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OP018. Identification of an anti-inflammatory protein from Faecalibacterium prausnitzii, a deficient commensal bacteria implicated in Crohn's disease

E. Quévrain1, M.-A. Maubert1, F. Chain2, R. Marquant3, P. Kharrat2, L. Carlier3, L. Bermudez2, B. Pigneur1, O. Lequin3, C. Bridonneau2, G. Thomas1, S. Lavielle3, J.-P. Grill1, G. Chassaing3, J. Masliah1, G. Trugnan1, R. Xavier4, P. Langella2, H. Sokol1, P. Seksik1, 1INSERM, UPMC, ERL U1057/UMR 7203, Paris, France, 2INRA, UMR 1319 Micalis, Jouy-en-Josas, France, 3UPMC, UMR 7203 UPMC, Paris, France, 4Harvard University, Center for Computational & Integrative Biology, Boston, United States

Background

Crohn's disease (CD) associated dysbiosis is characterized by a loss in the Firmicutes and especially Faecalibacterium prausnitzii. Culture supernatant of this major bacteria of gut microbiota exerts anti-inflammatory effects in vitro and in vivo involving inhibition of the NF κB pathway. We aimed to determine the chemical nature of the anti-inflammatory compounds produced by F. prausnitzii.

Methods

Signaling pathways of anti-inflammatory effect of the culture supernatant of F. prausnitzii A2–165 was studied by a multiplex quantification of several proteins of the NF κB pathway. The thermostable nature and molecular weight <15 kDa of bioactive molecules present in the bacterial supernatant led us to perform a peptidomic analysis. Using mass spectrometry analyses, we identified several molecules present only in the culture supernatant of F. prausnitzii. We searched for anti-inflammatory effect of these compounds on intestinal epithelial cells (Caco-2, HT29 and HT29-MTX) by measuring the secretion of IL-8 after stimulation by a pro-inflammatory cytokine (TNF-α or IL-1β). A molecular biology approach was also developed by gene transfection of molecules of interest in eukaryotic epithelial cells (HEK293, HT29) using a bioluminescent NF κB reporter system.

Results

Study of the NF κB signaling pathway in Caco-2 cell model modulated by the supernatant of F. prausnitzii A2–165 suggested an effect of bioactive molecules in the distal part of the pathway. Seven peptides were identified in the culture supernatant of F. prausnitzii. They are all derived from MAM, a single protein of 15 kDa (ZP05614546.1) of unknown function comprising 53% of nonpolar residues. This last feature has prevented the characterization of the putative anti-inflammatory activity of MAM-derived peptides. The gene transfection of MAM (MAM+) in epithelial cells (HEK293T, HT29) led to a significant decrease in the activation of the NF κB pathway with a dose-dependent effect (24±3 × AU activation in MAM cells vs. 4±1 × AU activation in MAM+ cells). This decrease in the activity of NF κB was also observed in the MAM+ cells after stimulation with IKKB (distal part of the NF κB pathway), confirming the results obtained with F. prausnitzii supernatant. Finally, immunofluorescence experiments showed that after expression in epithelial cells, MAM was localized in the perinuclear zone and colocalized with IKKB.

Conclusion

We identified a 15 kDa protein with anti-inflammatory properties produced by F. prausnitzii, a commensal bacterium involved in CD pathogenesis. This protein and/or derived peptides involved in the anti-inflammatory effect are able to inhibit NF κB pathway in intestinal epithelial cells.