Bacterial Stimulation Remodels Macrophage Extracellular Vesicle Lipids and Reveals iNOS as an Inflammatory Cargo

Abstract

Background: Inflammation drives the progression of acute and chronic diseases, including sepsis and peritonitis, yet the mechanisms by which bacterial stimuli alter intercellular inflammatory communication remain incompletely understood. Extracellular vesicles (EVs) are increasingly recognized as important mediators of immune signaling and may transmit stimulus-specific lipid and protein cargo between cells. We investigated whether bacterial stimulation remodels macrophage-derived EV composition and whether these changes are associated with altered EV uptake and inflammatory activity. Methods: EVs were isolated from RAW264.7 macrophages under basal conditions or following stimulation with bacterial lysate (BL) from Lacticaseibacillus rhamnosus CCM7091 or lipopolysaccharide (LPS). EV identity and quality were assessed according to MISEV recommendations. Lipidomic profiling was performed to define stimulus-associated lipid remodeling, while EV-associated inducible nitric oxide synthase (iNOS) and its enzymatic activity were evaluated using biochemical approaches. EV uptake and functional activity were assessed in macrophages and endothelial cells. EV-associated iNOS was further investigated in murine models of peritoneal inflammation and fibrosis and in EVs isolated from peritoneal exudates of patients with acute peritonitis. Results: Bacterial stimulation altered the molecular composition and biological activity of macrophage-derived EVs. BL-EVs exhibited enrichment of saturated fatty acids and ceramides, generating a distinct lipid signature associated with enhanced cellular uptake. Functionally, bacteria-stimulated EVs promoted macrophage activation, increasing nitric oxide and TNF production, while LPS-EVs additionally induced endothelial IL-6 and CCL5/RANTES production and ICAM-1 expression. Importantly, enzymatically active iNOS was identified in bacteria-stimulated EVs, demonstrating functional iNOS cargo in small EVs derived from a defined macrophage cell line. EV-associated iNOS was further detected in murine models of peritoneal inflammation and fibrosis and in patient-derived EVs from acute peritonitis, supporting its relevance beyond the in vitro system. Conclusions: Bacterial stimulation remodels macrophage-derived EV lipid and protein cargo and generates EV populations with altered uptake and inflammatory activity. These findings identify stimulus-dependent lipid remodeling as a feature of bacteria-stimulated EVs and establish enzymatically active iNOS as a previously unrecognized inflammatory EV cargo. The detection of EV-associated iNOS in experimental models and patients with peritoneal inflammation further supports its potential as a biomarker of inflammatory disease and for therapeutic modulation of EV-mediated immune signaling.