Epigenetic modification of CD4+ T cells into Tregs by 5-azacytidine as cellular therapeutic for atherosclerosis treatment

Recent research has explored the potential of the demethylating drug 5-azacytidine (Aza) as therapy for a range of diseases. However, the therapeutic efficacy of Aza for patients of atherosclerosis remains unclear. This study investigates the therapeutic application of Aza to atherosclerosis in order to elucidate the underlying mechanisms. We generated induced Tregs (iTregs) from CD4⁺ T cells by using Aza in vitro, and this was followed by the intravenous infusion of iTregs for the treatment of atherosclerosis. The adoptive transfer of Aza-iTreg significantly increased peripheral blood Treg cells, suppressed inflammation, and attenuated atherosclerosis in ApoE^-/- mice. Furthermore, we observed a notable demethylation of the Forkhead box P3 (Foxp3)-regulatory T cell-specific demethylated region (TSDR) and an upregulation of Foxp3 expression in the CD4⁺ T cells in the spleen of the ApoE^-/- mice following the transfer of Aza- iTregs. We also demonstrated that Aza converted naive CD4⁺ T cells into Tregs by DNA methyltransferase 1 (Dnmt1)-mediated Foxp3-TSDR demethylation and the upregulation of Foxp3 expression. Conversely, the overexpression of Dnmt1 in the CD4⁺ T cells attenuated the Aza-induced Foxp3-TSDR demethylation and upregulation of Foxp3 expression. Our results reveal that Aza converts naive CD4⁺ T cells into functional Tregs by inhibiting Dnmt1, and the transfer of Aza-iTregs suppresses atherosclerosis in mice.