P-219 Regulation of the Mucosal Phenotype in Dendritic Cells by PPARγ

Abstract

Mucosal dendritic cells (DCs) of the intestinal tract play a critical role in regulation of immune homeostasis in the gut. However, this immunoregulatory capacity is reduced during chronic inflammatory conditions, such as Inflammatory Bowel Diseases, and therapeutic approaches to restore this function are needed. A variety of stimuli which induce a mucosal phenotype in DCs have been identified; however, the molecular pathways which regulate appropriate gene expression for development of mucosal DC function are relatively unknown. The goal of this study was to determine if the ligand-activated transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ), contributed to the development of the “mucosal” phenotype in mouse mDCs. Bone marrow-derived dendritic cells (BMDCs). BMDCs were prepared by growing bone marrow cells from C57Bl/6 wildtype or PPARγΔDC mice for 10 days in the presence of GM-CSF. The CD11c+ fraction was isolated by differential adherence and/or flow cytometry. Splenic CD4+ T cells were isolated from normal mice by nylon wool adherence plus treatment with anti-CD8, anti-Ia and complement. Normal splenic B cells were isolated by magnetic sorting using a B220 positive selection kit. Flow cytometric analysis was performed on a iCyt-Sony Synergy cell sorter system using standard techniques for surface and intracellular staining. Cell proliferation was measured by incorporation of 3H-thymindine in 72 hour cell cultures. Mice with a targeted deletion of PPARg in dendritic cells were generated by breeding PPARg floxed mice with CD11c-Cre mice. Experiments indicated that activation of PPARγ in bone marrow-derived DCs (BMDCs) induced a more immunosuppressive phenotype in which BMDCs had reduced ability to induce proliferation of naïve CD4 T cells. This immunosuppressive function correlated with reduced expression of MHC II and costimulatory molecules and increased IL-10 secretion, which was shown to contribute to the reduced T cell proliferation. BMDCs with activated PPARγ interacted with CD4 T cells in a manner consistent with a mucosal phenotype. This included an enhanced ability to polarize naïve CD4 T cells toward iTregs and to induce expression of the mucosal homing receptor, CCR9, on T cells. Activation of PPARγ in BMDCs also increased their ability to induce T cell-independent IgA production in naïve B cells. Evaluation of BMDCs from mice with a Cre-Lox conditional deletion of PPARγ in CD11c+ cells (PPARγΔDC) confirmed a regulatory role for PPARγ on expression of the mucosal phenotype in DCs. PPARγΔDC BMDCs displayed enhanced steady-state expression of costimulatory molecules, enhanced proinflammatory cytokine production and decreased IL-10 synthesis. Contrary to the inflammatory BMDC phenotype in vitro, PPARγΔDC mice showed no change in the frequency or phenotype of myeloid DCs (mDC) in the colon. In contrast, mDCs in the lungs were significantly increased in PPARγΔDC mice. A modest increase in colitis severity was observed in DSS-treated PPARγΔDC mice compared to control. These results indicate that activation of PPARγ in mDCs induces a mucosal phenotype and that loss of PPARγ expression promotes a more inflammatory phenotype. However, the intestinal microenvironment in vivo can help to maintain the mucosal phenotype of mDCs via PPARγ-independent mechanisms.