TFEC对脓毒症中性粒细胞抑制性功能的调控作用和机制研究

Immunosupression is the main cause of high mortality of sepsis. Our previous study showed that neutrophils were inhibitory during sepsis. They could induce T lymphocyte apoptosis through PD-L1 expression. A recent study suggested that there was a subset of activated neutrophils which expressed higher PD-L1. The microarray analysis showed that the expression of TFEC are significantly upregulated on this subset. TFEC is a transcription factor, but its effect and activation mechanism have been seldom investigated. Our preliminary study showed that TFEC was upregulated in neutrophils during sepsis. Knockdown of TFEC by siRNA inhibited the expression of PD-L1. Therefore, we speculated that TFEC might be involved in the regulation of PD-L1 in neutrophils and mediate the inhibitory effect of neutrophils during sepsis. The present study will investigate the correlation of TFEC on neutrophil with the immunosuppression status and severity of sepsis. The role of TFEC on the inhibitory effect of neutrophils will be investigated in vitro. The mechanisms of the expression and activation of TFEC will be studied at the transcriptional and post-transcriptional levels. Finally, the in vivo effect of TFEC on sepsis will be confirmed by using the TFEC knockout mice. This projection may provide a new target for improving immunosupression of sepsis.

免疫抑制是脓毒症死亡率高的重要原因。我们前期研究发现，脓毒症时中性粒细胞表现出抑制性功能，可通过PD-L1诱导淋巴细胞凋亡。文献显示，活化的中性粒细胞中存在一个高表达PD-L1的亚群，基因芯片筛选发现该亚群中TFEC表达显著升高。TFEC是一种转录因子，但研究甚少，作用与活化机制不清。预实验显示脓毒症患者中性粒细胞TFEC表达升高，干扰TFEC后PD-L1表达下调，据此推测，TFEC可能参与了中性粒细胞PD-L1表达的调控，从而介导脓毒症时中性粒细胞的抑制性功能。因此，本课题拟探讨脓毒症患者中性粒细胞TFEC表达水平与患者免疫抑制和病情的相关性；离体研究TFEC对中性粒细胞抑制性功能的影响；从转录和转录后修饰两个水平探讨TFEC表达和活化的调控机制；最后通过利用基因敲除小鼠研究TFEC对脓毒症发生发展的在体影响，从而为改善脓毒症免疫抑制提供新靶点。

脓毒症因严重全身炎症反应导致机体处于一种免疫失衡的状态。中性粒细胞是机体免疫的重要组成之一，脓毒症时中性粒细胞凋亡延迟。本项目主要旨在脓毒症时TFEC对中性粒细胞功能抑制的调控进行相关机制方面的研究。首先从临床样本上证实脓毒症时中性粒细胞高表达TFEC，且在类中性粒细胞上利用TFEC siRNA干扰TFEC后PD-L1表达上调。进一步研究发现在转录水平，干扰TFEC使PD-L1的转录因子c-Jun表达升高；在转录后的修饰水平，由于ERK1/2和PI3K是PD-L1表达相关的两个关键激酶，利用ERK1/2抑制剂和PI3K抑制剂后，中性粒细胞PD-L1表达升高，且抑制TFEC的表达，提示TFEC通过ERK1/2和PI3K磷酸化来调控PD-L1的表达。PI3K的磷酸化水平不仅影响TFEC的表达而且也与细胞凋亡相关，我们前期研究发现，中性粒细胞上PD-L1的表达也与细胞凋亡相关。由此我们研究了TFEC在中性粒细胞凋亡中的作用，发现干扰TFEC后中性粒细胞凋亡延迟。进一步观察发现，TFEC干扰后可使凋亡蛋白Bad磷酸化增强，Bad转化为更多的p-bad后, Bad 不能与抗凋亡蛋白Bcl-xl结合，从而抑制凋亡。本项目有望将来利用TFEC为改善脓毒症免疫失衡状态作为新的靶点提供理论依据。