Elodie Quévrain

Identification of an anti-inflammatory protein from Faecalibacterium prausnitzii, a commensal bacterium deficient in Crohn’s disease

Background Crohn’s disease (CD)-associated dysbiosis is characterised by a loss of Faecalibacterium prausnitzii, whose culture supernatant exerts an anti-inflammatory effect both in vitro and in vivo. However, the chemical nature of the anti-inflammatory compounds has not yet been determined. Methods Peptidomic analysis using mass spectrometry was applied to F. prausnitzii supernatant. Anti-inflammatory effects of identified peptides were tested in vitro directly on intestinal epithelial cell lines and on cell lines transfected with a plasmid construction coding for the candidate protein encompassing these peptides. In vivo, the cDNA of the candidate protein was delivered to the gut by recombinant lactic acid bacteria to prevent dinitrobenzene sulfonic acid (DNBS)-colitis in mice. Results The seven peptides, identified in the F. prausnitzii culture supernatants, derived from a single microbial anti-inflammatory molecule (MAM), a protein of 15 kDa, and comprising 53% of non-polar residues. This last feature prevented the direct characterisation of the putative anti-inflammatory activity of MAM-derived peptides. Transfection of MAM cDNA in epithelial cells led to a significant decrease in the activation of the nuclear factor (NF)-κB pathway with a dose-dependent effect. Finally, the use of a food-grade bacterium, Lactococcus lactis, delivering a plasmid encoding MAM was able to alleviate DNBS-induced colitis in mice. Conclusions A 15 kDa protein with anti-inflammatory properties is produced by F. prausnitzii, a commensal bacterium involved in CD pathogenesis. This protein is able to inhibit the NF-κB pathway in intestinal epithelial cells and to prevent colitis in an animal model.

The presence of the anti-inflammatory protein MAM, from Faecalibacterium prausnitzii , in the intestinal ecosystem

We recently reported in your journal the discovery of an anti-inflammatory protein produced by Faecalibacterium prausnitzii , a commensal bacterium involved in Crohns disease (CD) dysbiosis.1 We wish to highlight and complete these recent data. The involvement of the intestinal microbiota in the pathogenesis of CD is well recognised. It has been shown that dysbiosis (an imbalance in the composition of the intestinal microbiota) could participate in chronic and inappropriate activation of the intestinal immune system and lead to inflammation. Dysbiosis is characterised by a deficiency of certain bacteria, such …

A Cell-Penetrant Manganese SOD-Mimic Is Able To Complement MnSOD and Exerts an Antiinflammatory Effect on Cellular and Animal Models of Inflammatory Bowel Diseases

Inorganic complexes are increasingly used for biological and medicinal applications, and the question of the cell penetration and distribution of metallodrugs is key to understanding their biological activity. Oxidative stress is known to be involved in inflammation and in inflammatory bowel diseases for which antioxidative defenses are weakened. We report here the study of the manganese complex Mn1 mimicking superoxide dismutase (SOD), a protein involved in cell protection against oxidative stress, using an approach in inorganic cellular chemistry combining the investigation of Mn1 intracellular speciation using mass spectrometry and of its quantification and distribution using electron paramagnetic resonance and spatially resolved X-ray fluorescence with evaluation of its biological activity. More precisely, we have looked for and found the MS signature of Mn1 in cell lysates and quantified the overall manganese content. Intestinal epithelial cells activated by bacterial lipopolysaccharide were taken as a cellular model of oxidative stress and inflammation. DNBS-induced colitis in mice was used to investigate Mn1 activity in vivo. Mn1 exerts an intracellular antiinflammatory activity, remains at least partially coordinated, with diffuse distribution over the whole cell, and functionally complements mitochondrial MnSOD.

High-resolution mass spectrometry and partial de novo sequencing constitute a useful approach for determining the profile of chemokine secretion following the stimulation of human intestinal epithelial cells.

