Establishment of a stableE. coli/F. prausnitzii-diassociated mouse model.

We first tried to obtainF. prausnitzii-monocolonized mice by intragastric inoculation of 2 × 10⁸ ± 1 × 10⁸ CFU into germfree (GF) mice. After 24 h, noF. prausnitzii was detected in the feces, indicating that this species was not able to stably colonize the GIT of GF mice after a single oral inoculation. We then tried to establishF. prausnitzii in GF mice by both intragastric and intrarectal inoculations, and as shown inFig. 1A, we succeeded since the amount ofF. prausnitzii A2-165 ranged from 1.5 to 5 × 10⁸ CFU/g of mouse feces. However, this colonization was obtained with a poor yield, with only 5 mice successfully colonized from 18 inoculated. Interestingly,F. prausnitzii exhibited an unusual morphology in the cecum of these 5F. prausnitzii monoassociated animals, with cell sizes ranging from 6 to 25 µm (Fig. 1B), which is very rarely seen inin vitro cultures.

We thus decided to useE. coli as a bacterial companion in order to get stableF. prausnitzii-colonized mice (hereafter named theE. coli/F. prausnitzii-diassociated mouse model).E. coli strain JM105 rapidly and stably colonized mice gut during at least 4 weeks after oral inoculation (seeFig. S1 in the supplemental material). By “priming” the GIT of mice withE. coli for at least 6 days, a durable and stable implantation ofF. prausnitzii was systematically obtained in all mice (n = 29) (Fig. 1C). The population ofF. prausnitzii was 10-fold lower (3.4 × 10⁸ to 2 × 10⁹ CFU/g of feces) than that ofE. coli (2 × 10⁹ to 1.2 × 10¹⁰ CFU/g of feces). Because of their different morphologies, the two bacterial speciesE. coli andF. prausnitzii could easily be distinguished in the cecal content (Fig. 1D). In additionF. prausnitzii, as a strict anaerobe species, was only mainly present from the cecum to the rectum, whereasE. coli was gradually abundant from the stomach to the colon (Fig. 1E).

Impact ofF. prausnitzii on the metabolite profile during inflammation in the gnotobiotic mouse model.

We compared 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis in eitherE. coli-monoassociated orE. coli/F. prausnitzii-diassociated mice in order to establish a metabolite profile under conditions allowingF. prausnitzii to display its anti-inflammatory properties (Fig. 2A). To decipher the impact ofF. prausnitzii, after TNBS induction of colitis, metabolite profiling was performed with ileum, cecum, and colon contents, feces, and serum samples using gas chromatography-time of flight (GC/TOF) mass spectrometry (seeText S1 in the supplemental material) (Fig. 2B). Using a system that combines mass spectrometric and biological metadata, 983 metabolic features were validated. The relational database system BinBase was used for automated metabolites annotation (19,20), with a criterion of presence in 50% or more of the biological replicates (seeText S1 andTable S1 in the supplemental material). Using a stringent 3-fold-intensity criterion, the number of metabolites distinguishingE. coli-monoassociated andE. coli/F. prausnitzii-diassociated animals greatly varied, depending on the GIT segment (Fig. 2B; seeTable S1). In the upper part of the GIT, 86 and 142 metabolites were overrepresented in the ileum and cecum, respectively, in theE. coli-monoassociated mice. Conversely, 187 and 482 metabolites were more abundant in the colon and feces fromE. coli/F. prausnitzii-diassociated mice, suggesting a metabolic imprinting correlated to the presence ofF. prausnitzii (Fig. 1E). A total of 279 metabolic signals were structurally identified and assigned to a compound involved in known chemical reactions (as shown by KEGG number inTable S1). Interestingly, the metabolites identified in the colon mapped onto distinct functional categories (amino acids, energy metabolism, SCFA, lipids, phenolic acid, and sugars), indicating a broad metabolic signature ofF. prausnitzii (Table 1). Compared to previous metabolomic data (21), ourE. coli/F. prausnitzii-diassociated model displayed many classes of molecules present in conventional animals but absent in axenic animals as energy metabolites, lipids and fatty acids, amino acids, and sugars. Moreover, three metabolites are common: palmitic acid, 3,4-dihydroxyhydrocinnamic acid, and glucuronic acid.

