Multimodal profiling of microglia during HIV infection and substance use disorder

HIV早期进入CNS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 在这些细胞中的表达与持续的神经精神损害的关系尚不清楚。非HIV相关 诸如物质使用障碍的合并症可使神经精神缺陷恶化。兴奋剂如可卡因 已经报道了改变HIV在巨噬细胞和小胶质细胞中的复制动力学，并改变它们的 炎症状态，并可能导致PWH的神经精神缺陷。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CD 4 + T细胞中更大的HIV潜伏库有关。低- 水平/残余炎症已显示在CD 4 + T细胞中促进持久的HIV储库。然而一个主要 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的长期HIV表达 以及炎症是否介导了这种效应。明确了解可卡因对艾滋病毒表达的影响， 复杂的多细胞环境是鉴定有效ART方案的新靶点的先决条件， 连续性神经保护疗法和潜伏期逆转策略。我们首要的假设是- 巨噬细胞和小胶质细胞中的长期HIV表达有助于神经精神损害， 可卡因加剧了病情解决这些关键问题需要能够确定 携带HIV DNA和RNA的细胞，同时将HIV DNA和RNA的存在与功能性 通过同一细胞中的转录组控制细胞结果。蛋白基因组学方法将联合收割机DNA和 RNA测序与表面标志物表达的分析，以确定细胞群提供了深入了解 HIV DNA和RNA在特定CD 4 + T细胞亚型中的表达。然而，重要的警告阻碍了他们的 适用于CNS细胞。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，dCas 9-Tn 5 标签化扩增整合在宿主基因组中的前病毒DNA，单核RNASeq使用经验证的 引物库，以检测特定的HIV RNA种类沿着检测所有宿主细胞中的RNA种类。 相同单元HIV前病毒DNA、HIV RNA和宿主细胞RNA使用分裂测序进行分子条形码化， 追踪DNA和RNA产物到单个细胞。在R61阶段（目标1），我们将开发HID-Seq， HIV DNA和RNA表达的多级索引，同时检查宿主细胞转录组 体外在R33阶段（目标2），我们将使用HID-Seq来确定可卡因对HIV DNA的影响， 体外人诱导多能干细胞衍生的小胶质细胞和巨噬细胞中的RNA表达（Aim 2a） PWH尸检标本外周血单个核细胞和中枢神经系统细胞（Aim 图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CNS中，以及（ii）确定可卡因使用如何改变 HIV DNA和RNA与宿主细胞转录组图谱之间的关系。

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.