NK Cell Activation and Function in HIV-1 Exposed Uninfected IV Drug Users

暴露于 HIV-1 且未感染静脉注射吸毒者的 NK 细胞激活和功能

• 批准号: 8044828
• 负责人: Luis J Montaner
• 金额: $ 66.55万
• 依托单位: WISTAR INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-15 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8044828
• 关键词: Activation Analysis; Acute; Antiviral Agents; Area; Autologous; Automobile Driving; Behavior; Biological Assay; Biological Markers; Blood; Cell Surface Proteins; Cell Surface Receptors; Cell physiology; Cells; Clinical; Clinical Research; Coculture Techniques; Communities; Cytolysis; Data; Dendritic Cells; Dendritic cell activation; Dependence; Disease Progression; Disease Resistance; Down-Regulation; Drug Addiction; Drug usage; Drug user; Epidemiology; Exhibits; Exposure to; Flow Cytometry; Fostering; Frequencies; Genomics; Genotype; HIV; HIV Infections; HIV-1; Immune; Infection; Injectable; Integration Host Factors; Interferon Type II; Interferon-alpha; Interferons; Intervention; KIR3DS1; Life; Ligands; Lytic; Measures; Mediating; Membrane; Membrane Proteins; Methods; Molecular; Monitor; NK Cell Activation; NK cell receptor NKB1; Natural Killer Cells; Needle Sharing; Opioid; Pathway interactions; Patient Care; Patients; Peripheral Blood Mononuclear Cell; Persons; Pharmaceutical Preparations; Phenotype; Philadelphia; Population; Proteins; Proteome; Proteomics; Recording of previous events; Recruitment Activity; Research Personnel; Resistance; Resistance to infection; Risk; Role; Signal Transduction; Stimulus; Stress; Study Subject; Testing; Vietnam; Work; base; beta-Chemokines; chemokine; cohort; cytokine; cytotoxic; cytotoxicity; disorder control; experience; gamma-Chemokines; immune function; improved; in vitro activity; innate immune function; interest; intravenous drug user; killer inhibitory receptor; killings; molecular phenotype; novel; programs; protein activation; protein expression; protein profiling; public health relevance; receptor; response

DESCRIPTION (provided by applicant): Multiply-exposed uninfected IV opioid drug users (EU-IVDU) remaining HIV sero-negative are one of very few clinical cohorts where live HIV exposure can be studied in conjunction with epidemiological teams that are studying risk of infection in these subjects. The described seronegative state of subjects known to be at risk of infection via shared needle behavior with persons of unknown status has heightened interest in identifying the mechanism(s) of immune control that may provide resistance to infection in association with HIV exposure and drug use. NK cell activation, increased constitutive degranulation and increased lytic activity, have been proposed in EU-IVDU as a critical feature associated with protection, yet it remains unknown what specific NK membrane protein profiles and functional responses best defines or identifies an NK or DC cell activation program in EU-IVDU. The effects of drug use on NK or PDC activation programs, even in the absence of repeated HIV exposure, are also unknown. We propose to analyze three groups identified by lack of HIV infection (serongative) with different histories of IV opioid drug use. Three groups of 50 subjects each will be recruited for this study as follows: Group 1: EU-IVDU for >3 yrs, Group 2: Clean users (not needle sharing)-IVU users and Group 3: uninfected-non IVU users. For each of the drug user groups a 1:1 recruitment will take place between exclusive opioid users and opioid and non-injectable drug use in absence of dependence to the latter. For all groups, we will collect blood and characterize circulating immune cell subsets by flow cytometry while preparing PBMC and isolated NK cell subsets for analysis with and without an acute stimulation with Interferon-alpha. Interferon-alpha is chosen as a reference activation stimuli based for its role in activating NK lysis of HIV-infected targets as shown in preliminary data. For Aim 1, we will complete an analysis defining the molecular phenotype of NK cells in the EU-IVDU by (1) measuring activation (i.e., CD69), regulatory (i.e., Np44, etc.), IFN-gamma/chemokine secretion, and STAT-1 signaling potential; (2) using proteomics-based methods to compare global and cell surface protein profiles on NK cells to identify key cellular pathways and cell surface receptors selectively associated with EU-IVDU (KIR3DL1, KIR3DS1). We anticipate to find unique differences in group 1 as compared to groups 2 and 3. Aim 2 will analyze NK and DC-dependent NK activation potential in conjunction with protein expression of KIR associated with disease control or the Plasmacytoid DC secretome after TLR-7 stimulation (associated with HIV-dependent PDC-mediated NK activation). Constitutive circulating NK CD107a and in vitro activity of PDC and cytotoxic function of NK cells when co-cultured with autologous HIV-1 infected targets (with and without IFN-1 stimulation) will be done anticipating to show greater NK activation and lysis in Group 1 as compared to 2 or 3. Taken together, the proposed work will test the hypothesis that a defined NK and pDC activation phenotype mediates both soluble and direct antiviral function in EU-IVDU, thereby decreasing HIV-1 infection efficiency upon IV exposure. PUBLIC HEALTH RELEVANCE: The inter-relationship between HIV-1 infection and drug addiction is poorly understood. By using global unbiased proteomic and genomic discovery approaches, we expect to discover novel biomarkers and molecular mechanisms that will improve patient care by helping to guide clinical research and interventions of patients based on their HIV-1 and drug user status.

