Neutrophil-secreted extracellular vesicles induce blood-brain barrier leakage and tight junction disruption.

Extracellular vesicles (EVs) are potent mediators in cell-cell communication that regulate diverse cell functions through the delivery of their bioactive cargo molecules to recipient cells. Previous work from our group demonstrated elevated plasma EV levels in patients and animals following septic or inflammatory insults, with a substantial proportion originating from neutrophils. As frontline defenders against bacterial infection, activated neutrophils release germicidal factors, some of which circulate systemically and inflict collateral tissue damage at a distance, including the brain. The role of neutrophil-derived EVs in regulating blood-brain barrier (BBB) structure and permeability after septic injury remains poorly defined. In this study, we first characterized EV production by mouse neutrophils stimulated with bacterial lipopolysaccharide (LPS) and subsequently investigated their functional and mechanistic effects on BBB integrity under in vivo and in vitro settings. Nanoparticle tracking analysis (NTA), immunoblotting, and transmission electron microscopy (TEM) revealed that LPS stimulation of neutrophils promoted EV secretion, indicated by increased particle number and protein content. Systemic administration of these EVs in mice induced cerebral microvascular leakage of plasma tracers (sodium fluorescein, at 376-Da; and dextran at 3-kDa) as quantified by near-infrared (NIR) nano-imaging and fluorometric assays. In cultured brain microvascular endothelial monolayers, EVs from naïve unstimulated neutrophils exerted minimal effects, whereas EVs from LPS-stimulated neutrophils caused a concentration-dependent reduction in transendothelial electrical resistance (TER) and a significant increase in solute permeability, indicative of paracellular hyperpermeability. Confocal microscopy revealed that tight junction proteins claudin-5 and zonula occludens-1 (ZO-1), which normally form continuous belt-like structures at endothelial cell–cell contacts, appeared discontinuous or fragmented upon EV internalization. Consistently, endothelial cells exposed to activated neutrophil-derived EVs exhibited reduced expression of tight junction proteins. Furthermore, TEM of brain capillaries from EV-injected mice provided ultrastructural evidence of tight junction disruption. Collectively, these findings suggest that neutrophil activation in response to infection promotes BBB leakage through the release of EVs capable of compromising endothelial tight junction integrity. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12987-025-00744-8.