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. Furthermore, we evaluated gene regulation in resident and inflammatory colon MPs and determined that a major, previously unappreciated level of control of inflammatory cytokine production by intestinal MPs is via post-transcriptional mechanisms, which differentially affect the production of inflammatory cytokines, such as TNFalpha, IL1beta, and IL6, as well as the expression of the inflammasome NLRP3, and regulatory cytokines, such as 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. In addition, 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 2025, using cell-specific gene knockout mice and experimental models of inflammatory bowel disease we have continued to explore the role of specific receptors that 1) have known regulatory roles on mononuclear phagocytes, 2) have been identified by GWAS to be susceptibility genes for Crohn's disease or ulcerative colitis, 3) have been identified on intestinal phagocytes in other studies, and 4) that regulate intestinal mononuclear phagocyte function and susceptibility to experimental colitis. We previously found that only certain MP populations are profoundly lost during acute intestinal inflammation in the DSS colitis model, and return during repair. During FY 2025 we actively investigating how the dynamics of macrophage population changes affects the induction and resolution of disease, as well as directing studies toward understanding the mechanisms of cell loss and replenishment. In addition, in a separate set of experiments we have continued to explore 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 dome region of the PP, and a separate role for IL-6 in the LP correlated with the enhanced IgA production with SFB colonization. We are currently further exploring the mechanisms by which this enhancement of the IgA response occurs in the presence of this commensal bacteria, and whether it is due to changes in antigen uptake and processing by intestinal myeloid cells. In FY2024 we characterized cell populations associated with the enhanced IgA responses in the presence of SFB, focusing on the details of both cDC and MP populations, and the clonality of B cells expressing IgA and IgG2b, which is also induced in the presence of SFB, to understand the contributions of memory B cells in the PP to humoral immunity in both the intestine and the peripheral immune system. In FY2025, we focused on identifying other cell populations and factors that may be important for IgA and IgG2b cell differentiation in the SFB-colonized PPs, including naive B cells recruitment, follicular T helper cell dependence and differentiation, further defining memory B cell localization and differentiation, and addressing factors for recruitment and differentiation using gene-knockout mice. Furthermore, during FY2025 we are continuing to evaluate as yet uncharacterized populations of myeloid cells in the mouse colon in SPF and germ-free mice to determine how specific microbes in the intestine can influence macrophage and DC differentiation and phenotypes. We are also evaluating the role of macrophage populations in the infection and pathogenesis of the intestine by via systemic spread from other tissues. Furthermore, in FY2025 we collaborated with Warren Leonard's laboratory to identify the role of BLIMP-1 in the induction of regulatory T cells in the intestine in a mouse model of inflammatory bowel disease. These studies have implications for understanding the pathogenesis of intestinal viral infections, the immunopathogenesis of inflammatory bowel disease, and immune responses to oral vaccines.