Lamina propria macrophages and dendritic cells differentially ... - Nature

© 2007 Nature Publishing Group http://www.nature.com/natureimmunology

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Lamina propria macrophages and dendritic cells differentially induce regulatory and interleukin 17–producing T cell responses Timothy L Denning1, Yi-chong Wang1, Seema R Patel2, Ifor R Williams2 & Bali Pulendran1,2 The intestinal immune system must elicit robust immunity against harmful pathogens but must also restrain immune responses directed against commensal microbes and dietary antigens. The mechanisms that maintain this dichotomy are poorly understood. Here we describe a population of CD11b+F4/80+CD11c– macrophages in the lamina propria that expressed several anti-inflammatory molecules, including interleukin 10 (IL-10), but little or no proinflammatory cytokines, even after stimulation with Toll-like receptor ligands. These macrophages induced, by a mechanism dependent on IL-10, retinoic acid and exogenous transforming growth factor-b, the differentiation of Foxp3+ regulatory T cells. In contrast, lamina propria CD11b+ dendritic cells elicited IL-17 production. This IL-17 production was suppressed by lamina propria macrophages, indicating that a dynamic interaction between these subsets may influence the balance between immune activation and tolerance.

A fundamental puzzle in immunology is how the intestinal immune system responds robustly to harmful pathogens yet remains tolerant to commensal organisms and food antigens. Antigen-presenting cells (APCs) such as dendritic cells (DCs) and macrophages are thought to be critical in maintaining this balance1. Many DC and macrophage subsets have been described and have been shown to be important in regulating immune responses. For example, CD8a+ and CD8a– DCs differentially regulate the balance between T helper type 1 (TH1) and TH2 responses in the spleen2–4, whereas immature DCs or DCs conditioned with interleukin 10 (IL-10) may drive the differentiation of regulatory T cells (Treg cells)5. Like DCs, macrophage populations may also contain subsets that promote TH1 or TH2 differentiation6. In addition, APCs can demonstrate functional plasticity depending on the type of organisms they encounter and the microenvironmental milieu in which they are located7. Although subsets of DCs and macrophages have been described in the intestinal microenvironments8–12, their relative functions in the induction of protective immunity versus immune tolerance remain poorly understood. Mucosal APCs are derived from unique blood monocyte precursors13–15, are located in the lamina propria, Peyer’s patches and mesenteric lymph nodes, and are among the first cells to sense and respond to endogenous bacterial flora16. DCs in Peyer’s patches can take up and process live bacteria and soluble antigens delivered orally17, and a subset of these DCs express the chemokine receptor CCR6 and can stimulate pathogen-specific T cells18. In steady-state conditions however, DCs in Peyer’s patches can secrete IL-10 and drive the differentiation of TH2 cells19 and B cells producing

immunoglobulin A (IgA)20,21. DCs in the mesenteric lymph nodes ‘preferentially’ induce T cell expression of the gut-homing molecules a4b7 and CCR9 (refs. 22–24) and may also be involved in the differentiation of CD4+ T cells producing IL-4, IL-10 and transforming growth factor-b (TGF-b). Less is known about lamina propria DCs, but evidence indicates that these cells are able to express tight junction proteins and extend dendrites into the intestinal lumen, where they can sample bacteria25. This process depends on expression of the chemokine receptor CX3CR1 (ref. 26) and receipt of signals derived from epithelial cells that also sense bacteria27. Much attention has been paid to intestinal DCs, but far less is known about other intestinal APCs, specifically mucosal macrophages. Because these cells are the most abundant population of phagocytic cells in the intestine9 and demonstrate inflammatory anergy11, we hypothesized that lamina propria macrophages may have a fundamental function in maintaining mucosal tolerance. Here we identify and study a previously uncharacterized population of lamina propria macrophages present in appreciable numbers in juxtaposition to CD4+ T cells. We distinguished these lamina propria macrophages from their peripheral counterparts by their expression of immunoregulatory molecules, hyporesponsiveness to Toll-like receptor (TLR) stimulation, spontaneous production of large quantities of IL-10, and ability to suppress the differentiation of TH1 and IL-17-producing T helper cells (TH-17 cells) and to promote the differentiation of Treg cells. In addition, these lamina propria macrophages counteracted the TH-17 differentiation induced by CD11b+ lamina propria DCs. Thus, coordination between lamina propria macrophages and

1Vaccine Research Center and Yerkes Regional Primate Research Center, Emory University, Atlanta, Georgia 30329, USA. 2Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia 30322, USA. Correspondence should be addressed to B.P. ([email protected]).

Received 10 May; accepted 15 August; published online 16 September 2007; doi:10.1038/ni1511

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RESULTS Abundant CD11b+F4/80+CD11c– macrophages in lamina propria Consistent with published reports, we detected CD11b+CD11chi DCs in the small intestinal lamina propria. We also identified an abundant population of CD11b+ cells with low expression of CD11c (Fig. 1a). These ‘CD11b+CD11cdull–neg ’ cells were present at much greater frequency than were conventional CD11chi DCs (9.5% versus 1.6%) in the lamina propria. Injection of Flt3 ligand, a cytokine that stimulates DC differentiation in vivo28, resulted in a profound increase in the frequency of CD11chi DCs but not of CD11b+CD11cdull–neg cells in the lamina propria, suggesting that CD11b+CD11cdull–neg lamina propria cells were not a subset of conventional Flt3 ligand–responsive DCs (data not shown). To assess the morphology of CD11b+CD11cdull–neg lamina propria cells, we assessed by scanning electron microscopy CD11b+CD11c– cells sorted from the spleen and lamina propria. Many CD11b+CD11cdull–neg lamina propria cells had ultrastructural characteristics typical of macrophages (such as eccentric nuclei, extensive villous processes, primary and secondary lysosomal granules, and phagocytic vacuoles; Fig. 1b). However, there was some heterogeneity, suggesting the existence of various subsets of macrophages in the lamina propria. Next we determined the microenvironmental location of these intestinal cells by immunohistology. Consistent with the flow cytometry data (Fig. 1a), we noted many CD11b+CD11c– cells in the

