补体C9基因缺陷对B6狼疮型小鼠自身免疫病发病的促进作用及机制研究

Compared to other early complement components, there is a lack of direct evidences revealing the exact role of complement C9 and MAC in lupus development. We previously established a novel congenic lupus prone mice train B6.lpr.C9-/- which confers both lpr mutation and complement C9 deficiency. Surprisingly, B6.lpr.C9-/- mice developed comparable lymphadenopathy, splenomegaly and anti-dsDNA autoantibodies, the mice also had marked increased atypical CD3+B220+T cells, CD4-CD8- DN T cells, and severe nephritis compared with B6.lpr mice. Our preliminary study indicates that an involvement of C9 deficiency in accelerating lupus development, yet its critical role and mechanisms still need to be clarified. In the present project, we aim to elucidate how C9 and/or MAC deficiency contributes to lupus through establishing 3 novel congenic strains. With our research, we will answer: 1) role of C9 deficiency on development of SLE and nephritis; 2) its relationship with MAC or not; 3) features of immune phenotypes and pathological charicteristics; 4) precise mechanisms of C9 deficiency contributing to SLE, if is due to loss of tolerance to self-antigens or enhance terminal organ injury. A more complete understanding of the intricacies of C9 and MAC involvement in the evolution of lupus from our model would lead to the revisit of C9 and MAC, and to more effective therapies or drug targeting. Data from this project will provide clear and crucial information for extensive researches in this field.

我们建立了补体C9基因敲除的B6.lpr.C9-/-小鼠，发现C9缺陷能加速lpr的作用，使小鼠淋巴结和脾脏肿大，淋巴结和脾脏CD3+B220+T细胞、CD4-CD8-双阴性T细胞等自身免疫亚群显著升高，肾脏炎症和病理损伤加重，初步推测C9缺陷可能促进SLE发病，其确切作用和机制急需探讨。本课题我们将建立3种新型同类系小鼠模型，通过监测小鼠SLE疾病进程和免疫病理损伤变化，确定以下问题：C9缺陷对于狼疮性自身免疫病的发病究竟发挥怎样的作用；C9的作用与MAC形成的依赖性关系如何；详细描述C9缺陷的新型模型小鼠体内主要的免疫表型特征和病理特征；较为清楚地证实C9缺陷的致病作用是增强末端器官的损伤还是打破免疫耐受。清晰地描述这些问题，将有助于更新我们对补体C9和MAC功能的全面认识，有利于帮助阐明人类SLE的发病机制，寻找有效的临床治疗手段或研制靶向药物，也将为后续更加深入研究提供关键信息。

狼疮性肾炎（lupus nephritis, LN）是SLE最严重和常见的并发症。研究发现补体系统在该疾病免疫病理机制扮演重要角色，但其确切作用仍不明确。补体三条活化途径最终都汇集于终末成分C9及其参与形成的膜攻击复合物MAC，然而C9及MAC在LN中发挥的作用的研究结论不一致。为了清晰地阐明补体C9在LN中的作用及机制，进一步丰富我们对狼疮型自身免疫机制的认识，我们构建了C9基因缺陷小鼠（B6.C9-/-），发现至12月龄时B6.C9-/-小鼠虽然其系统性免疫反应无明显变化，但肾脏出现狼疮样病变倾向，提示C9缺陷有LN发生的潜能。鉴于Yaa基因在自身免疫倾向遗传背景的小鼠中可诱导或加速SLE的进展的作用，我们构建了B6.Yaa.C9-/-小鼠并发现，从整体病变水平来看，C9缺陷与Yaa并存时脾脏、淋巴结重量显著增加、肾脏狼疮样病变显著加重、免疫应答轻微升高，提示C9缺陷小鼠具有狼疮样病变倾向。随即，在两种狼疮病变小鼠模型B6.lpr和B6.Sle1Yaa基础上构建了B6.lpr.C9-/-和B6.Sle1Yaa.C9-/-小鼠，结果证实C9缺陷后这两种小鼠的肾脏狼疮样损伤和全身性自身免疫发应均显著加剧，其主要机制是C9缺陷后CD4+T细胞分泌的IFN-γ升高，进而增强了脾脏B细胞活化和GC反应。综上提示，C9缺陷能够促进自身反应性B细胞应答加重LN病变，因而C9参与的功能是抑制狼疮样小鼠自身免疫进程。通过本项目，我们创建了SLE相关LN发病和进展机制研究的新型模型小鼠，我们的结果丰富了对补体系统在SLE尤其是LN中免疫病理机制的认识，这将引导我们重新审视补体活化与LN发病和损伤之间的关系，继续深度解析其中的机制，将为临床SLE疾病的发病机制阐释和新的治疗靶向策略研究提供重要依据。