抗Ro52自身抗体在SLE中致病机制的研究

SLE is a diffuse connective tissue disease characterized by multiple autoantibodies in serum and multiple organs and system involved. High level of IFN-α in serum may be one of the pathogenesis. The anti-Ro52 antibody was found to be an independent autoantibody different from the anti-Ro60 antibody recently, which has been found in a variety of autoimmune diseases. It is associated with the clinical manifestations such as sun sensitivity to skin in patients with lupus and interstitial fibrosis of idiopathic inflammatory myopathy. The Ro52 protein, as a target antigen, is involved in the immune regulation by mediating ubiquitination of IRFs in vivo. The immune complex containing autoantibody may mediate intracellular effect by TLR on pDCs and PBMCs and induce NETosis interfere the function of Ro52 protein.We hypothesize that: SLE patients in context of a variety of susceptible genes induce anti-Ro52 antibody with mutiple factors together. The antibody in the form of immune complexes may affect the activation of TLR on pDCs to interfere the immunocompetent cells ubiquitination IRFs, resulting in uncontrolled IFN-α which is involved with pathogenesis in SLE; and aggravate tissue damage as a consequence of the release of NETs in the environment of autophagy, chronic inflammation. If this hypothes is confirmed, it would contribute to revealing the pathogenesis of anti-Ro52 antibodies in SLE and developing novel molecular targeted agents.

SLE是以血清中出现多种自身抗体和多器官、多系统累及为主要特征的弥漫性结缔组织病，血液高IFN-α是发病原因之一。抗Ro52抗体是近年发现有别于抗Ro60抗体的独立自身抗体，该抗体可出现在多种自身免疫病，且与皮肤光敏、炎性肌病的肺间质纤维化等临床表现相关。作为靶抗原的Ro52蛋白介导多个IRF泛素化而参与机体的免疫调节；抗原抗体形成的复合物可作用于DC的TLR介导细胞内效应，同时可引起NETosis，进而影响T、B淋巴细胞功能而导致IFN-α水平增高。我们推测：SLE患者在易感基因背景下由多种因素共同作用产生抗Ro52抗体，抗体以免疫复合物的形式通过影响pDCs的TLR进而影响免疫活性细胞泛素化IRF，导致IFN-α产生失控引起SLE的发病；并在自噬、慢性炎症及必要的免疫环境下促进NETs释放加重组织损伤。如果这种假设成立，有助于揭示抗Ro52抗体的致病机制和开发新的SLE分子靶向药。

按课题设计，首先进行了不同抗Ro52抗体类型与临床表现的相关性研究，发现：SLE/SS/IIM患者血清中抗Ro52抗体主要是以IgG型为主，抗Ro52抗体阳性的SLE患者关节受累的概率高于阴性者，抗Ro52抗体IgG型IIM患者的ILD的发生率更高。在SLE患者抗Ro52抗体与NETs生成异常的相关性研究中没有观察到抗Ro52抗体对NETs水平影响；也不影响SLE患者血浆体外诱发NETs或PMA诱导中性粒细胞产生NETs。我们思考抗Ro52抗体可能不是致病的始动因素，而使其靶抗原Ro52蛋白即TRIM21蛋白异常的结果。接着研究了TRIM21、TLR9、NF-κB和炎症细胞因子IFN-γ、TNF-α、IL-6和IL-17A在SLE患者外周血中表达，结果发现SLE患者PBMC中TLR9、TRIM21和NF-κB较健康对照增高；抗Ro52抗体与血中IFN-γ、TNF-α的水平呈正相关；用sh-TRIM21转染THP-1/Jurkat细胞敲低TRIM21和空载质粒THP-1/Jurkat对照研究发现，在静息状态下，sh-TRIM21组较对照组NF-κB mRNA和IRF8mRNA表达水平升高（P<0.05），给予TLR9激动剂刺激后24小时，sh-TRIM21组与NC组比较，NF-κB mRNA转录水平没有出现组成性增强，提示TRIM21对NF-κB调控与细胞环境有关；分别用IFN-γ/TNF-α/IL-6处理THP-1/Jurkat细胞及sh-TRIM21细胞组，结果IFN-γ、TNF-α处理8h和24h时，sh-TRIM21及NC组TRIM21的表达均出现随时间推移的逐渐上调，基于这样的研究结果，我们推测SLE患者炎症环境下可诱导TRIM21过度表达，TRIM21通过泛素化选择性自噬受体p62，抑制其功能，导致p62与STING靶向结合障碍，引起STING自噬降解受阻而STING异常激活导致Ⅰ型IFNs大量产生。对此进行验证发现SLE患者IFN-β的mRNA水平和STING蛋白水平均增高，通过自噬溶酶体抑制剂氯喹和蛋白酶体抑制剂MG132处理后，氯喹下调了STING蛋白的峰值，而蛋白酶体抑制剂MG132则不能下调。而在TRIM21过表达组中，氯喹和MG132对STING表达的影响没有差异。提示TRIM21抑制了STING的自噬降解；进一步用cGAMP刺激过表达TRIM

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