自身免疫性疾病易感基因NCF1/NCF4调控B细胞发育和活化的分子机制研究

传统理论认为氧化应激产生的活性氧簇ROS是致炎因素之一，但我们发现细胞低或缺ROS会增加自身免疫性疾病的易感性和严重程度，其机制仍不清楚。本课题拟在与人类基因突变位点一致的Ncf190H/Ncf458A突变小鼠中诱导自身免疫疾病，明确NCF1/4的突变对B细胞表型及自身免疫病的影响；继而利用蛋白组学，免疫共沉淀等技术解析受ROS氧化调节的蛋白参与B细胞发育和活化的分子机制；而后利用比色和化学发光方法检测ROS下游抗氧化系统相关基因表达等方法获得抗氧化系统参与B细胞活化机制；再利用流式细胞仪分选具有抑制功能的调节性B细胞，通过转移及体外干预实验明晰ROS在调节性B细胞在骨髓获得功能活化的分子基础；最后验证ROS作用靶点作为干预关节炎治疗的可能性。研究结果将为阐明NCF1/4突变调控B细胞功能参与自身免疫病的进展提供理论和实验依据，也为自身免疫疾病的治疗提供全新治疗靶点。

A prevailing dogma is that reactive oxygen species (ROS) promotes inflammation. It, therefore, came as a surprise that the first genetic polymorphism controlling autoimmune disease was associated with low activity of Ncf1, a critical component of the NOX2 complex, which determines the ROS response in the immune system. Later, we and others showed that both NCF1 and NCF4 play the same role in human autoimmune diseases. A key mechanism on how ROS regulate autoimmune diseases is likely operating in B cells. B cells express the NOX2 complex and secreted ROS could interact with the antigen presentation to T cells. We have made a unique observation that antigen-specific regulatory B cells are selected in the bone marrow of mice. These Bregs are selected by cartilage proteins in the bone marrow trabecular and show a specific phenotype including that they suppress the development of autoimmune arthritis. We have now found that ROS regulate the function of these Bregs. In the present project, we aim to understand how these B cells are regulated by ROS. We have now made an Ncf190H mouse and got a gift for the Ncf458A mouse. Using these mice we plan to clarify the point mutation of Ncf1 and Ncf4 roles in systemic lupus erythematosus and rheumatoid arthritis mouse models and in particular use this humanized model to address the role of B cells. Using the proteomics to analyze the mechanism of ROS regulated protein and its function in B cells. We have made an Ncf1 conditional knock-in mouse and will use this to analyze the specific role of Ncf1 in B cells and its subtypes as well as B cells in specific tissues. We will isolate B cells of critical regulatory importance in autoimmunity to study the molecular pathways regulated by ROS and from this work identify new therapeutic targets. It is a unique opportunity to study the major gene associated with autoimmunity shared between models and humans and we hope to obtain insight in both basic mechanisms and to disclose new therapeutic targets.