This project focuses on the roles of different populations of dendritic cells (DC) and macrophages (MP) in immune responses in mucosal tissues. While it is clear that the normal outcome of mucosal antigen exposure can be positive, i.e., the development of intestinal IgA and effector T cell responses, and in some cases the induction of systemic immunity; and/or largely regulatory, i.e., the induction of mucosal tolerance, the details of why one or the other outcome occurs is complex and still poorly understood. Furthermore, the normal mucosal immune response to symbiotic/commensal bacteria, which allows for one to tolerate these organisms without the onset of inflammation, is essential for immune homeostasis, as a defect in this homeostasis results in inflammatory bowel disease (IBD), such as Crohn's disease and ulcerative colitis. Therefore, this project focuses on how immune responses are regulated in mucosal tissues with a focus on the roles of DCs and MPs in this regulation, and on factors that control inflammatory functions of these cells. Furthermore, we have addressed the role of IL-21, a factor important for T cell-dependent IgA B cell class-switching in the PPs, in generating IgA to populations of commensal bacteria, and how this consequently affects T cell response to oral antigens and intestinal pathogens. In prior studies, we defined antigen-presenting cell populations in the Peyer's patches (PP), and detailed their surface phenotype, function, and migration using in situ immunofluorescence microscopy and mRNA hybridization, flow cytometry, and in vitro assays of cytokine production and T cell differentiation. Furthermore, we delineated for the first time precise definitions of MPs and DCs in the colon lamina propria (LP) and isolated lymphoid follicles based on the use of a comprehensive array of surface markers, gene expression analysis, and development from defined circulating precursors; and demonstrated the dual capacity of Ly6Chi blood monocytes to differentiate into either regulatory MP or inflammatory DCs in the colon, and that the balance of these immunologically antagonistic cell types is dictated by micro-environmental conditions. Furthermore, we evaluated gene regulation in resident and inflammatory colon MPs. We determined that a major, previously unappreciated level of control of inflammatory cytokine production by intestinal MPs is via post-transcriptional mechanisms. From freshly isolated cells levels of mRNA for the pro inflammatory cytokines proIL-1-beta, TNF-alpha, and IL-6, together with the inflammasome NLRP3 were very high, while protein levels were low to non-existent. In contrast, mRNA and protein levels of IL-10, a major suppressive cytokine, were both high. Furthermore, activation of cMPs resulted in low levels of pro inflammatory cytokine production, and poor NLRP3 activation, but high production of IL-10. This distinct post-transcriptional regulation of IL-10 and pro-inflammatory cytokines was present in resting and activated cMPs in the steady-state, but lost during experimental colitis, indicating that environmental conditions present in the intestinal LP influence cMPs directly or their differentiation from blood monocytes to influence post-transcriptional gene regulation. Given that the production these pro inflammatory cytokines is essential for tissue inflammation in patients with IBD, these results suggested that the control of cytokines by post-transcriptional mechanisms is essential for controlling susceptibility to IBD. Furthermore, we demonstrated that the polyubiquitin/proteosome pathway is important for the control of both NLRP3 and pro-IL1-beta protein levels in cMPs. This was the first data showing that NLRP3 leaves can be controlled by degradation in a relevant cell type in vivo. During FY 2017, using a combination of genomic and proteomic approaches we further explored post-transcriptional regulation of cytokines in intestinal MPs and DCs. We developed new techniques to study intracellular cytokine and mRNA expression together in single cells, which allowed us to determine that the post-transcriptional regulation of cytokines occurs as monocytes differentiate into macrophages. Thus, the capacity of to produce inflammatory cytokines is intact in early infiltrating and differentiating monocytes, but lost as monocytes differentiae into macrophages. Surprisingly, this effect was only partially dependent on IL-10. Furthermore, we performed single cell mRNA analysis of intestinal myeloid cells in mice and determined a new level of heterogeneity amongst DC and monocyte/MP populations. Thus, we have defined 5 unique populations of monocyte/macrophages and 4 populations of DCs in normal mouse colon. Unique gene expression by these populations have allowed for developmental trajectory analysis resulting in the identification of two unique developmental pathways for the development of macrophages from monocyte precursors. These two unique populations were further characterized for unique surface markers, and localized in tissues by immunofluorescence. Ongoing studies are addressing the specific function of these unique macrophage populations, as well as further characterizing populations of macrophages and DCs by deep mRNA sequencing of isolated cell populations. In separate studies we established the role of IL-21 in immune regulation in the intestine with regard to B cell differentiation and T-cell responses to commensal bacteria and the consequences for the development of T cell responses to soluble oral antigens (oral tolerance) and to infection with Citrobacter rodentium, a model of enteropathogenic E. coli infection in humans. Using IL-21-reporter mice, we demonstrated that IL-21 is expressed by a large percentage of CD4+ T cells in the Peyer's patches and LP of the small intestine, but many fewer in the colon. In addition, using IL-21R-deficient mice with different endogenous microbiota, we showed a unique role of IL-21 in T cell dependent, but not independent, IgA responses to commensal bacteria. IL-21 effects on IgA levels in the intestine were limited to mice with certain unique commensal bacteria (termed pathobionts) that included segmented filamentous bacteria (SFB) and Helicobacter typhlonius. The absence of IL-21R-dependent IgA responses to these pathobionts resulted in in their expansion in the intestine, as well as increased numbers of both Th17 and regulatory T cells in lamina propria, due to their increased contact with the intestinal epithelium. The biological effects of these changes in responses to commensal bacteria resulted in dramatically enhanced Th17 responses to an innocuous oral antigen (ovalbumin), as well as altered immunopathology during an intestinal infection with C. rodentium. These data provide important basic data regarding the role of IL-21 in IgA responses, and highlight the ability of IgA production to certain commensal bacteria (pathobionts) by its ability to prevent contact with the intestinal epithelium, to influence T cell responses to oral antigens and subsequent infection with pathogenic microorganisms. These data are also relevant to human patients with IL-21R-signaling defects, who develop intestinal inflammation and susceptibility to intestinal infection with Cryptosporidium parvum, and have implications for the development of non-IgE-mediated food allergy.