Protective effect ofF. prausnitzii on TNBS-induced colitis is linked to specific metabolic signatures in the colon.

In theE. coli/F. prausnitzii-diassociated mice, all biological markers indicated a decrease of the inflammation level in the presence ofF. prausnitzii. After colitis induction, weight loss tended to decrease in the presence ofF. prausnitzii (−3.2% ± 1.9% compared to −5.2% ± 1.5%). The disease activity index (DAI), Wallace macroscopic scores, and Ameho microscopic scores (architectural derangements, goblet cell depletion, edema/ulceration, and degree of inflammatory cell infiltrates) were significantly (P < 0.001) decreased in the colons ofE. coli/F. prausnitzii-diassociated mice (Fig. 3A, B, and C). A severe disruption of the epithelial architecture was observed inE. coli-infected, TNBS-treated mice in contrast toE. coli/F. prausnitzii-diassociated, TNBS-treated mice (Fig. 3D).E. coli-infected TNBS-treated mouse colon epithelium was characterized by the absence of crypts and massive immune cell infiltrations. Such tissue erosions were less marked in the presence ofF. prausnitzii. All of these observations indicated a protective effect ofF. prausnitzii in our TNBS-induced-colitis model. Moreover, theF. prausnitzii population level was inversely correlated to the disease activity markers (weight loss, DAI, and Wallace and Ameho scores), suggesting that its anti-inflammatory effects could be dose dependent (seeFig. S2 in the supplemental material).

Regarding metabolic profiles, we obtained strong evidence of the magnitude of the effect of anE. coli/F. prausnitzii microbial context in the TNBS-treated animals. The rarefaction curve showed that 80% of the known metabolites were more represented in the colon content of dixenic mice (seeFig. S3 in the supplemental material). We searched for metabolites segregating the monoxenic and dixenic animals after induction of colitis (t test on the mean). For instance, in the colon, butyrate (4-hydroxybutyric acid) (P = 0.0006), shikimic acid (P = 0.017) related to the salicylic acid pathway (P = 0.2055), and indole-3-lactate (P = 0.0035) were overrepresented in theE. coli/F. prausnitzii-diassociated mice (Table 1 andFig. 3E). The metabolomic profiles sharply differed according the gut segment (Fig. 2B). For instance, raffinose was more discriminatory in the ileum (P = 0.0091) (data not shown) than in the colon (P = 0.1703) and was negatively related toF. prausnitzii abundance (Fig. 1E).

F. prausnitzii is a metabolically active speciesin vivo.

The beneficial mitigation effects, observed locally in the colon in the presence ofF. prausnitzii, were confirmed in the serum, where systemic inflammation markers were reduced. First, the degree of infiltration by polymorphonuclear neutrophils (estimated by tissue myeloperoxidase [MPO] activity) was significantly decreased during TNBS-induced colitis in the presence ofF. prausnitzii (P < 0.001) (Fig. 4A). In the presence ofF. prausnitzii after TNBS induction of colitis, the serum cytokine response was globally similar to the one measured without TNBS (seeTable S2 in the supplemental material). Interestingly, in the serum, the levels of proinflammatory cytokines, such as interleukin-1α (IL-1α), IL-2, and IL-5, were statistically decreased to a level corresponding to a noninflammatory status (Fig. 4B).

The high number of metabolites detected in the serum samples was noteworthy (Fig. 2B; seeTable S1 in the supplemental material). Nineteen metabolites were overrepresented in the serum ofE. coli/F. prausnitzii-diassociated mice. Seven were only found in the serum, and 11 were also found in the colon, whereF. prausnitzii was most prevalent. Four metabolites were present in ileum, cecum, colon, and feces, but they are so far unknown (BinBase no. ID436364, -269147, -200610, and -310063) (Fig. 4C). The serum metabolome shared 5 entities with both colon and feces (uric acid, elaidic acid, lyxose, and unknown compounds BinBase ID231544 and ID459916). As observed in the colon content, the presence ofF. prausnitzii is correlated to high levels of salicylic acid (P = 0.367) and shikimic acid (P = 0.4328) (Fig. 4D). Strikingly, α-ketoglutaric (AKG) acid was 10-fold more abundant in the serum ofE. coli/F. prausnitzii-diassociated mice (P = 0.028), together with the linked citric acid and uric acid pathway (P = 0.085 andP = 0.0733, respectively). AKG acid is important in ammonia recycling and is reported to be depleted during gastrointestinal dysbiosis (22). Its involvement in cell proliferation and differentiation through the activity of the AKG-acid-dependent enzymes has recently been reviewed (23). The concomitant analysis of salicylic, shikimic, and AKG acids as standard in the GC mix validated their identification.

