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 p53/CD4-Cre mice by cecal ligation and puncture (CLP) and evaluated the proportions of CD4CD25 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 p53/CD4-Cre mice.

RESULTS

The proportion of CD4CD25 Foxp3 Tregs was significantly reduced in p53/CD4-Cre mice compared to WT mice after CLP. The enhanced expression of Foxp3 in WT mice was downregulated in the p53/CD4-Cre group. We found decreased DMNT3a and increased TET2 levels after CLP. However, the dysregulation of DNMT3a and TET2 was significantly reversed in p53/CD4-Cre mice. TSDR underwent increased demethylation in p53/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 p53/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 p53/CD4-Cre CLP mice. Finally, the mortality rate of the p53/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.