This project focuses on how antigens are processed in the intestine of mice and presented by different populations of dendritic cells (DC) and macrophages influence immune responses in the intestine. While it is clear that the outcome of oral antigen exposure can be either positive, i.e., the development of mucosal IgA responses, and in some cases the induction of systemic immunity as well, or negative, i.e., the induction of oral tolerance, the details of why one or the other outcome occurs is complex and poorly understood. Furthermore, the normal intestinal immune response to symbiotic/commensal bacteria, which allows for one to tolerate these organisms without the onset of inflammation, is essential for immune homeostasis in the intestine, as a defect in this homeostasis results in inflammatory bowel disease. Furthermore, while it is known that the antigen formulation, the presence of adjuvants, and the antigen dose, as well as genetic factors, can affect mucosal immune responses, how these act together to influence immunity has never been established. Therefore, this project focuses on how immune responses are regulated in the intestine with a focus on the roles of dendritic cells and macrophages in this regulation, and on factors that control inflammatory functions of these cells. In prior studies we defined different antigen-presenting cell populations in the Peyer's patch (PP) and lamina propria and have detailed the surface phenotype, function, and migration of DCs in the PP using in situ immunofluorescence microscopy and in situ hybridization, flow cytometry of purified cells, and in vitro assays of cytokine production (ELISA and quantitative RT-PCR) and T cell differentiation. PP DCs have the unique capacity to induce the differentiation of T cells that produce high levels of IL-10, a cytokine important for the IgA B cell differentiation. These studies thus were some of the first to directly demonstrate that DCs from different tissues may be unique in their ability to induce tissue specific immunity. We also demonstrated that DCs in the subepithelial dome region of the PP process viral antigen from virally infected apoptotic epithelial cells following reovirus infection, the first direct demonstration of cross-processing of viral antigens by DCs. Furthermore, we have defined sub-populations of macrophages and DCs in the mouse colon and are exploring their role in maintaining immune homeostasis in steady-state conditions and during inflammation in murine models of inflammatory bowel disease. We demonstrated four populations of cells based on surface markers that correlate with either a macrophage (MP) or DC phenotype, and have begun to understand their function in vivo. Cells defined to be MPs constitutively released high levels of IL-10 at least partially in response to the microbiota via an MyD88-independent mechanism. In contrast, cells identified as DCs comprise three separate cell populations. In non-inflammatory conditions, Ly6Chi monocytes differentiated primarily into CD11c+, but not CD11c- MPs. In contrast, during colitis, Ly6Chi monocytes massively invaded the colon and differentiated into pro-inflammatory DC/macropahges. These findings 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. These studies delineated for the first time the precise definitions of macrophages and dendritic cells in the colon based on the use of a comprehensive array of surface markers, gene expression analysis, and development from defined circulating precursors. We extended our studies of the role of type-1 interferons in the regulation of intestinal immunity and found an essential role for type-1 interferons in preventing abnormal inflammation in mouse models of inflammatory bowel disease. We demonstrated that type 1 interferon signaling on non- T cells in the adoptive transfer model of colitis helps prevent inflammation via a mechanism that involves the induction of IL-10 production and the suppression of IFNbeta function, at least partially by enhancing the production IL-1RA, a natural inhibitor of IL-1 activity. Therefore, type-1 interferons have essential roles both in prevention of infection by some, but not all, intestinal viruses, as well as in controlling abnormal intestinal inflammation. This opens the possibility of looking further into the functional effects of type-1 interferon signaling pathways in the pathogenesis of IBD and in re-addressing therapy with type-1 interferons in selected patients. In FY2015, we evaluated the evaluated gene regulation in macrophages in the colon (cMPs) to determine the precise control of inflammatory and non-inflammatory cytokines by these cells. We determined that a major, previously unappreciated level of control of cytokine production 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 lamina propria influence cMPs directly or their differentiation from blood monocytes to influence post-transcriptionl gene regulation. Given that the production these pro inflammatory cytokines is essential for tissue inflammation in patients with inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis, these results suggest 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, as MG-132, a widely used proteosome inhibitor was able to significantly enhance NLRP3 and pro-IL1-beta levels in resting or activated cMPs in vitro. This is the first data showing that NLRP3 leaves can be controlled by degradation. Using a combination of genomic and proteomic approaches we plan on further exploring the mechanisms involved in post-transcriptional regulation of cytokines in intestinal macrophages and dendritic cells, and to develop models for how genes such as IL-10, IL-12 and TNF-alpha, which are important for intestinal homeostasis are controlled. In separate studies, in collaborative studies with Warren Leonards laboratory we have evaluated the regulation of intestinal immune responses by the cytokine IL-21, since patients with IL-21R deficiency are highly susceptible to the intestinal protozoan parasite Cryptosporidium parvum. Using basic approaches and infectious challenge models we will identify innate and adaptive immune defects that control this important human pathogen in an IL-21 dependent manner.
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