The four metabolites specifically associated with the presence ofF. prausnitzii possess anti-inflammatory capacitiesin vitro.

We tested the immunomodulatory properties of four candidate molecules in a human colon adenocarcinoma cell line model (HT-29) by usingF. prausnitzii supernatant, butyrate, and the 5-aminosalicylic acid (5-ASA) as controls. As shown inFig. 5, salicylic acid (10 µM) blocked IL-8 production, as did butyrate (10 mM),F. prausnitzii supernatant, and 5-ASA (20 mM). Shikimic acid, raffinose, and AKG acid (100 µM) did not block IL-8 production, and a mixture of the different molecules did not show any additional effect (data not shown).

Bacterial culture.

F. prausnitzii A2-165 (DSM 17677), isolated from human fecal stool (49), was grown at 37°C in YBHI medium (brain-heart infusion medium supplemented with 0.5% yeast extract [Difco]) supplemented with cellobiose (1 mg/ml [Sigma]), maltose (1 mg/ml [Sigma]), and cysteine (0.5 mg/ml [Sigma]) in an anaerobic chamber filled with 85% N₂, 10% CO₂, and 5% H₂.Escherichia coli JM105 was grown on LB medium (Difco) at 37°C.

Animals and experimental design.

Germfree 7- to 8-week-old BALB/c mice (male) were obtained from the germfree rodent breeding facilities of Anaxem-Micalis (INRA, Jouy-en-Josas, France). Animals were kept in flexible-film isolators (Getinge-La Calhène, Vendôme, France) in standard cages (5 mice/cage) with sterile wood shavings as animal bedding. Mice were given free access to autoclaved tap water and to a standard diet, R03-40 (Scientific Animal food and Engineering, Augy, France), sterilized by gamma irradiation at 45 kGy (IBA Mediris, Fleurus, Belgium). Isolators were maintained under controlled conditions of light (0730 to 1930 h), temperature (20 to 22°C), and humidity (45 to 55%). All procedures were carried out according to European Community Rules of Animal Care and with authorization 12-092 from the French Veterinary Services.

Inoculation of germfree mice withF. prausnitzii.

To obtainF. prausnitzii-monoassociated mice, germfree animals receive both intragastric and intrarectal inoculations of a 100× concentrated culture ofF. prausnitzii A2-165 (10⁹ CFU/inoculation). For intragastric gavages, mice were first treated with 0.1 ml of sodium bicarbonate (0.2 M) and then received 0.5 ml of the bacterial suspension 10 min later. For intrarectal inoculation, mice were first anesthetized by an intraperitoneal injection of a volume (1% of the body weight) of 10% ketamine hydrochloride (Imalgen 500 [Merial]) and 6% xylazine (Rompun [Bayer]) in 0.9% NaCl and then treated with 0.2 ml of the bacterial suspension.

To obtain dixenicE. coli/F. prausnitzii-diassociated mice, germfree mice were orally inoculated with a fresh culture ofE. coli JM105 (10⁸ to 10⁹ CFU /ml). Twenty-four hours later, whenE. coli was established in the GIT, 10⁹ CFU ofF. prausnitzii at 0.2 ml was administered to a donor mouse by intragastric gavage as described above. As soon asF. prausnitzii was established in the donor mouse, its feces were homogenized in water inside the isolator and immediately administered by intragastric gavage to other mice pretreated with sodium bicarbonate.

