Assessing the root causes of chronic inflammation in HIV-infected individuals using drugs of abuse

评估使用滥用药物的艾滋病毒感染者慢性炎症的根本原因

• 批准号: 9761514
• 负责人: Warner C. Greene
• 金额: $ 71.45万
• 依托单位: J. DAVID GLADSTONE INSTITUTES
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-15 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9761514
• 关键词: Affect; Aging; Apoptosis; Appearance; Attention; Biological Assay; CASP1 gene; CCL2 gene; CCL7 gene; CCR5 gene; CD34 gene; CD4 Positive T Lymphocytes; Cell Death; Cells; Cessation of life; Chronic; Cleaved cell; Clinical; Cocaine; Collaborations; Complex; Cytomegalovirus Infections; DNA; Dendritic Cells; Detection; Disease; Disease Progression; Drug usage; Event; Exhibits; Fire - disasters; Genes; Genome; Genomics; Guide RNA; HIV; HIV Infections; Hematopoietic stem cells; Human; Immune response; Implant; Individual; Infection; Inflammasome; Inflammation; Inflammatory; Inflammatory Response; Integrins; Intercept; Interferon Type II; Interleukin-1; Interleukin-1 beta; Interleukin-18; Knock-out; Lead; Link; Lymphoid; Measures; Membrane; Modeling; Molecular; Mus; Natural History; Nature; Opioid; Pathogenesis; Pathogenicity; Pathologic; Pathway interactions; Patients; Pharmaceutical Preparations; Plant Roots; Plasma; Process; Production; Proteins; Proviruses; Recombinants; Regulatory T-Lymphocyte; Reporting; Rest; Ribonucleoproteins; Sampling; Signal Pathway; Site; Spleen; System; T-Cell Depletion; T-Lymphocyte; Technology; Testing; Therapeutic; Time; Tissues; Tonsil; Toxic effect; Transplantation; Viral reservoir; Viremia; Virion; Virus; Virus Latency; antiretroviral therapy; chemokine; cocaine use; cohort; cytokine; drug abuser; drug of abuse; gastrointestinal epithelium; human tissue; humanized mouse; immune activation; inflammatory marker; interleukin-1beta-converting enzyme inhibitor; lymph nodes; memory CD4 T lymphocyte; microbial; molecular modeling; monocyte; mouse model; novel therapeutic intervention; reconstitution; recruit; response; viral DNA

Project Summary This study seeks to understand the molecular events that trigger the chronic inflammatory response during HIV infection and to better define how drugs of abuse enhance this response. We hypothesize that chronic inflammation is initiated by the pyroptotic death of resting CD4 T cells abortively infected with HIV. Pyroptosis is a highly inflammatory form of programmed cell death involving inflammasome assembly, caspase-1 activation, and membrane pore formation. Pyroptosis uniquely unites the two pathologic hallmarks of HIV infection––CD4 T cell depletion and chronic inflammation––in a single process. Pyroptosis also likely promotes inflammation-sustaining microbial translocation through damaging effects on the gut epithelium. Drugs of abuse like cocaine and opioids are known to increase the cellular expression of inflammasome-associated proteins including NLRP3 and pro-IL-1thus potentially priming CD4 T cells for heightened levels of pyroptosis. Further, these drugs up-regulate the expression of the CCL2 and CCL7 chemokines that recruit monocytes, memory CD4 T cells, and dendritic cells to the sites of pyroptosis thereby “adding cellular fuel to the fire.” The fact that central memory CD4 T cells displaying CCR5, CCR2, and the 47 integrin are recruited raises the intriguing possibility that initial seeding of the latent HIV reservoir occurs within zones of pyroptotic inflammation. Pyroptosis also persists in some subjects receiving antiretroviral therapy (ART) likely because this death pathway is self-amplifying through the release of ATP, an inducer of pyroptosis. From a therapeutic perspective, these various caspase-1 dependent inflammatory effects can be blocked with VX-765, a caspase-1 inhibitor already found safe and well tolerated in humans. To assess the extent to which pyroptosis is occurring HIV-infected drug abusers, we propose to measure levels of caspase-1 activation and other pyroptotic markers occurring in CD4 T cells isolated from HIV-infected patients using cocaine or opiates versus HIV-infected subjects not using these drugs. We will also assess plasma from these individuals for the presence of cleaved IL-18 and IL-1 plus other soluble inflammatory markers. Patients will be obtained through the UCSF SCOPE cohort (Aim 1). In parallel, humanized mice will be implanted with hematopoietic stem cells (HSCs) containing or lacking the CASP1 gene and infected with R5 transmitted-founder viruses. Recombinant Cas9 and caspase-1 specific guide RNAs will be nucleofected into HSCs to knockout CASP1 expression. These mice will be studied for levels of inflammation (and CD4 T cell depletion) following infection and studied to assess whether CASP1 deficiency alters inflammation induced by cocaine or opioids (Aim2). Finally, this mouse model will also be used to investigate whether pyroptotic inflammation propels initial seeding of the latent HIV reservoir (Aim 3). Through the use of different experimental approaches, we will explore caspase-1 driven pyroptosis as an initiator and driver of chronic inflammation in HIV infection and determine whether drugs of abuse enhance this signaling pathway.

