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. <p/>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. Furthermore, we determined that clearance of lethal experimental infection with a model mucosal virus infection, type 1 reovirus, is dependent on type-1 interferon production in the PP, that type-1 interferon production by dendritic cells within the PP is a primary determinant of whether this mucosal pathogen survives and is disseminated to other tissues. 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. <p/>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. In 2013, we have 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 separate studies, we furthered our understanding of intestinal virus infection by understanding the role of PPs in the generation of IgA to intestinal rotavirus infection. Rotavirus is an important human disease that kills an estimated 450,000 children under the age of 5 years. We demonstrated that PPs are a primary initial site for rotavirus infection in mice that PP dendritic cells capture viral antigens very similar to what we had shown with the related type-1 reovirus, and that lymphoid tissues, including PPs and MLNs are absolutely required for a primary IgA responses to rotavirus that then results in viral clearance. Importantly, systemic antibody responses were effective in clearing systemically disseminated virus after oral infection, but were insufficient for the clearance of intestinal virus. This was the first time that the importance of IgA generated in local lymphoid tissues has been shown to directly result in viral clearance, e.g., that the lack of intestinal IgA responses resulted in chronic rotavirus infection.We also addressed a longstanding issue in the laboratory, the enhanced susceptibility of RAG-deficient mice on the C57BL/10 background to inflammatory disease following adoptive transfer of nave CD4+ T cells.<p/> In prior studies, we have shown that RAG-deficient C57BL/10 mice have a more rapid progression and increased severity of colitis, as well as develop inflammatory skin and eye disease in particular environmental conditions. We found that adoptively transferred T cells proliferate at an increased rate, and have a defect in the de novo induction of CD4+Foxp3+ regulatory T cells in the MLNs of RAG-deficient C57BL/10 compared to in the MLNs of RAG-deficient C57BL/6 mice. We also demonstrated that this skewed differentiation was not likely due to differences in bacterial flora, but was likely dependent on enhanced IL-23 and IL-12 production by DCs from C57BL/10 mice following adoptive transfer.<p/> These studies linked prior studies showing a genetic propensity of C57BL/10 mice to produce higher levels of IL-12 and IL-23 (due to a polymorphism in the IL-12p40 gene) to those showing an enhanced susceptibility to colitis induction in this mouse model; and described one mechanism by which this propensity enhances disease. In collaborative studies with Warren Leonards laboratory, we demonstrated the functional consequences of controlling dendritic cell apoptosis by IL-21 and GM-CSF, by showing enhanced inflammation of IL-21-deficient RAG-/- mice to T cell transfer colitis; that was associated with increased dendritic cell numbers, as well as increased IFN-gamma production T cells. These studies showed that IL-21 can enhance and GM-CSF can reduce apoptosis of conventional dendritic cells that has consequences for the development of intestinal inflammation.
The overall focus of this project is to dissect at the cellular and molecular levels, the induction and regulation of innate and acquire immunity in the intestine under three physiological conditions: the normal, unstressed "steady-state", the successful eradication of a mucosal pathogen, and the abnormal maladaptive immune response that results in inflammatory bowel disease. Distinct populations of dendritic cells (DCs) are now known to be of primary importance in directing both innate and acquired immune responses. In addition, intestinal macrophages have been poorly defined and little is know regarding their function in the intestine with regard to inducing or maintaining immunological suppression, contributing to mucosal repair, or providing innate defense against commensal or pathogenic commensal microbes. Therefore, the current objectives in this long-term project are directed to defining and understanding the role of DC and macrophage populations in intestinal immune responses, and understand the factors that control their function, including the role played by regulatory T cells in defining their phenotype and fuunction. The primary long-term goals of this project are to develop novel methods for inducing immunity to oral vaccines and for the treatment of inflammatory bowel disease.