In differentiated HL-60 neutrophil-like cells, MRP1- (ABCC1-) mediated glutathione efflux stimulated by BzATP and P2X7 receptor signalling regulates exosome release through nSMase activity.

Exosomes are endosome-derived extracellular vesicles (EVs) playing key roles in immune regulation and inflammatory signalling, yet with poorly defined mechanisms of biogenesis. We investigated how purinergic signalling and redox regulation intersect, controlling exosome formation in NLCs differentiated from the HL-60 cell line. Differentiated NLCs displayed hallmark neutrophil features, including increased CD11b expression, reduced CD71, enhanced phagocytosis, robust reactive oxygen species generation, and cell-cycle arrest. CD63-positive exosomes released from NLCs fulfilled MISEV 2023 criteria, exhibiting typical morphology, density, and size distribution. Stimulation with the P2X7R agonist BzATP induced a rapid, dose-dependent increase in exosome release correlating strongly with depletion of intracellular glutathione (iGSH) and activation of neutral sphingomyelinase (nSMase). Pharmacological inhibition of nSMase abrogated BzATP-induced exosome release, implicating ceramide-dependent intraluminal vesicle formation. Mechanistically, BzATP activated PI3K/AKT signalling, leading to stimulation of the ABC transporter MRP1/ABCC1, enhanced efflux of GSH, and a concomitant rise in extracellular GSH. Inhibition of PI3K/AKT or MRP1 prevented iGSH efflux, reduced nSMase activity, and significantly attenuated exosome secretion. These findings define a previously uncharacterized pathway in Neutrophil-like cells (NLCs) whereby extracellular ATP acts as a danger signal triggering PI3K/AKT-MRP1-dependent redox gating of nSMase activity, thereby driving rapid exosome biogenesis. This mechanism provides insight into how neutrophils translate inflammatory cues into vesicle-mediated communication and highlights potential therapeutic targets to modulate pathological neutrophil-driven inflammation.