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lamina propria (Fig. 1c). These cells were scattered throughout the villus–tip axis and were present in all parts of the small and large intestine that we examined (data not shown). We next evaluated the phenotype of intestinal CD11b+CD11c– cells by flow cytometry. Like splenic macrophages, lamina propria CD11b+CD11c– cells did not express the lineage markers CD4, CD8a, TCRb, CD19, B220 and NK1.1, suggesting that they were not T cells, B cells or natural killer cells (Supplementary Fig. 1 online). In addition, lamina propria CD11b+CD11c– cells lacked the DC markers DEC-205 and 33D1, as well as the Flt3 receptor CD135. However, relative to their splenic counterparts, lamina propria CD11b+CD11c– cells had high expression of the macrophage marker F4/80, the class II major histocompatibility complex molecule I-Ab, and the coinhibitory molecule programmed death ligand 1 (PD-L1; Fig. 1d). In addition, lamina propria CD11b+CD11c– cells had negligible expression of Gr-1 (data not shown) and CD62L (Fig. 1d). These results collectively suggest that lamina propria CD11b+CD11c– cells are distinct from previously reported lamina propria DCs and probably represent an abundant population of macrophages. Lamina propria macrophage function and gene expression Next we used microarrays to analyze gene expression in sorted lamina propria and splenic macrophages to obtain insight into the putative biological functions of lamina propria macrophages (Fig. 2). Lamina propria macrophages had higher expression of genes encoding antiinflammatory molecules, including IL-10 (363-fold more), TGF-b1 (1.4-fold more), TGF-b3 (14.5-fold more), PD-L1 (2.5-fold more) and

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Figure 1 Phenotype, morphology and microenvironmental location of lamina propria macrophages. (a) Flow cytometry of cells isolated from the spleen and small intestine lamina propria of C57BL/6 mice. Numbers adjacent to outlined areas indicate percent CD11c+CD11bneg–pos cells (upper number) or CD11cdull–negCD11b+ cells (lower number). (b) Scanning electron microscopy of flow cytometry–sorted CD11b+CD11cdull–neg cells from the spleen or intestine, gated as in a. Original magnification, 2,500. (c) Immunofluorescence microscopy of frozen tissue section of the small intestine of a C57BL/6 mouse, fixed and stained with antibodies specific for mouse CD11c (red), CD11b (green) and E-cadherin (blue). Original magnification, 400. (d) Flow cytometry of gated CD11b+CD11cdull–neg cells isolated from the spleen and small intestine lamina propria (antibodies, below plots). Solid black lines, isotype control antibodies; shaded histograms, specific staining. Data are representative of one of more than five independent experiments (a) or one of two (b,c) or three (d) independent experiments.

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with the microarray data (Fig. 2a), freshly isolated lamina propria but not splenic macrophages spontaneously produced large quantities of IL-10; this IL-10 production was further augmented by exposure to TLR ligands, except for zymosan, which may have induced some cell death (Fig. 3a). In contrast, the TLR ligands lipopolysaccharide, tripalmitoyl cysteinyl lipopeptide, zymosan and CpG dinucleotide elicited little or no production of IL-12p40 and IL-12p70 by lamina propria macrophages (Fig. 3a). Notably, IL-10 produced by lamina propria macrophages seemed to act in an autocrine way to mediate their hyporesponsiveness to TLR ligands, as lamina propria macrophages from IL-10-deficient mice and wild-type cells treated with antibody to IL-10 (anti-IL-10) and antibody to the IL-10 receptor (IL-10R) produced more IL-12p40 and IL-12p70 after stimulation with TLR ligands than did cells not treated with antibodies (Fig. 3b and Supplementary Fig. 3a online). Splenic macrophages either isolated from IL-10-deficient mice or treated with IL-10- and IL-10R-neutralizing antibodies also produced more IL-12p40 and IL-12p70 (Supplementary Fig. 3b). These data collectively demonstrate that lamina propria macrophages spontaneously secrete IL-10 that renders them hyporesponsive to TLR ligands and unable to produce proinflammatory cytokines.

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PD-L2 (8-fold more), than splenic macrophages did. We confirmed the expression of several of these genes (Il10, Tgfb1 and Tgfb3) by quantitative real-time PCR (Supplementary Fig. 2 online). In addition, lamina propria macrophages had higher expression of genes encoding costimulatory molecules (CD80 and CD86), major histocompatibility complex–related molecules (H2-DM and I-E), chemokines (CCL7, CCL8, CCL11, CCL12, CCL17, CCL22, CCL24, CXCL4, CXCL7 and CXCL11) and chemokine receptors (CCR5, CCR9 and CX3CR1); some of these chemokines and chemokine receptors are known to regulate the homing of leukocytes to mucosal tissues29 (Fig. 2b). Next we evaluated the responsiveness of freshly isolated splenic and lamina propria macrophages to various TLR ligands. Consistent

