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. 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. In other studies, we localized and studied the function of plasmacytoid DCs (pDCs) from the Peyer's patch. 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, cells known to drive non-pathogenic Th2 responses, suggesting that treatments that enhance the number of plasmacytoid DCs in the blood may have therapeutic efficacy in Th1-mediated autoimmune disorders. In FY2012, 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. More specifically, we found that F4/80hi CX3CR1hi (CD11b+CD103-) cells account for 80% of mouse colonic lamina propria (cLP) MHC-IIhi cells. Both CD11c+ and CD11c- cells within this population were identified as MPs based on multiple criteria, including a MP transcriptome revealed by microarray analysis. These MPs constitutively released high levels of IL-10 at least partially in response to the microbiota via an MyD88-independent mechanism. In contrast, cells expressing low to intermediate levels of F4/80 and CX3CR1 were identified as DCs, based on phenotypic and functional analysis and comprise three separate CD11chi cell populations: CD103+CX3CR1-CD11b- DCs, CD103+CX3CR1-CD11b+ DCs and CD103-CX3CR1intCD11b+ DCs. 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 CD103-CX3CR1intCD11b+ DCs, which produced high levels of IL-12, IL-23, iNOS and TNFalpha. 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. These studies are important in that they delineate 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. They also demonstrate the primary role played by peripheral blood monocytes in contributing to intestinal inflammation, and thus indicate that blocking the trafficking of this cell type to the inflamed colon could provide a novel strategy for treatment of inflammatory bowel disease. Second, we have addressed 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. 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. We are currently delineating mechanisms by which type-1 interferons control intestinal immune homeostasis. In collaborative studies we helped demonstrate and essential role for NK-cell- derived IFN-gamma in both inducing the loss of resident monocyte/macrophage populations and driving the differentiation of newly recruited monocytes into inflammatory monocyte-derived dendritic cells in the peritoneal cavity during Toxoplasma gondii infection in mice. In addition, we conducted experiments to show the importance of tetramerization of STAT5 in driving functional regulatory T cell responses. In these experiments, regulatory T cells from Stat5a-Stat5b double knock-in (DKI) N-domain mutant mice in which STAT5 proteins form dimers but not tetramers were not able to control colitis in an adoptive transfer model of inflammatory bowel disease. These studies were important as they provided the first data that IFN-gamma, in particular produced by NK-cells was essential for orchestrating both the cellular dynamics and cytokine production capacities of antigen-presenting cells during infection by T. gondii, an important human pathogen, and demonstrated an essential molecular role for a STAT tetramerization in regulating immunity. Finally, in ongoing collaborative efforts with Dr. Berzofskys laboratory, we helped perform studies to demonstrate that IL-15 and TLR agonists included in a vaccine formulation for SIV infection, the macaque model of HIV in humans, resulted in the preservation of CD4+ T cells in the intestine. SIV and HIV normally target intestinal CD4+ T cells in the intestine and their loss results in susceptibility to opportunistic infections, loss of intestinal barrier function, and SIV/HIV progression.

Project Start
Project End
Budget Start
Budget End
Support Year
15
Fiscal Year
2012
Total Cost
$1,456,836
Indirect Cost
City
State
Country
Zip Code
Filardy, A A; He, J; Bennink, J et al. (2016) Posttranscriptional control of NLRP3 inflammasome activation in colonic macrophages. Mucosal Immunol 9:850-8
Veenbergen, S; van Berkel, L A; du Pré, M F et al. (2016) Colonic tolerance develops in the iliac lymph nodes and can be established independent of CD103(+) dendritic cells. Mucosal Immunol 9:894-906
Cho, Hyeseon; Kelsall, Brian L (2014) The role of type I interferons in intestinal infection, homeostasis, and inflammation. Immunol Rev 260:145-67
Lopatin, Uri; Blutt, Sarah E; Conner, Margaret E et al. (2013) Lymphotoxin alpha-deficient mice clear persistent rotavirus infection after local generation of mucosal IgA. J Virol 87:524-30
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Wan, Chi-Keung; Oh, Jangsuk; Li, Peng et al. (2013) The cytokines IL-21 and GM-CSF have opposing regulatory roles in the apoptosis of conventional dendritic cells. Immunity 38:514-27
Goldszmid, Romina S; Caspar, Pat; Rivollier, Aymeric et al. (2012) NK cell-derived interferon-γ orchestrates cellular dynamics and the differentiation of monocytes into dendritic cells at the site of infection. Immunity 36:1047-59
Rivollier, Aymeric; He, Jianping; Kole, Abhisake et al. (2012) Inflammation switches the differentiation program of Ly6Chi monocytes from antiinflammatory macrophages to inflammatory dendritic cells in the colon. J Exp Med 209:139-55
Lin, Jian-Xin; Li, Peng; Liu, Delong et al. (2012) Critical Role of STAT5 transcription factor tetramerization for cytokine responses and normal immune function. Immunity 36:586-99
Wilson, Mark S; Ramalingam, Thirumalai R; Rivollier, Aymeric et al. (2011) Colitis and intestinal inflammation in IL10-/- mice results from IL-13Rα2-mediated attenuation of IL-13 activity. Gastroenterology 140:254-64

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