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-beta, 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.

Although several subsets of intestinal antigen presenting cells (APCs) have been described, to date is no systematic evaluation of their phenotypes, functions and geographical localization. Here we used 10-color flow cytometry, to define the major APC subsets in the small and large intestine LP. LP APCs could be subdivided into CD11c+CD11b−, CD11c+CD11b+, and CD11cdullCD11b+ subsets. CD11c+CD11b− cells were largely CD103+F4/80− DCs, while the CD11c+CD11b+ subset comprised of CD11c+CD11b+CD103+F4/80− DCs versus CD11c+CD11b+CD103-F4/80+ macrophage-like cells. The majority of CD11cdullCD11b+ cells were CD103-F4/80+ macrophages. Although macrophages were more efficient at inducing Foxp3+ Treg cells than DCs, at higher ratios of T:APC cells all DC subsets efficiently induced Foxp3+ Treg cells. In contrast, only CD11c+CD11b+CD103+ DCs efficiently induced TH-17 cells. Consistent with this, the geographical distribution of CD11c+CD11b+CD103+ DCs correlated with that of TH-17 cells, with duodenum>jejunum>ileum>colon. Conversely, CD11c+CD11b−CD103+ DCs, macrophages and Foxp3+ Treg cells were most abundant in the colon and scarce in the duodenum. Importantly however, the ability of DC and macrophage subsets to induce Foxp3+ Tregs versus TH17 cells was strikingly dependent on the source of the mouse strain. Thus, DCs from C57BL/6 mice from Charles River, (that have segmented filamentous bacteria, which induce robust levels of TH-17 cells in situ (1, 2)), were more efficient at inducing TH-17 cells, and less efficient at inducing FoxP3+ Treg cells than DCs from B6 mice from Jackson Laboratories. Thus the functional specializations of APC subsets in the intestine is dependent on the T:APC ratios, regional localization, and the source of the mouse strain.

Peripheral mechanisms preventing autoimmunity and maintaining tolerance to commensal microbiota involve CD4+Foxp3+ regulatory T cells1,2 generated in the thymus (tTregs) or extrathymically by induction of naive CD4+Foxp3− T cells (iTregs). Prior studies suggested that the T cell receptor (TCR) repertoires of tTregs and iTregs are biased towards self and non-self antigens, respectively 3–6 but their relative contribution in controlling immunopathology, e.g. colitis and other untoward inflammatory responses triggered by different types of antigens, remains unresolved 7. The intestine, and especially the colon, is a particularly suitable organ to study this question, given the variety of self-, microbiota- and food-derived antigens to which Tregs and other T cell populations are exposed. Intestinal environments can enhance conversion to a regulatory lineage 8,9 and favor tolerogenic presentation of antigens to naive CD4+ T cells 10,11, suggesting that intestinal homeostasis depends on microbiota-specific iTregs 12–15. Here, to identify the origin and antigen-specificity of intestinal Tregs, we performed single cell as well as high-throughput (HT) sequencing of the TCR repertoires of CD4+Foxp3+ and CD4+Foxp3− T cells and analyzed their reactivity against specific commensal species. We show that tTregs constitute the majority of Tregs in all lymphoid and intestinal organs, including colon, where their repertoire is heavily influenced by the composition of the microbiota. Our results suggest that tTregs, and not iTregs, dominantly mediate tolerance to antigens produced by intestinal commensals.

Immune sensing of a microbe occurs via multiple receptors. How signals from different receptors are coordinated to yield a specific immune response is poorly understood. We demonstrate that the different pathogen recognition receptors, TLR2 and dectin-1, recognizing the same microbial stimulus, stimulate distinct innate and adaptive responses. TLR2 signaling induced splenic dendritic cells (DCs) to express the retinoic acid (RA) metabolizing enzyme Raldh2 and IL-10, and to metabolize vitamin A and stimulate Foxp3+ T regulatory cells (Treg cells). RA acted on DCs to induce Socs3 expression, which suppressed activation of p38 MAPK and pro-inflammatory cytokines. Consistent with this, TLR2 signaling induced Treg cells, and suppressed IL-23 and TH-17/ TH-1 mediated autoimmune responses in vivo. In contrast, dectin-1 signaling mostly induced IL-23 and pro-inflammatory cytokines, and augmented TH-17/ TH-1 mediated autoimmune responses in vivo. These data define a new mechanism for the systemic induction of RA and immune suppression against autoimmunity.

The two most common forms of inflammatory bowel disease (IBD), Crohn's disease and ulcerative colitis, affect approximately 1 million people in the United States. Uncontrolled APC reactivity toward commensal bacteria is implicated in the pathogenesis of the disease. A number of functionally distinct APC populations exist in the mucosal lamina propria (LP) below the intestinal epithelium, but their relative contributions to inflammation remain unclear. Here, we demonstrate in mice important roles for the chemokine receptor CX3CR1 in maintaining LP macrophage populations, preventing translocation of commensal bacteria to mesenteric lymph nodes (mLNs), and limiting colitogenic Th17 responses. CX3CR1 was found to be expressed in resident LP macrophages (defined as CD11b + F4/80 + ) but not DCs (defined as CD11c + CD103 + ). LP macrophage frequency and number were decreased in two strains of CX3CR1-knockout mice and in mice deficient in the CX3CR1 ligand CX3CL1. All these knockout strains displayed markedly increased translocation of commensal bacteria to mLNs. Additionally, the severity of DSS-induced colitis was dramatically enhanced in the knockout mice as compared with controls. Disease severity could be limited by either administration of neutralizing IL-17A antibodies or transfer of CX3CR1-sufficient macrophages. Our data thus suggest key roles for the CX3CR1/CX3CL1 axis in the intestinal mucosa; further clarification of CX3CR1 function will likely direct efforts toward therapeutic intervention for mucosal inflammatory disorders such as IBD.