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Lamina propria macrophages in CD4+ T cell anergy Given the anti-inflammatory ‘signature’ of lamina propria macrophages and the presence of many CD4+ T cells in juxtaposition to these cells (Fig. 4a), we postulated that lamina propria macrophages might exert regulatory effects on the differentiation of antigen-specific T cells. To test that hypothesis, we used an in vitro antigenpresentation assay in which we labeled OT-II CD4+ T cells, which express a transgenic T cell receptor specific for ovalbumin (OVA) peptide presented by major histocompatibility complex class II molecules, with carboxyfluorescein succinimidyl diester (CFSE) and cultured them together with lamina propria or splenic macrophages in the presence of OVA. After 4 d of culture, both lamina propria and splenic macrophages induced OT-II T cell proliferation, as demonstrated by CFSE dilution (Fig. 4b). We obtained a similar proliferation profile by [3H]thymidine incorporation (data not shown).

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5 4 Figure 3 Lamina propria macrophages spontaneously secrete IL-10 and are hyporesponsive to TLR 4 3 stimulation. (a) ELISA of IL-10, IL-12p40 and 3 IL-12p70 in supernatants of purified spleen or 2 2 intestinal CD11b+CD11c– cells cultured for 24 h with 1 medium alone (–), E. coli lipopolysaccharide (LPS; 1 1mg/ml), tripalmitoyl cysteinyl lipopeptide (Pam; 0 0 10 mg/ml), yeast zymosan (Zymo; 10 mg/ml) or CpG – LPS Pam Zymo CpG – LPS Pam Zymo dinucleotide (CpG; 1 mg/ml). (b) ELISA of the production of IL-12p40 and IL-12p70 by lamina propria macrophages from wild-type mice (Il10+/+) or IL-10-deficient mice (Il10–/–), stimulated as described in a. Data are representative of one of three independent experiments (error bars, s.d. of averages from triplicate cultures). IL-12p40 (ng/ml)


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Figure 4 IL-10 derived from lamina propria macrophages inhibits the TH1 polarization of CD4+ T cells. (a) Immunofluorescence microscopy of a frozen tissue section of the small intestine of a wild-type C57BL/6 mouse, fixed and stained with antibodies specific for mouse CD4 (red), CD11b (green) and E-cadherin (blue). Original magnification, 400. (b) Proliferation of CD11b+CD11c– cells isolated from the spleen or intestine, cultured for 4 d together with CFSElabeled naive OT-II T cells and OVA, assessed by CFSE dilution with gating on CD4+ T cells. (c) Cytokines in supernatants obtained after 72 h culture of splenic or lamina propria macrophages with OT-II T cells in the presence (OVA) or absence (–) of OVA. (d) ELISA of IFN-g production by OT-II T cells left unstimulated (–) or stimulated for 72 h with OVA alone (OVA) or in the presence of antibodies neutralizing IL-10 and IL-10R (OVA + a-IL-10 + a-IL-10R) or TGF-b (OVA + a-TGF-b) or control antibodies (OVA + control). (e) Flow cytometry of intracellular cytokine production by OT-II T cells primed as described in b, then allowed to ‘rest’ and restimulated for 6 h with anti-CD3 and anti-CD28. Numbers in outlined areas indicate percent cells in gate. TNF, tumor necrosis factor. (f) Proliferation OT-II T cells primed as described in b, then allowed to ‘rest’ and restimulated for 48 h with anti-CD3 and anti-CD28, assessed by [3H]thymidine incorporation. *, P o 0.05. Data are representative of one of two (a,f), three (b,d) or more than three (c,e) independent experiments.

We next assessed cytokine production by the OT-II T cell cultures described above. We detected more IL-2 and interferon-g (IFN-g) in cultures of splenic macrophages, and we did not detect the TH2 cytokines IL-4 and IL-5 in any of the culture conditions (Fig. 4c and data not shown). Cultures containing lamina propria macrophages had larger amounts of the anti-inflammatory cytokine IL-10; cultures containing OVA contained twofold more IL-10 than did cultures lacking OVA (Fig. 4c). These data collectively indicate that lamina propria macrophages trigger mainly IL-10 production by responding T cells. We confirmed the T cell production of IL-10 by using real-time PCR to quantify Il10 transcripts in purified T cells (data not shown). Next we evaluated the mechanism by which lamina propria macrophages suppressed IFN-g production by responding antigenspecific T cells. Because PD-L1, TGF-b and IL-10 are robustly expressed by lamina propria macrophages and are known to exert potent immunosuppressive effects on T cells, we assessed the effect of neutralizing these molecules in APC–T cell cultures. PD-L1-deficient and wild-type lamina propria macrophages elicited similar responses from OT-II T cells (data not shown), and neutralization of TGF-b did not result in any detectable change in the OT-II T cell response (Fig. 4d). However, neutralization of IL-10 or its receptor enhanced IFN-g production by OT-II T cells cultured with lamina propria macrophages to quantities similar to those of OT-II T cells cultured with splenic macrophages (Fig. 4d). This result suggested that IL-10 secreted by lamina propria macrophages is critical in regulating CD4+ T cell responses.

