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

• 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
• 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

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.

摘要 全世界约有3800万艾滋病毒携带者。尽管接受了抗逆转录病毒治疗(ART)，15%-40%的 艾滋病毒携带者(PWH)会出现艾滋病毒相关的神经认知障碍(HIV-NCI)。艾滋病毒进入大脑 早期，通常是在人们意识到自己的艾滋病毒状况或被诊断出来之前。中枢神经系统感染的一个机制是 HIV感染的中间CD14+CD16+单核细胞跨血脑屏障(BBB)的迁移。 CD14+CD16+或成熟单核细胞在PWH患者的血液中增加，是最容易感染HIV的人， 并优先跨越血脑屏障轮回迁徙。这种跨越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 介导HIV-NCI发展的环境和神经元损伤。因此，将成熟描述为 单核细胞对于理解它们在慢性粒细胞白血病发病机制中的作用至关重要。 在艾滋病毒存在的情况下的炎症性疾病，并确定阻断其病理影响的方法。至 研究成熟单核细胞的功能特性以及HIV感染对这些细胞的影响 之前对未感染和感染艾滋病毒的成熟细胞进行了单细胞RNA测序(scRNA-seq) 单核细胞。研究表明，这两个种群分为9个单核细胞群。基因的表达 从每个集群中涉及迁移、炎症或神经毒性功能的分子通路进行比较 在未感染和感染艾滋病毒的细胞中都发现了两组单核细胞簇， 在一个聚类组(组1)中，这些通路中的大多数基因的表达高于另一个聚类组(组1 (2)组。这种基因表达的差异是否表明移行性、炎症性和 组1中单核细胞与组2中单核细胞的神经毒性特性尚不清楚。我们 假设第1组中未感染和感染艾滋病毒的成熟单核细胞表达高水平的基因 在迁移和炎症功能方面，它们优先跨血脑屏障迁移到CCL2，并产生 与第二组相比，更多的炎性细胞因子、趋化因子和ROS导致HIV神经发病 单核细胞簇。目的1：鉴定第1组和第2组成熟单核细胞簇的转录组 确定它们导致炎症增加和血脑屏障迁移的机制。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 用流式细胞仪检测这些基因在第1组单核细胞簇中的表达，并用qRT-PCR方法证实这些基因的表达。 目的2：对比成熟组1和成熟组2的血脑屏障移行和炎症特性 单核细胞簇。我们将通过测定ROS来确定第1组单核细胞簇的功能特性， 细胞因子和趋化因子的产生通过流式细胞术，我们的体外血脑屏障模型的迁移倾向， 以及阻断针对第一组蛋白的抗体，看看转移性是否被抑制。ScRNA-seq也将被 对移植的细胞进行实验，以更好地表征和定义组1和组2单核细胞簇。