HIV Tat and morphine-mediated pyroptosis activates astrocytes: Role of NLRP6 inflammasome in HAND

项目摘要 尽管联合抗逆转录病毒疗法能够显著抑制病毒血症，但大脑仍在继续 成为HIV病毒低水平复制的储存库。使这一问题更加复杂的是药物滥用与 HIV相关的神经认知障碍和神经HIV。在几种滥用药物中，阿片类药物的使用是 在艾滋病毒感染者中非常普遍，既作为滥用药物，也用于疼痛管理。已经 表明HIV/HIV蛋白和滥用的药物如吗啡都会导致神经炎症， 进而涉及炎性小体的激活。越来越多的证据表明，非编码RNA 作为表观遗传和转录后调节因子，控制重要的细胞功能，包括激活， 从而改变各种生物过程的动态。虽然艾滋病毒蛋白和阿片类药物都已被证明 为了改变CNS细胞的基因表达谱，在详细的分子生物学方面存在知识空白。 HIV转录反式激活因子（达特）蛋白和 吗啡对星形胶质细胞活化的影响这个建议的中心假设是，艾滋病毒达特和吗啡，通过 不同的调节机制增强星形胶质细胞的活化，反过来导致 神经炎症我们对SIV额叶皮质全基因组亚硫酸氢盐测序的初步数据- 感染的恒河猴显示NLRP 6（NOD样受体家族，pyrin）的DNA低甲基化增加 含结构域蛋白6）启动子，伴随NLRP 6表达上调。此外，本发明还 暴露于HIV达特和吗啡的人和小鼠原代星形胶质细胞也导致了 microRNA-152的表达伴随着NLRP 6的表达增加。有趣的是，我们也 发现这些初级星形胶质细胞暴露于HIV达特或吗啡导致细胞凋亡增加， 通过焦亡激活并增加促炎性细胞因子（IL 1 β和IL 18）的表达。基于 根据中心假设和可靠的初步数据，这一建议导致了以下具体目标： 1：确定参与microRNA-152介导的NLRP 6炎性小体激活的分子机制 体外暴露于HIV达特和吗啡的星形胶质细胞中;特定目的2：确定表观遗传机制 参与体外HIV达特和吗啡暴露星形胶质细胞中NLRP 6启动子DNA低甲基化; 具体目的3：研究HIV达特和吗啡对NLRP 6炎性小体介导的免疫抑制作用的联合作用。 星形胶质细胞激活，在体内。了解HIV诱导星形胶质细胞活化的机制 吗啡将为未来开发旨在抑制艾滋病毒的新疗法奠定基础， 阿片介导的神经炎症反应。

Project Summary Despite the ability of combination antiretroviral therapy to dramatically suppress viremia, the brain continues to be a reservoir of HIV low-level replication. Adding further complexity to this is the co-morbidity of drug abuse with HIV-associated neurocognitive disorders and neuroHIV. Among several abused drugs, the use of opiates is highly prevalent in HIV-infected individuals, both as an abused drug as well as for pain management. It has been shown that both HIV/HIV proteins and abused drugs such as morphine contribute to neuroinflammation, which, in turn, involves activation of the inflammasomes. Mounting evidence has also shown that noncoding RNAs function as epigenetic and post-transcriptional regulators controlling vital cellular functions, including activation, thus altering the dynamics of various biological processes. While both HIV proteins and opioids have been shown to change the gene expression profiles of CNS cells, there is a gap of knowledge on the detailed molecular mechanism(s) underlying the co-operative effects of HIV Transactivator of transcription (Tat) protein and morphine on astrocyte activation. The central hypothesis of this proposal is that HIV Tat and morphine, via distinct regulatory mechanism(s) augment astrocyte activation leading, in turn, to exacerbated neuroinflammation. Our preliminary data on whole-genome bisulfite sequencing in the frontal cortices of SIV- infected rhesus macaques show increased DNA hypomethylation of the NLRP6 (NOD-like receptor family, pyrin domain-containing protein 6) promoter with a concomitant upregulation in NLRP6 expression. Additionally, exposure of human and mouse primary astrocytes to HIV Tat and morphine also resulted in decreased expression of microRNA-152 that was accompanied by increased expression of NLRP6. Interestingly, we also found that exposure of these primary astrocytes to either HIV Tat or morphine resulted in increased cellular activation with increased expression of proinflammatory cytokines (IL1β and IL18) via pyroptosis. Based on the central hypothesis and the reliable preliminary data, this proposal led to the following specific aims: Specific Aim 1: Determine the molecular mechanism(s) involved in microRNA-152 mediated NLRP6 inflammasome activation in HIV Tat and morphine-exposed astrocytes in vitro; Specific Aim 2: Determine the epigenetic mechanism(s) involved in promoter DNA hypomethylation of the NLRP6 in HIV Tat and morphine-exposed astrocytes in vitro; Specific Aim 3: Validate the combinatorial effects of HIV Tat and morphine on NLRP6 inflammasome mediated astrocyte activation, in vivo. Understanding the mechanisms responsible for astrocyte activation induced by HIV and morphine will set the stage for the future development of novel therapeutics aimed at dampening HIV and opiate-mediated neuroinflammatory responses.