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Antigen-specific T cells stimulated with lamina propria macrophages may be IL-10-producing Treg cells or tolerogenic T cells with impaired differentiation potential. The considerable difference in IFN-g production by T cells stimulated with splenic and lamina propria macrophages was particularly evident in secondary cultures in which OT-II T cells were restimulated with CD3- and CD28-specific antibodies. Approximately 15% of CD4+ T cells initially stimulated with splenic macrophages and restimulated with anti-CD3 and anti-CD28 produced IFN-g; in contrast, only 2.5% of T cells initially cultured with lamina propria macrophages produced IFN-g after restimulation (Fig. 4e). We noted similar patterns with the TH1 cytokine tumor necrosis factor and with IL-2; impaired IL-2 production supported the hypothesis that CD4+ T cells stimulated with lamina propria macrophages became anergic. Intracellular IL-4 and IL-10 did not accumulate in any conditions. In addition, compared with OT-II T cells initially stimulated with splenic macrophages, those initially cultured with lamina propria macrophages proliferated less after restimulation with anti-CD3 and anti-CD28 (Fig. 4f). To investigate the function of lamina propria macrophages in vivo, we transferred OT-II T cells (Thy-1.2+) into B6.PL mice (Thy-1.1+) that we then injected in the footpad with OVA-pulsed splenic or lamina propria macrophages. After 4 d, we analyzed the proliferation and cytokine production of draining lymph node OT-II T cells restimulated in vitro. OT-II T cells from mice that received OVA-pulsed splenic or lamina propria macrophages proliferated to a similar extent (Supplementary Fig. 4a online). However, consistent

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with our in vitro observations (Fig. 4c), OT-II T cells from mice that received OVA-pulsed lamina propria macrophages produced less IL-2 and IFN-g but more IL-10 after in vitro restimulation (Supplementary Fig. 4b). These data indicate that lamina propria macrophages act similarly in vitro and in vivo. Lamina propria macrophages induce Treg cell differentiation As lamina propria macrophages were able to induce T cell anergy, we determined whether anti-inflammatory cytokines present in the local intestinal milieu might further influence this process. For this, we set up T cell priming cultures similar to those described above (Fig. 4c), to which we added recombinant TGF-b. After 4 d of culture of splenic macrophages and naive CD4+ OT-II T cells together in the presence of antigen and TGF-b, approximately 5% of cells expressed the lineage marker Foxp3; identical cultures with lamina propria macrophages yielded over 36% Foxp3-expressing T cells (Fig. 5a). We investigated the involvement of lamina propria macrophage–derived IL-10 in driving Treg cell differentiation by neutralizing IL-10 and its receptor in these cocultures. IL-10- and IL-10R-specific antibodies but not

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isotype control antibodies almost completely suppressed the induction of Foxp3 in responding T cells (Fig. 5b). Thus, IL-10 produced spontaneously by lamina propria macrophages acted in synergy with TGF-b in the local milieu to promote the generation of Foxp3+ Treg cells. In addition, as has been demonstrated in other systems, the efficient generation of Foxp3+ Treg cells was abrogated by the addition of IL-6 (Fig. 5c). In the lamina propria, macrophages represent only one of several APC subsets; CD11c+ DCs are also present in appreciable numbers in this microenvironment (Fig. 1a). We thus determined whether such DCs functioned similarly to macrophages in promoting the differentiation of naive OT-II T cells into Foxp3+ Treg cells in vitro. Neither CD11b+ nor CD11b– lamina propria DCs were capable of efficiently generating Foxp3+ Treg cells (Fig. 5d). These data indicate that macrophages are unique among lamina propria APCs in their ability to promote the generation of Foxp3-expressing Treg cells. To further probe the mechanisms underlying the ability of lamina propria macrophages to efficiently generate Foxp3+ Treg cells, we investigated the function of the vitamin A metabolite retinoic acid

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Figure 6 OT-II T cell cytokine production induced by lamina propria macrophages and DCs. (a) Flow cytometry of the intracellular production of IL-17 and IFN-g by lamina propria CD11b+CD11c– macrophages or CD11c+ DCs cultured for 4 d together with OT-II T cells and OVA in the presence of control antibodies (Rat IgG) or antibodies neutralizing TGF-b (a-TGF-b) or IL-10 and IL-10R (a-IL-10 + a-IL-10R), then restimulated for 6 h with plate-bound anti-CD3 86 12 85 14 and anti-CD28. (b) Flow cytometry of the intracellular production of IL-17 and IFN-g by lamina IFN-γ propria CD11b+CD11c– macrophages and equal numbers of CD11c+ DCs cultured for 4 d together with OT-II T cells and OVA in the presence of control antibodies (–) or antibodies neutralizing IL-10 and IL-10R and then restimulated for 6 h with plate-bound anti-CD3 and anti-CD28. Numbers in quadrants indicate percent cells in each. Data are representative of one of three independent experiments. IL-17


Figure 5 Induction of Foxp3+ Treg cells by lamina propria macrophages but not by DCs. (a) Intracellular staining and flow cytometry to assess Foxp3 expression by CD4+ T cells among splenic or lamina propria CD11b+CD11c– cells cultured for 4 d with OT-II T cells in the presence (TGF-b) or absence (–) of TGF-b. (b) Flow cytometry of the induction of Foxp3 in OT-II T cells stimulated for 4 d with lamina propria macrophages with OVA in the presence or absence of TGF-b and either control antibodies (Rat IgG) or antibodies neutralizing IL-10 and IL-10R (a-IL-10 + a-IL-10R). (c) Flow cytometry of Foxp3 expression by OT-II T cells cultured and analyzed as described in a, but in the presence of TGF-b with or without IL-6. (d) Intracellular staining and flow cytometry to assess Foxp3 expression by lamina propria macrophages (MF), CD11b– DCs or CD11b+ DCs cultured with OT-II T cells as described in a. Numbers beside outline areas indicate percent Foxp3+CD4+ cells. Data are representative of one of three (a,b,d) or two (c) independent experiments.