描述（由申请人提供）：多次暴露且未感染的静脉注射阿片类药物使用者 (EU-IVDU) 仍保持 HIV 血清阴性，是极少数临床队列之一，可以与正在研究这些受试者感染风险的流行病学团队一起研究活体 HIV 暴露情况。已知由于与身份不明的人共用针头行为而面临感染风险的受试者所描述的血清阴性状态，引起了人们对确定免疫控制机制的兴趣，该机制可能提供与 HIV 暴露和药物使用相关的感染抵抗力。 NK 细胞活化、增加的组成性脱颗粒和增加的裂解活性已在 EU-IVDU 中被提出作为与保护相关的关键特征，但仍不清楚哪种特定的 NK 膜蛋白谱和功能反应最好地定义或识别 EU-IVDU 中的 NK 或 DC 细胞活化程序。即使没有反复接触 HIV，吸毒对 NK 或 PDC 激活程序的影响也是未知的。我们建议分析三个没有 HIV 感染（血清）且具有不同静脉阿片类药物使用史的群体。本研究将招募三组，每组 50 名受试者，如下：第 1 组：超过 3 年的 EU-IVDU，第 2 组：清洁使用者（非共用针头）-IVU 用户，第 3 组：未感染-非 IVU 用户。对于每个吸毒者群体，将在专门的阿片类药物使用者和阿片类药物及非注射药物使用者之间进行 1:1 的招募（如果不依赖后者）。对于所有组，我们将收集血液并通过流式细胞术表征循环免疫细胞亚群，同时制备 PBMC 和分离的 NK 细胞亚群，用于在有或没有干扰素-α 急性刺激的情况下进行分析。干扰素-α 被选为参考激活刺激物，因为如初步数据所示，其在激活 HIV 感染目标的 NK 裂解中发挥作用。对于目标 1，我们将通过 (1) 测量激活（即 CD69）、调节（即 Np44 等）、IFN-γ/趋化因子分泌和 STAT-1 信号传导潜力来完成定义 EU-IVDU 中 NK 细胞分子表型的分析； (2) 使用基于蛋白质组学的方法比较 NK 细胞的整体和细胞表面蛋白质谱，以确定与 EU-IVDU（KIR3DL1、KIR3DS1）选择性相关的关键细胞途径和细胞表面受体。我们预计会发现第 1 组与第 2 组和第 3 组相比的独特差异。目标 2 将分析 NK 和 DC 依赖性 NK 激活潜力，以及与疾病控制相关的 KIR 蛋白表达或 TLR-7 刺激后的浆细胞样 DC 分泌组（与 HIV 依赖性 PDC 介导的 NK 激活相关）。当与自体 HIV-1 感染靶标（有或没有 IFN-1 刺激）共培养时，将进行组成型循环 NK CD107a 和 PDC 的体外活性以及 NK 细胞的细胞毒性功能，预计第 1 组中与第 2 组或第 3 组相比会显示出更大的 NK 激活和裂解。综上所述，拟议的工作将检验以下假设：确定的 NK 和 pDC 激活表型介导可溶性 EU-IVDU 中的直接抗病毒功能，从而降低 IV 暴露后的 HIV-1 感染效率。 公共卫生相关性：人们对 HIV-1 感染与毒瘾之间的相互关系知之甚少。通过使用全球公正的蛋白质组学和基因组学发现方法，我们期望发现新的生物标志物和分子机制，通过根据患者的 HIV-1 和吸毒状况帮助指导临床研究和干预措施，从而改善患者护理。