Viable bacteria in the inoculum were enumerated before and after gavage by plating of appropriate dilutions on supplemented YBHI agar in the anaerobic chamber. To enumerate the A2-165 strain in feces, fresh samples were introduced into the anaerobic chamber, serially 10-fold diluted, and plated on supplemented YBHI agar. In order to selectively enumerate A2-165 in the feces of dixenic mice by eliminatingE. coli cells, fecal samples were treated with sodium azide (3%) for 5 min at 37°C and then plated on sodium azide (0.02%) YBHI agar.

TNBS-induced colitis.

Mice were weighed before TNBS administration and anesthetized by an intraperitoneal injection as described above. TNBS (Sigma-Aldrich) was dissolved in 0.9% NaCl–ethanol (50:50 vol/vol). Fifty microliters of TNBS (50 mg/kg body weight) was administered intrarectally (using a 3.5 French catheter [Solomon Scientific]) to mice maintained for 30 s in a vertical position. Control mice received 50 µl of 0.9% NaCl–ethanol solution intrarectally. Inflammation was monitored 48 h after TNBS administration.

Scanning electron microscopy.

Scanning electron microscopy analyses were performed at the MIMA2 platform (INRA, Massy, France) from 0.2 g of cecal samples as previously described (50).

Tissue preparation for histological analysis.

Flushed colons were opened longitudinally, cut into 2-cm sections, and rolled according to the Swiss roll procedure (51). The samples were fixed in 4% paraformaldehyde (4 h, room temperature), dehydrated, and embedded in paraffin according to standard histological protocols. Four-micrometer sections of distal colon were mounted on SuperFrost Plus slides. To assess TNBS-induced colitis, the sections were stained with hematoxylin-eosin and examined blindly according to the Ameho criteria (52).

Inflammation score assessment of TNBS-induced colitis.

The colon was removed without mesenteric fat and mesentery, carefully opened, and cleaned with phosphate-buffered saline (PBS). Colonic damage and inflammation were assessed blindly according to the Wallace score (53).

MPO activity.

MPO activity was measured using the method of Bradley et al. (54), modified as follows. Tissue samples were homogenized (50 mg/ml) in ice-cold 50-mM potassium phosphate buffer (pH 6) containing 5% hexadecyl trimethyl ammonium bromide (Sigma-Aldrich) and hydrogen peroxide. MPO is expressed in units per milligram of wet tissue.

Cytokine analysis.

Colonic samples were homogenized in 400 µl of Tris-HCl buffer containing protease inhibitors (Sigma-Aldrich) in a tissue lyser. Samples were centrifuged for 20 min, and the supernatant was frozen at −80°C until further analysis. Before sacrifice, blood samples were obtained from the retro-orbital venous plexus and centrifuged, and sera were stored at −80°C. Cytokine levels were determined by flow cytometry using Cytometric bead array analysis (mouse inflammation kit) (BD, NJ).

Metabolite extraction and analysis.

Fifty microliters of serum and feces, intestinal content, cecal content, and colonic content were lyophilized and subjected to 10 s of bead beating. Metabolite extraction was performed as previously described (20) with 700 µl of a precooled methanol-isopropanol-water (3:3:2) mixture in an ultrasonic water bath (5 min). After centrifugation, 650 µl of the supernatant was collected (5 min, 15,000 rpm) and dried before derivatization. Metabolite analysis was performed according to Fiehn et al. (55).

In vitro experiments with the HT-29 cell line.

The protocols used in this study were adapted from Kechaou et al. (56). The human colon carcinoma cell line HT-29 was cultured and stimulated with recombinant human tumor necrosis factor alpha (TNF-α) (5 ng/ml; PeproTech, NJ) as described previously. Cell incubations were done in the presence or not of different metabolites tested at the indicated concentration: butyric acid sodium salt, reference 303410, Sigma-Aldrich; shikimic acid, reference S5375, Sigma-Aldrich; salicylic acid, reference 84210, Sigma-Aldrich;d-(+)-raffinose pentahydrate, reference R0514, Sigma-Aldrich; α-ketoglutaric acid disodium salt dihydrate, reference 75892, Sigma-Aldrich; 5-aminosalicylic acid, reference 18858, Sigma-Aldrich. All samples were analyzed in triplicate. After coincubation, cell supernatants were collected and mixed with an antiprotease cocktail according to the manufacturer’s instructions (Complete EDTA-free protease inhibitor; Roche Applied Bioscience), and frozen at −80°C until further analysis of interleukin-8 (IL-8) concentrations by an enzyme-linked immunosorbent assay (ELISA) (BioLegend, San Diego, CA, and Mabtech, Sweden, respectively).

