Antigen Processing And Presentation In The Intestine

National Institute Of Allergy And Infectious Diseases

Investigators

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Abstract

This project focuses on the roles of different populations of dendritic cells (DC) and macrophages (MP) in immune responses in mucosal tissues. The primary focus in on the intestine, but we have also explored eye inflammation in a new model of dry-eye disease developed in the laboratory. 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, and as we now believe inflammatory eye disease. 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. 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-transcriptionl 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 2016, using a combination of genomic and proteomic approaches we further explored post-transcriptional regulation of cytokines in intestinal MPs and DCs. We have developed new techniques to study intracellular cytokine and mRNA expression together in single cells, which allowed us to confirm that many cytokines in intestinal MPs are controlled at the post-transcriptional level, including IL-1beta. We are currently exploring the post-transcriptional mechanisms that control these cytokines. Furthermore, we established the first single cell mRNA analysis of intestinal myeloid cells in mice. These data have established a new level of heterogeneity amongst DC and monocyte/MP populations and has led to new hypotheses regarding cytokine control in the intestine in the steady-state and in IBD. They will inform many future studies in the laboratory. In separate collaborative studies with Warren Leonard's laboratory, we established the role of IL-21 in immune regulation in the intestine with regard to B cell differentiation and Th17 responses to commensal bacteria and to infection with C. 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, we showed a unique role of IL-21 in T cell dependent, but not independent, IgA responses to commensal bacteria, the absence of which results commensal-dependent pathogenic Th17 responses to C. rodentium. Furthermore, we found that IL-21 was important for IgA induction to only specific commensal bacteria, including the clostridia species, segmented filamentous bacteria, consistent with the hypothesis that IgA against the majority of commensal bacteria is produced in a T cell-independent manner. 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 subsequent infection with pathogenic microorganisms, such as pathogenic E. coli. 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. Finally, we explored the pathogenesis of eye inflammation that developed in one colony of mice with experimental IBD. Dry eye disease (DED) affects one third of population worldwide. In prior studies, experimental autoimmune lacrimal keratoconjunctivitis (EALK) induced by desiccating stress in mice has been used as a model of DED. This model is complicated by a requirement for exogenous epithelial cell injury and the administration of anticholinergic agents that have broad immunological effects. We observed that in addition to colitis, EALK spontaneously developed in 60% of C57BL/10 RAG2-/- mice following adoptive of CD4+CD45RBhigh nave T cells characterized by the infiltration of CD4+ T cells, MPs, and neutrophils. In addition to lacrimal keratoconjunctivitis, mice also developed damage to the corneal nerve, which connects components of lacrimal functional unit (LFU). Pathogenic T cell differentiation was dependent on IL-23p40 and controlled by co-transferred CD4+CD45RBloCD25+ regulatory T cells (Tregs). Th17 rather than Th1 CD4+ cells were primarily responsible for EALK likely due to the ability of IL-17 to drive the expression of CXC chemokines within the cornea, and the subsequent influx of myeloid cells. Thus, we have described a novel model of spontaneous EALK that supports a role for Th17 cells in disease pathogenesis, and that will contribute to our understanding of autoimmune lacrimal keratoconjunctivitis in many human eye diseases, including DED. Finally, the fact that disease only occurred in some NIAID facilities suggests that eye-commensal bacteria may be essential for disease development and subsequent collaborative studies will address this possibility.