P053 Impact of N- acyl-homoserine lactone 3-oxo-C12:2, a major quorum sensing molecule from gut microbiota, on intestinal epithelial barrier function

Abstract

Gut microbiota imbalance (dysbiosis) is a key factor in inflammatory bowel diseases (IBD) physiopathology. N-acyl homoserine lactones (AHLs) are molecules involved in quorum sensing, a bacterial communication network, which can also impact the host. We recently showed the presence of AHLs in the human gut ecosystem and identified a new AHL, named 3-oxo-C12:2, which was decreased in IBD during flare and associated with normobiosis. In this study, we investigated how 3-oxo-C12:2 impacts the intestinal barrier function. Using human Caco-2/TC7 enterocytic cells grown on semi-permeable filters, we compared effects of 3-oxo-C12:2 AHL to a structurally close AHL, 3-oxo-C12, produced by the pathogen P. aeruginosa and whose deleterious effects on epithelial barrier function have been shown. Caco-2/TC7 cells were exposed on their apical side to 3-oxo-C12 or 3-oxo-C12:2 AHLs (150µM) for 48h, in the presence or absence of pro-inflammatory cytokines Interferon-γ et TNF-α (50 ng/ml) supplied in the basal compartment. Paracellular permeability to macromolecules was assessed via the passage of fluorescent tracer FITC-dextran 4 kDa across the cell monolayer. Tight junction (TJ) proteins (Occludin and Tricellulin) and the adherent junction (AJ) protein E-Cadherin were analysed by immunofluorescence staining. Upon 3-oxoC12 exposure, paracellular permeability was increased (x5; p < 0.001), the AJ protein E-Cadherin was mislocalised and the signal intensity of TJ proteins Occludin and Tricellulin was decreased (−15%), thus showing an impairment of intercellular junctions. In contrast, 3-oxo-C12:2 AHL modified neither paracellular permeability nor intercellular junction integrity. While 3-oxoC12 AHL potentiated the increase in paracellular permeability induced by pro-inflammatory cytokines (x25 vs. x5 upon cytokines exposure only), 3-oxo-C12:2 AHL did not modulate paracellular permeability when combined to pro-inflammatory cytokines. Deleterious effects of cytokines on TJ proteins Occludin (-43% vs. control, p < 0.001) and Tricellulin (-52% vs. control, p < 0.001) were reduced in the presence of 3-oxo-C12:2 (Occludin -29%, p = 0.24 vs. cytokines only; Tricellulin -24%, p < 0.01 vs. cytokines only). Our results show that 3-oxo-C12:2, a major AHL of the human gut ecosystem, does not impact paracellular permeability and mitigates deleterious effects of pro-inflammatory cytokines on TJ proteins. We previously demonstrated that 3-oxo-C12:2 AHL was decreased in IBD associated dysbiosis. Hence, the described hereinabove protective effects of 3-oxo-C12:2 on intestinal barrier function in a pro-inflammatory context lead to interesting perspectives to better understand host-microbiota interactions in IBD.