HIV infection-induced mitochondrial dysfunction and premature T cell aging

HIV感染引起的线粒体功能障碍和T细胞过早衰老

• 批准号: 10203459
• 负责人: Zhi Q. Yao
• 金额: $ 42.76万
• 依托单位: EAST TENNESSEE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-05 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10203459
• 关键词: 8-hydroxyguanosine; APEX1 gene; Aging; Apoptosis; Base Excision Repairs; Biomedical Research; Butylated Hydroxytoluene; CD4 Positive T Lymphocytes; CRISPR/Cas technology; Cardiovascular Diseases; Cell Aging; Cell physiology; Chronic; Complement; Cysteine; DNA; DNA Damage; DNA Maintenance; DNA Repair; DNA biosynthesis; DNA copy number; DNA polymerase gamma; Data; Disease; Ectopic Expression; Elderly; Environment; Enzymes; Exhibits; Exonuclease; Exposure to; Functional disorder; Funding; Genetic Recombination; Goals; HIV; HIV Infections; Health; Hydrogen Peroxide; Immune; Immune response; Immunocompetence; Impairment; Individual; Infection; Inflammatory; Institution; Knock-out; Lead; Maintenance; Malignant Neoplasms; Mediating; Mitochondria; Mitochondrial DNA; Mitochondrial Proteins; Morbidity - disease rate; Nerve Degeneration; Pathway interactions; Patients; Ploidies; Polymerase; Production; Protein Analysis; Proteins; Proteomics; Reactive Oxygen Species; Recovery; Research; Respiration; Role; Short Interspersed Nucleotide Elements; Signal Transduction; Small Interfering RNA; Students; Superoxides; T-Cell Depletion; T-Cell Receptor; T-Lymphocyte; Telomere Shortening; Testing; Viral; Virus Diseases; antiretroviral therapy; base; clinically relevant; endonuclease G; functional disability; graduate student; improved; knock-down; liquid chromatography mass spectrometry; mitochondrial dysfunction; mortality; novel; nuclease; nucleotide metabolism; overexpression; oxidative damage; peroxiredoxin I; premature; prevent; repaired; response; senescence; single molecule; student training; superoxide dismutase 1; undergraduate student

HIV infection-induced mitochondrial dysfunction and premature T cell aging HIV infection appears to drive premature T cell aging, evidenced by mitochondrial dysfunction. How CD4 T cells develop mitochondrial dysfunction during HIV infections is unclear. The objective of this proposal is to elucidate the mechanisms of mitochondrial dysfunction during chronic HIV infection, so as to develop effective means to rescue CD4 T cell depletion or functional impairment, the sine que non of HIV-infection. To elucidate the mechanisms underlying mitochondrial dysfunction in CD4 T cell aging, we analyzed the mitochondrial function of CD4 T cells derived from ART-controlled HIV patients. Our preliminary data show that HIV CD4 T cells have decreased mitochondrial DNA (mtDNA) content, mitochondrial respiration, and ATP production. To identify candidate proteins involved in dysregulating mtDNA copy numbers, we performed Liquid Chromatography Mass Spectrometry (LC-MS) on purified mitochondria from CD4 T cells of HIV patients and health subjects (HS). We found largest reduction of mitochondrial proteins (SOD1 and PRDX1) in destroying reactive oxygen species (ROS), and in repair of ROS- mediated DNA damage repair (APEX1), and elevation of proteins in mtDNA degrading (EXOG and ENDOG) and mtDNA replication (POLG and MGME1). Based on these and other preliminary data, we hypothesize that ROS- mediated mtDNA damage (via lower SOD1 and/or PDRX1 and APEX1) may cause higher mtDNA degradation (by EXOG and ENDOG), which may not be sufficiently complemented by mtDNA replication (through higher POLG and MGME1), leading to lower mtDNA copy number and impaired mitochondrial functions that we have seen in HIV- derived CD4 T cells. We propose two aims to define the mechanisms leading to mtDNA decrease and compromised function. In Aim 1, We will determine if ectopic expression of SOD1 and/or PRDX1 can reduce ROS level and oxidative mtDNA damage in CD4 T cells of HIV patients. In addition, ectopic expression of APEX1 will be performed to determine the involvement of APEX1 in repairing damaged mtDNA via the base excision repair (BER) pathway. siRNA knockdown of SOD1 and/or PRDX1, and APEX1 will also be performed in healthy CD4 T cells to confirm their roles in mtDNA damage and copy number maintenance, mitochondrial respiration, and ATP production. In Aim 2, we will use transient siRNA knockdown or Crisper/Cas9 knockout to reduce the EXOG and/or ENDOG nucleases in CD4 T cells from HIV patients, and to assess the levels of oxidative mtDNA damage and rescue of mtDNA copy number. We will perform single molecule analysis of replicated DNA (SMARD) on mtDNA in cultured CD4 T cells from HIV patients to comprehensively assess the status of mtDNA replication in response to T cell receptor (TCR) stimulation. Overall, this application is novel and strong in both concept and approach to answer clinically relevant questions: how chronic viral infection induces mitochondrial dysfunction, leading to premature T cells aging, and whether interfering those over-activated enzymes responsible for mtDNA copy number reduction and mitochondrial dysfunction can remodel T cell aging and function during HIV infection. Understanding such mechanisms is critical for developing approaches to improve immune responses in the setting of many infectious or inflammatory diseases.

