Regulation of B cell activation in lupus

项目总结 系统性红斑狼疮是一种以自身抗体为特征的免疫系统紊乱。 产生、高活性B细胞抗原受体(BCR)信号和B细胞高反应性；然而， 调节异常B细胞功能的机制还知之甚少。蛋白质和蛋白质的研究 在SLE中驱动BCR信号、B细胞过度激活和自身抗体产生的过程应该 提供机械性的见解，并导致更有效的治疗方法的开发。保持B细胞的激活 受到一条有效的抑制途径的抑制，其中一个关键成分是Src家族的蛋白激酶Lyn。人类 GWAS的研究表明，Lyn的低表达与SLE的发生率之间存在很强的相关性。老鼠 LYN基因缺失导致外周耐受性丧失，并表现出与人类有关的所有症状 系统性红斑狼疮，包括BCR信号加剧，B细胞过度激活，血清自身抗体水平高，以及 肾小球肾炎。因此，Lyn-/-小鼠代表了一种临床相关的研究分子的模型。 系统性红斑狼疮患者B细胞自身免疫功能的调节我们之前已经报道过膜-细胞骨架连接物 蛋白质Ezrin通过动态磷酸化调节B细胞功能的多个方面- 去磷酸化。有趣的是，我们观察到Ezrin在Lyn-/-B细胞中过度磷酸化，并且 Lyn-/-小鼠B细胞中Ezrin的条件性缺失导致B细胞活化显著降低， 肾脏的分化、自身抗体的产生和免疫复合体的沉积。我们的数据表明 在缺乏Lyn的情况下，Ezrin是B细胞过度激活的重要介质，因此是一个潜在的分子 目标。我们推测Ezrin通过促进分子水平促进LYN-/-小鼠B细胞的发病 以及依赖于膜-细胞骨架重组的细胞过程。我们的具体目标是：(1) 研究Ezrin缺失对体内免疫病理的影响，(2)确定Ezrin缺失的影响 (3)检测Ezrin缺失对BCR组织和B细胞的影响 体外激活。我们将利用遗传学、免疫学、蛋白质组学、分子生物学和高分辨率 活细胞成像技术来实现我们的目标。我们预期这些研究的结果将会确定 LYN-/-小鼠不同病期Ezrin缺失引起的SLE病理特征 进展，确定Ezrin是否通过增强生发中心B细胞来促进Lyn-/-小鼠的自身免疫 反应，并揭示了Ezrin促进高活性B细胞的空间和分子机制 在林恩不在的情况下的回应。意义和影响：我们预计创新的鼠标型号和 这里提出的实验分析将建立Ezrin依赖的膜细胞骨架重塑作为一种 LYN缺陷引起的B细胞自身免疫调节新模式此外，我们的结果将 通知未来评估Ezrin在其他小鼠模型疾病发病机制中作用的研究设计 在系统性红斑狼疮患者中也是如此。最终，我们的研究应该导致确定新的途径 系统性红斑狼疮治疗进展。

PROJECT SUMMARY Systemic lupus erythematosus (SLE) is a disorder of the immune system that is characterized by autoantibody production, hyperactive B cell antigen receptor (BCR) signaling and B cell hyperresponsiveness; however, the mechanisms that regulate aberrant B cell function are poorly understood. Investigation of proteins and processes that drive BCR signaling, B cell hyperactivation and autoantibody production in SLE should offer mechanistic insights and lead to development of more effective therapies. B cell activation is kept under check by a potent inhibitory pathway, a key component of which is the Src family kinase Lyn. Human GWAS studies have shown a strong association between lower expression of Lyn and incidence of SLE. Mice with genetic deletion of Lyn lose peripheral tolerance and display all the symptoms associated with human SLE, including exacerbated BCR signaling, B cell hyperactivation, high levels of serum autoantibodies, and glomerulonephritis. Therefore, Lyn-/- mice represent a clinically relevant model to investigate the molecular regulation of B cell autoimmunity in SLE. We have previously reported that the membrane-cytoskeleton linker protein Ezrin regulates multiple facets of B cell function by undergoing dynamic phosphorylation- dephosphorylation. Interestingly, we observed that ezrin is hyperphosphorylated in Lyn-/- B cells, and that conditional deletion of ezrin in B cells of Lyn-/- mice leads to a significant decrease in B cell activation, differentiation, autoantibody production and immune complex deposition in the kidneys. Our data suggest that ezrin is an important mediator of B cell hyperactivation in the absence of Lyn, and thus a potential molecular target. We hypothesize that ezrin facilitates B cell pathogenesis in Lyn-/- mice by promoting molecular and cellular processes that rely on membrane-cytoskeletal reorganization. Our specific aims are, (1) to investigate the impact of ezrin deletion on immunopathology in vivo, (2) to determine the effect of ezrin deletion on B cell differentiation in vivo, and (3) to examine the effect of ezrin deletion on BCR organization and B cell activation in vitro. We will employ genetic, immunological, proteomic, molecular biology, and high-resolution live-cell imaging techniques to accomplish our aims. We expect that the results of these studies will identify the pathological features of SLE that are altered by loss of ezrin in Lyn-/- mice at different stages of disease progression, ascertain if ezrin promotes autoimmunity in Lyn-/- mice by enhancing germinal center B cell response, and reveal the spatial and molecular mechanisms employed by ezrin to facilitate hyperactive B cell responses in the absence of Lyn. Significance & Impact: We anticipate that the innovative mouse models and experimental analyses proposed here will establish ezrin-dependent membrane-cytoskeletal remodeling as a novel mode of regulating B cell autoimmunity arising from the deficiency of Lyn. Moreover, our results will inform the design of future studies assessing the role of ezrin in disease pathogenesis in other mouse models of SLE, as well as in SLE patients. Ultimately, our studies should lead to identification of new avenues for therapeutic development in SLE.