系统性红斑狼疮新易感基因WDFY4在B细胞发育中的作用及其致病机制研究

Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by pathogenic autoantibody formation, immune complex deposition, and end organ damage. A growing body of evidence supports the key role of the B cell in SLE pathogenesis. Our previous GWAS identified WDFY4 as a novel susceptibility gene of SLE. Although its function is unknown, it predominantly expressed in immune tissues such as lymph node, spleen, thymus and tonsil. Its closest paralog is WD repeat and FYVE domain containing 3 (WDFY3), which was reported to be involved in autophagy. Similar to WDFY3,WDFY4 does contain WD40 domains and a BEACH (Beige and chediak-kigashi) domain. WD40 domain is found in a number of eukaryotic proteins that over a wide variety of functions including adaptor/regulatory modules in signal transduction, pre-mRNA processing and cytoskeleton assembly, while the BEACH domains are implicated in membrane trafficking. Also, our previous results demonstrated that WDFY4 expressed highly in B cells and implicated in autophagy, cell apoptosis and proliferation. Based on these preliminary findings, we hypothesize that WDFY4 might plays an essential role in SLE pathogenesis by affecting B cell development or differentiation through involving in autophagy process. To test the hypothesis, we are constructing the conditional knock out mouse model. In vitro and in vivo studies are employed to investigate the potential role of WDFY4 in B cell behaviour and the mechanisms in SLE pathogenesis. Our proposed study might help elucidate the biological function of WDFY4 and identify the potential SLE therapy target.

申请者利用全基因组关联研究发现WDFY4基因是系统性红斑狼疮（SLE）的易感基因，进一步分析发现该基因在病例与对照中表达有差异，且主要在脾脏、骨髓等组织的B细胞中高表达，可能影响了细胞的自噬、增殖与凋亡，但具体的机制及其在SLE发生中的作用尚不清楚。由于其同家族基因是自噬相关基因，据此我们提出假说：WDFY4可能通过参与自噬而影响了B细胞的发育分化进而参与了SLE的发生发展。为验证此假说，申请者启动了WDFY4条件性基因敲除小鼠的构建，拟与EIIa-Cre杂交得到全身敲除小鼠，系统分析小鼠表型。与CD19-Cre小鼠杂交得到B细胞条件性敲除Wdfy4的小鼠，分析其对B细胞的自噬、发育的影响，深入揭示该基因的生物学功能。同时，将Wdfy4敲除小鼠与MRL/lpr狼疮鼠杂交，通过分析小鼠的表型，明确其在SLE发生中的作用，为理解其生物学功能及寻找新的治疗靶点提供理论依据进行积极的探索。

系统性红斑狼疮（SLE）是一种典型的自身免疫性疾病，以自身抗原抗体复合物沉积于各器官形成的组织损伤为临床特点。遗传因素在SLE发病中起重要所用，多个全基因组关联研究（GWAS）指出WDFY4基因在多个人群中与SLE发病相关，并已知其在免疫系统中高表达，但其影响SLE发病的具体机制还没有研究。WDFY4能够编码一个含有3184个氨基酸的巨大蛋白，但其功能还未见报道。本研究通过Loxp-Cre系统首次构建了B淋巴细胞中条件性敲除Wdfy4的小鼠模型，对WDFY4导致SLE易感的机制进行了探究。通过对B细胞及其各亚群细胞进行流式细胞术分析，我们发现敲除Wdfy4后，B淋巴细胞数量减少。利用人工免疫方法对对照及Wdfy4 CKO小鼠胸腺依赖性（TD）及胸腺非依赖性（TI）抗原免疫反应情况进行检测，发现敲除Wdfy4后小鼠TD免疫反应受到抑制，B淋巴细胞抗体分泌量减少，抗体亲和力成熟受阻，B淋巴细胞功能受损。为了研究Wdfy4在系统性红斑狼疮发病中的作用，我们利用降植烷（Pristane）对小鼠进行狼疮表型的化学诱导，发现经狼疮诱导后，对照小鼠出现了脾脏肿大，脾脏结构紊乱，红髓白髓分界不清，同时出现肾小球肾小球增大，肾小球毛细血管基底膜增厚，明显炎性细胞浸润，尿蛋白增多的狼疮肾炎表现，此外血清中也检测到自身反应性的抗双链DNA抗体，而B细胞敲除Wdfy4的小鼠在这些方面的狼疮表型均轻于对照小鼠。接下来我们对敲除Wdfy4所产生表型的分子机制进行了研究，在体外水平，敲低WDFY4后，B细胞自噬水平升高，Western blot及免疫荧光均显示LC3II发生沉积，同时发现这种沉积是非经典的自噬活动引起的。我们将Wdfy4 CKO小鼠与对照小鼠脾脏中B淋巴细胞分选出来，对体外结果进行了验证，结果同样显示敲除Wdfy4后，B淋巴细胞表现出LC3II蛋白累积，同时免疫荧光也显示其LC3II荧光焦点增多。总结一下，我们的研究证实了WDFY4能够通过非经典的自噬通路影响B细胞的数量及功能，从而影响SLE的表型。