferentially expressed IL-10-related genes. LMf obtained from mice lacking IL-10 or Stat3 showed hyperproduction of tumour necrosis factor (TNF)-a and IL-6 in response to LPS. IL-10 production in the large intestine was mainly induced by LMf and regulatory T cells and was dependent on the presence of commensal microbiota. Accordingly, LMf from germ-free mice showed less production of IL-10 and increased levels of LPS-induced TNF-a and IL-6 production. Taken together, these results demonstrate that the activity of LMf to produce pro-inammatory cytokines is negatively regulated through commensal microbiota-dependent IL-10 production in the large intestine. Keywords: inammation, innate immunity, intestinal mucosa

Introduction The gastrointestinal tract is a unique site, covered by a single layer of epithelial cells that are exposed to a variety of commensal bacteria as well as dietary and environmental antigens. The intestine is also a main target for entry of pathogenic microorganisms into the host tissues (1, 2). Therefore, the intestinal immune system is unique in that it maintains homeostasis by discriminating between invasive pathogens and non-pathogenic microbiota (3, 4). The innate immune system plays an important role in the recognition of microorganisms through pattern recognition receptors, which trigger host immune responses to these microorganisms (5, 6). In the intestine, where the innate immune system has close contact with microorganisms, there are several unique subsets of innate immune cells (7, 8). These intestinal innate immune cells exhibit these unique functions through the interaction with intestinal microbiota and dietary components (9). Of these innate immune cells, two subsets of dendritic cells (DCs) have recently been characterized. One comprises CD11bCD11c+CD103+ DCs, which are derived from classical DC precursors and induce regulatory T (Treg) cells. In addition to the induction of Treg cells, CD103+ DCs possess several unique properties, such as inducing the expression of gut homing receptors (a4b7 and CCR9) in T cells and the proliferation of CD8+ T cells (10). These unique functions are conserved between humans and mice (11); however, they are only observed in the small intestine. This is especially true for the induction of CCR9 expression by T cells (11). The other comprises CX3CR1+CD11b+CD11c+ cells, which are derived from Ly6Chigh monocytes and mediate inammatory responses through induction of Th1 and Th17 cells (12, 13). In addition to these intestinal DC subsets, intestinal macrophages also have unique properties. Characterization of intestinal macrophages in mice has mainly been performed using isolated CD11b+ cells, including CD11b+CD11c+ DCs. Intestinal CD11b+ cells are hyporesponsive to Toll-like