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Lamina propria DCs induce IL-17 production Having noted a distinct difference in the ability of lamina propria macrophages and DCs to induce the differentiation of Foxp3+ Treg cells, we next investigated whether lamina propria DCs functioned differently from lamina propria macrophages in stimulating the differentiation of inflammatory CD4+ T cells in vitro. Whereas lamina propria macrophages were inefficient in inducing T cells producing IFN-g (1.4%) and IL-17 (0.2%), total intestinal DCs induced greater frequencies of T cells producing IL-17 (10%) and IFN-g (6.8%; Fig. 6a). The ability of intestinal DCs to promote T cell IL-17 production depended on the production of TGF-b by intestinal DCs, as neutralization of TGF-b reduced the percentage of IL-17-producing T cells generated from 10% to 1.9% (Fig. 6a). We obtained similar results when we neutralized IL-10 function by adding antibodies specific for IL-10 and IL-10R (Fig. 6a). The addition of IL-10 to intestinal cocultures did not substantially affect the frequency of IL-17-producing T cells (Supplementary Fig. 6 online). Notably, neutralizing TGF-b or IL-10 diminished IL-17 production but increased the frequency of IFN-g-producing T cells, suggesting a reciprocal balance between TH1 and TH-17 differentiation induced by intestinal DCs that depends on the availability of TGF-b and/or IL-10. The addition of lamina propria macrophages to intestinal DC–T cell cocultures (at a ratio of 1:1 with DCs) abrogated TH-17 differentiation but did not alter the differentiation of IFN-g-producing T cells (Fig. 6b). This effect was independent of IL-10 production by macrophages, as neutralization of IL-10 in cocultures had no apparent effect.

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in this process. Aldh1a1 and Aldh1a2, which encode retinal dehydrogenases responsible for the conversion of retinal to retinoic acid, had higher expression in lamina propria macrophages than in splenic macrophages (Supplementary Fig. 5a online). To assess whether retinoic acid was involved in the differentiation of TGFb-dependent Foxp3+ Treg cells induced by lamina propria macrophages, we added the synthetic retinoic acid receptor antagonist LE540 to APC–T cell cocultures. Neutralization of retinoic acid reduced the frequency of Foxp3+ Treg cells generated by lamina propria macrophages from 33% to less than 10% (Supplementary Fig. 5b). Therefore, the mechanisms by which lamina propria macrophages induced the generation of Treg cells involved both IL-10 and retinoic acid.

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Figure 7 Intestinal DC subsets differentially generate IL-17-producing T cells. (a) Flow cytometry of the intracellular production of IL-17 and IFN-g by lamina propria macrophages or CD11b– or CD11b+ CD11c+ DCs stained with anti-CD103 or anti-CX3CR1 (below) or cultured for 4 d with OT-II T cells and OVA and then restimulated for 6 h with plate-bound anti-CD3 and anti-CD28 (above). (b) ELISA of cytokines in supernatants obtained after 90 h of culture of CD11b– or CD11b+ DCs with OT-II cells in the presence of OVA. (c) Flow cytometry of lamina propria CD11b+CD11c+ DCs cultured for 4 d alone (left) or with CD11b+CD11c– macrophages at a ratio of 6:1 with OT-II T cells and OVA; OT-II T cells were then restimulated and analyzed as described in a. Numbers in quadrants (a,c) indicate percent cells in each. Data are representative of one of two (a,b) or three (c) independent experiments.

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Because the lamina propria contains two main DC subsets distinguished by their expression of CD11b (Figs. 1a and 7a), we investigated whether these subsets differentially supported the generation of IL-17-producing T cells. Notably, these DC subsets showed distinct expression of the integrin CD103 (aEb7) and the fractalkine receptor (CX3CR1); CD11b– DCs had universally high expression of CD103 and low expression of CX3CR1, whereas CD11b+ DCs and macrophages had lower expression of CD103 and higher expression of CX3CR1 (Fig. 7a). Few naive OT-II T cells stimulated in vitro with CD11b– DCs expressed IL-17 (2%) or IFN-g (1.4%); in contrast, CD11b+ DCs elicited a high frequency of IL-17-producing T cells (25%; Fig. 7a). We confirmed those results by enzyme-linked immunosorbent assay (ELISA; Fig. 7b). Whereas T cells stimulated with lamina propria CD11b+ DCs produced mainly IL-17, CD11b– DCs elicited the secretion of mainly IL-10 (Fig. 7b), suggesting that the CD11b– DCs may exert a regulatory function. As shown above (Fig. 6b), lamina propria macrophages added to T cell–DC cocultures at a physiological ratio of 6:1 counteracted the TH-17 production induced by CD11b+ DCs (Fig. 7c). To investigate the effect of macrophage or DC depletion on T cell responses in vivo, we attempted to achieve macrophage depletion by using clodronate liposomes and to achieve DC depletion by using CD11c-DTR mice, in which CD11c+ cells express the diphtheria toxin receptor. Although intraperitoneal injection of clodronate liposomes efficiently depleted the mice of splenic F4/80+ macrophages, there was also substantial depletion of splenic DCs (data not shown). Notably, with this methodology, we did not find reproducible depletion of intestinal macrophages (data not shown). However, oral administration of diphtheria toxin for 10 d (every 2 d) resulted in robust depletion of lamina propria DCs from CD11c-DTR mice (Supplementary Fig. 7a online). Depletion of lamina propria DCs slightly enhanced the presence of Foxp3-expressing Treg cells and substantially reduced the differentiation of lamina propria CD4+ T cells that produced IL-17 after restimulation ex vivo (Supplementary Fig. 7b). These data collectively suggest that lamina propria macrophages and DCs are distinct in their ability to modulate T cell responses and that lamina propria macrophages limit the ability of intestinal CD11b+ DCs to promote TH-17 differentiation. We have summarized a model of the proposed function of APCs in the lamina propria (Supplementary Fig. 8 online).

