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3 800万人感染艾滋病毒。尽管有抗逆转录病毒疗法，15-40%的 HIV感染者（PWH）会出现HIV相关的神经认知障碍（HIV-NCI）。艾滋病毒进入大脑 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。CNS感染的一种机制是 HIV感染的中间CD 14 + CD 16+单核细胞穿过血脑屏障（BBB）的迁移。 CD 14 + CD 16+，或成熟单核细胞在PWH的血液中增加，是最容易感染HIV的， 并优先穿过BBB。这种穿过BBB的迁移有助于 CNS病毒储库的建立和再接种以及导致神经毒性的慢性神经炎症 环境和神经元损伤介导的HIV-NCI发展。因此，描述成熟 单核细胞是至关重要的了解机制，他们有助于发病机制， 在HIV存在下的炎症性疾病，并确定阻断其病理作用的方法。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 先前进行的未感染和HIV感染的成熟细胞的单细胞RNA测序（scRNA-seq）， 单核细胞这项研究表明，两个群体分为9个单核细胞簇。的基因表达 从分子途径参与迁移，炎症，或神经毒性功能，在每个集群比较 在未感染和HIV感染的细胞中发现了两组单核细胞簇， 与另一组相比，一个簇组（组1）中这些途径中大多数基因的表达增加 （第二组）。基因表达的这种差异是否表明转迁、炎症和 与来自组2的那些相比，来自组1的簇中的单核细胞的神经毒性性质是未知的。我们 假设第1组群中未感染和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CCL 2，并产生 与第2组相比，导致艾滋病毒神经发病机制的炎性细胞因子、趋化因子和活性氧更多 单核细胞簇。目的1：表征组1和组2成熟单核细胞簇的转录组， 确定它们对增加炎症和BBB迁移的作用机制。我们将验证 第1组聚类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。 目的2：与第2组相比，表征第1组的BBB迁移和炎症特性。 单核细胞簇。我们将通过测定ROS， 通过流式细胞术测定细胞因子和趋化因子的产生，我们的体外BBB模型的迁移倾向， 以及针对第1组蛋白质的阻断抗体，以观察是否抑制了迁移。scRNA-seq也将是 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。