Abstract

Prevotella copri (Pc) is an abundant Gram-negative bacterium of the human intestinal microbiota whose enrichment has been associated with disease-promoting effects on rheumatoid arthritis, diabetes mellitus, liver disease, gastrointestinal infections, and cancer. It has been reported that Pc induces a pro-inflammatory cytokine profile in murine antigen presenting cells which favors the induction of Th17 cell responses. During the last decade, outer membrane vesicles (OMVs) secreted by Gram-negative bacteria have gained interest due to their role in delivering virulence factors to immune cells and distant organs, thereby promoting systemic diseases. Thus, the aim of this study was to explore Pc OMVs, its internalization, and effects on human macrophages. We characterized Pc OMVs in terms of size, morphology and composition using nanoparticle tracking analysis, transmission electron microscopy and proteomics. To analyze endocytosis, we monitored uptake of DiO-labelled Pc OMVs into macrophages by confocal microscopy and flow cytometry using inhibitors of endocytic pathways. Moreover, we analyzed the effect of Pc OMVs on phenotype and cytokine secretion of M0, M1 and M2 monocyte-derived macrophages by flow cytometry and ELISA, respectively. We found that Pc OMVs are readily internalized by macrophages, mainly via clathrin-mediated mechanisms. Moreover, Pc OMVs promoted a dose-dependent (re-)polarization of M0 and M2 macrophages towards a pro-inflammatory M1 phenotype and cytokine profile. The results highlight the important role of Pc OMVs in promoting inflammatory macrophage responses, which might contribute to the development and progression of systemic diseases. The authors thank ANID-Chile for financial support (FONDECYT 11220882).