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ARTICLES DISCUSSION Here we have shown that macrophages abundant in the lamina propria can induce Treg cell differentiation and limit the generation of proinflammatory immune responses. Spontaneous IL-10 production by lamina propria macrophages controlled their reactivity to various TLR ligands and promoted the generation of tolerogenic IL-10-producing T cells and, when combined with TGF-b, Foxp3+ Treg cells. In addition, lamina propria macrophages counteracted the ability of CD11b+ lamina propria DCs to induce the differentiation of IL-17-producing T cells. These findings emphasize the complexity of the intestinal APC network that differentially modulates mucosal immune reactivity. Several studies have described intestinal DCs in mucosal tissues and have detailed the function of these cells in the induction of intestinal tolerance and immunity in vitro30. However, there are very few reports of the phenotype or function of resident mucosal macrophages8,31, even though they constitute a numerically greater subset of intestinal APCs9. We have demonstrated here that lamina propria macrophages have a unique anti-inflammatory gene expression ‘signature’ and surface profile; they express PD-L1 (ref. 32) and secrete the immunoregulatory cytokine IL-10, as well as retinoic acid, which can induce the mucosal homing of lymphocytes21,33 and induction of Treg cell responses34–37. Consistent with a report on human intestinal macrophages11, we found that lamina propria macrophages failed to generate proinflammatory responses to TLR ligands. Unlike human intestinal macrophages and mouse intestinal DCs11,19, however, mouse lamina propria macrophages spontaneously produced copious IL-10; this IL-10 was mostly responsible for the immunosuppressive function of lamina propria macrophages. IL-10 production by intestinal CD11b+ macrophages may also contribute to the maintenance of intestinal tolerance in vivo, as mice depleted of colonic macrophages by means of clodronate liposomes38 show enhanced intestinal inflammation in response to dextran sodium sulfate, and mice that lack the transcription factor STAT3 in myeloid cells and thus are unable to respond to IL-10 develop spontaneous intestinal inflammation39. In addition, a report has shown that CD11b-deficient mice have defective oral tolerance40, suggesting that CD11b-expressing cells may be critical to this process. In addition to immunoregulatory genes, genes encoding several chemokine and chemokine receptors involved in mucosal homing and functions41 were ‘preferentially’ expressed by lamina propria macrophages. For example, CCR9 is selectively expressed on mucosal homing lymphocytes42 and was also modestly enriched on lamina propria macrophages. The fractalkine receptor CX3CR1, which endows lamina propria DCs with the ability to extend dendrites into the intestinal lumen, where they can access bacteria and lead to their clearance26, was also enriched in lamina propria macrophages. The failure of lamina propria macrophages to promote TH1 and TH-17 differentiation and their capacity to induce Foxp3+ Treg cell differentiation depended on IL-10 derived from lamina propria macrophages. Our data suggest that lamina propria macrophages induce the differentiation of IL-10-producing tolerogenic or Foxp3+ CD4+ Treg cells depending on the availability of local TGF-b during the T cell stimulation. Consistent with the idea that macrophages promote the differentiation of Treg cells in situ, analysis of a mouse expressing an Il10-driven reporter gene has shown that the intestine is a unique site for IL-10-producing T cells43. In addition, mice lacking IL-10 (ref. 44) and mice expressing a dominant negative form of the receptor for TGF-b on CD4+ T cells45 develop spontaneous intestinal inflammation. Thus, local secretion of TGF-b by intestinal stromal cells46 combined with IL-10 produced by lamina propria

