Amplifying Inflammation: Lipid Remodeling and Cargo Transfer by Macrophage-Derived Extracellular Vesicles

Inflammation drives pathology in acute and chronic diseases, including sepsis and peritonitis, where dysregulated immune activation leads to serious disease progression. As extracellular vesicles (EVs) are emerging carriers of inflammatory signals, defining how bacterial stimuli reshape macrophage-derived EVs is of direct clinical importance. Here, we show that bacterial lysate (BL) from Lacticaseibacillus rhamnosus CCM7091 induces marked lipid remodeling in EVs from RAW264.7 macrophages. Lipidomics revealed enrichment of saturated fatty acids and ceramides, forming a pro-inflammatory lipid signature and corresponding with enhanced cellular uptake of BL-EVs. Functionally, bacteria-stimulated EVs (BL-treated alongside the standard LPS model) promoted macrophage activation, increasing nitric oxide and TNF production, while LPS-EVs additionally induced endothelial activation through IL-6, RANTES, and ICAM-1 upregulation. We further identified inducible nitric oxide synthase (iNOS) within bacteria-stimulated EVs, the first demonstration of its incorporation into small EVs and EVs from a defined cell line and confirmed its presence in EVs from murine models of peritoneal inflammation and fibrosis. These findings identify lipid remodeling as a defining feature of bacteria-stimulated EVs that correlate with their pro-inflammatory activity and highlight iNOS as a novel inflammatory cargo. Together, our results establish bacteria-stimulated EVs as active mediators of immune activation with potential biomarker and therapeutic relevance.