Antigen Processing And Presentation In The Intestine

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications & trials

Abstract

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. 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 2020, we completed studies of 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 defined 6 populations of monocyte/macrophages and 5 populations of DCs in normal mouse colon. Unique gene expression by these populations 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. We found that these two discrete developmental pathways resulted in cells that are either near the lumen of the intestine and thus more exposed to epithelial cell and commensal microbial products, or near the base of the lamina propria near blood vessels and lymphatic drainage. We further identified the ability of these two macrophage populations to differentially sample antigens from the blood, indicating a novel function of macrophages in the intestine, to sample blood derived products. We have further generated novel hypotheses for the functions of these discrete macrophage populations based on their expressions of specific proteins that have unique functions. Finally, we showed that the intestinal microbiota are essential for the differentiation of several but not all macrophage populations, and for not for DC differentiation. We have now made progress in identifying unique markers for both macrophage and DC populations that will allow us to further localize these cells and allow for their isolation to help understand thrive functions. During the current FY 2021, We explored role of specific receptors that have known regulatory roles on mononuclear phagocytes, have been identified by GWAS to be susceptibility genes for Crohn's disease or ulcerative colitis, and have been identified on intestinal phagocytes in other studies, on intestinal mononuclear phagocyte function and susceptibility to experimental colitis using cell-specific gene knockout mice and experimental models of inflammatory bowel disease. We evaluated cell-specific deficiency in EP4, a receptor for prostaglandin E2, in two models of inflammatory bowel disease, the DSS colitis model and the T cell transfer colitis model. We found that in both models EP4 deficiency in CX3CR1+ cells results in more severe disease, and in the former has major early effects on the intestinal barrier. We are currently exploring the mechanisms of this enhanced disease. In addition, in a separate set of experiments we explored the role of specific commensal bacteria in the generation of IgA responses to model intestinal vaccines. We found that the presence of a particular bacteria, segmented filamentous bacteria (SFB) is able to enhance IgA responses to vaccination with cholera toxin or infection with reovirus, a model intestinal infection in mice. We used single cell mRNA analysis combined with analysis of cell populations using flow-cytometry, and both mRNA and protein expression by isolated cells identified in scRNAseq studies. We found an increase in the proportions of certain conventional dendritic cell (cDC) populations that influence early IgA B cell class switching in the subepithelial cell dome region of the PP, and a separate role for IL-6 in the LP to correlate with the enhanced IgA production with SFB colonization. We are currently further exploring the mechanisms by which this enhancement of IgA responses occurs in the presence of this commensal bacteria, and whether it is due to changes in antigen uptake and processing by intestinal myeloid cells. Finally, we have characterized the cDC populations in the the colon of mice by scRNAseq analysis, and identified unique populations that appear to represent key developmental intermediates for cDC1 and cDC2 populations, as well as potential receptors for environmental factors driving their differentiation. We are testing these hypotheses using mouse models of infections that drive cDC1 or cDC2 differentiation in wild-type mice, and mice deficient in candidate developmental factors. These studies have implications for understanding the immunopathogenesis of inflammatory bowel disease and immune responses to oral vaccin