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.

总结 系统性红斑狼疮（SLE）是一种破坏性的自身免疫性疾病，病因不明 全球每10万人中就有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，自身抗体的过度产生，失调的细胞因子产生，免疫复合物沉积， 和增加的氧化应激。了解导致疾病的免疫和生化途径， SLE发病机制不仅对治疗这种疾病至关重要，而且对预防和潜在治愈SLE也至关重要。我们 研究HRES-1/Rab 4（Rab 4A），一种参与细胞表面蛋白（如 CD 4，参与T细胞与抗原之间免疫突触的形成和维持 提呈细胞（APC）。Rab 4A还参与线粒体膜电位的维持，因为其 这种破坏与SLE患者T细胞中观察到的线粒体超极化有关。损伤 肝脏是人体最大的代谢器官，约20%的患者存在肝脏，然而，我们的研究中存在差距。 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。 初步研究揭示了在狼疮倾向的C57 Bl/6 lpr（B6.lprQ72L）小鼠中Rab 4A的组成性激活 促进肝脏中线粒体活性氧（ROS）的产生和炎症。删除 T细胞中的Rab 4A进一步促进B6.lprCD4KO小鼠肝脏中的功能障碍。值得注意的是，我们观察到， 在这些小鼠的肝脏的分离的线粒体中的异柠檬酸和减少的NADPH。初步研究 还显示调节性T细胞（Treg）效应蛋白CTLA-4（CD 152）的表达显著增加 在具有Rab 4A组成性激活的CD 4 + T细胞上，然后用T细胞特异性免疫抑制剂将Rab 4A降低至基线。 删除Rab 4A。增加的氧化应激与Rab 4A的组成性激活和Rab 4A的表达的结合， Rab 4A的T细胞特异性缺失可能降低TcB的活性，这表明Rab 4A可能调节 通过调节动力蛋白相关蛋白1实现肝细胞线粒体自噬和调节性T细胞功能 （Drp 1）和CTLA-4（CD 152）的再循环。在目标1中，我们将检验以下假设： Rab 4A通过以Drp 1依赖的方式抑制线粒体自噬，增加肝细胞中的氧化应激， 阻止促进ROS积累的IDH 2的周转。在目标2中，我们将检验T Rab 4A的细胞特异性缺失通过阻断CTLA-4的运输来抑制Treg活性。我们将使用一个回收 为了评估Rab 4A将CTLA-4运输至细胞表面的能力，使用Treg抑制测定来评估Rab 4A的细胞增殖。 检测分离培养的Treg细胞的抑制功能，并使用高通量成像流式细胞术， 评估Treg在抑制免疫突触中的功能。这些研究将在SUNY Upstate Medical进行 大学，并将帮助提供密集的培训，奠定基础，在学术医学的职业生涯。这些 这些研究将阐明Rab 4A与肝细胞线粒体自噬和Treg之间的一种新的简洁机制 功能，解释SLE小鼠模型中Rab 4A介导的肝功能障碍的发病机制。