TThis project focuses on how antigens are processed in the intestine of mice, and how antigens 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 commensal bacteria which allows for one tolerate these organisms without the onset of inflammation is essential for immune homeostasis in the intestine, as an 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 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 immunofluorecense 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. Most importantly, we demonstrated that 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. We also demonstrated that DCs 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 PP DCs and not epithelial cells at this site are major producers of type-1 interferon during infection. This indicated that type-1 interferon production by bone-marrow derived cells, and most clearly by dendritic cells within the PP is a primary determinant of whether this mucosal pathogen survives and is disseminated to other tissues. In other studies, we localized and studied the function of plasmacytoid DCs (pDCs) from the Peyer's patch. PP pDCs were localized to the interfollicular and subepithelial dome region of the PP by immunohistochemistry, exressed typical surface markers for pDCs from other tissues, as examined by flow cytometry, and produced IL-12 but little type-1 interferon following stimulation with either influenza virus, or bacterial DNA. The lack of type-1 interferon production by PP pDCs is significant because this is different from pDCs from other tissues. Furthermore, we determined that factors present in the microenvironment of the PP, in particular PGE(2), TGFbeta, and IL-10 were able to suppress type-1 interferon production by pDCs from the spleen, suggesting that microenvironmental factors may influence type-1 interferon production by pDCs. The lack of type-1 interferon producton by PP pDCs may help control untoward immune responses to commensal bacteria, and thus be pivotal in regulating intestinal inflammation, as occurs in inflammtory bowel diseases. Furthermore, we have performed studies in humans the ability of granulocyte-macrophage colony stimulating factor (GM-CSF), a growth factor that is commonly used to treat organ transplant patients to improve their production of neutrophil and monocyte after immunosuppression, to treat patients with inflammatory bowel disease. Treatment efficacy was correlated with changes in peripheral blood T cell production of IL-10, and sustained changes in the numbers of plasmacytoid DCs. As plasmacytoid DCs have been shown to drive non-pathogenic Th2 responses, these studies suggest that treatmetns that enhance the number of plasmacytoid DCs in the blood, may have therapeutic efficacy in Th1-mediated autoimmune disorders. In FY2010, we have focused our efforts on two areas. First, 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 or DC phenotype, and have begun to understand their function in vivo. These studies are under consideration for publication and will be described in more detail in next years report. Second, we have demonstrated a fundamental role of the chemokine receptor CCR6 in the migration and function of induced regulatory T cells in a murine model of colitis. Specifically, we showed that CCR6 is a marker for a particular subtype of regulatory T cell in the intestine that produces IL-10, a cytokine with suppressive function on DCs and macrophages and which has been shown to be fundamental for intestinal immune homeostasis. Furthermore, T cells from mice deficient in CCR6 induced a higher degree of colitis when transferred to RAG-deficient mice that lack an adaptive immune system, and that this enhanced disease was due to poor migration and function of regulatory T cells that are induced to differentiate in this model. These studies are important in that demonstrate that CCR6 is an important surface marker for colon-specific regulatory T cells and show an important role for CCR6 in the function of these cells in the colon, specifically in regulating the onset of colitis. Furthermore, they provide direction for future studies of CCR6+ regulatory T cell differentiation, and function, including their direct effect on DCs and macrophages, and suggest that immunotherapeutic approaches that induce such cells may be a new avenue to explore for control of inflammatory bowel disease.
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