项目概要 本研究旨在了解在艾滋病毒感染期间引发慢性炎症反应的分子事件 感染并更好地确定滥用药物如何增强这种反应。 炎症是由感染 HIV 的静息 CD4 T 细胞焦亡引发的。 是一种高度炎症性的程序性细胞死亡，涉及炎症小体组装、caspase-1 激活和膜孔形成独特地结合了 HIV 的两个病理特征。 感染——CD4 T 细胞耗竭和慢性炎症——在一个过程中也可能促进细胞焦亡。 通过对肠道上皮细胞的破坏作用维持炎症的微生物易位。 已知可卡因和阿片类药物等滥用会增加炎症小体相关细胞的表达 包括 NLRP3 和 pro-IL-1在内的蛋白质，因此可能启动 CD4 T 细胞以达到 此外，这些药物上调招募的 CCL2 和 CCL7 趋化因子的表达。 单核细胞、记忆 CD4 T 细胞和树突状细胞到达焦亡部位，从而“为细胞焦亡添加细胞燃料” 事实上，显示 CCR5、CCR2 和 α4α7 整合素的中央记忆 CD4 T 细胞是 招募提出了一种有趣的可能性，即潜伏艾滋病毒储存库的最初播种发生在以下区域内： 焦亡性炎症也可能在一些接受抗逆转录病毒治疗（ART）的受试者中持续存在。 因为这种死亡途径通过释放 ATP（细胞焦亡的诱导剂）而自我放大。 从治疗的角度来看，这些各种 caspase-1 依赖性炎症效应可以用 VX-765 阻断， 已发现一种 caspase-1 抑制剂在人体中安全且耐受性良好。 细胞焦亡是 HIV 感染者吸毒者发生的，我们建议测量 caspase-1 激活水平并 使用可卡因或阿片类药物从 HIV 感染者中分离出的 CD4 T 细胞中出现的其他焦亡标记物 我们还将评估感染艾滋病毒的受试者与不使用这些药物的受试者的血浆。 将获得存在裂解的 IL-18 和 IL-1 以及其他可溶性炎症标志物的患者。 通过 UCSF SCOPE 队列（目标 1），人源化小鼠将被植入造血系统。 含有或缺乏 CASP1 基因并感染 R5 传播病毒的干细胞 (HSC)。 重组 Cas9 和 caspase-1 特异性引导 RNA 将被核转染到 HSC 中以敲除 CASP1 将研究这些小鼠感染后的炎症水平（和 CD4 T 细胞耗竭）。 并研究评估 CASP1 缺陷是否会改变可卡因或阿片类药物引起的炎症 (Aim2)。 最后，该小鼠模型还将用于研究焦亡炎症推进剂是否最初 潜在 HIV 储存库的播种（目标 3） 通过使用不同的实验方法，我们将 探索 caspase-1 驱动的细胞焦亡作为 HIV 感染中慢性炎症的引发剂和驱动因素 确定滥用药物是否会增强该信号通路。