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

项目摘要 这项研究试图了解在HIV感染过程中触发慢性炎症反应的分子事件 并更好地界定滥用药物如何增强这一反应。我们假设慢性病 炎症是由感染艾滋病毒的静止的CD4T细胞的嗜热性死亡引起的。上睑下垂 是一种高度炎症性的程序性细胞死亡，涉及炎症体组装，caspase-1 活化，膜孔形成。上睑下垂独一无二地统一了艾滋病毒的两个病理特征 感染--CD4T细胞耗尽和慢性炎症--在一个过程中。上睑下垂也可能促进 通过对肠道上皮的破坏作用维持炎症的微生物移位。毒品问题 已知滥用可卡因和阿片类药物会增加炎症体相关的细胞表达 因此，包括NLRP3和前IL-1在内的蛋白质可能会激活CD4T细胞，使其升高 上睑下垂。此外，这些药物上调CCL2和CCL7趋化因子的表达 单核细胞、记忆CD4T细胞和树突状细胞到下垂部位，从而增加细胞燃料 大火。“显示CCR5、CCR2和47整合素的中央记忆CD4T细胞是 被招募提出了一种耐人寻味的可能性，即潜伏的艾滋病毒储备库的初始播种发生在 嗜热性炎症。在接受抗逆转录病毒治疗(ART)的一些受试者中，下垂也会持续存在 因为这条死亡途径是通过释放三磷酸腺苷自我放大的，三磷酸腺苷是上睑下垂的诱因。从一个 从治疗的角度来看，这些不同的caspase-1依赖的炎症效应可以被VX-765阻断， 一种caspase-1抑制剂已经被发现在人类中安全且耐受性良好。评估在多大程度上 上睑下垂是发生HIV感染的药物滥用者，我们建议检测caspase-1的激活水平和 从使用可卡因或阿片类药物的HIV感染者分离的CD4T细胞中出现的其他焦链标志物 与不使用这些药物的艾滋病毒感染者的对比。我们还将对这些人的血浆进行评估 存在裂解的IL-18和IL-1以及其他可溶性炎性标志物。患者将获得 通过加州大学旧金山分校的观察组(目标1)。同时，人源化的小鼠将被植入造血细胞 含或不含CASP1基因并感染R5传播的创始病毒的干细胞(HSCs)。 重组的Cas9和Caspase-1特异性引导RNA将被核重组到HSC中以敲除CASP1 表情。这些小鼠将被研究感染后的炎症水平(和CD4T细胞耗尽) 并研究评估CASP1缺乏是否改变可卡因或阿片类药物引起的炎症(AIM2)。 最后，这个小鼠模型也将被用来研究嗜热性炎症是否推动了最初的 播种潜伏的艾滋病毒蓄水池(目标3)。通过使用不同的实验方法，我们将 探讨caspase-1驱动的下垂作为HIV感染和慢性炎症的启动者和驱动者 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.