Role of Tat and SIRT1 in the T-cell Hyperactivation Syndrome Induced by HIV-1

Tat 和 SIRT1 在 HIV-1 诱导的 T 细胞过度激活综合征中的作用

• 批准号: 8206497
• 负责人: Melanie Maria Ott
• 金额: $ 47.27万
• 依托单位: J. DAVID GLADSTONE INSTITUTES
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-01 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8206497
• 关键词: AIDS/HIV problem; Acquired Immunodeficiency Syndrome; Address; Aging; Antibodies; Antiviral Agents; Apoptotic; Attenuated; Binding; Blood Circulation; CD28 gene; CD3 Antigens; CD4 Positive T Lymphocytes; CD8B1 gene; Cell Culture Techniques; Cell Death; Cell Line; Cells; Chronic; Consensus; Coupled; Cytomegalovirus; Data; Deacetylase; Deacetylation; Development; Disease; Disease Progression; Engineering; Equilibrium; Flow Cytometry; Gene Expression; Generations; Genes; Green Fluorescent Proteins; HIV; HIV Infections; HIV-1; HLA-DR Antigens; Health; Helper-Inducer T-Lymphocyte; Highly Active Antiretroviral Therapy; Histone Deacetylase; Human; Immune; Immune response; Immune system; Individual; Infection; Infectious Agent; Inflammation; Inflammatory; Interleukin-1; Interleukin-2; Interleukin-6; Interphase Cell; Investigation; Laboratories; Lentivirus Vector; Link; Longevity; Measures; Mediating; Microbe; Modeling; Molecular; Molecular Biology; Molecular Profiling; Nature; Nicotinamide adenine dinucleotide; Outcome; Participant; Patients; Peripheral; Phenotype; Plasma; Play; Population; Process; Production; Proteins; Regulation; Regulatory T-Lymphocyte; Reporting; Research; Role; SIV; Shapes; Signal Transduction; Subfamily lentivirinae; Surface; Symptoms; Syndrome; T-Cell Activation; T-Lymphocyte; TNF gene; Testing; Therapeutic; Trans-Activators; Viral; Virus Diseases; cytokine; design; genetic regulatory protein; genome-wide; immune activation; immune function; insight; interest; lymph nodes; microbial; novel; novel strategies; novel therapeutic intervention; p300/CBP-Associated Factor; p65; programs; receptor expression; research study; response; small hairpin RNA; small molecule; tat Protein; transcription factor

DESCRIPTION (provided by applicant): Symptoms of T-cell hyperactivation shape the course and outcome of HIV-1 infection, but the molecular mechanism(s) underlying this chronic immune activation are not well understood. We have identified a novel mechanism by which the HIV-1 Tat protein leads to chronic immune activation. We find that Tat hyperactivates T cells by blocking the deacetylase activity of SIRT1 (Kwon et al, Cell Host Microbe, 2008). SIRT1 is a nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylase and an important regulator of the transcription factor NF-B. Tat directly interacts with the deacetylase domain of SIRT1 and blocks the ability of SIRT1 to deacetylate the RelA/p65 subunit of NF-B. Because acetylated p65 is more active as a transcription factor, Tat hyperactivates the expression of NF-B-responsive genes, such as interleukin-2 (IL-2). These results support a model where the normal function of SIRT1 as a negative regulator of T-cell activation is suppressed by Tat during HIV infection. We propose to define the novel role of SIRT1 as a regulator of immune activation in HIV-infected T cells. We have new and unpublished findings that link the SIRT1 deacetylase activity with immune-suppressive functions of regulatory T cells (Tregs). Special focus of this proposal lies therefore on the characterization of the function of Tat and SIRT1 in Tregs and on the identification of novel SIRT1 substrates in effector and regulatory T cells populations. The importance of these studies is highlighted by their possible impact on the development of novel therapeutic interventions. Potent small molecule activators of SIRT1 have recently become available with great promise in diseases associated with aging (Milne et al, Nature, 2007). We will use these activators to test the hypothesis that activating the suppressor function of SIRT1 will counterbalance the immune stimulatory function of Tat and may represent a novel strategy to treat chronic immune activation in HIV-infected patients. Our specific aims are: 1) we will study the role of SIRT1 in HIV-infected T cells. We will infect Jurkat T cell lines in which SIRT1 expression is down regulated via shRNAs with a GFP-tagged infectious clone of HIV-1 and study intracellular IL-2 production in infected (GFP+) and uninfected (GFP-) cells in response to activation with anti-CD3 and CD28 antibodies. We will also infect primary CD4+ T cell cultures with HIV-1 and test whether treatment with SIRT1 activators can reverse hyperstimulation of IL-2 production. 2) We will identify novel targets of the SIRT1 deacetylase activity in infected T cells. We have preliminary evidence that Tat by inhibiting SIRT1 induces hyperacetylation of several SIRT1 targets including proapoptotic factors p53 and Foxo3A. Since both factors are important regulators of T-cell death during HIV infection, we will study how Tat manipulates the transcriptional activities and proapoptotic functions of both factors. We will also perform genome-wide expression profiling and identify gene programs that are modulated by Tat expression or SIRT1 knockdown. 3) We will examine how the SIRT1/Tat interaction influences the development and function of Tregs. We have preliminary results showing that SIRT1 promotes TGF--induced Treg development and is involved in deacetylation of the transcription factor FoxP3. We propose to study how SIRT1 regulates expression and function of FoxP3 in Tregs and will characterize how Tat expression during HIV infection can interfere with this process. These studies will bring novel insight into the molecular biology of HIV-induced immune activation and identify yet undefined mechanisms of HDAC-mediated control of the immune response. PUBLIC HEALTH RELEVANCE: We seek to identify and characterize novel regulatory mechanisms controlling T cell hyperactivation during HIV-1 infection. T cell hyperactivation is a hallmark of pathogenic HIV-1 infection and the strongest predictor of the progression to AIDS in infected individuals. Our proposed studies characterize novel mechanisms by which the HIV-1 Tat protein hyperactivates T cells. These studies are directly relevant to HIV/AIDS and may contribute to the development of novel antiviral drugs that will address public need.

