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.

中文翻译：

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p53 通过脓毒症中 DNMT3a 和 TET2 介导的 Foxp3 表达促进调节性 T 细胞的扩增

背景 免疫抑制是脓毒症的一个重要特征，与不良预后密切相关。调节性 T 细胞 (Treg) 通过抑制效应 T 细胞 (Teff) 增殖和分化来抑制免疫。我们的目的是研究 p53 在脓毒症后 Treg 扩增中的作用。方法通过盲肠结扎穿刺（CLP）方法构建野生型（WT）和p53f/f/CD4-Cre+小鼠脓毒症模型，并通过流式细胞术评估CD4+CD25+ Foxp3+ Tregs的比例。使用定量实时 PCR 和蛋白质印迹分析检查叉头/翼状螺旋转录因子 p3 (Foxp3)、DNA 甲基转移酶 (DMNT)3a 和 10-11 易位 (TET)2 的表达水平。通过亚硫酸氢盐测序 PCR 分析细胞中 Treg 特异性去甲基化区域 (TSDR) 甲基化位点。此外，使用荧光素酶测定说明了 p53 与 Dnmt3a 和 TET2 启动子的直接结合。通过对细胞因子水平和共培养 Teff 增殖的酶联免疫吸附测定分析表明了 Tregs 的抑制能力。最后，比较了 WT 和 p53f/f/CD4-Cre+ 小鼠 CLP 后的死亡率。结果 CLP 后，与 WT 小鼠相比，p53f/f/CD4-Cre+ 小鼠中 CD4+CD25+ Foxp3+ Tregs 的比例显着降低。WT 小鼠中 Foxp3 的增强表达在 p53f/f/CD4-Cre+ 组中下调。我们发现 CLP 后 DMNT3a 水平降低，TET2 水平升高。然而，DNMT3a 和 TET2 的失调在 p53f/f/CD4-Cre+ 小鼠中显着逆转。TSDR 在 p53f/f/CD4-Cre+ 小鼠中经历了更多的去甲基化。荧光素酶活性表明 p53 与 DNMT3a 和 TET2 的启动子区域直接结合以调节其转录。因此，p53f/f/CD4-Cre+ CLP 小鼠的 Tregs 表现出有限的抑制能力，转化生长因子-β 和白细胞介素 10 (IL-10) 的产生减少表明了这一点。在共培养系统中，在从 p53f/f/CD4-Cre+ CLP 小鼠中分离出的 Tregs 存在的情况下，Teffs 显示出保留的 IL-2 产生、分化为 Th1 细胞和增殖。最后，与WT组相比，CLP后p53f/f/CD4-Cre+组的死亡率显着降低。结论 p53 通过调节 DNMT3a 和 TET2 的诱导，从而导致脓毒症中 Foxp3-TSDR 去甲基化，对 Foxp3 表达和随后的 Treg 扩增至关重要。