This project focuses on how antigens are processed in the intestine of mice. 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. 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. In prior studies we have established the presence of 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. We determined that there are 3 separate subpopulations of immature DCs in the PP, lymphoid (CD8+), myeloid (CD11b+), and double negative (DN) Dcs that express neither CD8 or CD11b. These separated DC subpopulations are located in distinct sites in the PP, and are capable of inducing the differentiaion of T cells into cells that produce unique cytokine profiles. Most importantly, we demonstrated that PP DCs have the unique capacity to induce the differntiation 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 have also recently demonstrated that DN DCs in the subepithelial dome region of the PP process viral antigen from virally infected apoptotic epithelial cells. Furthermore, we previously 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 indicates 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. Furthermore, 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 signfifcant 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 studied the mechanisms by which TGFbeta and IL-10 regulate type-1 IFN prodution by pDCs in response to CpG oligonucleotides. We determined that TGFbeta appears to act on the initial CpG signal mediated by toll-like receptor 9 (TLR9), which IL-10 appears to act on both intital TLR9 signals as well as dominantly on the autocrine feedback that type-1 IFN proovides though the IFNalpha/beta receptor. The suppression by IL-10 is mediated through suppression STAT-1-phosphorylation in response to type-1 IFN. We have also begun to understand the subpopulations DCs in the colon of mice and their progenitors. In particular we find that there is one particular population so of cells that is replenished by peripheral blood monocytes, while the others appear to be derived from other blood precursors. Furthermore, we have begun to study the requirement for certain chemokine recpetors on T cells that are important for trafficing into the colon during inflammation in mouse models of inflammatory bowel disease. Finally, we have studied 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.
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