Multimodal profiling of microglia during HIV infection and substance use disorder

HIV 感染和物质使用障碍期间小胶质细胞的多模式分析

• 批准号: 10813965
• 负责人: Cagla Akay Espinoza
• 金额: $ 54.79万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-30 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10813965
• 关键词: Address; Anatomy; Anti-Retroviral Agents; Autopsy; Bar Codes; Bioinformatics; Blood Vessels; CD4 Positive T Lymphocytes; Cell Line; Cell physiology; Cells; Characteristics; Cocaine; Complex; DNA; DNA Integration; DNA sequencing; Detection; Exhibits; Fostering; Gene Expression; Gene Expression Profile; Genetic Transcription; Genome; HIV; HIV Infections; Human; In Vitro; Individual; Inflammation; Inflammatory; Knowledge; Libraries; Life Expectancy; Link; Location; Macrophage; Mediating; Methods; Microglia; Modeling; Molecular; Molecular Profiling; Myelogenous; Myeloid Cells; National NeuroAids Tissue Consortium; Neurologic; Nuclear; Outcome; Pathway interactions; Peripheral Blood Mononuclear Cell; Persons; Pharmaceutical Preparations; Phase; Physiological; Population; Printing; Productivity; Proviruses; RNA; Recording of previous events; Recrudescences; Regimen; Reporting; Residual state; Resolution; Special Equipment; Specimen; Stimulant; Substance Use Disorder; Surface; T-Lymphocyte; Technology; Tissues; Viral; Virus; antiretroviral therapy; cell type; cocaine exposure; cocaine use; comorbidity; examination questions; improved; in vivo; indexing; induced pluripotent stem cell; insight; latent HIV reservoir; multimodality; neuroprotection; neuropsychiatry; novel; novel strategies; novel therapeutic intervention; prevent; proteogenomics; tool; transcriptome; transcriptome sequencing; transcriptomics

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.

艾滋病毒的早期中枢神经系统进入促进其在血管周围巨噬细胞和小胶质细胞中的表达。但是，是否艾滋病毒 这些细胞中的表达与持续的神经精神损伤有关。非HIV相关 诸如药物使用障碍之类的合并症可能会恶化神经精神病缺陷。可卡因等兴奋剂 据报道会改变巨噬细胞和小胶质细胞中的HIV复制动力学，并改变其 炎症状态，并可能导致PWH中的神经精神缺陷。最近的预印本提供了唯一的 CD4+ T细胞中使用可卡因常常与较大的HIV潜在储层大小有关的证据。低的- 已证明水平/残留炎症可在CD4+ T细胞中促进持续的HIV储量。但是，一个专业 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 以及炎症是否介导这种作用。对可卡因对艾滋病毒表达的影响的清晰了解 复杂的多细胞上下文是确定有效艺术方案的新目标的先决条件， 辅助神经保护疗法和潜伏期逆转策略。我们的总体假设是长期 巨噬细胞和小胶质细胞中的术语HIV表达有助于神经精神损害 可卡因加剧。解决这些关键问题需要可以确定的方法 具有HIV DNA和RNA的细胞，并简单地将HIV DNA和RNA的存在连接到功能 细胞结局通过同一细胞中的转录组。结合DNA和 通过分析表面标记表达以鉴定细胞群的RNA测序提供了洞察力 HIV DNA和RNA在特定CD4+ T细胞亚型中的表达。但是，重要的警告阻碍了他们 CNS细胞中的适用性。我们将开发一种新型的分子，高分辨率的单细胞水平方法 HIV综合临时DNA（HID）/单核RNASEQ（HID-SEQ）。在这种组合方法中，dcas9-tn5 标记放大器在宿主基因组中集成的前病毒DNA，单核RNASEQ使用经过验证的 引物文库检测特定的HIV RNA种类以及检测所有宿主细胞RNA种类 相同的单元。 HIV前病毒DNA，HIV RNA和宿主 - 细胞RNA使用分裂序列进行分子测量， 它跟踪DNA和RNA产物到单个细胞。在R61阶段（AIM 1），我们将为 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（AIM 2），我们将使用HID-SEQ来确定可卡因对HIV DNA和 人类诱导多能干细胞衍生的小胶质细胞和巨噬细胞的RNA表达体外（AIM 2A） 在外周血单核细胞和CNS细胞中，从PWH的尸检标本获得（AIM 2b-c）。生物信息学分析将（i）确认具有HIV DNA和RNA的细胞的身份和特征 在PWH的中枢神经系统中，可卡因使用史和（ii）确定可卡因的使用如何改变关联 在HIV DNA和RNA以及宿主细胞转录组谱之间。