Despite the success of antiretroviral therapy (ART), cardiovascular (CVD), liver (LD), and other chronic diseases increasingly replace AIDS-related complications as the most common causes of morbidity and mortality in HIV-1-infected patients. Gut mucosal damage leading to increased intestinal permeability and ongoing chronic microbial translocation is a central factor in persistent systemic immune activation and inflammation and represents a critical driving mechanism of HIV-1 pathogenesis. However, the specific mechanisms and mediators of these processes are not well understood. Neutrophils, the most abundant immune cell population in the body, are specifically geared for sensitive detection of invading microbial and viral pathogens and represent the first and most robust innate immune population responding to microbial translocation. Neutrophil NETosis results in the formation of neutrophil extracellular traps (NETs) that promote endothelial damage, atherosclerosis, and atherothrombosis. NETs provide the stimulus and the scaffold for thrombus formation, prime macrophages for the production of cytokines that amplify immune cell recruitment in atherosclerotic plaques, and induce endothelial damage. We show that neutrophils from HIV-1-infected patients display a high capacity for NETosis and the production of NETs. The goal of this application is to identify the specific subpopulations of neutrophils responsible for NETosis in HIV-1-infected individuals. The central hypothesis of this proposal is that HIV-1 infection is associated with the induction and expansion of specific neutrophilic subpopulations with increased capacity to undergo NETosis. Reactive oxygen species (ROS) and NETs released from activated neutrophils promote organ damage and contribute to the progression of CVD, LD, and other chronic conditions. This hypothesis has been formulated on the basis of our own preliminary data and recently published reports demonstrating the critical role of neutrophils in HIV-1 infection and other chronic inflammatory conditions. The overall objective of the proposed studies is to elucidate the mechanisms responsible for chronic neutrophilic activation and production of NETs in HIV-1 infection in order to reveal the specific checkpoints for intervention. In preliminary studies, we optimized the methods for detailed neutrophil characterization and showed that neutrophils from HIV-1-infected individuals display an activated phenotype, immunosuppressive properties, specific transcriptional profile, increased rate of degranulation, and a high capacity to undergo NETosis. Using a novel method based on cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq), we identified specific neutrophil subpopulations in chronic inflammatory conditions. Specific properties of the newly identified neutrophil subpopulations strongly indicate that they play a critical role in the pathogenesis of HIV-1 infection. The objectives of this proposal will be a...