P780 3-oxo-C12: 2-HSL, a new N-acyl-homoserine lactone identified in gut ecosystem exerts an anti-inflammatory effect and does not modify paracellular permeability

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Background: It is now recognized that IBD patients exhibit imbalance in gut microbiome. In this setting, the fact that bacteria can use quorum sensing small signal molecules for cell-cell communication including inter-kingdom (prokaryote-eukaryote) communication is an interesting way to tackle functional impact of dysbiosis on host epithelial cells. In various ecosystems, one of the most studied quorum sensing system relies on amphiphilic molecules called N-acyl-homoserine lactones (AHLs). In a previous study, fecal samples from 49 IBD patients in remission (n=24) and during flare (n=25) and from 26 healthy subjects were analyzed. AHLs profile was determined for each sample using HPLC coupled with tandem mass spectrometry. We detected 10 different AHLs in human gut microbiota and identified a prominent and never described AHL: 3-oxo-C12:2-HSL. Its presence was significantly associated with normobiosis. We now aimed to look for the impact of the 3-oxo-C12:2-HSL on intestinal inflammatory pathways and the intestinal permeability. To note 3-oxo-C12:2-HSL is close to the well-known quorum sensing molecule 3-oxo-C12-HSL from pseudomonas aeruginosa. This latest has anti-inflammatory properties and is known to increase paracellular permeability.

Methods: Caco-2/TC7 cells were cultured until confluence and then stimulated by IL1B at 25 ng/mL during 18h with increasing concentration of 3-oxo-C12:2-HSL (0, 1, 5, 10, 25, 50, 100 and 200 μM). The inflammatory response was measured by the level of IL-8 in the supernatant using ELISA. The impact of the 3-oxo-C12:2-HSL (200 μM) on the paracellular permeability was measured by the passage of a fluorescein-dextran 4kDa (FD4-FITC) tracer, from the apical to the basal side of a monocellular layer of Caco-2/TC7 cells, cultured on Transwell filters. In these experiments, 3-oxo-C12:2-HSL effects were compared to 3-oxo-C12-HSL.

Results: After IL1B stimulation of Caco-2 cells, 3oxoC12:2-HSL (10–50 μM) as well as 3-oxo-C12-HSL (5 μM) were able to lower levels of IL-8 (p<0.05). Furthermore, 3-oxo-C12:2-HSL (200 μM) did not modify paracellular permeability after 4 or 20-hours exposure whereas 3-oxo-C12-HSL (200 μM) increases paracellular permeability after 4-hour (×2) or 20-hour (×10) exposure compared to controls (p<0.0001).

Conclusions: 3-oxo-C12:2-HSL exerts an anti-inflammatory effect on enterocyte like Caco-2/TC7 cells. This anti-inflammatory effect is not associated with an increase of paracellular permeability contrary to what we observed with the 3-oxo-C12-HSL. These results support the hypothesis of a protective role of the 3-oxo-C12:2-HSL in gut ecosystem.

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