描述（由申请人提供）：T 细胞过度激活的症状决定了 HIV-1 感染的过程和结果，但这种慢性免疫激活的分子机制尚不清楚。我们已经确定了 HIV-1 Tat 蛋白导致慢性免疫激活的新机制。我们发现 Tat 通过阻断 SIRT1 的脱乙酰酶活性来过度激活 T 细胞（Kwon 等人，Cell Host Microbe，2008）。 SIRT1 是一种烟酰胺腺嘌呤二核苷酸 (NAD+) 依赖性组蛋白脱乙酰酶，也是转录因子 NF-B 的重要调节因子。 Tat 直接与 SIRT1 的脱乙酰酶结构域相互作用，并阻断 SIRT1 使 NF-B 的 RelA/p65 亚基脱乙酰化的能力。由于乙酰化 p65 作为转录因子更加活跃，Tat 会过度激活 NF-B 反应基因的表达，例如白介素 2 (IL-2)。这些结果支持这样一种模型，即 SIRT1 作为 T 细胞激活的负调节因子的正常功能在 HIV 感染期间被 Tat 抑制。我们建议定义 SIRT1 作为 HIV 感染 T 细胞免疫激活调节剂的新作用。我们有未发表的新发现，将 SIRT1 脱乙酰酶活性与调节性 T 细胞 (Treg) 的免疫抑制功能联系起来。因此，该提案的特别重点在于 Tregs 中 Tat 和 SIRT1 功能的表征，以及效应 T 细胞和调节 T 细胞群中新型 SIRT1 底物的鉴定。这些研究的重要性因其对新型治疗干预措施的发展可能产生的影响而凸显。最近，SIRT1 的有效小分子激活剂在与衰老相关的疾病中具有巨大的前景（Milne 等人，Nature，2007）。我们将使用这些激活剂来检验以下假设：激活 SIRT1 的抑制功能将抵消 Tat 的免疫刺激功能，并可能代表治疗 HIV 感染患者慢性免疫激活的新策略。我们的具体目标是：1）我们将研究SIRT1在HIV感染的T细胞中的作用。我们将用带有 GFP 标记的 HIV-1 感染性克隆感染 Jurkat T 细胞系，其中 SIRT1 表达通过 shRNA 下调，并研究受感染 (GFP+) 和未感染 (GFP-) 细胞响应激活的胞内 IL-2 产生具有抗 CD3 和 CD28 抗体。我们还将用 HIV-1 感染原代 CD4+ T 细胞培养物，并测试 SIRT1 激活剂治疗是否可以逆转 IL-2 产生的过度刺激。 2) 我们将确定受感染 T 细胞中 SIRT1 脱乙酰酶活性的新靶标。我们有初步证据表明，Tat 通过抑制 SIRT1 诱导几个 SIRT1 靶标的过度乙酰化，包括促凋亡因子 p53 和 Foxo3A。由于这两个因子都是 HIV 感染期间 T 细胞死亡的重要调节因子，因此我们将研究 Tat 如何操纵这两个因子的转录活性和促凋亡功能。我们还将进行全基因组表达谱分析并鉴定受 Tat 表达或 SIRT1 敲低调节的基因程序。 3）我们将研究SIRT1/Tat相互作用如何影响Tregs的发育和功能。我们的初步结果表明，SIRT1 促进 TGF 诱导的 Treg 发育，并参与转录因子 FoxP3 的脱乙酰化。我们计划研究 SIRT1 如何调节 Tregs 中 FoxP3 的表达和功能，并将描述 HIV 感染期间 Tat 表达如何干扰这一过程。这些研究将为 HIV 诱导的免疫激活的分子生物学带来新的见解，并确定 HDAC 介导的免疫反应控制尚未明确的机制。公共卫生相关性：我们寻求识别和表征 HIV-1 感染期间控制 T 细胞过度激活的新调控机制。 T 细胞过度激活是致病性 HIV-1 感染的标志，也是感染者进展为 AIDS 的最强预测因子。我们提出的研究描述了 HIV-1 Tat 蛋白过度激活 T 细胞的新机制。这些研究与艾滋病毒/艾滋病直接相关，可能有助于开发满足公众需求的新型抗病毒药物。