PD-1调控有氧糖酵解抑制脓毒症早期中性粒细胞NET形成的作用机制研究

The formation of neutrophils extracellular trap(NET) plays an important role in controlling infection in early sepsis. It is well known that enhanced aerobic glycolysis is needed for the formation of NET, while our research found that aerobic glycolysis in neutrophil was inhibited and related to hampered NET formation in early sepsis. We have previously revealed that in the early stage of sepsis, PD-1 was involved in the immunosuppression of monocytes and lymphocyte. Furthermore, we found that the expression of PD-1 on neutrophil was increased in early sepsis, and an increase in aerobic glycolysis and NET formation was seen when PD-1 was blocked. To elucidate the specific role and mechanism of PD-1 in the regulation of neutrophil aerobic glycolysis, and its role in the formation of NET in early sepsis, we will conduct the research project to ① clarify the expression of PD-1 on neutrophil and its relationship with aerobic glycolysis level and NET formation; ② elucidate the role of PD-1 on aerobic glycolysis and NET formation using myeloid-specific PD-1 knockout mice and sPD-L1 (to activate PD-1); ③ clarify the molecular mechanism that PD-1 inhibit neutrophil aerobic glycolysis via mTOR at the transcriptional, translational and metabolic levels. The project will ultimately refine the mechanism of immunosuppression in early sepsis, and provide a potential target for the clinical prevention and treatment for sepsis.

中性粒细胞诱捕网（NET）的形成在控制脓毒症早期感染中发挥重要作用。已知有氧糖酵解水平正向调控NET形成，本课题组发现脓毒症早期中性粒细胞（PMN）存在有氧糖酵解受抑导致NET形成减少。我们既往证实程序性细胞死亡因子-1（PD-1）参与脓毒症早期免疫功能受损，并发现PMN的PD-1表达升高，阻断PD-1后有氧糖酵解水平增加，NET形成增多。我们推测脓毒症早期PMN的PD-1表达升高通过抑制有氧糖酵解调控NET形成，本课题拟①明确脓毒症早期患者PMN的PD-1表达及其与有氧糖酵解水平、NET形成的相关性；②利用髓系特异性PD-1敲除小鼠及sPD-L1过度激活PD-1，从正反论证PD-1抑制PMN有氧糖酵解及该作用对NET形成的影响；③在转录、翻译及代谢层面明确脓毒症早期PD-1经mTOR调控下游转录因子进而抑制PMN有氧糖酵解的分子机制。力图完善脓毒症免疫抑制的发生机制，为临床防治提供靶点。

有效去除病原菌是改善脓毒症预后的关键。中性粒细胞是固有免疫的最重要组成部分，在清除病原菌中起着至关重要的作用。然而，脓毒症中性粒细胞的代谢特征和免疫调节机制尚不清楚。本项目纳入脓毒症患者、非脓毒症感染患者和健康志愿者，分离中性粒细胞进行代谢组学分析，结果表明脓毒症患者的 中性粒细胞糖酵解水平发生显著改变。对从健康志愿者和脓毒症患者中分离的中性粒细胞进行转录组学分析，提示脓毒症中性粒细胞糖酵解和 mTOR/HIF-1α 信号通路发生明显变化。本课题组进一步通过体外细胞实验，明确脓毒症中性粒细胞存在趋化及吞噬功能抑制，并且糖酵解水平调控其趋化及吞噬功能；进一步发现PI3K/Akt-HIF-1α 通路参与调控 LDHA 表达水平，进而影响中性粒细胞的趋化和吞噬功能。本研究为脓毒症患者的诊治提供了新的理论依据，并在此基础上探索治疗靶点，为脓毒症的临床防治提供新的理念。