CD4+ Treg细胞的PD-1表达调控肠缺血再灌后肠免疫屏障损伤的机制研究

Ischemia/reperfusion (I/R) injury of the intestine is associated with high morbidity and mortality in surgical and traumatic patients. Intestinal I/R injury leads to the destruction of intestinal mucosal barrier and subsequently multiple organ dysfunction syndrome. However, the mechanism of intestinal I/R-induced barrier injury is poorly understood. It has been demonstrated that CD4+ regulatory T cell (Treg) plays an important role in maintaining immune and inflammatory responses by regulating transforming growth factor (TGF)-β and interleukin (IL)-10 production. Our previous study demonstrated that, in peyer’s patches, the expression of programmed cell death-1 (PD-1) on CD4+ T cells significantly decreased following intestinal I/R. And the decreased PD-1 expression contributes to the death and dysfunction of Treg cell. The results of our preliminary experiment showed that, following intestinal I/R, activation of PD-1 promoted the number of CD4+ Treg cell and increased the production of TGF-β1 and IL-10, and then improved the intestinal immunoglobulin A synthesis. Therefore, we hypothesized that down-regulation of PD-1 may hinder the secretion of TGF-β1 and IL-10 from CD4+ Treg, lead to the death of lymphocytes and the disrupted mucosal immune responses in the intestine, and subsequently impair gut immune barrier after intestinal I/R injury. In this study, through using the techniques of Treg induction and gene knockout, we plan to investigate the effect of decreased PD-1 expression of CD4+ Treg cells on gut mucosal immunity after intestinal I/R, and elucidate the molecular mechanism of I/R-induced gut barrier injury. The data of this study will demonstrate the pathogenesis of intestinal I/R injury and provide new therapeutic targets for protecting gut mucosal immune barrier.

肠缺血再灌注(I/R)常导致危重患者的死亡。肠道免疫屏障破坏是肠I/R损伤的核心环节，但其机制尚不清楚。CD4+调节性T细胞（Treg）是调节免疫和炎症反应的关键细胞。既往我们发现肠缺血后肠内CD4+ T细胞的程序性死亡受体1（PD-1）表达降低，PD-1受抑被证实干扰了Treg细胞的功能和生成。我们的预实验首次发现肠I/R后激活PD-1可增加CD4+ Treg细胞数量并促进白介素（IL）-10和转化生长因子（TGF）-β1的生成，改善免疫球蛋白A的分泌。因此，我们假设肠I/R后PD-1的下调引起CD4+ Treg细胞生成IL-10和TGF-β1减少，造成肠道粘膜免疫反应障碍和淋巴细胞死亡，进而破坏了肠道免疫屏障。本项目拟使用Treg细胞诱导和基因干预等技术，探索肠I/R后CD4+ Treg细胞PD-1下调对肠道免疫屏障的影响及其分子机制，以期深入阐明肠I/R损伤的机制并寻找新的防治靶点。

背景： CD4+调节性T细胞（Treg）是调节免疫和炎症反应的关键细胞。既往发现肠缺血后肠内CD4+T细胞的程序性死亡受体1（PD-1）表达降低，PD-1受抑被证实干扰了Treg细胞的功能和生成。我们的预实验首次发现肠I/R后激活PD-1可增加CD4+Treg细胞数量并促进白介素（IL）-10的生成，改善免疫球蛋白A的分泌。因此，我们假设肠I/R后PD-1的下调引起CD4+Treg细胞功能丧失，IL-10减少造成肠道粘膜免疫反应障碍和淋巴细胞死亡，破坏了肠道免疫屏障。研究内容： 1. 构建小鼠肠缺血（IR）模型，实验小鼠分为6组，分别为对照组（假手术组），肠I/R组，PD-L1 Ig 组 (PD-L1)； 对照 IgG 组 ；PD-L1 Ig + Anti-IL-10 组；PD-L1 Ig+ MiR-155 agomir 组。检测指标：肠道PP结中CD4+细胞 PD-1 expression, IL-10 mRNA, IL-10表达；肠粘膜组织中的 miR-155 水平, (TNF)-α，IL-1β 浓度, 和AID的表达；肠道灌洗液中IgA 的细菌结合能力。2.使用野生型雄性C57/BL6小鼠和PD-1基因敲除小鼠构建肠IR模型，每组中均分别给予PD1阻断及IL-10中和，检测指标： 肠内IgA细菌结合能力、肠内菌群高通量、肠内细菌移位免疫荧光、远隔器官病理、生存分析。.重要结果：激活PD-1改善肠IR后IgA和IgM的抗菌能力，减轻肠IR后肠PP内炎症反应；激活PD-1通过CD4+T细胞/IL-10/miR-155信号轴调控肠IR后AID表达；PD-1阻断治疗造成肠道菌群紊乱、加重肠IR后肠、肝、肾损伤；PD-1基因敲除小鼠IL-10+Treg和Tfh较野生型小鼠增加。PD-1抗体处理组和PD-1基因敲除组小鼠肠PP和脾脏Treg细胞IL-10 mRNA表达均较对照组明显增加，PP和血清内IL-10含量亦升高。.结论及科学意义：肠道 CD4+T 细胞PD-1 激活可通过 IL-10/miR-155途径调控肠IR后肠道粘膜 IgA 生成和炎症反应，从而减少肠道免疫防御功能破坏。阻断PD-1信号通路可造成肠道菌群失调，并且加重肠IR后多器官损伤。

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