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954 Gut ora induce LPS refractoriness in colonic macrophage receptor (TLR) stimulation, and the breakdown of this hyporesponsiveness correlates with the pathogenesis of intestinal inammation (14, 15). Human intestinal macrophages are also refractory to TLR-dependent inammatory responses, despite normal phagocytic activity, and the aberrant activity of these intestinal macrophages contributes to the pathogenesis of inammatory bowel diseases such as Crohns diseases (16 18). In a recent study, CD11b+CD11c macrophages in the murine small intestine were found to induce Treg cell development (19). This property of macrophages in the murine small intestine is similar to that observed in CD11bCD11c+CD103+ DCs in the murine small intestine (20, 21). Thus, although both CD11b+CD11c macrophages and CD103+ DCs in the small intestine have the ability to induce Treg cells, the function of macrophages in the large intestine is largely uncharacterized. It is also unclear how the activity of macrophages and DCs within the large intestine is regulated by commensal bacteria, which reside in close proximity to intestinal innate immune cells (2). In this study, we characterized the function of intestinal CD11b+CD11c macrophages residing in the murine large intestine, by comparing their functions with colonic CD11bCD11c+ DCs. Unlike CD11bCD11c+ DCs, CD11b+CD11c macrophages residing in the large intestine were defective in the TLR-dependent induction of inammatory cytokines. This was due to suppression mediated by the anti-inammatory cytokine, IL-10, produced in the intestine in a commensal microora-dependent manner. The breakdown of this refractory response to bacterial stimulation through the microbiota/IL-10-axis resulted in increased inammatory responses by large intestinal macrophages. Methods Mice C57BL/6 mice, BALB/c mice and BALB/c germ-free mice were purchased from CLEA Japan (Osaka, Japan). Myd88/, Il10/ and LysM-cre; Stat3ox/ox mice backcrossed onto C57BL/6 for eight or more generations were used. All animal experiments were performed according to the guidelines of the Institutional Animal Care and Use Committees of the Graduate School of Medicine, Osaka University. Isolation of lamina propria macrophages and DCs The entire large and small intestine, excluding the cecum, was removed, opened longitudinally and washed to remove fecal content. The intestine was shaken in HBSS containing 5 mM EDTA for 15 min at 37C. The tissues were washed with PBS and the epithelial cells and muscle layers were removed with tweezers. The intestine was then cut into small pieces and incubated with RPMI 1640 containing 4% fetal bovine serum, 1 mg ml1 collagenase D (Roche Diagnostics), 1 mg ml1 dispase (Invitrogen) and 40 lg ml1 DNase I (Roche Diagnostics) for 1 h at 37C in a shaking water bath. The digested tissues were washed with HBSS containing 5 mM EDTA and ltered through a 40-lm cell strainer. The obtained cells were incubated with FITC-conjugated anti-CD11b and PEconjugated anti-CD11c (BD Biosciences) after blockade of Fc receptors. M/ and DC were sorted using a FACS Aria (BD Biosciences). The purity of the sorted cells was routinely > 95%. Isolation of bone marrow-derived macrophages and DCs Bone marrow (BM) cells were isolated from the femurs of mice. After separation of BM mononuclear cells, bone marrowderived macrophages (BMM/) were obtained by culture of BM cells in 30% macrophage colony-stimulating factor (M-CSF) (supernatant from L929 cells) at 37C in 5% CO2. On Day 3 of culture, 5 ml of medium was gently removed and replaced with fresh medium containing 30% M-CSF. On Day 5, CD11b+ cells were puried using a magnetic cell separation system (MACS; Miltenyi Biotec) with anti-mouse CD11b microbeads. Bone marrow-derived DCs (BMDCs) were obtained by culture of BM cells in 10 ng ml1 recombinant mouse granulocyte macrophage colony-stimulating factor (R&D systems) and 10 ng ml1 recombinant mouse IL-4 (R&D systems) at 37C in 5% CO2. On Day 3 of culture, 5 ml of medium was gently removed and replaced with fresh medium and cytokines. On Day 5, CD11c+ cells were puried using MACS and anti-mouse CD11c microbeads. Isolation of naive CD4+ T cells To prepare single-cell suspensions from spleens, the collected organs were ground between glass slides and the cells passed through a 40-lm nylon mesh and suspended in HBSS. Splenocytes were treated with RBC lysis buffer (0.15 M NH4Cl, 1 mM KHCO3 and 0.1 mM EDTA) for 5 min and washed. For FACS sorting, cells were stained with PerCP/ Cy5.5-conjugated anti-CD4 (Biolegend), allophycoerythrinconjugated anti-CD62L, FITC-conjugated anti-CD25 and PEconjugated anti-CD44 (BD Biosciences). Naive CD4+ T cells were isolated by FACS sorting for CD4+CD62LhighCD25CD44low (FACS Aria; BD Bioscience). The purity of the sorted cells was routinely >95%. Intracellular cytokine staining and ow cytometry The intracellular expression of IL-10 and IFN-c in CD4+ T cells was analyzed using Cytox/Cytoperm Kit Plus with Golgistop (BD Biosciences) according to the manufacturers instructions. In brief, lymphocytes obtained from intestinal lamina propria were incubated with 50 ng ml1 phorbol myristate acetate (Sigma), 5 lM calcium ionophore A23187 (Sigma) and Golgistop in complete media at 37C for 4 h. Surface staining was performed with PerCP/Cy5.5conjugated anti-CD4 (Biolegend) for 20 min at 4C and intracellular cytokine staining was performed with PE-conjugated anti-IL-10 (BD Biosciences) and FITC-conjugated anti-IFN-c (BD Biosciences) for 20 min. Data were acquired using a FACS Canto II (BD Biosciences) and analyzed using FlowJo software (Tree Star). T-cell differentiation assay M/ and DC from the large intestine and naive T cells were sorted as described above. LM/ and LDC (5 3 104) were co-cultured with 1.5 3 105 naive T cells in the presence of soluble anti-CD3 antibody (1 lg ml1) and transforming growth factor (TGF)-b (1 ng ml1). After 5 days, expression of Foxp3 was analyzed using a FACS Canto II. Intracellular

