Foxp3 DNA去甲基化在脓毒症时调节性T细胞增殖分化中的作用及调控机制

Sepsis-induced immunosuppression is the major cause of poor prognosis and even death in critically illness. Recently, it has been reported a marked activation of regulatory T cell (Treg) proliferative activity in the setting of sepsis, which may contribute to the development of shifting effector T cells together with host immune dysfunction. The key role of Foxp3 in mediated Treg proliferation as well as differentiation has been well documented, however, the underlying mechanism of its expression is still unknown. Our previous study suggested a critical role of p53 on sepsis induced Th2 shift and immunosuppression. The current project will focus on the effect and regulatory mechanism of p53 on modulating Foxp3 expression following septic challenge. We will investigate expressions of DNMT3a and TET2, and Foxp3 DNA demethylation level in CD4-specific p53 knockout mice model underwent sepsis. Meanwhile, the potential role of DNMT3a and TET2 in demethylation of Foxp3 will be identified by mRNA or shRNA interference, in which the effect of p53 on promoting Treg proliferation by DNMT3a- or TET2-mediated Foxp3 demethylation and stable expression will be illustrated, in turn resulting in Th2 shift and abnormal immune response. In addition, we will confirm the clinical significance of p53/DNMT3a or TET2/Foxp3 DNA demethylation in sepsis-induced immune depression in patients, which might provide reliable clinical prognosis markers and new target for immune modulation therapies in septic complications.

近年来资料提示，脓毒症时调节性T细胞（Treg）免疫反应异常，诱导效应T细胞向Th2型分化，造成机体免疫应答障碍。Foxp3是影响Treg增殖分化和功能的关键因子，但其表达调控机制尚未阐明。前期研究发现，脓毒症状态下p53诱导Th2漂移。本课题将深入探讨p53对Foxp3表达的影响及分子机制。拟采用CD4-p53-/-小鼠复制脓毒症模型，观察p53对甲基化调节蛋白—DNMT3a和TET2的表达调控，以及对Foxp3 DNA去甲基化的影响。进一步通过慢病毒干扰DNMT3a和TET2表达，阐明p53/DNMT3a或TET2/Foxp3 DNA去甲基化在Foxp3表达、诱导Treg增殖分化，进而导致Th2漂移的关键机制。同时结合临床试验澄清Foxp3 DNA去甲基化在脓毒症免疫失调中的重要地位及病理生理意义。该研究将为深刻揭示脓毒症免疫抑制的分子调控机制开拓新思路，并为其免疫调理治疗提供新方向。

免疫抑制是脓毒症时的重要特征之一，与不良预后紧密相关。脓毒症时，调节T细胞通过抑制效应性T细胞增殖和分化发挥免疫抑制作用。.本项目旨在前期研究基础上深入探讨p53在脓毒症时调控Treg增殖的作用及其机制。采用野生型和CD4特异性p53敲除（p53f/f/CD4-Cre+）小鼠，通过盲肠结扎穿孔术制作脓毒症模型。利用流式细胞仪分析小鼠脾脏CD4+CD25+ Foxp3+ Treg比例的差异；利用qPCR和Western blot法检测Foxp3、DNMT3a和TET2的表达水平；利用BSP法分析TSDR区甲基化水平；利用双荧光酶素法验证p53和DNMT3a或TET2基因的结合作用；在阐明机制的基础上，通过ELISA法检测细胞因子的水平，分析Treg的免疫抑制功能；最后观察两组小鼠在CLP术后的生存率。.实验结果发现，野生型小鼠在CLP术后CD4+CD25+ Foxp3+ Treg比例显著升高，与之相较p53f/f/CD4-Cre+小鼠脾脏CD4+CD25+ Foxp3+ Treg比例较低；CLP术后，野生型小鼠脾脏CD4+ T细胞中Foxp3表达增加，伴随着DNMT3a表达减少和TET2的表达增多，p53f/f/CD4-Cre+小鼠CD4+ T细胞中上述蛋白表达变化较之不明显；CLP术后，野生型小鼠CD4+CD25- T细胞中Foxp3 TSDR区的去甲基化水平有较大程度增加，相较之，p53f/f/CD4-Cre+小鼠CD4+CD25- T细胞Foxp3 TSDR区的去甲基化水平维持在较低水平；经过双荧光酶素法检验，p53与DNMT3a和TET2的启动子区域存在结合作用，并抑制DNMT3a的转录，促进TET2转录； CLP术后的野生型小鼠体内Treg分泌的抑制因子TGF-β和IL-10水平明显高于p53f/f/CD4-Cre+小鼠Treg；将CLP术后小鼠体内分离的Treg和野生型Teff共培养后发现，野生型Treg对Teff的抑制作用更加明显，表现为IL-2分泌减少，向Th2漂移明显和增殖抑制；最终，CLP术后p53f/f/CD4-Cre+小鼠的生存率显著高于野生型小鼠。.本项目研究结果证实，脓毒症后p53通过直接的转录激活和转录抑制作用分别调控DNMT3a表达减少和TET2表达增加，进而影响Foxp3 TSDR区的去甲基化水平，促进Foxp3的稳定表达，进而诱导T