Extracellular Vesicles Secreted by Human Microglia Support Blood–Brain Barrier Function Under Both Physiological and Inflammatory Conditions

Brain endothelial cells (BECs) are essential for maintaining central nervous system homeostasis through the formation of the blood-brain barrier (BBB), whose integrity depends on continuous signaling from neighboring cells of the neurogliovascular unit. Although microglia are positioned in close proximity to cerebral vessels, their contribution to BBB regulation remains poorly defined. Here, we investigated the impact of paracrine signals from human microglia on BBB function under homeostatic and inflammatory conditions. Using a non-contact co-culture system with human induced pluripotent stem cell-derived BECs, we found that unstimulated microglia enhanced BBB integrity, as evidenced by increased transendothelial electrical resistance (TEER), upregulation of the tight junction proteins claudin-5, occludin, and ZO-1, and promotion of BEC proliferation. In contrast, TNFα-activated microglia did not support barrier integrity. Conditioned media from unstimulated microglia recapitulated the TEER-enhancing effects, whereas extracellular vesicle (EV)-depleted conditioned media failed to do so, implicating EVs as key mediators. Purified EVs from both unstimulated and TNFα-activated microglia promoted proliferation, increased TEER, elevated tight junction protein expression, and, notably, mitigated TNFα-induced barrier disruption by preserving TEER, endothelial proliferation, and tight junction continuity. Together, these findings identify microglia-derived EVs as potent positive regulators of BBB integrity and demonstrate that, even under inflammatory activation, microglial EVs retain the capacity to support endothelial barrier function. This work reveals a previously underappreciated mechanism by which microglia contribute to vascular homeostasis and suggests microglial EVs as potential therapeutic targets for preserving BBB integrity in neuroinflammatory diseases.