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

Abstract

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-β and 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.

Keywords

Sepsis / Immunosuppression / p53 / Regulatory T cells / Foxp3 / DNMT3a / TET2

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