Wednesday, December 4, 2024
12/4/2024
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.
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