P051 The N- acyl-homoserine lactone 3-oxo-C12, an inter-bacterial signaling molecule (involved in quorum sensing), exerts effects on the host: thus implicating quorum sensing in inflammatory bowel disease.
C. Landman*1, M. Clément1, H. Nsiri1, E. Quévrain1, T. Bazin1, L. Brot1, J.-P. Grill1, M.-A. Maubert1, L. Humbert1, H. Sokol1, 2, G. Trugnan1, D. Rainteau1, P. Seksik1, 2
1UPMC, ERL INSERM 1157 / UMR7203, PARIS, France, 2APHP Saint-Antoine, Service de Gastroentérologie et nutrition, Paris, France
Inflammatory bowel disease (IBD) associated dysbiosis causes profound changes in the gut ecosystem. We have shown that the profile of inter-bacterial signaling molecules, the N-acyl-homoserine lactones (AHLs), is altered during the course of dysbiosis. In particular, we observed a decrease in 3-oxo-C12:2, the dominant gut ecosystem AHL. These Results prompted us to study the functional role of this AHL on the gut microbiota and the host. As synthesized 3-oxo-C12:2 was unavailable, we focused our research on the structurally similar AHL, 3-oxo-C12. Existing knowledge of the host enzymes (the paraoxonases) involvement in quorum quenching and their deficiency in IBD, rendered it interesting to explore the functional role of 3-oxo-C12 on the inflammatory pathway. The aim of this study was to explore 3-oxo-C12 ability to interact with eukaryotic cells, such as intestinal epithelial cells and immune cells.
Firstly, the sub cellular localization of 3-oxo-C12, was visualized, in a human epithelial cell model, by confocal microscopy. We examined the effect of 3-oxo-C12, compared with the effect of a short chain AHL (C4), on macrophage cell line. Experiments for both AHLs were carried out under a range of conditions including: presence of 1-10 µM DMSO, with/without the paraoxanase inhibitor, 2-hydroxyquinoline (2HQ) (100µM) and with/without INF- γ (20UI/mL) and LPS (50ng/mL) stimulation. Macrophage cell response was quantified by measurement of TNF- α and IL-6 levels.
Confocal microscopy showed intracellular localization of 3-oxo-C12 (1µM) after 1 hour. We observed co-localization with euchromatin suggesting a transcriptional factor role for 3-oxo-C12 within the host cells. Fluorescence diminished after addition to the media of non-fluorescent 3-oxo-C12 in excess (20 µM). No intracellular fluorescence was observed after treatment with fluorescein alone (negative control). When examining the effect of 3-oxo-C12 on raw cells, we observed a significant decrease in IL-6, 6 hours post stimulation with IFN- γ and LPS, in the presence of both 3-oxo-C12 (5µM) and 2HQ compare to the negative control (DMSO +2HQ): 23208 +/- 4639 vs 95373 +/-12875 pg/mg protein, p = 0.03. Decrease was also observed for TNF- α under the same conditions (non-significant). Finally, no significant decrease in IL-6 or TNF- α was observed 6 hours post stimulation with C4 (5µM).
We have demonstrated that 3-oxo-C12, an AHL structurally close to 3-oxo-C12:2 present in the human gut ecosystem, interacts with eukaryotic cells by penetration of intestinal epithelial cells. Additionally, 3-oxo-C12 exerts an anti-inflammatory effect, at low concentrations, in the presence of paraoxonases inhibitors in a murine macrophage model.