Phenotype Of Myeloid Dendritic Cells Research Articles

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

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.

Increased immunopotency of monocyte derived dendritic cells from patients with optic neuritis is inhibited in vitro by simvastatin

Multiple sclerosis (MS) is an autoimmune disease where myelin-reactive lymphocytes and their activation depend on interactions with antigen presenting cells (APCs). Dendritic cells (DC) are professional APCs dependent on maturation to attain full T-cell priming capacity. The immunomodulatory properties of simvastatin influence the function of both T cells and APCs and could thus be a potential therapy for MS. The phenotype of myeloid DC in untreated patients with monosymptomatic optic neuritis (ON) was determined by flow cytometry and the impact of simvastatin on the function of myeloid DC derived from peripheral blood mononuclear cells (PBMC) was analysed in vitro. DC from ON patients had more mature phenotype compared with healthy controls (HC). Particularly the fraction of DC expressing CD1a and CD80 was significantly higher in ON than in HC ( P < 0.05). Addition of 10 μΜ simvastatin significantly inhibited the maturation of DC in the ON group. Furthermore, ON derived DC induced stronger T-cell proliferation in the mixed leukocyte reaction (MLR), and simvastatin solely inhibited this proliferation of T-cells in the ON group and not in HC. In conclusion DC from ON patients have a more mature phenotype and an increased stimulatory capacity. Simvastatin has an inhibitory effect on the differentiation and maturation of DC, and selectively reduce the T-cell proliferation induced by DC from patients with ON. The results from these in vitro assays suggest potential beneficial inhibitory effects of Simvastatin in the inflammation in ON and early MS, but we need more clinical trials to confirm it.

Effects of cyclosporine on human dendritic cell subsets

Cyclosporine is widely used as an immunosuppressive agent after solid organ transplantation. Limited data are available on the modulation of human dendritic cells by cyclosporine. We investigated the effects of cyclosporine on the phenotype and function of human dendritic cell (DC) subsets. DCs were isolated from peripheral blood using magnetic bead-conjugated antibodies. Cyclosporine did not alter the ability of myeloid and plasmacytoid dendritic cells to take up antigens. Expression of the co-stimulatory molecule CD80 but not CD86 increased on both DC subsets when stimulated with cyclosporine. The ability of cyclosporine treated myeloid DCs to stimulate proliferation of allogenic PBMC was significantly reduced. Similarly, stimulation of memory CD8+ T cells by dendritic cells was impaired by cyclosporine pretreatment. In conclusion, cyclosporine differentially alters function and phenotype of myeloid dendritic cells leading to a partially impaired capacity to stimulate allogenic and autologous T cells.

Autoantigen-specific protection of non-obese diabetic mice from cyclophosphamide-accelerated diabetes by vaccination with dendritic cells

Dendritic cells (DCs) are professional antigen presenting cells involved in the initiation of primary immune responses and the preservation of peripheral tolerance. The aim of this study was to develop a DC vaccine for autoantigen-specific prevention of autoimmune diabetes. Splenocytes from diabetes-prone NOD mice were cultured in conditioned media to obtain a homogeneous DC sub-population for vaccination experiments. These cells were used to modulate autoimmune responses in NOD mice after synchronization of diabetes with cyclophosphamide. After immunisation with insulin-pulsed DCs the incidence of diabetes, the insulitis grade and the cytokine production was examined. The long-term culture of splenocytes resulted in the generation of a cell line, termed NOD-DC1, which have a phenotype of myeloid DCs (CD11c, CD11b, DEC-205), express MHC class II and co-stimulatory molecules (CD40, CD80, CD86). The NOD-DC1 cells have preserved functional activity shown by the detection of a high antigen uptake capacity, the induction of a mixed lymphocyte reaction and stimuli-dependent IL-6 and TNF-alpha secretion. Vaccination with insulin-pulsed NOD-DC1 cells results in an antigen-specific prevention of diabetes. This was mediated by a reduction of the severity of insulitis and a decrease of T helper 1 effector cells. We describe the generation of a DC line which confers protection from diabetes in an antigen-specific way. Our data shows that autoantigen-loaded DCs can induce strong immunoregulatory effects supporting the hypothesis that DCs are promising candidates to develop novel vaccines for the prevention of autoimmune diabetes.