Comparative analysis of extracellular vesicles released from resting neutrophils under distinct pharmacological inhibition

Cells in resting conditions spontaneously release extracellular vesicles (EVs). Comparative studies for pharmacological EV inhibition, targeting multiple biogenesis elements within the same cell type, remain scarce. Spontaneously released neutrophil-derived EVs are important mediators of immunity that exhibit anti-inflammatory properties. Here, we present a systematic investigation of seven pharmacologic inhibitors (calpeptin, D-pantethine, Y27632, cytochalasin D, GW4869, R5421, and Nexinhib20) on the quantitative and qualitative properties of the selected EV population (16,100 g pellet obtained by filtration, size‑exclusion chromatography, and differential centrifugation) derived from resting neutrophils. We measured EV numbers, size distribution, total lipid, protein, and RNA content. We also analyzed EV morphology by cryogenic electron microscopy and EV cargo composition qualitatively by mass spectrometry and RNA sequencing. The results revealed differential profiles of proteins, mRNAs, and miRNAs depending on the inhibitor applied, as well as changes in the physical characteristics of the EVs. Inhibition of a single biogenesis pathway often led to compensatory upregulation of alternative vesicle formation mechanisms, as reflected in preserved EV numbers but altered cargo composition. These findings underscore the necessity of carefully selecting and reporting EV-inhibiting pharmacologic agents, as each compound uniquely shapes both the quantity and biological cargo of released EVs, and may have physiological consequences.