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Gut ora induce LPS refractoriness in colonic macrophage 955 staining of Foxp3 was performed using a Foxp3 Mouse Regulatory T cell Staining Kit (eBioscience). T-cell proliferation assay LM/, LDC and naive T cells were sorted as described above. LM/ and LDC (8 3 103) were co-cultured with 8 3 103 naive T cells in the presence of soluble anti-CD3 antibody (1 lg ml1). After 48 h, cells were pulsed with [3H] thymidine (1 lCi per well) and cultured for 15 h. Then, the cells were harvested with a Packard Micromate cell harvester and thymidine incorporation into DNA was measured using a MicroBeta2 plate counter (PerkinElmer) as counts per minute (c.p.m.). Microarray analysis M/ and DC were collected from colonic lamina propria. Total RNA was extracted with an RNeasy kit (Qiagen). RNA quality was assessed with an Agilent Bioanalyser 2100 and only RNA with minimal degradation and distinct 18S and 28S rRNA bands were used for analysis. Fragmented and biotinlabeled cDNA was synthesized from 100 ng puried mRNA with GeneChip 3IVT Express Kit Assay (Affymetrix). The cDNA was hybridized to Mouse Genome 430A 2.0 Array (Affymetrix). Hybridized chips were stained and washed and were scanned with GeneChip Scanner 3000 (Affymetrix). Genespring software (Silicon Genetics) was used for data analysis. Real-time reverse transcriptionPCR cDNAs were synthesized from RNA samples prepared with the RNeasy Mini Kit (Qiagen) using ReverTra Ace (Toyobo). cDNAs were analyzed by real-time reverse transcription (RT)PCR using GoTaq qPCR Master Mix (Promega) in an ABI 7300 real-time PCR system (Applied Biosystems). Serial dilutions of a standard were included for each gene to generate a standard curve and allow calculation of the input amount of cDNA for each gene. Values were then normalized against the amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in each sample. The primer sets were designed with Primer Express Version 3.0 (Applied Biosystems). The following primer sets were used: Gapdh, 5#-CCTCGTCCCGTAGACAAAATG-3# and 5#-TCTCCACTTTGCCACTGCAA-3#; Il10, 5#-ATTTAGAGACTTGCTCTTGCACTACCA-3# and 5#-GGCATGATGGAGCTCTCTTTTC-3#; Irf8, 5#-GCATGAGCGAAGTTCCTGAGAT-3# and 5#-CCATGTACTCATCCACAGAAGGTT-3#; Cebpb, 5#-TGACGCAACACACGTGTAACTG-3# and 5#-AACAACCCCGCAGGAACA-3#; Mafb, 5#-AACGCGTCCAGCAGAAACAT-3# and 5#-AGCTGCTCCACCTGCTGAAT-3#; Cd163, 5#-CAGTGCCCCTCGTCACCTT3# and 5#-TATGCCCTTCCTGGAGTCTTATTT-3#; S100a9, 5#-AACATCTGTGACTCTTTAGCCTTGAG-3# and 5#-GCTGCGCTCCATCTGAGAAG-3#; Il10ra, 5#-TTCTCAGAACTAAAGAATGCAACCA-3# and 5#-GGGCAGCACCTTGACACAA-3#; Il10rb, 5#-GCTTTCCCCAAAACGAACCT-3# and 5#-CAGTGCGCTTGCAGTGATCT-3# Enzyme-linked immunosorbent assay The concentrations of IL-6, IL-12p40 and tumour necrosis factor (TNF)-a in the culture supernatants were measured using an ELISA Ready-SET-Go (eBioscience) according to the manufacturers instructions. The concentration of IL-10 was determined using the DuoSet ELISA Development System (R&D Systems). Statistical analysis Differences between control and experimental groups were evaluated using the Students t-test. A P value < 0.05 was considered signicant. Results Comparison of intestinal CD11b+CD11c macrophages and CD11bCD11c+ DCs In order to characterize the macrophages in the large intestinal lamina propria, we rst analyzed the surface expression of CD11b and CD11c in the lamina propria cells from the small and large intestines (Fig. 1A). In both the large and the small intestines, three subsets comprising CD11bCD11c+, CD11b+CD11c and CD11b+CD11c+ cell populations were observed, although the CD11b+CD11c+ cell population was smaller in the large intestine. The CD11b+CD11c+ cell population contains several heterogeneous subsets of DCs with unique characteristics (19, 22, 23). Therefore, we used the CD11bCD11c+ population as representative DCs from the small and large intestines (SDC and LDC) and compared their surface marker expression with that of CD11b+CD11c macrophages from the small and large intestines (SM/ and LM/) (Fig. 1B). Both SDC and LDC showed moderate expression of CD40, CD80 and CD86 and high expression of MHC class II and CD103. This pattern is similar to that observed in CD103+ DCs (10). SDC and LDC showed similar patterns of surface expression for several markers. However, only slight expression of CX3CR1 was observed in LDC and CX3CR1 was not expressed in SDC. A different expression pattern for CX3CR1 was also observed between LM/ and SM/. LM/ expressed high levels of CX3CR1, but its expression was weak in SM/. However, molecules other than CX3CR1 were similarly expressed in both SM/ and LM/. Expression of F4/ 80 and Ly6C was higher in LM/ and SM/ than in LDC and SDC. Thus, DCs and M/s from the large intestine had a similar phenotype to those from the small intestine. We next compared the function of LM/ and LDC. LDC enhanced the proliferation of co-cultured CD4+ T cells (Fig. 1C). In contrast, LM/ did not enhance CD4+ T-cell proliferation. Because both SDC and SM/ have been shown to induce Treg cells (1921), we examined whether LDC and LM/ also induce Treg. Naive CD4+ T cells were co-cultured with these intestinal cells for 5 days in the presence of TGF-b and then analyzed for expression of Foxp3 (Fig. 1D). Both SDC and SM/ induced expression of Foxp3 in co-cultured T cells, but SM/ cells were more potent. LDC and SDC showed a similar capacity to induce Foxp3. LM/ also induced Foxp3 in T cells, albeit at lower levels than SM/. Thus, both LDC and LM/ induced Treg, although this ability was less in LM/. Distinct patterns of LPS-induced responses in LM/ and LDC We next examined the LPS-induced response in LM/ and LDC. Both BMM/ and BMDC produced inammatory