Statistical analysis.

Data are reported as means ± standard errors of the means (SEM) and for figures using box-and-whisker plots with the interquartile range, where the top portion of the box represents the 75th percentile and the lower portion represents the 25th percentile. The horizontal bar within the box represents the median, with the mean shown as “+.” The upper horizontal bar outside the box represents the 95th percentile, and the lower horizontal bar outside the box represents the 15th percentile. Statistical analysis was performed using one-way analysis of variance followed by Student-Newman-Keuls multiple comparisonpost hoc analysis, and with aP value of <0.05 considered significant. For metabolomic analysis, the resulting data sets were imported into the MatLab software for univariate and multivariate statistical analyses. Univariate statistics evaluated the number and percentage of features that are either unique or vary significantly betweenE. coli-monoassociated andE. coli/F. prausnitzii-diassociated animals. Venn diagrams were constructed as described in reference57.

• 1.Qin J, Li R, Raes J, Arumugam M, Burgdorf KS, Manichanh C, Nielsen T, Pons N, Levenez F, Yamada T, Mende DR, Li J, Xu J, Li S, Li D, Cao J, Wang B, Liang H, Zheng H, Xie Y, Tap J, Lepage P, Bertalan M, Batto JM, Hansen T, Le Paslier D, Linneberg A, Nielsen HB, Pelletier E, Renault P, Sicheritz-Ponten T, Turner K, Zhu H, Yu C, Li S, Jian M, Zhou Y, Li Y, Zhang X, Li S, Qin N, Yang H, Wang J, Brunak S, Dore J, Guarner F, Kristiansen K, Pedersen O, Parkhill J, Weissenbach J, Bork P, Ehrlich SD, Wang J. 2010. A human gut microbial gene catalogue established by metagenomic sequencing. Nature464:59–65. doi: 10.1038/nature08821. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 2.Tomas J, Wrzosek L, Bouznad N, Bouet S, Mayeur C, Noordine ML, Honvo-Houeto E, Langella P, Thomas M, Cherbuy C. 2013. Primocolonization is associated with colonic epithelial maturation during conventionalization. FASEB J27:645–655. doi: 10.1096/fj.12-216861. [DOI] [PubMed] [Google Scholar]
• 3.Miquel S, Martín R, Rossi O, Bermúdez-Humarán LG, Chatel JM, Sokol H, Thomas M, Wells JM, Langella P. 2013.Faecalibacterium prausnitzii and human intestinal health. Curr Opin Microbiol16:255–261. doi: 10.1016/j.mib.2013.06.003. [DOI] [PubMed] [Google Scholar]
• 4.Rajilić-Stojanović M, Biagi E, Heilig HG, Kajander K, Kekkonen RA, Tims S, de Vos WM. 2011. Global and deep molecular analysis of microbiota signatures in fecal samples from patients with irritable bowel syndrome. Gastroenterology141:1792–1801. doi: 10.1053/j.gastro.2011.07.043. [DOI] [PubMed] [Google Scholar]
• 5.Sobhani I, Tap J, Roudot-Thoraval F, Roperch JP, Letulle S, Langella P, Corthier G, Tran Van Nhieu J, Furet JP. 2011. Microbial dysbiosis in colorectal cancer (CRC) patients. PLoS One6:e16393. doi: 10.1371/journal.pone.0016393. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 6.Swidsinski A, Loening-Baucke V, Vaneechoutte M, Doerffel Y. 2008. Active Crohn’s disease and ulcerative colitis can be specifically diagnosed and monitored based on the biostructure of the fecal flora. Inflamm Bowel Dis14:147–161. doi: 10.1002/ibd.20330. [DOI] [PubMed] [Google Scholar]