HIV感染引起的线粒体功能障碍和T细胞过早衰老 HIV 感染似乎会导致 T 细胞过早衰老，线粒体功能障碍就是证明。 CD4 T细胞如何 HIV 感染期间线粒体功能障碍的发生尚不清楚。该提案的目的是阐明 探讨慢性HIV感染过程中线粒体功能障碍的机制，从而制定有效的挽救手段 CD4 T 细胞耗竭或功能受损是 HIV 感染的必要条件。阐明机制 CD4 T 细胞衰老中潜在的线粒体功能障碍，我们分析了 CD4 T 细胞的线粒体功能 源自 ART 控制的 HIV 患者。我们的初步数据显示，HIV CD4 T 细胞有所减少 线粒体 DNA (mtDNA) 含量、线粒体呼吸和 ATP 产生。鉴定候选蛋白质 由于 mtDNA 拷贝数失调，我们进行了液相色谱质谱分析 (LC-MS) 来自 HIV 患者和健康受试者 (HS) 的 CD4 T 细胞的纯化线粒体。我们发现最大的减少 线粒体蛋白（SOD1 和 PRDX1）破坏活性氧 (ROS) 和修复 ROS- 介导的 DNA 损伤修复 (APEX1)，以及 mtDNA 降解中蛋白质的升高（EXOG 和 ENDOG）和 mtDNA 复制（POLG 和 MGME1）。基于这些和其他初步数据，我们假设 ROS- 介导的 mtDNA 损伤（通过降低 SOD1 和/或 PDRX1 和 APEX1）可能会导致更高的 mtDNA 降解（通过 EXOG 和 ENDOG），这可能无法通过 mtDNA 复制（通过更高的 POLG 和 MGME1），导致线粒体 DNA 拷贝数降低和线粒体功能受损，正如我们在 HIV 中看到的那样 衍生的 CD4 T 细胞。我们提出两个目标来定义导致 mtDNA 减少和受损的机制 功能。在目标 1 中，我们将确定 SOD1 和/或 PRDX1 的异位表达是否可以降低 ROS 水平并 HIV患者CD4 T细胞的线粒体DNA氧化损伤。此外，将进行 APEX1 的异位表达 确定 APEX1 通过碱基切除修复 (BER) 途径参与修复受损 mtDNA。 SOD1 和/或 PRDX1 和 APEX1 的 siRNA 敲低也将在健康 CD4 T 细胞中进行，以确认 它们在 mtDNA 损伤和拷贝数维持、线粒体呼吸和 ATP 产生中的作用。瞄准 2、我们将使用瞬时 siRNA 敲低或 Crisper/Cas9 敲除来减少 EXOG 和/或 ENDOG 核酸酶 HIV 患者的 CD4 T 细胞，并评估线粒体 DNA 氧化损伤水平和线粒体 DNA 拷贝的拯救 数字。我们将对培养的 CD4 T 细胞中的 mtDNA 进行复制 DNA (SMARD) 的单分子分析 从 HIV 患者身上全面评估 mtDNA 复制对 T 细胞受体 (TCR) 的反应状态 刺激。总体而言，该应用程序在概念和方法上都新颖且强大，可以回答临床相关问题 问题：慢性病毒感染如何引起线粒体功能障碍，导致 T 细胞过早衰老，以及 是否干扰那些导致 mtDNA 拷贝数减少和线粒体的过度激活的酶 功能障碍可以重塑 HIV 感染期间 T 细胞的衰老和功能。了解此类机制至关重要 开发方法来改善许多传染病或炎症性疾病中的免疫反应。