Multimodal profiling of microglia during HIV infection and substance use disorder

HIV早期进入中枢神经系统促进其在血管周围巨噬细胞和小胶质细胞中的表达；但是，HIV是否 这些细胞的表达与持续性的神经精神损害有关，目前尚不清楚。与艾滋病毒无关 物质使用障碍等并存疾病会加重神经精神缺陷。可卡因等兴奋剂 已被报道改变巨噬细胞和小胶质细胞中的HIV复制动力学，并改变它们的 炎症状态，并可能导致PWH的神经精神障碍。最近的预印本提供了唯一的 有证据表明，频繁使用可卡因与CD4T细胞中较大的HIV潜伏储存库大小有关。低- 水平/残留炎症已被证明在CD4T细胞中培养了一个持久的HIV储存库。然而，一个主要的 知识差距是可卡因暴露是否支持巨噬细胞和小胶质细胞中HIV的长期表达 以及炎症是否介导了这一效应。清楚了解可卡因对艾滋病病毒表达的影响 复杂的多细胞环境是识别有效ART方案的新靶点的先决条件， 辅助性神经保护疗法和潜伏期逆转策略。我们最重要的假设是- 巨噬细胞和小胶质细胞中HIV的长期表达有助于神经精神损害，这是 因可卡因而加重。解决这些关键问题需要能够确定身份的方法 携带HIV DNA和RNA并同时将HIV DNA和RNA的存在与功能 通过转录组在同一个细胞中产生的细胞结果。结合DNA和蛋白质组学的蛋白质组学方法 RNA测序与表面标记表达的分析为识别细胞群体提供了洞察力 HIV DNA和RNA在特定的CD4T细胞亚型中的表达然而，重要的警告阻碍了他们的 在中枢神经系统细胞中的适用性。我们将开发一种新的分子、高分辨率、单细胞水平的方法，我们称之为 HIV整合前病毒DNA(HID)/单核RNAseq(HID-Seq)。在这种组合方法中，dCas9-tn5 标记化扩增整合在宿主基因组中的前病毒DNA，单核RNAseq使用经过验证的 用于检测特定HIV RNA物种的引物库，以及检测所有宿主细胞RNA物种的 同一间牢房。使用分裂测序对HIV前病毒DNA、HIV RNA和宿主细胞RNA进行分子条形码， 它追踪DNA和RNA产物到单个细胞。在R61阶段(目标1)，我们将为 同时检测宿主细胞转录组时HIV DNA和RNA表达的多水平索引 在试管中。在R33阶段(目标2)，我们将使用HID-Seq来确定可卡因对艾滋病毒DNA和 体外诱导人多能干细胞来源的小胶质细胞和巨噬细胞的RNA表达(AIM 2a) 从PWH尸检标本中获得的外周血单核细胞和中枢神经系统细胞(AIM 2b-c)。生物信息学分析将(I)确认携带艾滋病毒DNA和RNA的细胞的身份和特征 在有可卡因使用史的PWH的中枢神经系统，以及(Ii)确定可卡因的使用如何改变这种联系 HIV DNA和RNA与宿主细胞转录图谱之间的关系。

Early CNS entry of HIV fosters its expression in perivascular macrophages and microglia; but, whether HIV expression in these cells is associated with persistent neuropsychiatric damage is unclear. Non-HIV-related comorbidities such as substance use disorders can worsen neuropsychiatric deficits. Stimulants such as cocaine have been reported to alter HIV replication dynamics in macrophages and microglia and change their inflammatory state, and may contribute to neuropsychiatric deficits in PWH. A recent preprint provides the only evidence that frequent cocaine use is associated with larger HIV latent reservoir size in CD4+ T cells. Low- level/residual inflammation has been shown to foster a persistent HIV reservoir in CD4+ T cells. However, a major knowledge gap is whether cocaine exposure supports long-term HIV expression in macrophages and microglia and whether inflammation mediates this effect. A clear understanding of cocaine’s impact on HIV expression in complex multicellular contexts is a prerequisite for the identification of novel targets for effective ART regimens, adjunctive neuroprotective therapies, and latency reversal strategies. Our overarching hypothesis is that long- term HIV expression in macrophages and microglia contributes to neuropsychiatric damage which is exacerbated by cocaine. Addressing these key questions requires methods that can determine the identity of cells that harbor HIV DNA and RNA and simultaneously link the presence of HIV DNA and RNA to functional cellular outcomes via the transcriptome in the same cell. Proteogenomic approaches that combine DNA and RNA sequencing with the analysis of surface marker expression to identify cell populations provide insight into the expression of HIV DNA and RNA in specific CD4+ T cell subtypes. However, significant caveats hinder their applicability in CNS cells. We will develop a novel molecular, high-resolution, single-cell level method we call HIV integrated proviral DNA (HID)/single-nuclear RNASeq (HID-Seq). In this combined approach, dCas9-Tn5 tagmentation amplifies proviral DNA integrated in the host genome, and single-nuclear RNASeq uses validated primer libraries to detect specific HIV RNA species along with the detection of all host-cell RNA species in the same cell. HIV proviral DNA, HIV RNA, and host-cell RNA are molecularly barcoded using split-sequencing, which tracks DNA and RNA products to individual cells. In the R61 phase (Aim 1), we will develop HID-Seq for multilevel indexing of HIV DNA and RNA expression while simultaneously examining the host cell transcriptome in vitro. In the R33 phase (Aim 2), we will use HID-Seq to determine the impact of cocaine on HIV DNA and RNA expression in human induced pluripotent stem cell-derived microglia and macrophages in vitro (Aim 2a) and in the peripheral blood mononuclear cells and CNS cells obtained from autopsy specimens of PWH (Aim 2b-c). Bioinformatic analyses will (i) confirm the identity and characteristics of cells harboring HIV DNA and RNA in the CNS of PWH with history of cocaine use and (ii) determine how cocaine use alters the associations between HIV DNA and RNA and host-cell transcriptomic profile.