MPO-ANCA相关性血管炎肾损害的新机制：中性粒细胞GSDMD膜孔结构的核心作用

中性粒细胞是抗中性粒细胞胞浆抗体(ANCA)相关小血管炎(AAV)的发病核心，MPO-ANCA激活中性粒细胞NETosis、释放组织因子和中性粒细胞胞外诱捕网(TF/NETs)、进而损伤血管内皮和激活凝血系统是MPO-AAV肾小球损害的重要环节。近年发现GSDMD被切割后、产生的N端剪切体可在中性粒细胞膜上打孔，形成GSDMD膜孔、激活释放TF/NETs，但中性粒细胞GSDMD的切割激活机制与“焦亡”并不完全相同。“GSDMD膜孔”是否为MPO-ANCA激活NETosis的核心结构及调控机制尚不清楚。本研究拟通过MPO-AAV患者和小鼠模型，采用基因敲除GSDMD、点突变酶切位点、条件性敲入膜孔修复基因，串联免疫共沉淀与质谱分析蛋白相互作用，探明MPO-AAV微环境精准切割GSDMD的蛋白酶及切割位点，揭示GSDMD膜孔驱动的中性粒细胞NETosis在MPO-AAV肾小球损害中的分子机制。

Neutrophils play a crucial role in the pathogenesis of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). NETosis is a neutrophil cell death pathway accompanied with release of chromatin decorated with specific proteins including tissue factor (TF) and neutrophil extracellular traps (NETs). Myeloperoxidase (MPO)-ANCA mediates activation of neutrophil NETosis to release TF/NETs, which in turn damages the endothelium and activates the coagulation system leading to glomerular damage. Gasdermin D (GSDMD), a pore-forming protein and an executor of pyroptosis, also plays a vital role in the generation of TF/NETs. In recent years, it has been found that after GSDMD is cleaved, the resulting N-terminal GSDMD fragments translocate to the membrane and form membrane pores (GSDMD pores) to induce TF/NETs activation and release. However, the mechanism by which GSDMD is being cleaved and activated within neutrophils may not be exactly same as that of "pyroptosis". In fact, whether “GSDMD membrane pores” is the core structure for MPO-ANCA-induced neutrophil NETosis and its underlying regulatory mechanisms remain largely unknown. We will purify neutrophils from MPO-AAV patients, establish a mouse model of MPO-AAV-associated glomerular damage, take approaches of genetic engineering techniques including gene knockout of GSDMD, point mutation of GSDMD cleavage site, and conditional gene knockin of CHMP4 for membrane repair and GSDMD in neutrophils, as well as employ immunoprecipitation and mass spectrometry to identify the protein-protein interaction systems. In short, the overall goals of this project will be (1) to investigate the core platform of GSDMD pores in MPO-AAV-associated glomerular damage; (2) to identify the proteases responsible for cleaving GSDMD and specific cleavage sites on GSDMD to unveil the molecular mechanisms of MPO-ANCA-induced neutrophil NETosis.