2gene manifestation was observed in spleens and lymph nodes of type II collagen-immunized mice treated with decitabine (Fig. the peripheral blood of patients following treatment with azacytidine (14C16). In addition, nucleoside analog demethylating providers have been reported to promote the generation and suppressive function of induced Treg (iTreg) cells in vitro (14, 17, 18) and to protect against experimental autoimmune encephalomyelitis and allogeneic cardiac transplant rejection in vivo (19). Against this background, we compared the ability of nucleoside- and nonnucleoside-based DNA-demethylating providers to promote induction of Treg cells in animal models of RA. We found that short-term treatment with the cytosine analog decitabine depleted pathogenic Teff cells and advertised Treg cell reactions, leading to enduring disease remission. Results DNA-Demethylating Medicines Promote Generation of Treg Cells In Vitro. We 1st assessed the ability of nucleoside- and nonnucleoside-based DNA-demethylating medicines to promote the generation of iTreg cells by revitalizing naive CD4+ T cells with anti-CD3 antibody under G6PD activator AG1 Treg cell-inducing conditions (Table 1). Treatment with decitabine, psammaplin A, or zebularine resulted in a dose-dependent increase in the percentage and total number of iTreg cells in vitro, as well as improved FoxP3 and CD25 manifestation (= 10). (= 7). (= 10). (= 3). * 0.05, ** 0.01, *** 0.001. To establish its mechanism of action, type II collagen-immunized mice were treated with decitabine or vehicle for 4 d as in the previous experiment. Measurement of T cell subsets exposed a profound reduction in numbers of Th1 (IFN+CD4+ and Tbet+CD4+) and Th17 (IL-17+CD4+ and RoRt+CD4+) cells in decitabine-treated mice (Fig. 1and gene manifestation in bone marrow-derived dendritic cells (BMDCs) in vitro, confirming earlier findings (23) (Fig. 2gene manifestation was observed in spleens and lymph nodes of type II collagen-immunized mice treated with decitabine (Fig. 2and wild-type mice, which were then treated with decitabine or vehicle for 4 d, as explained above. In the beginning, decitabine experienced the same restorative effect in both organizations but disease rapidly relapsed in mice but not wild-type mice (Fig. 2 Tm6sf1 and mice compared with wild-type mice (Fig. 2 and gene manifestation of BMDCs of C57BL/6 mice treated with IFN and/or decitabine was determined by qPCR. Values are the mean SEM (= 3). (gene manifestation was determined by qPCR. Values are the mean SEM (= 3). (and and were culled on day time 20. Lymph node cells were stained with lineage-specific transcription factors ((0.05, **0.01, ***0.001. Decitabine Selectively Focuses on ENT1+ T Cells. Decitabine is known to enter cells via the equilibrative nucleoside transporter 1 (ENT1), G6PD activator AG1 which is also known to be up-regulated on proliferating cells (13). We reasoned that this could explain the selective action of decitabine on Teff cells (Fig. 1). Indeed, ENT1 manifestation was higher in CD4+ T cells from type II collagen-immunized mice with active arthritis versus immunized mice without arthritis (Fig. 3and and and and 0.05, **0.01, ***0.001. Depletion of Teff Cells by Decitabine Is Dependent on ENT1. We next set out to address the mechanism by which decitabine depletes Teff cells. We 1st showed that proliferative reactions of FoxP3?CD4+ T cells from arthritic mice were significantly more sensitive to the inhibitory effects of decitabine than those of nonarthritic mice (Fig. 4 0.05, ** 0.01, *** 0.001. To determine the mechanism by which decitabine reduces numbers of proliferating T cells, we looked for evidence of DNA fragmentation and apoptosis in the T cell human population using the comet assay and annexin G6PD activator AG1 V/propidium iodide (PI) staining, respectively. Decitabine improved DNA fragmentation and annexin V staining of CD4+ T cells inside a dose-dependent manner (Fig. 4and and = 5). Mice were given an intraarticular injection of mBSA 15 d after immunization. Knee swelling was monitored for 5 d (. (was quantified based on MFI. *0.05, **0.01, ***0.001. Decitabine-Induced Treg Cells Possess Antiarthritic Activity. Finally, we confirmed the antiarthritic activity of decitabine-induced Treg cells by adoptive transfer from decitabine-treated donor mice to untreated, arthritic recipient mice. Treg cells were collected from spleens and lymph nodes of mBSA-immunized donor mice that had been treated previously with decitabine or vehicle only. Treg cells (3 105 cells per mouse) were then injected intravenously into mBSA-immunized recipient mice prior G6PD activator AG1 to intraarticular injection of mBSA. Treg cells from decitabine-treated mice, but not control mice, significantly reduced inflammatory cell infiltration with significant reductions in CD4+ T cells and Th1, Th2, and Th17 cells (Fig. 6 and and = 6). Knee swelling was monitored for 5 d (and and the nTreg cell marker Helios in was quantified based on MFI. *0.05, **0.01, ***0.001. Conversation Chronic inflammation is definitely associated with loss of Treg cell function due, at least in part, to DNA methylation at regulatory regions of functionally important genes, including FoxP3 and CTLA-4 (1C5). This led us to query whether DNA-methylation inhibitors would be effective in reestablishing tolerance in autoimmunity by augmenting Treg.