Particle Phagocytosis Amplifies Secretion of Small Extracellular Vesicles in Macrophages

ABSTRACT Macrophage-derived extracellular vesicles (EVs) are critical mediators of immune communication and tissue homeostasis, yet the exogenous factors that regulate their release and composition remain poorly defined. Here, we identify phagocytosis of particulate materials as a direct and general driver of macrophage small EV production through a lysosome-linked mechanism. Across primary and engineered murine and human macrophage cultures, phagocytic uptake robustly increased small EV secretion, an effect that was recapitulated in vivo following orotracheal instillation that resulted in elevated small EV abundance in bronchoalveolar lavage fluid. This phagocytosis-driven small EV amplification depended on particle size (hydrodynamic diameter >200 nm) and residence time within the phagolysosome. Electron microscopy and proteomic analyses revealed increased multivesicular body formation following particle uptake, establishing a mechanistic link between phagocytosis and small EV biogenesis. Notably, this pathway regulated both protein and particulate cargo in secreted macrophage small EVs. Together, these findings expand the canonical framework of macrophage EV biogenesis to include phagocytosis-driven regulation and establish a single step process for controlling small EV production and cargo loading, with implications for understanding particulate exposures and for developing macrophage- and EV-based immunotherapies.