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Fig. 1. Characterization of M/ and DC in the small and large intestines. (A) Flow cytometric analysis of cells isolated from the small and large intestinal lamina propria of C57BL/6 mice. SM/ and LM/ are CD11b+CD11c macrophages from the small and large intestines, respectively. SDC and LDC are CD11c+CD11b DCs. (B) Flow cytometry of gated M/ and DC isolated from the small and large intestinal lamina propria (antibodies, below plots). Solid black lines = isotype control antibodies; shaded histograms = specic staining. (C) Proliferation of CD4+ T cells responding to LM/ and LDC as determined by [3H] thymidine incorporation. (D) Foxp3 expression by CD4+ T cells cultured with M/ or DC for 5 days in the presence of TGF-b.

Gut ora induce LPS refractoriness in colonic macrophage 957 cytokines, such as TNF-a, IL-6 and IL-12p40, in response to LPS. In contrast, and consistent with previous studies using intestinal CD11b+ cells (14), LM/ showed impaired LPSinduced production of TNF-a, IL-6 and IL-12p40 (Fig. 2AC). Thus, LM/ were globally unresponsive to LPS. Although LDC also showed impaired LPS-induced production of TNFa and IL-6, they produced high amounts of IL-12p40 in a similar manner to BMDC (Fig. 2AC). We then analyzed LPS-induced IL-10 production further (Fig. 2D). Both BMM/ and BMDC produced IL-10 in response to LPS. LDC showed a similar pattern of LPS-induced IL-10 production, but in LM/, IL-10 production was constitutive and increased in response to LPS. Thus, LM/ were hyporesponsive to the LPSinduced production of many pro-inammatory cytokines with high levels of IL-10. In contrast, LDC did respond to LPS and produced IL-10 and IL-12p40, although a reduced response was observed in the context of LPS-induced production of TNF-a and IL-6. Gene expression proles of LM/ and LDC The different responses to LPS observed in LM/ and LDC prompted us to investigate the gene expression proles these cell types. Therefore, we isolated RNA from LM/ and LDC for DNA microarray analysis (Fig. 3A and Supplementary Table 1, available at International Immunology Online). We found that many genes were differentially expressed in LM/ and LDC. The expression of several of these genes was analyzed using real-time RTPCR (Fig. 3B). The transcription factors (Mafb and Cebpb) that determine differentiation into M/ were selectively expressed in LM/, whereas Irf8, which encodes a transcription factor that mediates DC function, was preferentially expressed in LDC. Among these genes, we focused on those that were highly expressed in LM/ and found that many genes selectively expressed in LM/ are reportedly induced by IL-10 treatment of monocytes/M/, including genes encoding CD163 and

