P066 Crohn's disease-associated adherent-invasive Escherichia coli induce secretion of exosomes with pro-inflammatory activity by intestinal epithelial cells
J. Carrière*, A. Bretin, N. Barnich, H.T.T. Nguyen
UMR 1071 Inserm/University of Auvergne; USC-INRA 2018, Microbes, Intestine, Inflammation and Susceptibility of the host, Clermont-Ferrand, France
Crohn's disease (CD) is a chronic inflammatory bowel disease of which the etiology involves environmental, genetic and microbial factors. Our group and others have shown a high prevalence of the invasive Escherichia coli strains, designated adherent-invasive E. coli (AIEC), in the intestinal mucosa of CD patients. Exosomes are small endosomal-derived vesicles involved in cell to cell communication and have been implicated in various diseases including cancer and infectious disorders. It has been reported that mammalian cells infected with pathogens can release exosomes containing microbial compounds. Here, we investigated the capacity of CD-associated AIEC bacteria to induce secretion of exosomes by intestinal epithelial cells and to determine the inflammatory characteristics of the released exosomes.
Human intestinal epithelial T84 cells were infected with the AIEC reference strain LF82. Exosomes were purified using the ExoQuick exosome precipitation reagent. Exosomes released by LF82-infected T84 cells were tested for their ability to promote pro-inflammatory responses in naïve macrophagic cells. Identification of exosomal proteins was performed by mass spectrometry.
Electron microscopy and immunogold-labeling analyses for CD63, an exosomal marker, showed that differentiated T84 cells infected with AIEC LF82 secreted an increased amount of exosomes compared to uninfected cells. This was confirmed by increased levels of CD63 as assessed by Western blot. Stimulation of human macrophages with exosomes secreted by LF82-infected T84 cells, but not by uninfected cells, significantly induced production of the pro-inflammatory cytokines TNF-alpha and IL-6, and this was not due to the presence of lipopolysaccharide, known to induce a pro-inflammatory response. Mass spectrometry analysis revealed that exosomes released by T84 cells upon LF82 infection carried microbial antigens such as the outer membrane protein C, known to be involved in AIEC adhesion and invasion.
Our study shows that in response to CD-associated AIEC infection, intestinal epithelial cells release exosomes that can trigger pro-inflammatory responses in naïve macrophagic recipient cells.