Sox2作为细菌DNA传感器介导中性粒细胞自噬在ANCA相关性血管炎中的作用及机制研究

Neutrophil extracellular traps (NETs) formation induced by bacterial infection contributes to the key role in pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Our previous data had shown that autophagy is involved in ANCA-induced NET formation in human neutrophils. New evidence indicates that transcription factor Sox2 as a cytosolic sequence-specific DNA sensor in neutrophils directly recognized microbial DNA through its high-mobility-group domain. Sox2 binds to a repressive region on the mTOR promoter and recruits the NuRD complex to mediate transcriptional repression. The NuRD complex epigenetically regulates the mTOR promoter temporal regulation of autophagy as a key step in cellular reprogramming processes. Based previous data that apoptosis might function as a backup program for NETosis when autophagy is prevented, we therefore hypothesize that the interplay between autophagy, dependent pathway promotes the collapse of both nuclear and granular membranes and mediates intracellular chromatin decondensation, while inhibiting the NETs machinery hopefully elucidate in detail the interplay between these processes and the essential enzymatic activities involved. Accordingly, the overall aim of this research project will be to determine that if bacterial DNA triggers Sox2-depended nucleosome remodeling and deacetylase (NuRD) complex-induced mTOR mediate autopahgy play key role in NETs formation. In order to fully study this aim, the abnormal death mode of neutrophils will be investigated based our preliminary experiment results in AAV pateients. Using immunized WKY rats model with purified FimH fusion protein emulsified in Titermax Gold, further combined specific inhibitor for histone deacetylase (HDAC1) and autophagy, or inhibition of mammalian target of rapamycin (mTOR) induce autophagy in vivo. The proteins of Sox2, NuRD complex (Mi-2b, LSD1, MTA2 and HDAC1) and mTOR will be tested by liquid chip, immunoblotting and Chromatin Immunoprecipitation (ChIP) assay. The DNA methylation and histone modification will be investigated by commercial kits. Findings from this study may eventually lead to the development of safe and rational therapy to ameliorate vasculitis and abrogate the autoimmunity in individuals suffering from AAV.

感染等诱发中性粒细胞胞外捕网(NETs)形成释放自身抗原，是中性粒细胞胞浆抗体相关性血管炎(AAV)的关键。根据自噬可调节凋亡、NETs转换的特性，及转录因子Sox作为细菌DNA传感器致mTOR表观遗传学修饰引发自噬的最新进展，结合AAV患者NETs增加及NETs形成依赖自噬的前期发现，推测感染时Sox2通过识别细菌DNA，募集NuRD复合物，抑制mTOR启动子活性，增强自噬诱导NETs形成。拟进一步通过临床分析，联合前期FimH融合蛋白成功诱导大鼠血管炎模型的基础上，通过含全基因组DNA大肠杆菌再感染，联合组蛋白去乙酰化酶（HDAC1）抑制剂及自噬阻断剂，通过液相芯片、免疫印迹、ChIP、DNA甲基化、组蛋白修饰等方法，认识Sox2-NuRD-mTOR-自噬途径对NETs的作用及调控机制，以期从表观遗传学角度，一定程度实现中性粒细胞程序性死亡的“重编程”，诱导免疫耐受，寻求治疗新靶点。

抗中性粒细胞胞浆抗体相关性血管炎(AAV)是以中性粒细胞异常死亡，导致血管发生坏死性炎症的一组自身免疫疾病。中性粒细胞胞外捕网(NETs)形成释放自身抗原是AAV的关键。课题组发现AAV患者外周血中性粒细胞NETs形成增加，并释放MPO、PR3、LAMP-2、LL37、DNA及组蛋白等自身抗原组分，阻断自噬可减少NETs形成及凋亡增加，进一步发现组蛋白去乙酰化酶(HDACs)协同PAD4介导的NETs相关组蛋白瓜氨酸化是肾小球内皮细胞损伤的重要机制。NETs-肾小球血管内皮细胞(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。Sox2作为转录因子，在AAV外周血特异性在中性粒细胞中表达，在其PBMC中不表达。意外发现Sox2在肾组织中与肾小球壁层上皮细胞标记物Claudin 1共表达，并与新月体形成相关。采用Luminex液相芯片技术检测发现在AAV患者及健康志愿者CXCL16、CTACK、MIP-1δ及YKL40的差异性表达，通过Kaplan-Meier生存分析显示，这些差异性表达的趋化因子有望成为判断AAV预后的新型血清学标志物。随后体外实验发现AAV血清可增加ciGEnC CXCL16表达并促进中性粒细胞的迁移。同时在临床部分实施过程中，根据标准进行纳入和排除患者时，探寻了基于伯明翰评分（BVAS）的评价系统是临床评估ANCA致病性和非致病性的简单、有效、实用的方法，并进一步筛选了D-2聚体、TNF-及α1球蛋白是判断AAV活动性新的炎症标志物及判断预后的独立危险因素。在探索动物模型的优化及中性粒细胞富集手段中，采用小鼠皮下琼脂糖明胶埋植法，成功富集、分离具有自发 NETs 倾向的小鼠中性粒细胞。不同表面积的明胶能够分离出106-107数量级细胞，具有中性粒细胞典型的分叶核形态学特征，流式细胞分析鉴定为Gr-1+CD11b+的中性粒细胞，纯度可达95.7%，为NETs的进一步研究提供了充足数量细胞来源，并为模式动物的建立奠定了基础。