Synergistic Mechanisms of chronic Innate Immune Activation in Microglia by Opiates and HIV Infection

摘要 持续的免疫激活是体内HIV-1感染的定义特征，也是HIV-1感染进展至终末期的驱动因素。 阶段艾滋病。艾滋病毒感染者的全身免疫激活被假设是导致艾滋病毒感染的原因。 慢性炎症性疾病的发病率，包括HIV相关的神经认知障碍（HAND）。 重要的是，许多研究表明，患有阿片类药物使用障碍（OUD）的人群 合并HIV感染的发生率更高，这表明潜在的社会经济和神经认知 影响力的社会OUD和HIV的这种共病也被证明会增加神经炎症， HIV感染和HAND的进展。虽然一些研究调查了OUD的交互作用， 尽管在少数细胞类型中存在HAND和HAND，但连接这两种条件的分子机制仍然在很大程度上是未知的。 未知，特别是在原代人类小胶质细胞、神经元和星形胶质细胞中。因此，我们认为， 通过建立原代人类神经元细胞培养物和无偏倚的系统性 在阿片类药物和HIV-1感染共同暴露的背景下，对这些培养物进行单细胞转录组学调查。 在本提案中，我们将建立由诱导多能干细胞（iPSC）衍生的3D球体培养物 人小胶质细胞、星形胶质细胞和神经元，来源于在BUMC建立的10个独立供体系， 将重现大脑中细胞间相互作用的空间复杂性，并询问艾滋病毒的影响 感染和吗啡暴露对持续先天免疫激活的影响。我们将利用最先进的单细胞 转录组学分析，以确定促进促炎性细胞因子诱导的小胶质细胞内源性通路。 共同暴露于HIV和阿片类药物后的反应以及导致神经元损伤的神经退行性途径 细胞损伤最后，来自这些转录组学研究的信息将被用来为基于CRISPR/Cas的研究提供信息。 敲除策略选择性地删除iPSC-小胶质细胞中的关键通路以减轻神经炎症。我们 相信这些研究的结果将为未来预防艾滋病毒和阿片类药物的治疗发展提供信息， 诱导神经炎症和预防HIV+物质使用人群中HAND的发展。

ABSTRACT Persistent immune activation is the defining feature of HIV-1 infection in vivo and a driver of progression to end- stage AIDS. Systemic immune activation in people living with HIV has been hypothesized to account for higher incidence of chronic inflammatory diseases, including HIV-associated neurocognitive disorders (HAND). Importantly, numerous studies have demonstrated that populations living with opioid-use disorder (OUD) have a higher occurrence of comorbid HIV infection, suggesting both potential socio-economic and neurocognitive influence. This comorbidity of OUD and HIV has also been shown to increase neuroinflammation and exacerbate the progression of HIV infection and HAND. While some studies have investigated the interactive effects of OUD and HAND in few cell types in isolation, the molecular mechanisms linking the two conditions is still largely unknown, especially in primary human microglia, neurons and astrocytes. Hence, we believe that this proposal fulfills a critical unmet need by establishing primary human neuronal cell cultures and an unbiased, systematic single cell transcriptomic survey of these cultures in the context of co-exposure of opiates and HIV-1 infection. In this proposal, we will establish 3D spheroid cultures consisting of induced pluripotent stem cell (iPSC)-derived human microglia, astrocytes and neurons, derived from 10 independent donor lines established at BUMC, that will recapitulate spatial complexity of cell-to-cell interactions in the brain, and interrogate the effect of HIV infection and morphine exposure on persistent innate immune activation. We will utilize cutting-edge single cell transcriptomics analysis to identify the microglia-intrinsic pathway that promotes induction of pro-inflammatory responses upon co-exposure to HIV and opiates and the neurodegenerative pathways contributing to neuronal cell injury. Finally, information from these transcriptomic studies will be leveraged to inform CRISPR/Cas-based knock-out strategies to selectively delete key pathways in iPSC-microglia to alleviate neuroinflammation. We believe that results from these studies will inform future therapeutic development to prevent HIV and opiate- induced neuroinflammation and prevent development of HAND in HIV+ substance use population.