The Impact on Virally Stimulated Airways Extracellular Vesicles on Brain Endothelial Cells

Recent biobank studies have identified a link between peripheral viral infections and neurodegeneration, yet the mechanisms underlying this connection remain poorly defined. This study examines whether extracellular vesicles (EVs) released from virally stimulated airway cells contribute to vascular and blood-brain barrier (BBB) dysfunction, thereby bridging a critical gap in our understanding of how peripheral viral infections drive neuroinflammation. Specifically, we evaluate the effects of EVs derived from airway epithelial cells stimulated with either polyinosinic-polycytidylic acid (poly I:C) or the SARS-CoV-2 receptor-binding domain (RBD) on hCMEC/D3, a human cerebral microvascular endothelial cell line that serves as an in vitro model of the BBB. Human bronchial epithelial cells (HBEC3-KT) were cultured in AlphaMEM medium, supplemented with 10% FBS, grown to ~90% confluency, and treated with either poly IC (12€ ŵg/mL, 2 h) or the RBD (100 ng/mL, 48 h), while untreated cells were used as controls EVs were isolated from conditioned cell culture medium supplemented with exosome-depleted serum using the prExo (Exolitus) precipitation-based isolation kit and characterised by nanoparticle tracking analysis (NTA) for size distribution and concentration, and cryogenic transmission electron microscopy (cryoTEM) for vesicle morphology. hCMEC/D3 were seeded and allowed to form confluent monolayers (4 days post-seeding) before treatment with EVs for 48 h. Endothelial cells metabolic activity was assessed using the PrestoBlue cell viability assay, mitochondrial function via Seahorse XF Mito Stress Test, BBB integrity by transendothelial electrical resistance (TEER), and endothelial migration by scratch wound healing assay. Statistical analyses were performed in GraphPad Prism. All EV isolation and characterisation procedures were conducted in accordance with the latest MISEV 2023 guidelines. Isolated EVs, mostly, were 100-150 nm diameter and had lipid bilayer. EVs from poly I:C- and RBD-stimulated airway cells significantly reduced hCMEC/D3 metabolic activity at 5A-10à particles/20000 cells, compared to treatment of EVs from healthy untreated airway cells. Seahorse analysis revealed decreased maximal respiration and spare respiratory capacity, with increased non-mitochondrial oxygen consumption in both treatment groups in endothelial cells. TEER measurements showed that both poly I:C-EVs and RBD-EVs impaired BBB integrity; however, only RBD-EVs significantly reduced endothelial migration in the scratch assay. EVs released from virally stimulated airway epithelial cells impair BBB endothelial metabolism, mitochondrial function, and barrier integrity, with RBD-EVs exerting additional inhibitory effects on endothelial migration. These findings highlight a potential mechanism by which peripheral viral infections may promote neurovascular dysfunction and contribute to neuroinflammation.