Abstract 15540: Single Cell Transcriptional-Epigenetic Profiling and Sub-Cellular Proteomics Demonstrate That HIV-Nef Extracellular Vesicles Modulate Human Macrophage Heterogeneity to Promote Atherosclerosis

Background: In the era of modern Anti-Retroviral Therapy, people living with HIV continue to remain at a higher risk than the general population for cardiovascular diseases including atherosclerosis. We hypothesized that circulating extracellular vesicles (EV) containing HIV-Nef protein promote inflammation by switching the pattern of macrophage heterogeneity towards a pro-inflammatory state that underlies cardiovascular disease. Methods & Results: Human primary macrophages were stimulated with EV engineered to contain HIV-Nef. Macrophage heterogeneity was characterized by simultaneous single cell mRNA-seq and single cell ATAC-seq integrated with WNN mapping in 50,931 cells. Among 16 clusters (Fig. A), pseudotime (Slingshot), gene regulatory (DoRothEA and SCENIC) and transcription factor motif accessibility (ChromVar) analyses demonstrated that HIV-Nef regulates 3 inflammatory clusters as gauged by differentially active and accessible transcription factors, e.g., JUN, YY1 and STAT1. Sub-cellular proteomics identified differentially abundant proteins associated with DNA (662 proteins), nucleus (261), cytosol (461), cell surface (72) and EV (132). Pathway enrichment analysis of these proteins revealed changes in biological processes in immune responses, cytoskeletal regulation, and metabolism. Functional phenotyping showed HIV-Nef shifts macrophages towards a pro-inflammatory phenotype by suppressing atheroprotective efferocytosis, promoting chemotaxis and altering metabolic state. Furthermore, short-term injection of HIV-Nef EV into WT mice induced inflammatory chemokine secretion, while long-term injection into Ldlr -/- mice enhanced atherogenesis (Fig.B). Conclusions: Persistent Nef in EV of HIV survivors may modulate macrophage heterogeneity to promote chronic inflammation. These findings may help identify patients at risk for cardiovascular complications and develop novel atheroprotective therapies.