Small extracellular vesicles from Salmonella typhimurium-infected macrophages overexpress proteolytically active Matrix Metalloproteinase 9 mediated by Toll-like Receptor 4 signaling

Extracellular vesicles (EVs) are a heterogenous group of lipid bilayer particles originating from the endosomal system or shed by the plasma membrane. EVs contain a wide variety of cargo, from nucleic acids to entire proteins, affecting diverse physiological and pathophysiological pathways in health and disease. EVs have been studied primarily as signaling particles, and very little is known about their capacity to proteolytically modify the extracellular environment. Our aim is to shed light on the composition and function of EVs secreted from Salmonella typhimurium-infected macrophages compared with uninfected controls. We further study EVs as bearers of bioactive proteolytic enzymes capable of modifying the extracellular environment. We established an in vitro model of Salmonella typhimurium infection in a murine macrophage cell line and isolated and characterized a population of small EVs with size range of 40 to 160 nm in diameter. We found that EVs from infected macrophages demonstrated significantly increased expression of proteolytically active Matrix Metalloproteinase 9 (MMP-9) capable of cleaving components of the extracellular matrix and the basement membrane. We further identified that the pathway responsible for MMP-9 upregulation in EVs is selective to immune cells and is regulated by the Toll-like Receptor 4 (TLR-4) mediated response to bacterial lipopolysaccharide. These findings indicate that EVs secreted from infected macrophages overexpress proteolytically active MMP-9, capable of modifying the extracellular environment as a part of a selective immune response to infection by gram negative bacteria.