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.

抽象的 全世界约有3800万人患有艾滋病毒。尽管抗逆转录病毒疗法（ART），但15-40％ 艾滋病毒（PWH）患者发展与HIV相关的神经认知障碍（HIV-NCI）。艾滋病进入大脑 早期，通常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种机制是 跨血脑屏障（BBB）的HIV感染的中间CD14+ CD16+单核细胞的迁移。 CD14+CD16+或成熟的单核细胞在PWH的血液中增加，最容易受到HIV感染， 并优先跨BBB传播。 BBB的这种移民有助于 CNS病毒储存库和慢性神经炎症的建立和恢复，导致神经毒性 介导HIV-NCI发育的环境和神经元损伤。因此，表征成熟 单核细胞对于理解它们有助于其发病机理的机制至关重要 在艾滋病毒存在下的炎症性疾病，并定义阻断其病理作用的方法。到 表征成熟单核细胞的功能特性以及这些细胞中HIV感染的作用，我们 先前对未感染和HIV感染的成熟的单细胞RNA测序（SCRNA-SEQ） 单核细胞。这项研究表明，两个种群分为9个单核细胞簇。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途径比较 在其他簇中显示了未感染和HIV感染细胞中的两组单核细胞簇， 与另一个集群组中，大多数基因在这些途径中的表达增加（第1组）相比 （第2组）。基因表达的这种差异是否表明迁移，炎症和 与第2组的单核细胞相比，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，它们优先跨BBB迁移到CCL2，并产生 与第2组相比 单核细胞簇。目标1：表征第1组和第2组成熟单核簇的转录组 确定其对增加炎症和BBB传播的贡献的机制。我们将验证 该组1簇显示出与基因相关的蛋白质的中值荧光强度增加 通过流式细胞仪在第1组单核细胞簇中表达，并通过QRT-PCR确认这些基因的表达。 AIM 2：与第2组成熟相比，第1组的BBB传播和炎症特性表征 单核细胞簇。我们将通过确定ROS， 细胞因子和流式细胞仪的趋化因子产生，通过我们的体外BBB模型进行迁移倾向， 并阻止靶向1组蛋白的抗体，以查看是否抑制了迁移。 scrna-seq也将是 在转移的细胞上进行，以更好地表征和定义第1组和第2组单核簇。