NETosis相关的组蛋白瓜氨酸化修饰在ANCA相关性血管炎中参与血管损伤的机制研究

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a group of autoimmune diseases characterized by fibrinoid necrosis of the vessel walls. We have found that enhanced neutrophil extracellular trap (NET) formation, which results the release of nuclear histones to the extracellular space, was observed in kidney biopsies and neutrophils from AAV patients. More recently, it was reported that extracellular histones exhibit toxicity on glomerular endothelia cells and cause vascular necrosis. Considering NET formation dependents on PAD4-mediated histone citrullination, we assume that aberrant citrullination contributes to the toxicity of extracellular histones. Recent data demonstrated that histone deacetylases (DHACs) associated withPAD4, and the corresponding activities, led to histone citrullination. This suggests that DHACs may serve as potential regulator and therapeutic target in AAV. Therefore, in this study, our aims are to analysis the toxicity of citrullinated extracellular histones related to NETosis in AAV using ex vivo, in vitro and in vivo experiment. Then we will evaluate the role of DHACs in NETosis-related histone citrullination using methods such as chromatin immunoprecipitation assay and RNA interference. At last, we will test the protection of PAD4 and HDACs inhibitors in “NETs-mDC” AAV mice. The results will not only reveal the mechanism of AAV-associated vascular injury, but also provide new targets for AAV treatment.

血管纤维素样坏死是中性粒细胞胞浆抗体相关性血管炎(AAV)的典型病理特征。基于课题组前期发现AAV患者肾组织及外周血中性粒细胞胞外捕网(NETs)形成增多、释放组蛋白的临床证据，及组蛋白损伤肾小球内皮细胞(GEC)的最新发现，结合NETs形成依赖肽酰基精氨酸脱亚胺酶4(PAD4)介导的组蛋白瓜氨酸的表观遗传学现象，推测：组蛋白去乙酰化酶(HDACs)协同PAD4介导的NETs相关组蛋白瓜氨酸化是GEC损伤的重要机制。拟通过分析AAV患者组蛋白瓜氨酸化水平、中性粒细胞-GEC共培养体系及瓜氨酸化组蛋白小鼠单侧肾动脉注射，了解组蛋白瓜氨酸化状态及效应；通过免疫共沉淀及shRNA干扰，明确HDACs是否协同PAD4参与组蛋白瓜氨酸化修饰；进一步通过“NETs-mDC”建立AAV小鼠模型，PAD4失活剂氯脒及经典HDACs抑制剂曲古抑菌素A干预，探讨组蛋白瓜氨酸化机制，以寻求AAV潜在的治疗靶点。

抗中性粒细胞胞浆抗体相关性血管炎（ANCA-associated vasculitis，AAV）是以中性粒细胞异常死亡，导致血管发生坏死性炎症的自身免疫疾病。中性粒细胞是AAV最主要的效应细胞及发病的核心。中性粒细胞胞外捕网 (neutrophil extracellular traps, NETs)是新近发现的，中性粒细胞除吞噬功能以外的另一种杀菌方式，直接与炎症及自身免疫相关。NETs形成伴随着PAD4介导的组蛋白瓜氨酸化修饰，据此我们推测，AAV中NETs相关的组蛋白瓜氨酸化是胞外组蛋白产生血管内损伤的重要原因，通过对组蛋白瓜氨酸化过程的干预和调控，有望为AAV的治疗提供新的策略。本项目采用激光共聚焦、免疫荧光、流式细胞检测等方法，初步明确组蛋白瓜氨酸化修饰参与AAV中NETs形成及血管内皮细胞损伤。主要结果：1.AAV患者外周血及肾组织有大量NETs形成并暴露ANCA靶抗原LAMP-2、MPO、PR3；2.AAV 患者肾组织组蛋白瓜氨酸化水平较健康对照组明显增高；3.NETs-肾小球血管内皮细胞(glomerular endothelial cells，GEnCs)共培养，导致GEnCs凋亡增多, PAD4失活剂Cl-amidine及HDACs抑制剂Trichostatin A可减轻NETs导致的GEnCs凋亡。4.成功采用琼脂糖明胶皮下埋植法富集、分离具有自发NETs倾向的小鼠中性粒细胞，可为中性粒细胞及NETs的进一步研究提供数量充足稳定的细胞来源。结论：本课题初步阐明了组蛋白瓜氨酸化参与AAV中血管损伤的机制，为进一步探讨AAV治疗新靶点提供理论依据。