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Characterizing subsets of HIV-infected and uninfected CD14+CD16+ monocytes that contribute to neuropathogenesis

表征导致神经发病的 HIV 感染和未感染 CD14 CD16 单核细胞亚群

基本信息

批准号：
10619848
负责人：
Vanessa Ruiz
金额：
$ 5.18万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-01-16 至 2027-01-15
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10619848
关键词：
ALCAM gene Acute Antibodies Atherosclerosis Awareness Blocking Antibodies Blood Blood - brain barrier anatomy Brain CCL2 gene CD14 gene Cell Separation Cell Surface Proteins Cell physiology Cells Central Nervous System Infections Characteristics Chronic Demyelinations Development Diagnosis Disease Environment FCGR3B gene Flow Cytometry Fluorescence Gene Expression Genes HIV HIV Infections HIV-associated neurocognitive disorder Heart failure Human In Vitro Individual Inflammation Inflammatory Laboratories Longevity Mediating Modeling Molecular Morbidity - disease rate Myocardial Infarction Myocardial Ischemia Neuronal Injury Neuropathogenesis Pathogenesis Pathologic Pathway interactions Penetrance Persons Population Predisposition Production Property Proteins Quality of life Quantitative Reverse Transcriptase PCR RNA Tissue-Specific Gene Expression Viral reservoir age related antiretroviral therapy blood-brain barrier crossing blood-brain barrier function chemokine comorbidity cytokine effective therapy fibroglycan gene network medication compliance migration monocyte mortality mortality risk neuroinflammation neurotoxic novel single-cell RNA sequencing synaptic pruning therapeutic development transcriptome

项目摘要

ABSTRACT Approximately 38 million people worldwide are living with HIV. Despite antiretroviral therapy (ART), 15-40% of people with HIV (PWH) develop HIV-associated neurocognitive impairments (HIV-NCI). HIV enters the brain early, often before people are aware of their HIV status or diagnosed. One mechanism for CNS infection is the transmigration of HIV-infected intermediate CD14+CD16+ monocytes across the blood-brain barrier (BBB). CD14+CD16+, or mature monocytes are increased in the blood of PWH, are the most susceptible to HIV infection, and preferentially transmigrate across the BBB. This transmigration across the BBB contributes to the establishment and reseeding of CNS viral reservoirs and chronic neuroinflammation that results in a neurotoxic environment and neuronal injury that mediates HIV-NCI development. Therefore, characterizing mature monocytes is critical to understanding the mechanisms by which they contribute to the pathogenesis of inflammatory diseases in the presence of HIV, and to define ways to block their pathological effects. To characterize functional properties of mature monocytes and the effects of HIV infection in these cells, we previously performed single-cell RNA sequencing (scRNA-seq) of uninfected and HIV-infected mature monocytes. This study showed that both populations separated into 9 monocyte clusters. Expression of genes from molecular pathways involved in migratory, inflammatory, or neurotoxic functions in each cluster compared to other clusters revealed two groups of monocyte clusters in both uninfected and HIV-infected cells, with increased expression of most genes in these pathways in one cluster group (Group 1) compared to the other (Group 2). Whether this difference in gene expression indicates increased transmigratory, inflammatory, and neurotoxic properties of monocytes in clusters from Group 1 compared to those from Group 2 is unknown. We hypothesize that uninfected and HIV-infected mature monocytes in Group 1 clusters express high levels of genes in migratory and inflammatory functions, they preferentially transmigrate across the BBB to CCL2, and produce more inflammatory cytokines, chemokines, and ROS that contribute to HIV neuropathogenesis than Group 2 monocyte clusters. Aim 1: Characterize the transcriptome of Group 1 and Group 2 mature monocyte clusters to identify mechanisms of their contributions to increased inflammation and BBB transmigration. We will validate that group 1 clusters show increased median fluorescence intensity of proteins that correlate with genes expressed in Group 1 monocyte clusters by flow cytometry, and confirm expression of these genes by qRT-PCR. Aim 2: Characterize BBB transmigration and inflammatory properties of Group 1 compared to Group 2 mature monocyte clusters. We will determine functional properties of Group 1 monocyte clusters by determining ROS, cytokine, and chemokine production by flow cytometry, transmigration propensity with our in vitro BBB model, and blocking antibodies targeting Group 1 proteins to see if transmigration is inhibited. scRNA-seq will also be performed on transmigrated cells to better characterize and define Group 1 and Group 2 monocyte clusters.

抽象的全球大约有 3800 万人感染艾滋病毒。尽管进行了抗逆转录病毒治疗 (ART)，仍有 15-40% 的患者 HIV 感染者 (PWH) 会出现与 HIV 相关的神经认知障碍 (HIV-NCI)。艾滋病毒进入大脑尽早，通常是在人们意识到自己的艾滋病毒感染状况或确诊之前。中枢神经系统感染的一种机制是 HIV感染的中间CD14+CD16+单核细胞穿过血脑屏障(BBB)的迁移。 CD14+CD16+，或成熟单核细胞在感染者血液中增加，最容易感染艾滋病毒，并优先穿越BBB。这种跨 BBB 的轮回有助于中枢神经系统病毒库的建立和重新播种以及导致神经毒性的慢性神经炎症介导 HIV-NCI 发展的环境和神经元损伤。因此，成熟的特征单核细胞对于理解它们促进疾病发病机制的机制至关重要艾滋病毒存在下的炎症性疾病，并确定阻止其病理影响的方法。到为了表征成熟单核细胞的功能特性以及 HIV 感染对这些细胞的影响，我们之前对未感染和 HIV 感染的成熟细胞进行了单细胞 RNA 测序 (scRNA-seq) 单核细胞。这项研究表明，两个群体都分为 9 个单核细胞簇。基因表达从每个簇中涉及迁移、炎症或神经毒性功能的分子途径进行比较对其他簇的研究揭示了未感染和 HIV 感染细胞中的两组单核细胞簇，与其他组相比，一个簇组（第 1 组）中这些途径中大多数基因的表达增加（第 2 组）。基因表达的这种差异是否表明轮回、炎症和炎症的增加与第 2 组相比，第 1 组簇中的单核细胞的神经毒性特性尚不清楚。我们假设第 1 组簇中未感染和 HIV 感染的成熟单核细胞表达高水平的基因在迁移和炎症功能中，它们优先穿过 BBB 迁移到 CCL2，并产生与第 2 组相比，导致 HIV 神经病变的炎症细胞因子、趋化因子和 ROS 更多单核细胞簇。目标 1：表征第 1 组和第 2 组成熟单核细胞簇的转录组，以确定它们对炎症增加和血脑屏障迁移的作用机制。我们将验证第 1 组簇显示与基因相关的蛋白质的中值荧光强度增加通过流式细胞术检测在第 1 组单核细胞簇中表达的基因，并通过 qRT-PCR 确认这些基因的表达。目标 2：与第 2 组成熟相比，表征第 1 组的 BBB 迁移和炎症特性单核细胞簇。我们将通过确定 ROS 来确定第 1 组单核细胞簇的功能特性，通过流式细胞术检测细胞因子和趋化因子的产生，使用我们的体外 BBB 模型进行迁移倾向，并阻断针对第 1 组蛋白的抗体，看看迁移是否受到抑制。 scRNA-seq 也将对迁移的细胞进行，以更好地表征和定义第 1 组和第 2 组单核细胞簇。