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Fig. 2. LPS refractoriness of the large intestine M/. M/ (LM/) and DC (LDC) were isolated from the large intestine. BMM/ or BMDC was obtained by culture with M-CSF or granulocyte macrophage colonystimulating factor, respectively. LM/, LDC, BMM/ and BMDC were cultured for 48 h in the presence or absence of 100 ng ml1 LPS. Production of TNF-a (A), IL-6 (B), IL-12p40 (C) and IL-10 (D) in the supernatants was analyzed by ELISA. Data are representative of three independent experiments.

Fig. 3. DNA microarray analysis of LM/ and LDC. (A) Microarray analysis of the gene expression by LM/ and LDC isolated from the large intestine. The left and right panels indicate 1177 and 1105 genes, respectively. (B) Mafb, Cebpb, Irf8, Il10, Cd163 and S100a9 expression in LM/ and LDC was analyzed by real-time RTPCR. Data were normalized relative to the expression of Gapdh. Data are representative of two independent experiments.

958 Gut ora induce LPS refractoriness in colonic macrophage S100a9 (24, 25). The gene encoding IL-10 was also preferentially expressed in LM/. Thus, LM/ highly expressed genes regulated by IL-10, which is known to downmodulate the function of M/. IL-10/Stat3-dependent induction of LPS refractoriness in LM/ Since IL-10-related genes were preferentially expressed in LM/, we next analyzed the involvement of the IL-10 pathway in LPS-induced responses. In LM/ derived from Il10/ or LysM-cre; Stat3 ox/ox mice, LPS stimulation resulted in robust production of TNF-a and IL-6 (Fig. 4A and B). Even in LM/ derived from Il10/ mice, IL-10 pre-treatment markedly reduced the LPS-induced production of TNF-a and IL-6 (Fig. 4C). These ndings indicate that the refractoriness of LM/ to LPS is induced by the IL-10/Stat3 axis. In sharp contrast, LPS-induced production of TNF-a and IL-6 was very low even in LDC from Il10/ or LysM-cre; Stat3 ox/ox mice (Fig. 4A and B). As the expression of IL-10-inducible genes was lower in LDC than LM/, we analyzed the expression of the IL-10 receptor by real-time RTPCR (Fig. 4D). Il10ra was similarly expressed between LM/ and LDC, but expression of Il10rb was profoundly decreased in LDC compared with LM/. These ndings indicate that LDC are less responsive to IL-10 compared with LM/ and that

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Fig. 4. Enhanced LPS response of LM/ in the absence of IL-10 or Stat3. (A) LM/ and LDC were isolated from LysM-cre; Stat3 ox/ox mice. LPS-induced production of IL-6 and TNF-a was analyzed by ELISA. (B) LM/ and LDC were isolated from Il10/ mice, and the LPS-induced production of IL-6 and TNF-a was analyzed by ELISA. (C) LM/ isolated from Il10/ mice were cultured for 18 h in the presence or absence of 10 ng ml1 recombinant IL-10 and then stimulated with LPS for 24 h. Production of IL-6, TNF-a and IL-12p40 in the supernatants was analyzed by ELISA. Data are representative of three independent experiments. (D) Expression of Il10ra and Il10rb in LM/ and LDC was analyzed by real-time RTPCR. Data were normalized relative to the expression of Gapdh. Data are representative of two independent experiments.

