P1341 Probing the interaction of probiotic bacterial extracellular vesicles with primary colonic epithelium using an ex-vivo organoid platform

Abstract Background Bacterial extracellular vesicles (BEV) are increasingly recognized as a relevant mode of contactless host-microbiota communication. BEV are nanoparticles pinched off from the membrane of multiple bacteria and carry nucleic acid snippets and functional proteins inherited from their parental bacteria. While evidence suggests that BEV act as transmitters to modulate the immune response, only scarce data exist as to whether and how efficiently BEV are internalized by colonic epithelium as the host‘s front line in the gut. This study aims to quantify BEV internalization into primary colonic epithelium and explore structural and physicochemical properties of BEV potentially influencing uptake. Methods BEV were isolated from Limosilactobacillus reuteri (LR) and Lactiplantibacillus plantarum (LP) and incubated with either colonoids or colonic epithelial monolayers from C57BL/6 mice. BEV were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), zeta potential (ZP) analysis, as well as lipidomic and proteomic analysis. Uptake of BEV into colonic epithelium was assessed by confocal microscopy and flow cytometry. Results BEV of either species displayed a uniform appearance with a lipid bilayer on TEM. NTA showed similar spectra of sizes with a dominant peak around 100 nm whereas with DLS particle size readings were higher and showed a bimodal distribution. The ZP of BEV measured in buffer with physiological salinity did not differ between LR and LP. Gas chromatographic mass spectrometry analysis revealed neither qualitative nor quantitative differences in the composition of long chain fatty acid in BEV membranes. As opposed to LP, LR pellets showed proteomic enrichment of pathways linked to glycerolipid metabolism, fatty acid biosynthesis, and terpenoid backbone synthesis, being precursor steps of lipoteichoic acid synthesis (LTA). LTA could be detected in BEV of both species, but appeared to more abundant in LR-BEV. A dose-dependent increment in BEV uptake rates was noted in both species, with LR-BEV being more readily internalized by colonic epithelium. Epithelial cells exposed to LR-BEV were characterized by irregular nuclei and partial detachment from the surface whilst cells incubated with LP-BEV appeared morphologically normal. The quantity of viable cells was reduced in colonoids treated with LR-BEV, but not LP-BEV. In the presence of LR-BEV, colonoids were less likely to undergo apoptosis. Conclusion LR- and LP-BEV differ in their ability to enter colonic epithelium and interfere with cellular homeostasis. A variable degree of LTA decoration of BEV membranes might account for differential uptake into the host cell.