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

项目摘要 本研究旨在了解HIV感染过程中引发慢性炎症反应的分子事件 感染和更好地确定药物滥用如何加强这种反应。我们假设慢性的 炎症是由感染HIV的静息CD 4 T细胞的自燃性死亡引发的。Pyroptosis 是一种高度炎症性的程序性细胞死亡形式，涉及炎性小体组装，半胱天冬酶-1 活化和膜孔形成。尸解独特地结合了艾滋病毒的两个病理标志 感染-CD 4 T细胞耗竭和慢性炎症-在一个单一的过程。焦亡也可能促进 通过对肠道上皮的破坏作用，炎症维持微生物移位。药物 滥用如可卡因和阿片类药物已知会增加炎性小体相关的细胞表达， 因此，包括NLRP 3和pro-IL-1 β在内的蛋白质潜在地引发CD 4 T细胞的升高水平。 焦亡此外，这些药物上调CCL 2和CCL 7趋化因子的表达， 单核细胞、记忆性CD 4 T细胞和树突状细胞转移到细胞凋亡的位点，从而“为细胞凋亡添加细胞燃料”。 火”。事实上，中央记忆CD 4 T细胞展示CCR 5，CCR 2和CCR 4 β 7整合素， 招募的人提出了一种有趣的可能性，即潜伏的艾滋病毒库的最初播种发生在 自燃性炎症在接受抗逆转录病毒治疗（ART）的一些受试者中， 因为这种死亡途径是通过释放ATP（一种引起细胞凋亡的物质）而自我放大的。从 从治疗的角度来看，这些不同的半胱天冬酶-1依赖性炎症作用可以用VX-765阻断， 一种半胱天冬酶-1抑制剂，已被发现在人类中安全且耐受良好。评估在多大程度 在HIV感染的药物滥用者中，我们建议测量caspase-1活化水平， 从使用可卡因或阿片类药物的HIV感染患者中分离的CD 4 T细胞中出现的其他热变性标志物 与未使用这些药物的HIV感染者相比。我们还将评估这些个体的血浆， 存在裂解的IL-18和IL-1 β以及其他可溶性炎症标志物。患者将获得 通过UCSF SCOPE队列（目标1）。与此同时，人源化小鼠将被植入造血干细胞。 干细胞（HSC）含有或缺乏CASP 1基因，并感染R5传播的创始人病毒。 将重组Cas9和半胱天冬酶-1特异性引导RNA核转染到HSC中以敲除CASP 1 表情将研究这些小鼠感染后的炎症水平（和CD 4 T细胞耗竭 并研究评估CASP 1缺乏是否会改变可卡因或阿片类药物诱导的炎症（Aim 2）。 最后，该小鼠模型也将用于研究是否pyroptotic炎症推动初始的 接种潜伏的HIV宿主（目标3）。通过使用不同的实验方法，我们将 探索caspase-1驱动的细胞凋亡作为HIV感染慢性炎症的启动和驱动因素， 确定滥用药物是否会增强这一信号通路。

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.