Rab4A Control of Liver Dysfunction in Mouse Models of Systemic Lupus Erythematosus.

摘要 系统性红斑狼疮(SLE)是一种病因知之甚少的破坏性自身免疫性疾病 它影响到全球每10万人中有20-150人。免疫系统功能障碍导致T细胞 自身反应性，自身抗体的过度产生，细胞因子的失调，免疫复合体的沉积， 并增加氧化应激。了解免疫和生物化学途径导致 SLE的发病机制不仅对治疗这种疾病至关重要，而且对预防和潜在地治愈SLE也是至关重要的。我们 研究HRES-1/Rab4(Rab4A)，一种参与细胞表面蛋白内体运输的GTPase，如 参与T细胞和抗原之间免疫突触的形成和维持的CD4 递呈细胞(APC)。Rab4A还参与线粒体膜电位的维持，因为它的 破坏与SLE患者T细胞中的线粒体超极化有关。伤势严重。 肝脏是人体最大的代谢器官，约有20%的患者存在肝脏，然而，在我们的 了解其在SLE疾病发病机制中的作用及其与T细胞功能障碍的关系。 初步研究揭示了Rab4A在狼疮易感C57BL/6 LPR(B6.1prQ72L)小鼠体内的结构性激活 促进肝脏线粒体活性氧(ROS)的生成和炎症。删除 T细胞中的Rab4A进一步促进B6.lprCD4KO小鼠肝脏功能障碍。值得注意的是，我们观察到 并降低小鼠肝脏线粒体中的NADPH。初步研究 也显示调节性T细胞(Treg)效应蛋白CTLA-4(CD152)的表达显著增加 在具有Rab4A结构性激活的CD4+T细胞上，然后用T细胞特异性降低到基线 删除Rab4A。氧化应激增加与Rab4A的结构性激活和 T细胞特异性缺失的Tregs活性降低提示Rab4A可能调节 肝细胞有丝分裂与动力蛋白相关蛋白1调控的T细胞功能 (DRP1)和CTLA-4的回收(CD152)。在目标1中，我们将检验这样的假设，即结构性激活 Rab4A通过抑制依赖于Drp1的有丝分裂吞噬增加肝细胞的氧化应激，从而 防止IDH2的周转，从而促进ROS的积累。在目标2中，我们将检验T的假设 细胞特异性缺失的Rab4A通过阻断CTLA-4的运输来抑制Treg的活性。我们将使用回收利用 评估Rab4A将CTLA-4运输到细胞表面的能力，使用Treg抑制试验 检测分离和培养的Treg细胞的抑制功能，并使用高通量成像流式细胞术 评估Treg抑制免疫突触的功能。这些研究将在纽约州立大学北部医疗中心进行 并将帮助提供强化培训，为在学术医学领域的职业生涯奠定基础。这些 研究将阐明一种新的、简洁的机制，将Rab4A与肝细胞有丝分裂和Treg联系起来 解释在系统性红斑狼疮小鼠模型中Rab4A介导的肝功能障碍的发病机制。

Summary Systemic Lupus Erythematosus (SLE) is a devastating autoimmune disease with a poorly understood etiology which affects 20-150 per 100,000 people worldwide. Dysfunction of the immune system leads to T cell autoreactivity, overproduction of autoantibodies, dysregulated cytokine production, immune complex deposition, and increased oxidative stress. Understanding both the immunological and biochemical pathways that lead to SLE pathogenesis is critical to not only treating this disease, but also to prevent, and potentially cure, SLE. We study HRES-1/Rab4 (Rab4A), a GTPase involved in the endosomal trafficking of cell surface proteins such as CD4, which is involved in the formation and maintenance of the immune synapse between T cells and antigen presenting cells (APCs). Rab4A is also involved in the maintenance of mitochondrial membrane potential, as its disruption is linked to mitochondrial hyperpolarization that is seen in the T cells of SLE patients. Injury to the liver, the largest metabolic organ of the body, is present in about 20% of patients, however, a gap exists in our knowledge in understanding its role in disease pathogenesis and its relationship to T cell dysfunction in SLE. Preliminary studies unveiled that constitutive activation of Rab4A in lupus-prone C57Bl/6 lpr (B6.lprQ72L) mice promoted mitochondrial reactive oxygen species (ROS) generation and inflammation in the liver. Deletion of Rab4A in T cells further promoted dysfunction in the livers of B6.lprCD4KO mice. Notably, we observed increased isocitrate and decreased NADPH in the isolated mitochondria of the livers of these mice. Preliminary studies also show a significant increase in the expression of the regulatory T cell (Treg) effector protein CTLA-4 (CD152) on CD4+ T cells with constitutive activation of Rab4A, which is then reduced to baseline with T cell-specific deletion of Rab4A. The combination of increased oxidative stress with constitutive activation of Rab4A and the potentially decreased activities of Tregs with T cell-specific deletion of Rab4A suggest that Rab4A may regulate hepatocellular mitophagy and regulatory T cell function through the modulation of dynamin related protein 1 (Drp1) and the recycling of CTLA-4 (CD152). In Aim 1, we will test the hypothesis that the constitutive activation of Rab4A increases oxidative stress in hepatocytes by inhibiting mitophagy in a Drp1-dependent manner, thus preventing the turnover of IDH2 which promotes the buildup of ROS. In Aim 2, we will test the hypothesis that T cell-specific deletion of Rab4A inhibits Treg activity by blocking the trafficking of CTLA-4. We will use a recycling assay to assess the ability of Rab4A to traffic CTLA-4 to the surface of the cell, use a Treg suppression assay to test the inhibitory functions of isolated and cultured Treg cells, and use high throughput imaging flow cytometry to assess Treg function in inhibiting the immune synapse. These studies will take place at SUNY Upstate Medical University and will help provide intensive training to lay the groundwork for a career in academic medicine. These studies will elucidate a novel and concise mechanism linking Rab4A with hepatocellular mitophagy and Treg function that explains the pathogenesis of Rab4A-mediated liver dysfunction in mouse models of SLE.