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macrophages may create an ideal milieu for the generation of IL-10and/or Foxp3-expressing T cells that promote tolerance and prevent intestinal inflammation. Although we used splenic macrophages as a reference population during our initial characterization of lamina propria macrophages, we also noted several differences between lamina propria macrophages and DCs in their ability to modulate T cell responses. Lamina propria macrophages triggered the differentiation of Foxp3+ Treg cells in the presence of TGF-b, whereas intestinal CD11b+ and CD11b– DCs were less efficient at doing so. However, a study using higher ratios of T cells to DCs than we used here has reported that lamina propria DCs can induce the generation of Foxp3+ Treg cells35. In addition, whereas lamina propria macrophages induced anti-inflammatory T cell responses, intestinal CD11b+ DCs efficiently induced the generation of IL-17-producing T cells; this process depended on the production of TGF-b by intestinal DCs. TH-17 cells are present in appreciable numbers in the lamina propria47, and intestinal CD11b+ DCs may be responsible for driving the differentiation of these cells in vivo. In support of that idea, depletion of intestinal DCs resulted in a substantial reduction in the frequency lamina propria CD4+ T cells that produced IL-17 after ex vivo restimulation. Lamina propria CD11b– DCs were unique among the APC subsets investigated here in their high expression of CD103. Thus, they may form part of the regulatory DC population48 that promotes the homing of T cells to the intestine49 and Treg cell differentiation36,37. Although we did not note Treg cell differentiation induced by lamina propria CD11b–CD103+ DCs here, it is possible that these APCs contribute to the maintenance of intestinal Treg cell function in vivo. When we compared the ability of intestinal macrophages and DCs to modulate T cell differentiation, an interesting dichotomy emerged: macrophages promoted Treg cell differentiation, whereas CD11b+ DCs drove TH-17 development. Both processes depended on the presence of TGF-b. Lamina propria macrophages and DCs may produce different types and/or amounts of proinflammatory cytokines (IL-6, IL-23 and so on) in response to bacterial flora. Lamina propria DCs may secrete more proinflammatory cytokines, which, in combination with TGF-b, promote TH-17 responses. In contrast, lamina propria macrophages, by secreting copious IL-10 and retinoic acid and remaining hyporeseponsive to TLR stimulation, may create a milieu ideal for Treg cell generation. Indeed, in the presence of TGF-b, IL-6 seems to be a key cytokine in promoting the switch from the Foxp3+ Treg cell lineage to the TH-17 lineage50. Thus, IL-6 production in the intestine may require tight regulation51 to control the balance between the proinflammatory effects of IL-17 and the anti-inflammatory effects of Foxp3-expressing T cells. Intestinal macrophages, by secreting large amounts of IL-10 and retinoic acid, may limit their own production of IL-6 and IL-23, so that in the TGF-b-rich intestinal microenvironment, lamina propria macrophages would continuously favor the generation of Treg cells. Lamina propria macrophages may express additional immunoregulatory factors or cell surface molecules that suppress the induction of IL-17-producing T cells by DCs, as this effect seems to be IL-10 independent. IL-10 is a pleiotrophic cytokine capable of suppressing proinflammatory cytokine production by APCs and other cells and of limiting TH1 differentiation52. However, little is known about how IL-10 influences TH-17 differentiation. As lamina propria DCs can generate both TH-17 and TH1 responses, it is possible that IL-10 regulates these pathways in a complex way. For example, IL-10 may be most potent at inhibiting TH1 differentiation53,54 and may exert modest or even

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ARTICLES counter-regulatory effects on TH-17 responses. In addition, as noted here, neutralization of IL-10 activity may release the restriction on TH1 differentiation and in turn limit TH-17 differentiation. Although T cell priming in the intestine requires the acquisition of antigen by APCs and subsequent presentation to antigen-specific T cells, T cells may encounter antigen in many locations throughout the intestine. Antigen acquisition by intestinal APCs can take place by means of ‘M’ cells overlying Peyer’s patches, isolated lymphoid follicles, specialized villous ‘M’ cells, direct luminal sampling or breaches in the intestinal epithelial layer, or through the absorption of dying intestinal epithelial cells16. Although the traditional view holds that most T cell stimulation takes place in mesenteric lymph nodes16, evidence suggests that Peyer’s patches18 and the lamina propria55 serve as sites in which T cells are activated. Thus, macrophages may modulate T cell responses in situ in the lamina propria or Peyer’s patches, and/or may be endowed with the ability to migrate to mesenteric lymph nodes. Our results collectively identify the lamina propria macrophage as a principal, previously unappreciated component of the intestinal APC network. The function of these cells in maintaining innate and adaptive immune tolerance demonstrates the importance of further understanding of intestinal macrophages, as they may influence the control of inflammatory bowel disease and the development of oral vaccines. METHODS Mice. C57BL/6 male mice 6–10 weeks of age were from Charles River Laboratory. OT-II (Rag1+/+Rag2+/+) and male B6.PL mice were from Jackson Laboratories or were bred onsite. CD11c-DTR and Il10–/– mice (B6.129P2Il10tm1Cgn/J) were from Jackson Laboratories. Mice were maintained in specific pathogen–free conditions in the Emory Vaccine Center vivarium. All animal protocols were reviewed and approved by the Institute Animal Care and Use Committee of Emory University. Isolation of CD11b+ or CD11c+ cells. For splenocytes, spleens were dissected and cut into small fragments and then were digested for 30 min at 37 1C with collagenase type 4 (1 mg/ml) in complete DMEM plus 2% (vol/vol) FBS. Splenocyte samples were enriched for CD11b+ cells by negative selection with microbeads coated with anti–mouse CD11c, followed by positive selection with microbeads coated with antibody to human or mouse CD11b, on LS MACS columns (Miltenyi Biotec). In some experiments, these samples enriched for CD11b+ cells were stained with fluorescein isothiocyanate–conjugated anti-CD11b (clone M1/70), phycoerythrin- or allophycocyanin-conjugated antiCD11c (clone HL3) (PharMingen) and, in some experiments, allophycocyaninconjugated F4/80 (clone BM8) (eBioscience). For cells from the small intestine, small intestines were removed and were carefully cleaned of their mesentery, then Peyer’s patches were excised and the intestines were opened longitudinally and washed of fecal contents. Intestines were then cut into pieces 0.5 cm in length, which were transferred into 50-ml conical tubes and shaken at 250 r.p.m. for 20 min at 37 1C in Hanks’ balanced-salt solution (Life Technologies) supplemented with 5% (vol/vol) FBS (CellGro) containing 2 mM EDTA. This process was repeated two additional times. Cell suspensions were passed through a strainer and the remaining intestinal tissue was washed and then minced, transferred to a 50-ml conical tube and shaken for 20 min at 37 1C in Hanks’ balanced-salt solution plus 5% (vol/vol) FBS and type VIII collagenase (1.5 mg/ml; Sigma). Cell suspensions were collected and passed through a strainer and were pelleted by centrifugation at 300g. Samples were enriched for CD11b+ or CD11c+ cells by positive selection with CD11b or CD11c microbeads, respectively. In some experiments, intestinal samples enriched for CD11b+ or CD11c+ cells were stained with fluorescein isothiocyanate–conjugated anti-CD11b (M1/70), phycoerythrin- or allophycocyanin-conjugated anti-CD11c (HL3; both from PharMingen) or allophycocyanin-conjugated F4/80 (BM8; eBioscience). Stained cells were sorted on a MoFlo (Dako Cytomation) at The Emory Vaccine Center Flow Cytometry Core Facility for the purification of various populations. The in vivo depletion of lamina propria DCs is described in the Supplementary Methods online.

