GSDMD介导的中性粒细胞死亡对宿主抗菌功能的影响

Neutrophils play a critical role in host defense against invading microbial pathogens. They are terminally differentiated cells and normally have a very short life-span. Neutrophils readily undergo programmed cell death and this death program needs to be well controlled to maintain the normal neutrophil count and to provide a balance between their immune functions and their safe clearance. The ultimate goal of the proposed research is to identify and characterize biochemical events that mediate neutrophil death during infection and inflammation. Several recent studies showed that cleavage of Gasdermin D (GSDMD) by caspase-1 and caspase-11 is essential for pryoptosis and cell survival of macrophages. Generally pyroptosis of macrophage leads to release of intracellular bacteria, which is beneficial to host defense against bacteria. However, we found that the GSDMD knockout mice displayed stronger bacteria killing capability compared to WT mice. .Our preliminary data found that GSDMD did not affect neutrophil bactericidal ability, but changed the death process of neutrophil. So we inferred that the neutrophil death mediated by GSDMD could affect the host defense against bacteria. To verify this hypothesis, we have constructed Gsdmd-/- model mice. Firstly we will examine the host defense against Escherichia coli (E.coli) and Staphylococcus aureus (S.aureus) of Gsdmd-/- mice. Then we will analyze the phagocytosis、killing、ROS release、chemotaxis and death process of neutrophil from Gsdmd-/- mice. Finally we will further explore the expression and activation of GSDMD in neutrophils. In conclusion, this study expect to find the effect of neutrophil death process mediated by GSDMD on host defense. And we hope the research could provide idea for treatment of disease with neutropenia and infection.

中性粒细胞在机体防御微生物病原体感染中起到关键作用。细胞焦亡是一种重要的程序性细胞死亡方式，研究发现Gasdermin D（GSDMD）的剪切为细胞焦亡过程所必须，影响细胞生存。一般认为巨噬细胞焦亡释放细胞内细菌有利于宿主对微生物的抵抗。但是我们发现GSDMD敲除小鼠对大肠杆菌的清除能力反而增强，且GSDMD并不影响中性粒细胞的杀菌功能，而是改变了中性粒细胞的死亡状态。我们推测，GSDMD介导的中性粒细胞死亡影响宿主对细菌的抵御能力。因此，本研究将基于GSDMD敲除小鼠模型，分析GSDMD对小鼠抵御大肠杆菌和金黄色葡萄球菌能力的影响，探究GSDMD对中性粒细胞功能以及死亡进程的改变；本文还将进一步分析GSDMD在中性粒细胞中表达和活化的调节机制。本研究拟揭示GSDMD介导的中性粒细胞死亡进程改变对宿主防御能力的影响，以期为临床上因中性粒细胞减少及功能紊乱导致感染发生的疾病治疗提供新的思路。

中性粒细胞是血液中最多的一种白细胞，在机体防御微生物病原体感染中起到关键作用。细胞焦亡是一种重要的程序性细胞死亡方式，研究发现Gasdermin D（GSDMD）的剪切为细胞焦亡过程所必须，影响细胞生存。一般认为巨噬细胞焦亡释放细胞内细菌有利于宿主对微生物的抵抗。但是我们的研究发现小鼠敲除GSDMD后粒细胞死亡延迟，进而增强小鼠对胞外菌大肠杆菌（Escherichia coli，E.coli）的清除能力。我们的研究首次证实GSDMD在机体抵御感染中也会充当负调节因子。.我们首次检测了GSDMD基因敲除对中性粒细胞功能的影响，发现GSDMD并不影响粒细胞对细菌的吞噬和杀伤功能。随后我们发现GSDMD敲除能够延迟中性粒细胞自发性死亡。进一步，我们发现中性粒细胞中弹性蛋白酶（NE，Neutrophil Elastase）能够独立剪切GSDMD，产生能够诱导粒细胞死亡的活性片段N-GSDMD，在粒细胞衰老过程中NE由胞质颗粒释放到胞浆。另外我们发现NE剪切GSDMD的位点位于caspase-1剪切GSDMD位点的上游。最后，我们构建了小鼠腹腔炎症模型，即小鼠腹腔注射大肠杆菌。如前所述，敲除GSDMD小鼠清除E.coli的能力增强，同时我们检测到感染后敲除小鼠腹腔粒细胞数目显著多余正常小鼠，且粒细胞死亡明显降低。该部分研究已经于2018年发表于cell reports杂志。.随后，我们的进一步研究发现，临床患者粪便来源的E.coli中，有些菌株可以迅速诱导粒细胞死亡。通过生物信息学技术比较分析细菌α-hemolysin可能是诱导粒细胞死亡的致病因子，随后的敲除实验证实细菌α-hemolysin确实为诱导粒细胞死亡的致病因子。我们还发现，含有α-hemolysin的E.coli通过激活粒细胞GSDMD，诱导GSDMD剪切引起粒细胞死亡，即GSDMD同样参与α-hemolysin诱导的粒细胞死亡。该部分研究还在进行中。.综合以上，本研究揭示了GSDMD介导的中性粒细胞死亡进程改变对宿主抵御大肠杆菌感染的影响，本研究将为临床上因中性粒细胞减少及功能紊乱导致感染发生的疾病治疗提供新的思路和潜在靶点。