Gut ora induce LPS refractoriness in colonic macrophage 959 the reduced production of TNF-a and IL-6 in LDC is regulated independently of IL-10. MyD88-dependent gene expression in LM/ We then investigated how the expression of IL-10 and IL-10inducible genes in LM/ was regulated. In innate immune cells, TLRs play a crucial role in microorganism-dependent gene expression (5). Therefore, we analyzed the involvement of TLR signaling. In mice lacking MyD88, a critical component for the TLR signaling (26), the number of LM/ was not altered (Fig. 5A). Therefore, we isolated LM/ from these mice and analyzed their gene expression proles (Fig. 5B). LM/ derived from Myd88/ mice showed severely reduced expression of Il10 and Cd163. These ndings indicate that expression of IL-10 and IL-10-inducible genes in LM/ is regulated by the TLR signaling pathway. Commensal microbiota-dependent induction of LPS refractoriness in LM/ Commensal microbiota are closely related to immune functions in the intestine (2, 27, 28). Therefore, we analyzed the involvement of commensal microbiota in the modulation of LM/ activity. In germ-free mice lacking gut microbiota, LM/ were normally present (Fig. 6A). However, IL-10 production was severely reduced compared with that in LM/ derived from specic-pathogen-free (SPF) mice (Fig. 6B). In addition to M/, IL-10 is produced by inducible regulatory T (iTreg) cells in the intestine and iTreg-derived IL-10 limits intestinal inammation (29). Therefore, we also analyzed IL-10 production by colonic lamina propria CD4+ T cells. The number of IL-10-producing CD4+ T cells was markedly decreased in germ-free mice, indicating that gut microbiota were responsible for IL-10 production by CD4+ T cells, as well as M/, in the large intestine (Fig. 6C and D). Therefore, we analyzed the LPS-induced production of inammatory cytokines in LM/ isolated from germ-free mice (Fig. 6E). We found that the LPS-induced production of both TNF-a and IL-6 was markedly increased compared with that in LM/ from SPF mice. These ndings indicate that commensal microbiota mediate IL-10 production in the large intestine, thereby inducing the reduced response to LPS seen in LM/. Discussion In this study, we showed that colonic macrophages were hyporesponsive to LPS in terms of inammatory cytokine production, whereas colonic DCs responded to LPS and produced IL-12p40 and IL-10. IL-10-inducible genes were highly expressed in colonic macrophages but not in colonic DCs. These ndings prompted us to investigate the role of the IL-10/Stat3 pathway in the function of colonic macrophages. Colonic macrophages derived from Il10/ and LysM-cre; Stat3 ox/ox mice showed markedly increased production of cytokines in response to LPS. However, colonic DCs derived from Il10/ and LysM-cre; Stat3 ox/ox mice showed only slightly increased response to LPS. IL-10 was abundantly produced by Tregs and macrophages in the large intestine, mediated by commensal microbiota. Accordingly, colonic macrophages derived from germ-free mice
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Fig. 5. Impaired expression of IL-10-related genes in Myd88/ LMu. (A) Flow cytometry analysis of cells isolated from the large intestinal lamina propria of wild-type and Myd88/ mice. Numbers adjacent to outlined areas indicate the percentage of CD11b+CD11c LMu. (B) LMu were isolated from the large intestine of wild-type and Myd88/ mice. Gene expression of Il10 and Cd163 was analyzed by real-time RTPCR. Data are representative of two independent experiments.

showed higher levels of cytokine production in response to LPS. These ndings demonstrate that commensal microbiota induce IL-10 production in the large intestine and thereby negatively regulate the innate inammatory responses of intestinal macrophages. Commensal microbiota regulate host intestinal immune responses (28). Indeed, several lines of evidence indicate that commensal bacteria induce IgA production in the small intestine (30, 31). In addition to intestinal IgA responses, development of helper T cells is also controlled by gut microbiota (32). Furthermore, a specic microbiota (segmented lamentous bacteria) has recently been shown to induce Th17 cell development in the small intestine (3335). More recently, polysaccharide A derived from Bacteroides fragilis has been shown to mediate the development of IL-10producing Tregs in the intestine (36). Thus, the gut microbiota has a great impact on intestinal adaptive immune responses by inducing T-cell and IgA responses. Our present study clearly indicates that gut microbiota play a role in the negative regulation of innate immune responses. Commensal microbiota-dependent suppression of LPS responses in LM/ was dependent on the IL-10/Stat3 pathway. These ndings are consistent with previous studies showing that LPS-induced inammatory cytokine production by macrophages is enhanced in the absence of IL-10 or Stat3 (3739). IL-10 is produced in the intestine by several cell types, such as macrophages and Tregs (4042). The present study indicates that intestinal IL-10 production was

