Regulation of B cell activation in lupus

狼疮中 B 细胞活化的调节

• 批准号: 9242562
• 负责人: Neetu Gupta
• 金额: $ 38.05万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9242562
• 关键词: Affect; Age; American; Anti-Inflammatory Agents; Anti-inflammatory; Antibody Formation; Antigen-Antibody Complex; Autoantibodies; Autoimmune Diseases; Autoimmune Process; Autoimmunity; B cell differentiation; B-Cell Activation; B-Lymphocytes; Biological Assay; Body part; Bone Marrow; Bone Marrow Cells; Cell membrane; Cell physiology; Cellular Infiltration; Cellular biology; Chimera organism; Clonality; Complement; Cytoskeletal Modeling; Cytoskeleton; Data; Deposition; Development; Disease; Disease Progression; Enzyme-Linked Immunosorbent Assay; Exhibits; Flow Cytometry; Frequencies; Functional disorder; Future; Generations; Genetic; Glomerulonephritis; Homing; Human; Hyperactive behavior; Image; Imaging Techniques; Immune System Diseases; Immunoglobulin Class Switching; Immunoglobulin Somatic Hypermutation; Immunohistochemistry; Immunologics; In Vitro; Incidence; Inflammation; Investigation; Kidney; Kidney Glomerulus; Knock-out; Lead; Life; Ligands; Lupus; Lymphoid; Measures; Mediating; Mediator of activation protein; Membrane; Microscopy; Modeling; Molecular; Molecular Biology; Molecular Target; Monitor; Mus; Myeloid Cells; Organ; Output; Pain; Pathogenesis; Pathogenicity; Pathologic; Pathology; Pathway interactions; Patients; Peripheral; Phosphorylation; Plasma Cells; Population; Process; Production; Property; Protein Dephosphorylation; Proteins; Proteomics; Reaction; Receptor Signaling; Receptors, Antigen, B-Cell; Regulation; Reporting; Research; Resolution; Role; Serum; Signal Pathway; Signal Transduction; Structure of germinal center of lymph node; Syndrome; Systemic Lupus Erythematosus; T-Lymphocyte; Testing; Western Blotting; associated symptom; cell motility; clinically relevant; crosslink; cytokine; design; differentiated B cell; ds-DNA; effective therapy; experimental analysis; experimental study; ezrin; genome wide association study; human disease; immunopathology; in vivo; innovation; insight; live cell imaging; migration; mouse model; new therapeutic target; next generation sequencing; novel; novel strategies; peripheral tolerance; response; small molecule inhibitor; src-Family Kinases; therapeutic development; treatment strategy

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.

项目摘要 系统性红斑狼疮（SLE）是一种以自身抗体为特征的免疫系统疾病 产生、高活性B细胞抗原受体（BCR）信号传导和B细胞高反应性;然而， 调节异常B细胞功能的机制知之甚少。研究蛋白质和 SLE中驱动BCR信号传导、B细胞过度活化和自身抗体产生的过程应 提供机械的见解，并导致更有效的治疗方法的发展。保持B细胞活化 在一个有效的抑制途径的检查下，其中一个关键组成部分是Src家族激酶林恩。人类 GWAS研究显示林恩的低表达与SLE的发病率之间有很强的相关性。小鼠 具有林恩基因缺失的人丧失外周耐受性并显示与人类相关的所有症状 SLE，包括BCR信号传导加剧、B细胞过度活化、高水平血清自身抗体， 肾小球肾炎因此，林恩-/-小鼠代表了临床相关的模型来研究分子生物学。 SLE中B细胞自身免疫的调节。我们以前曾报道，膜细胞骨架连接器， Ezrin蛋白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在林恩-/- B细胞中过度磷酸化， 林恩-/-小鼠的B细胞中ezrin的条件性缺失导致B细胞活化的显著降低， 肾脏中的分化、自身抗体产生和免疫复合物沉积。我们的数据表明 ezrin是林恩缺乏时B细胞过度活化的重要介质，因此是一种潜在的分子生物学活性调节剂。 目标我们假设ezrin通过促进分子水平的表达，促进林恩-/-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） 探讨ezrin缺失对体内免疫病理学的影响，（2）确定ezrin缺失的影响 研究ezrin基因缺失对B细胞分化的影响;（3）研究ezrin基因缺失对BCR结构和B细胞分化的影响 体外活化。我们将采用遗传学、免疫学、蛋白质组学、分子生物学和高分辨率技术， 活细胞成像技术来实现我们的目标。我们希望这些研究的结果将确定 林恩-/-小鼠在疾病不同阶段由于ezrin缺失而改变的SLE病理学特征 进展，确定埃兹林是否通过增强生发中心B细胞来促进林恩-/-小鼠的自身免疫 反应，并揭示空间和分子机制所采用的埃兹蛋白，以促进过度活跃的B细胞 林恩不在时的反应意义和影响：我们预计，创新的小鼠模型和 本文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 调节由林恩缺乏引起的B细胞自身免疫的新模式。此外，我们的结果将 为未来评估ezrin在其他小鼠模型中疾病发病机制中作用的研究设计提供信息 以及SLE患者。最终，我们的研究应该导致确定新的途径， SLE的治疗进展