• 7.Sokol H, Pigneur B, Watterlot L, Lakhdari O, Bermúdez-Humarán LG, Gratadoux JJ, Blugeon S, Bridonneau C, Furet JP, Corthier G, Grangette C, Vasquez N, Pochart P, Trugnan G, Thomas G, Blottière HM, Doré J, Marteau P, Seksik P, Langella P. 2008.Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients. Proc Natl Acad Sci U S A105:16731–16736. doi: 10.1073/pnas.0804812105. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 8.Sokol H, Seksik P, Furet JP, Firmesse O, Nion-Larmurier I, Beaugerie L, Cosnes J, Corthier G, Marteau P, Doré J. 2009. Low counts ofFaecalibacterium prausnitzii in colitis microbiota. Inflamm Bowel Dis15:1183–1189. doi: 10.1002/ibd.20903. [DOI] [PubMed] [Google Scholar]
• 9.Miquel S, Martin R, Bridonneau C, Robert V, Sokol H, Bermúdez-Humarán L, Thomas M, Langella P. 2014. Ecology and metabolism of the beneficial intestinal commensal bacteriumFaecalibacterium prausnitzii. Gut Microbes5:146–151. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 10.Hold GL, Schwiertz A, Aminov RI, Blaut M, Flint HJ. 2003. Oligonucleotide probes that detect quantitatively significant groups of butyrate-producing bacteria in human feces. Appl Environ Microbiol69:4320–4324. doi: 10.1128/AEM.69.7.4320-4324.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 11.Li M, Wang B, Zhang M, Rantalainen M, Wang S, Zhou H, Zhang Y, Shen J, Pang X, Zhang M, Wei H, Chen Y, Lu H, Zuo J, Su M, Qiu Y, Jia W, Xiao C, Smith LM, Yang S, Holmes E, Tang H, Zhao G, Nicholson JK, Li L, Zhao L. 2008. Symbiotic gut microbes modulate human metabolic phenotypes. Proc Natl Acad Sci U S A105:2117–2122. doi: 10.1073/pnas.0712038105. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 12.Louis P, Flint HJ. 2009. Diversity, metabolism and microbial ecology of butyrate-producing bacteria from the human large intestine. FEMS Microbiol Lett294:1–8. doi: 10.1111/j.1574-6968.2009.01514.x. [DOI] [PubMed] [Google Scholar]
• 13.Macfarlane GT, Macfarlane S. 2011. Fermentation in the human large intestine: its physiologic consequences and the potential contribution of prebiotics. J Clin Gastroenterol45(Suppl):S120–S127. doi: 10.1097/MCG.0b013e31822fecfe. [DOI] [PubMed] [Google Scholar]
• 14.Martín R, Miquel S, Ulmer J, Kechaou N, Langella P, Bermúdez-Humarán LG. 2013. Role of commensal and probiotic bacteria in human health: a focus on inflammatory bowel disease. Microb Cell Fact12:71. doi: 10.1186/1475-2859-12-71. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 15.Wrzosek L, Miquel S, Noordine ML, Bouet S, Joncquel Chevalier-Curt M, Robert V, Philippe C, Bridonneau C, Cherbuy C, Robbe-Masselot C, Langella P, Thomas M. 2013.Bacteroides thetaiotaomicron andFaecalibacterium prausnitzii influence the production of mucus glycans and the development of goblet cells in the colonic epithelium of a gnotobiotic model rodent. BMC Biol11:61. doi: 10.1186/1741-7007-11-61. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 16.Duncan SH, Louis P, Thomson JM, Flint HJ. 2009. The role of pH in determining the species composition of the human colonic microbiota. Environ Microbiol11:2112–2122. doi: 10.1111/j.1462-2920.2009.01931.x. [DOI] [PubMed] [Google Scholar]
• 17.Lopez-Siles M, Khan TM, Duncan SH, Harmsen HJ, Garcia-Gil LJ, Flint HJ. 2012. Cultured representatives of two major phylogroups of human colonicFaecalibacterium prausnitzii can utilize pectin, uronic acids, and host-derived substrates for growth. Appl Environ Microbiol78:420–428. doi: 10.1128/AEM.06858-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 18.Hopkins MJ, Macfarlane GT, Furrie E, Fite A, Macfarlane S. 2005. Characterisation of intestinal bacteria in infant stools using real-time PCR and Northern hybridisation analyses. FEMS Microbiol Ecol54:77–85. doi: 10.1016/j.femsec.2005.03.001. [DOI] [PubMed] [Google Scholar]
