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 系统性红斑狼疮 (SLE) 是一种毁灭性的自身免疫性疾病，其病因尚不清楚 全球每 10 万人中有 20-150 人受到影响。免疫系统功能障碍导致T细胞 自身反应性、自身抗体过度产生、细胞因子产生失调、免疫复合物沉积、 和氧化应激增加。了解导致的免疫学和生化途径 SLE 发病机制不仅对于治疗这种疾病至关重要，而且对于预防和潜在治愈 SLE 也至关重要。我们 研究 HRES-1/Rab4 (Rab4A)，一种参与细胞表面蛋白内体运输的 GTP 酶，例如 CD4，参与T细胞和抗原之间免疫突触的形成和维持 呈递细胞（APC）。 Rab4A 还参与线粒体膜电位的维持，因为它 破坏与 SLE 患者 T 细胞中可见的线粒体超极化有关。伤害到 肝脏是人体最大的代谢器官，约 20% 的患者存在肝脏，然而，我们的肝脏却存在差距。 了解其在疾病发病机制中的作用及其与 SLE 中 T 细胞功能障碍的关系。 初步研究揭示了狼疮易发性 C57Bl/6 lpr (B6.lprQ72L) 小鼠中 Rab4A 的组成型激活 促进线粒体活性氧（ROS）的产生和肝脏炎症。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，我们观察到增加 异柠檬酸并降低了这些小鼠肝脏分离线粒体中的 NADPH。初步研究 还显示调节性 T 细胞 (Treg) 效应蛋白 CTLA-4 (CD152) 的表达显着增加 通过 Rab4A 组成型激活作用于 CD4+ T 细胞，然后通过 T 细胞特异性将其降低至基线 Rab4A 的缺失。氧化应激增加与 Rab4A 的组成型激活相结合 Rab4A 的 T 细胞特异性缺失可能会降低 Tregs 的活性，这表明 Rab4A 可能调节 通过动力相关蛋白 1 的调节实现肝细胞线粒体自噬和调节性 T 细胞功能 (Drp1) 和 CTLA-4 (CD152) 的回收。在目标 1 中，我们将检验本构激活的假设 Rab4A 通过以 Drp1 依赖性方式抑制线粒体自噬来增加肝细胞的氧化应激，因此 防止 IDH2 的更新，从而促进 ROS 的积累。在目标 2 中，我们将检验假设 T Rab4A 的细胞特异性缺失通过阻断 CTLA-4 的运输来抑制 Treg 活性。我们将使用回收 测定法评估 Rab4A 将 CTLA-4 运输到细胞表面的能力，使用 Treg 抑制测定法 测试分离培养的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学中心进行 大学并将帮助提供强化培训，为学术医学职业奠定基础。这些 研究将阐明一种新颖而简洁的机制，将 Rab4A 与肝细胞线粒体自噬和 Treg 联系起来 该功能解释了 SLE 小鼠模型中 Rab4A 介导的肝功能障碍的发病机制。