瓜氨酸蛋白促发类风湿性关节炎肺间质病变致病机理研究

Interstitial Lung Disease (ILD) is a leading cause of morbidity and mortality in rheumatoid arthritis(RA). Because subclinical forms of RA-ILD likely represent precursors of more severe parenchymal lung disease, establishment of readily obtainable, non-invasive biomarkers that can identify ILD at an earlier and more treatable stage is critical. Previous immunohistochemical studies that have demonstrated citrullinated proteins in the lung parenchyma of individuals with RA-ILD. The parenchymal lung abnormalities and enrichment of anti-citrullinated protein antibody( ACPAs) in the lungs (bronchoalveolar lavage fluid, BALF) occur in early RA, suggesting that the lung might be a site of initiation of immunity to citrullinated proteins and that early targeting of the immune reactions in the lung might be a new approach to modulate disease. Another study that anti-citrullinated HSP90(citHSP90) profiles between BALF and serum indicate that the lung plays a direct role in shaping the immune repertoire of RA-ILD. ..Hypothesis: Citrullinated antigens including citHSP90 formed in response to smoking/injurious stimuli and targeted by the humoral immune response and cell immune response that collectively contribute to the pathogenesis of RA-ILD...Aim 1. Autoreactive T cells to citrullinated target proteins associated with RA-ILD .Our previous study found citHSP90β stimulated and induced significantly higher levels of IFN-γ levels in RA-ILD compared to the RA-no ILD and CTD-ILD. The production of IFN-γ by T cells stimulated with citHSP90β reflects a TH1 bias that is consistent with earlier observations linking pulmonary infiltration of CXCR3+ T cells to RA-ILD. Specific T cell responses and T cell subtypes involved when citHSP90β stimulate BALF from RA -ILD patients with different stages will be investigated next...Aim2. To identify altered/novel protein interactions and signaling properties of citrullinated target proteins associated with RA-ILD .Our preliminary study shown citHSP90-induced signaling pathways. In vitro stimulation assays indicated that citHSP90 activate HEK293 cells transfected with Toll-like receptor 4 (TLR4), increased phosphorylation/activation of NF-κB and secretion of IL-8, suggesting that various citrullinated proteins (as well as HSP90) are able to directly/indirectly activate innate immune signaling pathways. We will continually investigate citHSP90 activate HEK293 cells and PBMC-derived macrophages in the presence/absence of exogenous TLR ligands. ..To clarify the pathogenesis of citrullinated antigens induced RA-ILD, to find biomarkers that can identify RA- ILD at an earlier and more treatable stage and set up the therapeutic theory basis on RA-ILD are our goals.

肺间质病变（ILD）是类风湿关节炎(RA)患者主要死因，目前发病机理不明。最新研究显示肺是RA患者瓜氨酸蛋白产生的重要场所，肺部蛋白瓜氨酸化可能诱发自身免疫应答，促发RA-ILD发生。我们假设吸烟等刺激后形成瓜氨酸蛋白抗原-抗瓜氨酸抗体反应，这些体液、细胞免疫反应导致RA-ILD发生。我们前期发现瓜氨酸抗原citHSP90可刺激RA-ILD患者血清产生特异性T细胞应答；建立了citHSP90介导支气管肺泡上皮细胞TLR4信号通路方法。本项目拟研究：瓜氨酸抗原citHSP90刺激RA-ILD患者血清、支气管肺泡灌洗液的特异性IgG应答、特异性T细胞应答、应答细胞亚群及相互间关系；citHSP90抗原介导的细胞内蛋白质间相互作用、信号转导，最终形成促炎、促纤维化的关键因素。阐明瓜氨酸蛋白促发RA-ILD发生的固有、被动免疫致病机理，提供可鉴定RA-ILD的生物标志物，奠定靶向免疫治疗理论依据。

目的：大量研究表明，瓜氨酸蛋白是导致RA,RA-ILD发生发展的重要原因之一，有研究发现除CCP2之外，还有许多其他致病性的瓜氨酸蛋白尚未发现。因此，本研究希望能够发现一些新型的标志物并研究其致病机制。由于目前商品化的抗瓜氨酸抗体具有识别偏好性，因此本研究通过制备特异性识别瓜氨酸残基的高亲和力抗瓜氨酸单克隆抗体，使它在瓜氨酸蛋白检测中成为一个有力的工具。并利用现有的质谱技术对新型瓜氨酸标志物进行筛选，评价并分析其意义。与此同时，本研究对一组结缔组织病人的血清进行了可能的肺间质病变生物标志物MUC5AC,MUC5B的检测，并评价分析该标志物的意义。.方法：1.通过设计瓜氨酸相关抗原免疫BALB/c小鼠，采用杂交瘤技术制备抗瓜氨酸单克隆抗体，利用间接ELISA等方法，以确定该抗体是否特异性识别瓜氨酸残基，并与商品化的抗瓜氨酸抗体进行对照，进行抗体对瓜氨酸蛋白反应性的检测分析。. 2.通过构建化学衍生物的方法进行质谱检测，将2，3-丁二酮（2，3-BD）加入RA患者血清中，在37℃水浴锅中反应16h，使2，3-BD同血清中的瓜氨酸蛋白特异性结合形成咪唑酮衍生物，反应结束后立即进行质谱检测。. 3.检测分析一组结缔组织病间质性肺疾病患者血清的MUC5AC,MUC5B蛋白水平与ILD严重程度之间的相关性。.结果：1.本研究成功制备出只识别瓜氨酸残基的单克隆抗体，并且该抗体对于瓜氨酸蛋白的反应性要高于商品化的抗瓜氨酸抗体。. 2.本研究利用构建化学衍生物进行质谱检测的方法成功在RA患者体内发现具有高水平的cit-4#短肽的自身抗体，并且本研究认为构建化学衍生物进行标志物的检测在人血清中具有可行性。. 3.本研究通过检测MUC5AC,MUC5B蛋白水平发现，在结缔组织病间质性肺疾病（CTD-ILD）患者中，随着ILD严重程度增加，MUC5AC蛋白水平也随之升高，具有较强的相关性。.结论：1. 现已制备出能够适用于不同的免疫分析平台的单克隆抗体，将有助于进一步推动蛋白瓜氨酸化的相关研究。. 2. cit-4#短肽有望成为RA、RA-ILD新靶标之一。. 3.本研究认为结缔组织病病人血清中MUC5AC蛋白水平与ILD级别具有较强的相关性。