• 19.Skogerson K, Wohlgemuth G, Barupal DK, Fiehn O. 2011. The volatile compound BinBase mass spectral database. BMC Bioinformatics12:321. doi: 10.1186/1471-2105-12-321. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 20.Lee do Y, Bowen BP, Nguyen DH, Parsa S, Huang Y, Mao JH, Northen TR. 2012. Low-dose ionizing radiation-induced blood plasma metabolic response in a diverse genetic mouse population. Radiat Res178:551–555. doi: 10.1667/RR2990.1. [DOI] [PubMed] [Google Scholar]
• 21.Velagapudi VR, Hezaveh R, Reigstad CS, Gopalacharyulu P, Yetukuri L, Islam S, Felin J, Perkins R, Borén J, Oresic M, Bäckhed F. 2010. The gut microbiota modulates host energy and lipid metabolism in mice. J Lipid Res51:1101–1112. doi: 10.1194/jlr.M002774. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 22.Hamer HM, De Preter V, Windey K, Verbeke K. 2012. Functional analysis of colonic bacterial metabolism: relevant to health?Am J Physiol Gastrointest Liver Physiol302:G1–G9. doi: 10.1152/ajpgi.00048.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 23.Cairns RA, Mak TW. 2013. Oncogenic isocitrate dehydrogenase mutations: mechanisms, models, and clinical opportunities. Cancer Discov3:730–741. doi: 10.1158/2159-8290.CD-13-0083. [DOI] [PubMed] [Google Scholar]
• 24.Ahmed S, Macfarlane GT, Fite A, McBain AJ, Gilbert P, Macfarlane S. 2007. Mucosa-associated bacterial diversity in relation to human terminal ileum and colonic biopsy samples. Appl Environ Microbiol73:7435–7442. doi: 10.1128/AEM.01143-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 25.Rigottier-Gois L.2013. Dysbiosis in inflammatory bowel diseases: the oxygen hypothesis. ISME J7:1256–1261. doi: 10.1038/ismej.2013.80. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 26.Aguirre de Cárcer D, Cuív PO, Wang T, Kang S, Worthley D, Whitehall V, Gordon I, McSweeney C, Leggett B, Morrison M. 2011. Numerical ecology validates a biogeographical distribution and gender-based effect on mucosa-associated bacteria along the human colon. ISME J5:801–809. doi: 10.1038/ismej.2010.177. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 27.Callery PS, Geelhaar LA. 1984. Biosynthesis of 5-aminopentanoic acid and 2-piperidone from cadaverine and 1-piperideine in mouse. J Neurochem43:1631–1634. doi: 10.1111/j.1471-4159.1984.tb06088.x. [DOI] [PubMed] [Google Scholar]
• 28.Yan HD, Ishihara K, Serikawa T, Sasa M. 2003. Activation byN-acetyl-l-aspartate of acutely dissociated hippocampal neurons in rats via metabotropic glutamate receptors. Epilepsia44:1153–1159. doi: 10.1046/j.1528-1157.2003.49402.x. [DOI] [PubMed] [Google Scholar]
• 29.Carlsson AH, Yakymenko O, Olivier I, Håkansson F, Postma E, Keita AV, Söderholm JD. 2013.Faecalibacterium prausnitzii supernatant improves intestinal barrier function in mice DSS colitis. Scand J Gastroenterol48:1136–1144. doi: 10.3109/00365521.2013.828773. [DOI] [PubMed] [Google Scholar]
• 30.Martín R, Chain F, Miquel S, Lu J, Gratadoux JJ, Sokol H, Verdu EF, Bercik P, Bermúdez-Humarán LG, Langella P. 2014. The commensal bacteriumFaecalibacterium prausnitzii is protective in DNBS-induced chronic moderate and severe colitis models. Inflamm Bowel Dis20:417–430. doi: 10.1097/01.MIB.0000440815.76627.64. [DOI] [PubMed] [Google Scholar]
