Role of extracellular vesicles derived from HIV-infected T cells and microglial cells in neuroinflammation and neuropathogenesis using a blood–brain barrier and cerebral organoid co-culture model

Despite effective antiretroviral therapy (ART) in suppressing HIV replication and reducing viral loads, latent reservoirs within the central nervous system (CNS) may contribute to persistent immune dysfunction, inflammation, and neurocognitive impairment in people living with HIV. Extracellular vesicles (EVs) isolated from serum and cerebrospinal fluid have been reported to contain HIV-1 RNAs, with their abundance associated with neurocognitive outcomes in ART-treated individuals. However, the mechanistic contribution of HIV-associated EVs in CNS pathology remains incompletely understood. In this study, we characterized EVs derived from latently HIV-1-infected T cells and microglial cells and identified HIV RNAs, including transactivation response element (TAR) RNA, Nef, and Tat RNA, within these particles. These EVs disrupted the integrity of a triple-cell blood–brain barrier (BBB) model by inducing apoptosis in BBB-associated cells. HIV-associated EVs were also able to traverse the BBB and promote apoptosis in cultured neuronal cells. In cerebral organoids derived from human induced pluripotent stem cells, these EVs induce production of pro-inflammatory cytokines, including IL-1β and TNF-α. Single-cell RNA sequencing of cerebral organoids exposed to microglial cell-derived EVs revealed upregulation of genes involved in double-stranded RNA signaling, apoptosis, inflammation, and microglial activation in response to HIV-associated EVs. Additionally, these EVs induced increased expression and phosphorylation of protein kinase R (PKR) in cerebral organoids. Plasma-derived EVs containing HIV RNA from individuals with HIV, but not control EVs from uninfected donors, induced apoptosis in both cerebral organoids and neuronal cells. These findings suggest that HIV-associated EVs may contribute to neuroinflammatory processes and cellular injury in CNS models, potentially informing mechanisms underlying HIV-related neurocognitive impairment that persist despite ART.