RATIONALE Intestinal epithelial cells (IEC) secrete many chemokines in response to proinflammatory stimuli. We investigated their role in the mucosal inflammatory response in the intestine, by developing a non-targeted approach for analyzing the profile of peptides secreted by stimulated IEC, based on differential mass spectrometry analysis. METHODS Lipopolysaccharide (LPS) was incubated with IEC as a proinflammatory stimulus. Differential peptidomic analysis was then carried out, comparing the profiles of IEC with and without LPS stimulation. A mass spectrometry procedure was developed, based on a liquid chromatography/tandem mass spectrometry (LC/MS/MS) approach without enzymatic pretreatment of the peptides. Partial de novo sequencing was carried out by Fourier transform ion cyclotron resonance (FTICR), and the native peptides in the culture media were identified. RESULTS A major ion (m/z 7862.51) detected after stimulation was identified as GRO alpha and a minor ion (m/z 8918.17) was identified as IL-8. ELISA-based comparisons gave results consistent with those obtained by MS. Surprisingly, GRO alpha was secreted in amounts 5 to 15 times higher than those for IL-8 in our cellular model. The truncated form of IL-8, resulting from activation, was detected and distinguished from the native peptide by MS, whereas this was not possible with enzyme-linked immunosorbent assay (ELISA). CONCLUSIONS Mass spectrometric analysis of culture media can be used to identify the principal peptides produced in response to the stimulation of IEC, and their metabolites. Mass spectrometry provides a comprehensive view of the chemokines and peptides potentially involved in gut inflammation, making it possible to identify the most appropriate peptides for further quantification.

Inter-kingdom effect on epithelial cells of the N-Acyl homoserine lactone 3-oxo-C12:2, a major quorum-sensing molecule from gut microbiota

Background and aims N-acyl homoserine lactones (AHLs), which are autoinducer quorum-sensing molecules involved in the bacterial communication network, also interact with eukaryotic cells. Searching for these molecules in the context of inflammatory bowel disease (IBD) is appealing. The aims of our study were to look for AHL molecules in faecal samples from healthy subjects (HS) and IBD patients to correlate AHL profiles with the microbiome and investigate the effect of AHLs of interest on epithelial cells. Methods Using mass spectrometry, we characterised AHL profiles in faecal samples from HS (n = 26) and IBD patients in remission (n = 24) and in flare (n = 25) and correlated the presence of AHLs of interest with gut microbiota composition obtained by real-time qPCR and 16S sequencing. We synthesised AHLs of interest to test the inflammatory response after IL1β stimulation and paracellular permeability on Caco-2 cells. Results We observed 14 different AHLs, among which one was prominent. This AHL corresponded to 3-oxo-C12:2 and was found significantly less frequently in IBD patients in flare (16%) and in remission (37.5%) versus HS (65.4%) (p = 0.001). The presence of 3-oxo-C12:2 was associated with significantly higher counts of Firmicutes, especially Faecalbacterium prausnitzii, and lower counts of Escherichia coli. In vitro, 3-oxo-C12:2 exerted an anti-inflammatory effect on Caco-2 cells. Interestingly, although 3-oxo-C12, the well-known AHL from Pseudomonas aeruginosa, increased paracellular permeability, 3-oxo-C12:2 did not. Conclusions We identified AHLs in the human gut microbiota and discovered a new and prominent AHL, 3-oxo-C12:2, which correlates with normobiosis and exerts a protective effect on gut epithelial cells.

Mo1814 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

Background Exclusive enteral nutrition (EEN) is a first-line induction therapy in pediatric Crohns disease (CD). Though unclear, the mode of action of EEN is proposed to involve changes in gut microbiome structure and function. Characterization of the microbiome in IBD has largely focused on the assessment of diversity and the identification of protective and disease-associated species. More recently, metagenomic approaches to IBD microbiome investigation have facilitated predictive mapping of microbiome function. Treatment-induced changes to microbiome function may contribute to the strong therapeutic effect of EEN. Our aims were to compare microbial community structure and functional assessments of microbial metabolic pathways in pediatric CD patients before and after induction of remission by EEN treatment. Methods Metagenomic sequences from stool samples obtained from 4 pediatric Crohns disease patients who underwent EEN treatment were obtained using MiSeq whole-metagenome sequencing. Sequences were searched against the SILVA database to obtain microbial composition profiles based on 16S ribosomal RNA genes. To obtain functional assignment, sequences were searched against 28 representative KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways. Samples collected prior to EEN treatment were compared to samples collected after 8 to 12 weeks of EEN treatment. All participants achieved clinical remission (PCDAI<10) following EEN treatment. Results Changes in CD patient microbial community structure before and after EEN were variable. However, functional profiling of CD patient microbiota before and after EEN treatment revealed a significant increase in metabolic functions related to biodegradation and metabolism of xenobiotics, such as benzoate (p<0.05). Conclusions The microbiome of CD patients is functionally altered by EEN treatment, specifically increasing metabolic potential for xenobiotic biodegradation and metabolism relative to pre-treatment. Further investigations are warranted to investigate the role of altered xenobiotics metabolism in the therapeutic modality of EEN, as well as CD etiopathogenesis.