960 Gut ora induce LPS refractoriness in colonic macrophage

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Fig. 6. Reduced IL-10 production and enhanced LPS responses of LM/ from germ-free mice. (A) Flow cytometry analysis of cells isolated from the large intestinal lamina propria of SPF and germ-free (GF) mice. Numbers adjacent to outlined areas indicate the percentage of CD11b+CD11c LM/. (B) LM/ were isolated from SPF and GF mice and analyzed for LPS-induced production of IL-10 by ELISA. Data are representative of four independent experiments. (C) Lamina propria cells were isolated from the large intestine of SPF and GF mice and stimulated with phorbol myristate acetate and ionophore for 4 h. Intracellular IL-10 and IFN-c production in the CD4+ cell population was analyzed by ow cytometry. Numbers indicate the percentage of cells within each quadrant. Data are representative of two independent experiments. (D) The numbers of IL-10 producing CD4+ cells in the large intestinal lamina propria were shown. (E) CD11b+ LM/ from SPF and germ-free (GF) mice were stimulated with LPS for 24 h and analyzed for production of IL-6 and TNF-a by ELISA. Data are representative of two independent experiments.

tightly regulated by commensal microbiota. IL-10 production by LM/ was dependent on the TLR signaling pathway; however, IL-10 production from intestinal T cells was independent of TLR signaling since IL-10-producing CD4+ T cells are normally observed in the large intestinal lamina propria of Myd88/ mice (Y. U. and H. K. unpublished data). This study also demonstrates that LPS responses were differentially regulated between LM/ and LDC. The LPS hyporesponsiveness of LM/ was totally dependent on the IL-10/Stat3 pathway, whereas LDC responded to LPS by producing IL-12p40 and IL-10. LDC were defective in the production of TNF-a and IL-6, and this was independent of the IL-10/Stat3 pathway. This defective production of inammatory cytokines may be developmentally controlled. Alternatively, LDC might become hyporesponsive to LPS through another anti-inammatory cytokine, TGF-b. The LDC population includes CD103+ DC that mediate T-cell responses in the intestine (10, 20, 21). Since IL-10 negatively modulates T-cell responses by affecting DC functions (43), LDC might

acquire defective responses to IL-10, along with the defective production of innate inammatory mediators, so that they can effectively induce intestinal T-cell-mediated immune responses without inducing innate inammation. A recent study demonstrated that TLR2 is responsible for B. fragilismediated induction of IL-10-producing Foxp3+ Treg cells in the intestine (36). Therefore, the responsiveness of LDC to TLR stimulation might be required for effective induction of IL-10-producing Treg cells. In this study, we demonstrated that commensal microbiota negatively regulates innate inammatory responses by the IL-10/Stat3 axis. The altered composition of commensal microbiota has recently been shown to be correlated with several immune disorders, such as inammatory bowel diseases and type I diabetes (44, 45). It would be interesting to analyze whether altered innate immune responses are also involved in the development of these immune disorders. This study also indicates that aberrant inammatory responses by intestinal macrophages to commensal

Gut ora induce LPS refractoriness in colonic macrophage 961 microbiota in the absence of IL-10 correlate with intestinal inammation. Indeed, ablation of intestinal CD11b+ M/ reduces the severity of colitis in Il10/ mice (46). Therefore, modulation of intestinal M/ activity may be a promising therapeutic target for the treatment of patients with inammatory bowel disease. Supplementary data Supplementary data are available at International Immunology Online. Funding This work was supported by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology; the Ministry of Health, Labor and Welfare; the Osaka Foundation for the Promotion of Clinical Immunology, Takeda Science Foundation. Acknowledgements
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