GW4869 Depletes Macrophages and Increases Number of Extracellular Vesicles in Murine Peritoneal Cavity Fluid

Pharmacological strategies to modulate extracellular vesicle (EV) release in vivo are gaining traction, yet their broader effects on local immune microenvironments remain unclear. In this study, we assessed how repeated intraperitoneal (i.p.) dosing of the neutral sphingomyelinase inhibitor GW4869 reshapes the peritoneal cavity. GW4869 administration triggered a strong local inflammatory reaction, was accompanied by a selective depletion of resident peritoneal macrophages and was associated with higher EV counts 24 h after the final injection. Targeted macrophage depletion in the absence of GW4869 produced a comparable increase in EV levels, implicating shifts in cellular composition and inflammatory state as key drivers of EV accumulation. By applying single-particle flow cytometry, we differentiated small EV subsets from ApoB+ lipoproteins and uncovered substantial heterogeneity within CD9+ particles, ruling out lipoprotein co-detection as the main explanation for the elevated EV signal. Overall, our results highlight that GW4869 treatment can reshape the local immune landscape and that such context-dependent changes must be considered when using this compound to infer EV biogenesis in vivo.