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Microarray analysis. Total RNA was extracted from freshly isolated, flow cytometry–sorted splenic or lamina propria macrophages (CD11b+CD11c–) 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 was used for analysis. Microarray processing was done by the Vanderbilt Microarray Shared Resource on a ‘fee-for-service’ basis. Fragmented and biotin-labeled cDNA was synthesized from 100 ng purified mRNA with the Ovation Biotin System (Nugen). The cDNA was hybridized to Affymetrix Murine Genome 430 2.0 microarray chips (Affymetrix). Hybridized chips were stained and washed and were scanned with a GeneArray scanner (Affymetrix). GeneSpring software (Silicon Genetics) was used for data analysis. TLR stimulation of APCs. Splenic or lamina propria macrophages (1 106 cells/ml) were cultured for 24 h with Escherichia coli lipopolysaccharide (1 mg/ml), tripalmitoyl cysteinyl lipopeptide (100 mg/ml), zymosan (10 mg/ml) or CpG dinucleotides (1 mg/ml; all from Sigma). Supernatants were collected and cytokines were measured by ELISA. In some experiments, anti-IL-10 and anti-IL-10R (1 mg/ml) were added for the duration of the stimulation. Stimulation of lymphocytes. For in vitro stimulation, purified splenic or lamina propria macrophages or DCs (1 105) were cultured together with CFSE-labeled or unlabeled naive CD4+CD62L+ OT-II CD4+ T cells (1 105) and OVA (ISQVHAAHAEINEAGR; 10 mg/ml) in 200 ml RPMI complete medium in 96-well round-bottomed plates. In coculture experiments, lamina propria macrophages were added to T cell–DC cocultures at a ratio of 1:1 or 6:1. Supernatants were analyzed after 72 h or 90 h and cells were collected and analyzed directly or were restimulated after 90 h. For measurement of proliferation by means of [3H]thymidine incorporation, 3H-TdR (Amersham) was added to cells during the final 16 h of culture. In some experiments, IL-10 or IL-6 (Peprotech) and/or TGF-b (R&D) were added to cultures at a final concentration of 1 ng/ml. Anti–mouse IL-10 (JES5-16E3; BD), anti-IL-10R (1B1.3a; BD), anti–mouse PD-L1 (MIH5; eBioscience), anti–human TGF-b (MAB1835; R&D Systems) or rat IgG isotype control antibody (A95-1; Pharmingen) was added to cultures at a final concentration of 1 mg/ml. LE540 was added to some cultures at a concentration of 1 mM. For secondary restimulation, cells were collected after 90 h of primary culture, then were restimulated for 6 h with plate-bound anti-CD3 (10 mg/ml; 145.2C11) and antiCD28 (2 mg/ml; 37.51; both from Pharmingen) for intracellular cytokine detection or were allowed to ‘rest’ for 48 h and then were restimulated for 48 h for analysis of proliferation and cytokine production in cell supernatants. Small intestine lamina propria lymphocytes were stimulated for 6 h with phorbol 12-myristate 13-acetate and ionomycin in the presence of GolgiStop. Additional procedures for the stimulation of lymphocytes are in the Supplementary Methods. Additional methods. Real-time PCR, electron microscopy, cytokine detection, flow cytometry and immunofluorescence analysis of frozen sections are described in the Supplementary Methods; PCR primers are in Supplementary Table 1 online. Statistics. The statistical significance of differences in the means ± s.d. of cytokines released by cells of various groups was calculated with the Student’s t-test (one-tailed). P values of less than 0.05 were considered statistically significant. Accession code. GEO: microarray data, GSE8868. Note: Supplementary information is available on the Nature Immunology website.

ACKNOWLEDGMENTS We thank M. Hulsey and S. Aguilar Mertens for assistance with cell sorting; T. Querec for assistance with gene array data analysis; R. Mittler (Emory University School of Medicine) for recombinant Flt3 ligand; A. Garcia for electron microscopy; M. Heffernan, S. Pai Kasturi and N. Murthy for synthesis of clodronate liposomes; W. Cao for genotyping CD11cDTR mice; and R. Ahmed (Emory University School of Medicine) and A. Sharpe (Harvard Medical School) for PD-L1 deficient mice. Supported by the National Institutes of Health (AI0564499, AI048638, AI05726601, DK057665, AI057157 and AI-50019 to B.P.; and DK007771-06A1 (Pathobiology of Mucosal/Epithelial Disease) to T.L.D.) and the Crohn’s and Colitis Foundation of America (T.L.D.).

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ARTICLES AUTHOR CONTRIBUTIONS T.L.D. and B.P. designed the experiments; T.L.D. did the experiments; Y.W., S.R.P. and I.R.W. did the immunohistology; and T.L.D. and B.P. wrote the manuscript.


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