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. 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. 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; 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. In FY2015-16, 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 collaborative studies with Warren Leonard's 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 demonstrated that IL21 is expressed by a large percentage of CD4+ T cells in the Peyer's patches and lamina propria of the small intestine, but many fewer in the colon. IL21R-deficient mice have low levels of IgA and paradoxically higher numbers of Th17 cells in intestinal lamina propria and have much less inflammation in response to C. rodentium infection, a mouse model of enteropathogenic and enterohemorrhagic E. coli infection in humans. The details of this phenomenon and under investigation.
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