FHR1/FHR5参与抗中性粒细胞胞浆抗体相关小血管炎发病的机制研究

Anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) is a group of potentially life-threatening autoimmune diseases，with kidneys frequently involved. The pathogenesis of AAV has not been fully elucidated. ANCA-induced neutrophil activation, complement activation via the alternative pathway, and activation of coagulation system are crucial for the development of AAV. Complement factor H (FH) is a key regulator of the alternative complement pathway. Moreover, we recently found that in addition to serving as a complement regulator, FH inhibits ANCA-induced neutrophil activation and protects against glomerular endothelial injury by interacting with neutrophils. It indicated an important role of FH in AAV. However, its functional activity in regulating alternative complement activation and inhibiting ANCA-induced neutrophil activation were impaired in AAV patients, and associated with the disease activity. The mechanism of impaired functional activity of FH in AAV has not been fully understood. Factor H related proteins (FHRs) share high sequence similarity to FH. Interestingly, It was revealed by recent findings that FHR1 and FHR5 function as competitive antagonists of FH for the binding to C3b thus inhibiting the complement regulatory activity of FH, termed as FH deregulation. Increasing evidence has implicated an important role of FHR1 and FHR5 in autoimmune diseases. Moreover, recent discoveries and our preliminary results suggest the involvement of FHR1/FHR5 in the pathogenesis of AAV. Therefore, we hypothesized that FHR1/FHR5 may contribute to the development of AAV by antagonize the functional activity of FH. Thus, in our current project, we plan to detect the plasma levels of FHR1/FHR5 in AAV and further analyze the association between plasma FHR1/FHR5 levels and disease activity. The effect of FHR1/FHR5 on the regulatory activity of FH, including its regulation on ANCA mediated neutrophil activation and glomerular endothelial injury, as well as platelet activation will be explored through in vivo assays. The effect of FHR1/FHR5 on complement activation through the activation of neutrophils and platelet will be further investigated. Furthermore, the role of FHR1/FHR5 in disease development will be explored using the hMPO induced necrotizing crescentic glomerulonephritis rat model. The effect of recombinant FHR11-2FH1-5 ^18-20 in treating experimental rat AAV will be further explored. The data will further extend the understanding of the pathogenesis of AAV, and provide potential clues for intervention strategies.

抗中性粒细胞胞浆抗体(ANCA)相关小血管炎(AAV)是临床常见急危重症，ANCA、中性粒细胞、补体及凝血系统是其发病机制的核心。申请者前期发现H因子蛋白家族中H因子(FH)在AAV中具有重要病生理作用，AAV患者FH功能异常，但具体机制不清。近期研究发现该家族中H因子相关蛋白(FHRs)包括FHR1及FHR5与FH功能密切相关，且可能在AAV中发挥重要致病作用。因此我们提出假说：AAV中存在FHR1、FHR5异常，通过竞争抑制FH对补体、中性粒细胞及血小板活化的调控参与AAV发病。本研究拟：分析AAV患者血浆FHR1、FHR5水平与疾病活动及预后的相关性；体外研究FHR1、FHR5对FH调控AAV中中性粒细胞活化、内皮细胞损伤和血小板活化的影响，验证FHR1、FHR5是否通过促进中性粒细胞和血小板活化进一步促进补体活化；并通过动物实验进一步验证，以期深入揭示AAV发病机制及探索治疗靶点。

抗中性粒细胞胞浆抗体(anti-neutrophil cytoplasmic autoantibody, ANCA) 相关小血管炎(ANCA-associated vasculitis, AAV) 是临床常见的急危重症，其发病机制尚不十分清楚。我们前期研究发现补体H因子（FH）参与AAV中补体旁路途径及中性粒细胞活化调控。H因子相关蛋白(factor H related proteins, FHRs)，与FH具有相当的同源性，国外的研究证据及申请者的前期结果均提示FHR1和FHR5可能与AAV发病密切相关，但具体作用及机制尚不明确。本研究通过检测FHR1和FHR5水平及分析其与AAV疾病活动及肾脏损伤的相关性，发现活动期AAV患者血浆中FHR1及FHR5水平均显著高于缓解期患者及健康对照。并且FHR1与患者eGFR呈显著负相关，与肾脏纤维素样坏死比列呈显著正相关，与血沉、CRP水平呈显著正相关，提示FHR1与AAV患者肾脏损伤程度、疾病活动相关。FHR5与患者eGFR呈显著负相关，与血肌酐、肾脏细胞新月体比例、肾脏纤维素样坏死比列呈显著正相关，与补体活化产物C3a呈显著正相关。提示FHR5水平与AAV患者疾病活动水平及肾脏损伤程度相关。基于FHR1与FH序列高度同源且其与患者预后更相关，我们通过细胞实验进一步探讨了FHR1参与AAV发病的机制。我们发现FHR1能明显促进TNF-α预刺激的ANCA介导的中性粒细胞呼吸爆发和脱颗粒水平，并且FHR1能明显竞争抑制FH对中性粒细胞活化的保护作用。利用中性粒细胞和内皮细胞的共培养体系，我们发现FHR1能促进中性粒细胞向内皮细胞黏附，并促进中性粒细胞在内皮细胞表面被ANCA活化及其介导的内皮细胞损伤，同时“去调控”FH对中性粒细胞活化及内皮损伤的保护作用。进一步我们重组表达了由FH功能域及FHR1聚合区组成的融合蛋白FH 1-5 ^18-20 FHR1 1-2，并在体外验证了其具有抑制补体旁路途径活化的功能，提示该融合蛋白可能在AAV中具有一定的治疗前景。