Outer membrane vesicles from Escherichia coli are efficiently internalized by macrophage cells and alter their inflammatory response

The release of extracellular vesicles (EV) by pathogenic microbes is considered an alternative cell-to-cell transport of macromolecules transport mechanism. In Gram-negative bacteria, EVs may be formed by outer membrane budding, so-called outer membrane vesicles (OMVs). Previous studies have revealed E. coli consti-tutively release nano-sized OMVs, which can be potent activators of cellular functions without live bacteria. But the immunomodulatory activity of E. coli OMVs is still relatively poorly understood. Here we investigated the morphological characterization and composition of E.Coli OMVs, kinetics of internalization by Raw 264.7 macrophage cells, and their immunomodulatory activity on cells. By transmission electron microscopy and dynamic light scattering, E.Coli OMVs were identified as typical cup-shaped, bilayered membranous structures, mainly distributed between 72.5 and 212.5 nm. We also demonstrated by confocal fluorescence microscopy that exposure of Raw 264.7 cells to E.Coli OMVs resulted in internalization of these nanoparticles and decreased mitochondrial membrane potential. In addition, E. Coli OMVs treatment induced the production of ROS, iNOS, IL-1 beta, IL-6, IL-10 and up-regulation of CD86 and CD206. Taken together, our results indicated that E.Coli OMVs are immunobiologically active, can directly interact with macrophage and participate in immune responses.