TWEAK/Fn14信号激活在抗dsDNA抗体致病机制中的关键作用

As an important serum marker in systemic lupus erythematosus (SLE), anti-dsDNA antibodies have been demonstrated to be responsible for the specific renal damage in the patients with lupus nephritis. However, the mechanism by which the pathogenic anti-dsDNA antibodies penetrate the infiltration slit and then deposit along glomerular basement membrane is not fully elucidated to date. Previously, we found that a novel pathway of TWEAK may induce regional tissue injury by engaging its exclusive receptor of Fn14. An interesting finding is that, the knock out of Fn14 gene in MRL/lpr lupus-prone mice does not alter the serum level of anti-dsDNA antibodies, but significantly decreased both the deposition of IgG in glomeruli and proteinuria. The discrepancy between anti-dsDNA antibodies in sera and amelioration of renal damage strongly indicates that the upregulation of TWEAK/Fn14 signaling might contribute to the desfunction of glomerular filtration units, which definitely increases immune complex depostion in glomerlar basement membrane. To verify this hypothesis that TWEAK/Fn14 pathway play a key role in the nephritogenicity of anti-dsDNA antibodies, we plan to study the effect of TWEAK/Fn14 signaling on the cell cycles and junction protein expression of both glomerular endothelial cells and podocytes in vitro. An in vitro permeability will be assessed by constructing the monolayer of either glomerular endothelial cells or podocytes. Moreover, the treatment with either anti-TWEAK/Fn14 mAb or in vivo transfection of Fn14 siRNA will be applied to the murine models of lupus-specific renal inury induced by pathogenic anti-dsDNA antibodies. The resuts from present study will contribute to the development of novel therapeutic approaches in the management of fatal organic damage in the patients with SLE.

抗dsDNA抗体是SLE患者血清中标志性自身抗体之一，可直接导致肾脏损害，但其如何穿透滤过膜、沉积于基底膜的机制不明。我们前期研究证实，一种新型信号通路TWEAK/Fn14的异常激活可导致炎症性局灶组织损害；且意外发现，Fn14基因敲除未改变MRL/lpr狼疮小鼠血清中抗dsDNA抗体等滴度，却明显降低肾脏基底膜IgG沉积与尿蛋白水平，强烈提示TWEAK/Fn14信号激活可破坏肾小球滤过膜结构。为证实假说，我们拟构建体外肾小球滤过膜模型，研究TWEAK/Fn14信号激活对内皮细胞、足细胞生存周期的影响及如何调控滤过膜连接蛋白表达的分子机制；筛选高致病性抗dsDNA抗体亚型并建立肾损害小鼠模型，运用TWEAK或Fn14中和性抗体、体内转染Fn14 siRNA等技术阻抑TWEAK/Fn14信号激活，观察对狼疮特异性肾损害的治疗效应。本研究将为高效、选择性阻断SLE重要靶器官损害建立新途径。

抗dsDNA抗体是SLE患者血清中标志性自身抗体之一，可直接导致肾脏损害，但其如何穿透滤过膜、沉积于基底膜的机制不明。我们前期发现，一种新型信号通路TWEAK/Fn14的异常激活可导致炎症性局灶组织损害；而且Fn14基因敲除未改变MRL/lpr狼疮小鼠血清中抗dsDNA抗体等滴度，却明显降低肾脏基底膜IgG沉积与尿蛋白水平，这提示TWEAK/Fn14信号激活可破坏肾小球滤过膜结构。. 在本课题，我们利用细胞因子刺激和质粒载体技术，构建了5种CDR区相同的抗dsDNA抗体杂交瘤克隆（IgG1、IgG2b、IgG2a、IgG3、IgM）。采用表面等离子共振技术等技术，定量检测了各亚型抗dsDNA抗体与肾组织相关抗原分子的特异性结合力，发现IgG3亚型结合能力较强。通过体外培养肾小球血管内皮细胞（GENC）、肾小球足细胞（GVEC），构建体外肾小球滤过膜模型，然后施加TWEAK刺激，发现TWEAK/Fn14信号激活诱导肾脏固有细胞凋亡或坏死的新机制。我们观察到TWEAK/Fn14信号激活诱导GENC与GVEC出现同时出现凋亡及坏死，Fn14 siRNA转染后细胞死亡率明显降低。在不同TWEAK/Fn14信号水平下，NF-κB下游信号相关蛋白Caspase-8（介导细胞凋亡）、cathepsin B（介导细胞坏死）表达变化，导致这些细胞凋亡、坏死及nephrin、podocin等连接蛋白表达降低。. 我们还利用CRISPR/Cas9技术，获得了Fn14基因敲除SCID小鼠模型。对该模型静脉注射抗dsDNA抗体，证实不同亚型抗dsDNA抗体存在体内差异化致病作用，Fn14信号抑制可以有效缓解这些致病性抗体在动物模型的效应，包括肾小球区域抗dsDNA抗体沉积减少，小鼠尿蛋白等指标改善。这些结果不仅阐明了抗dsDNA抗体诱导红斑狼疮肾损害的机制，也为今后探索阻断其致病效应的新疗法奠定了基础。