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

Rab4A 对系统性红斑狼疮小鼠模型肝功能障碍的控制。

• 批准号: 10537786
• 负责人: Akshay Patel
• 金额: $ 5.18万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10537786
• 关键词: Affect; Antigen-Antibody Complex; Antigen-Presenting Cells; Autoantibodies; Autoimmune Diseases; Biochemical Pathway; Biological Assay; CD4 Positive T Lymphocytes; CTLA4 gene; Cell Lineage; Cell Surface Proteins; Cell physiology; Cell surface; Cells; Citric Acid; Data; Deposition; Development; Disease; Dynamin; Dynamin I; Enzymes; Eragrostis; Etiology; FRAP1 gene; Flow Cytometry; Functional disorder; Generations; Guanosine Triphosphate Phosphohydrolases; Hepatic; Hepatocyte; Heterogeneity; Human; Image; Immune; Immune System Diseases; Immune checkpoint inhibitor; Immunologics; Impairment; Inflammation; Inflammatory; Investigation; Isocitrate Dehydrogenase; Isocitrates; Knowledge; Lead; Link; Liver; Liver Dysfunction; Liver Mitochondria; Liver diseases; Lupus; Maintenance; Mediating; Medical; Medicine; Membrane Potentials; Metabolic; Metabolic dysfunction; Mitochondria; Mus; NADP; Organ; Organism; Oxidation-Reduction; Oxidative Stress; Pathogenesis; Pathologic; Pathway interactions; Patients; Persons; Play; Pristane; Process; Production; Proteins; Reactive Oxygen Species; Recycling; Regulatory T-Lymphocyte; Role; Stable Isotope Labeling; Surface; Systemic Lupus Erythematosus; T-Cell Receptor; T-Lymphocyte; Testing; Training; Universities; autoreactive T cell; career; cytokine; effector T cell; immunological synapse; immunological synapse formation; immunoregulation; liquid chromatography mass spectrometry; liver inflammation; liver injury; mitochondrial membrane; mouse model; novel; prevent; reactive oxygen intermediate; response; small molecule; trafficking

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.

概括 全身性红斑狼疮（SLE）是一种毁灭性的自身免疫性疾病，病因不足 全球每10万人影响20-150人。免疫系统的功能障碍导致T细胞 自动反应性，自身抗体过量，细胞因子产生失调，免疫复合沉积， 并增加氧化应激。了解导致的免疫学和生化途径 SLE发病机理不仅对治疗这种疾病至关重要，而且对于预防和可能治愈，SLE至关重要。我们 研究HRES-1/RAB4（RAB4A），一种参与细胞表面蛋白内体运输的GTPase，例如 CD4参与T细胞和抗原之间免疫突触的形成和维持 呈现细胞（APC）。 Rab4a还参与了线粒体膜电位的维护，因为它 破坏与SLE患者T细胞中看到的线粒体超极化有关。伤害 肝脏是人体最大的代谢器官，大约有20％的患者存在，但是，我们 了解其在疾病发病机理中的作用及其与SLE中T细胞功能障碍的关系的知识。 初步研究揭示了Rab4a在狼疮易受的C57BL/6 LPR（B6.LPRQ72L）小鼠中的组成型激活 肝脏中促进线粒体活性氧（ROS）的产生和炎症。删除 T细胞中的Rab4a进一步促进了B6.LPRCD4KO小鼠肝脏中的功能障碍。值得注意的是，我们观察到增加 这些小鼠的肝脏分离的线粒体中的异位酸盐和NADPH降低。初步研究 还显示了调节T细胞（TREG）效应蛋白CTLA-4（CD152）的表达显着增加 在CD4+ T细胞上具有组成型RAB4A的活化，然后将其还原为基线，并用T细胞特异 删除Rab4a。氧化应激与本构的激活的组合结合在一起 tregs treg的活性降低了Rab4a的T细胞特异性缺失，这表明Rab4a可能会调节 肝细胞线细胞和调节性T细胞功能通过元素相关蛋白1的调节 （DRP1）和CTLA-4的回收（CD152）。在AIM 1中，我们将测试本构激活的假设 Rab4a的of通过以DRP1依赖性抑制线粒体来增加肝细胞中的氧化应激，从而增加 防止IDH2的营业额促进ROS的积累。在AIM 2中，我们将检验以下假设。 Rab4a的细胞特异性缺失通过阻止CTLA-4的运输来抑制Treg活性。我们将使用回收 测定评估RAB4A的能力CTLA-4到细胞表面的能力，使用TREG抑制测定法 测试孤立和培养的Treg细胞的抑制作用，并使用高吞吐量影像流式细胞仪 评估Treg在抑制免疫突触中的功能。这些研究将在SUNY UPSTATE Medical进行 大学，将有助于提供深入的培训，为学术医学的职业奠定基础。这些 研究将阐明一种新颖而简洁的机制，将Rab4a与肝细胞线索和Treg联系起来 解释了RAB4A介导的肝功能障碍在SLE的小鼠模型中的发病机理。