p53 promotes the expansion of regulatory T cells via DNMT3a-and TET2-mediated Foxp3 expression in sepsis

Background:Immunosuppression is an important characteristic of sepsis and is closely related to poor outcomes.Regulatory T cells(Tregs)contribute to immune suppression by inhibiting effector T cell(Teff)proliferation and differentiation.We aimed to investigate the role of p53 in Treg expansion after sepsis.Methods:We constructed a sepsis model in wild-type(WT)and p53f/f/CD4-Cre+mice by cecal ligation and puncture(CLP)and evaluated the proportions of CD4+CD25+Foxp3+Tregs by flow cytometry.The expression levels of forkhead/winged helix transcription factor p3(Foxp3),DNA methyltransferase enzyme(DMNT)3a and ten-eleven translocation(TET)2 were examined using quantitative real-time PCR and Western blot analysis.Treg-specific demethylation region(TSDR)methylation sites in cells were analyzed by bisulfite-sequencing PCR.Furthermore,the direct binding of p53 to the Dnmt3a and TET2 promoters was illustrated using a luciferase assay.The suppressive ability of Tregs was indicated by enzyme-linked immunosorbent assay analysis of cytokine levels and the proliferation of cocultured Teffs.Finally,mortality rates after CLP were compared among WT and p53f/f/CD4-Cre+mice.Results:The proportion of CD4+CD25+Foxp3+Tregs was significantly reduced in p53f/f/CD4-Cre+mice compared to WT mice after CLP.The enhanced expression of Foxp3 in WT mice was downregulated in the p53f/f/CD4-Cre+group.We found decreased DMNT3a and increased TET2 levels after CLP.However,the dysregulation of DNMT3a and TET2 was significantly reversed in p53f/f/CD4-Cre+mice.TSDR underwent increased demethylation in p53f/f/CD4-Cre+mice.Luciferase activity indicated direct binding of p53 to the promoter regions of DNMT3a and TET2 to regulate their transcription.Consequently,Tregs from p53f/f/CD4-Cre+CLP mice exhibited limited suppressive ability,as indicated by the reduced production of transforming growth factor-βand interleukin 10(IL-10).In the coculture system,Teffs showed preserved production of IL-2,differentiation into Th1 cells and proliferation in the presence of Tregs isolated from p53f/f/CD4-Cre+CLP mice.Finally,the mortality rate of the p53f/f/CD4-Cre+group after CLP was significantly reduced in comparison to that of the WT group.Conclusion:p53 appears to be critical for Foxp3 expression and consequent Treg expansion by regulating the induction of DNMT3a and TET2,thereby resulting in Foxp3-TSDR demethylation in the context of sepsis.