• 31.Sonnenberg A, Collins JF. 2006. Vicious circles in inflammatory bowel disease. Inflamm Bowel Dis12:944–949. doi: 10.1097/01.mib.0000231577.19301.95. [DOI] [PubMed] [Google Scholar]
• 32.Human Microbiome Project Consortium 2012. Structure, function and diversity of the healthy human microbiome. Nature486:207–214. doi: 10.1038/nature11234. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 33.Plöger S, Stumpff F, Penner GB, Schulzke JD, Gäbel G, Martens H, Shen Z, Günzel D, Aschenbach JR. 2012. Microbial butyrate and its role for barrier function in the gastrointestinal tract. Ann N Y Acad Sci1258:52–59. doi: 10.1111/j.1749-6632.2012.06553.x. [DOI] [PubMed] [Google Scholar]
• 34.Fusunyan RD, Quinn JJ, Fujimoto M, MacDermott RP, Sanderson IR. 1999. Butyrate switches the pattern of chemokine secretion by intestinal epithelial cells through histone acetylation. Mol Med5:631–640. [PMC free article] [PubMed] [Google Scholar]
• 35.Böcker U, Nebe T, Herweck F, Holt L, Panja A, Jobin C, Rossol S, Sartor RB, Singer MV. 2003. Butyrate modulates intestinal epithelial cell-mediated neutrophil migration. Clin Exp Immunol131:53–60. doi: 10.1046/j.1365-2249.2003.02056.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 36.Moore WE, Moore LV, Cato EP, Wilkins TD, Kornegay ET. 1987. Effect of high-fiber and high-oil diets on the fecal flora of swine. Appl Environ Microbiol53:1638–1644. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 37.Messori A, Brignola C, Trallori G, Rampazzo R, Bardazzi G, Belloli C, d’Albasio G, De Simone G, Martini N. 1994. Effectiveness of 5-aminosalicylic acid for maintaining remission in patients with Crohn’s disease: a meta-analysis. Am J Gastroenterol89:692–698. [PubMed] [Google Scholar]
• 38.Lee JS, Park SY, Thapa D, Choi MK, Chung IM, Park YJ, Yong CS, Choi HG, Kim JA. 2010.Grifola frondosa water extract alleviates intestinal inflammation by suppressing TNF-alpha production and its signaling. Exp Mol Med42:143–154. doi: 10.3858/emm.2010.42.2.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 39.Bochkov DV, Sysolyatin SV, Kalashnikov AI, Surmacheva IA. 2012. Shikimic acid: review of its analytical, isolation, and purification techniques from plant and microbial sources. J Chem Biol5:5–17. doi: 10.1007/s12154-011-0064-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 40.Xing J, You C, Dong K, Sun J, You H, Dong Y, Sun J. 2013. Ameliorative effects of 3,4-oxo-isopropylidene-shikimic acid on experimental colitis and their mechanisms in rats. Int Immunopharmacol15:524–531. doi: 10.1016/j.intimp.2013.02.008. [DOI] [PubMed] [Google Scholar]
• 41.LeBlanc JG, Silvestroni A, Connes C, Juillard V, de Giori GS, Piard JC, Sesma F. 2004. Reduction of non-digestible oligosaccharides in soymilk: application of engineered lactic acid bacteria that produce alpha-galactosidase. Genet Mol Res3:432–440. [PubMed] [Google Scholar]
• 42.Dawson DJ, Lobley RW, Burrows PC, Notman JA, Mahon M, Holmes R. 1988. Changes in jejunal permeability and passive permeation of sugars in intestinal biopsies in coeliac disease and Crohn’s disease. Clin Sci (Lond)74:427–431. [DOI] [PubMed] [Google Scholar]
• 43.Laval L, Martin R, Natividad J, Chain F, Miquel S, de Maredsous CD, Capronnier S, Sokol H, Verdu E, van Hylckama Vlieg J, Bermúdez-Humarán L, Smokvina T, Langella P. 2015.Lactobacillus rhamnosus CNCM I-3690 and the commensal bacteriumFaecalibacterium prausnitzii A2-165 exhibit similar protective effects to induced barrier hyper-permeability in mice. Gut Microbes6:1–9. doi: 10.4161/19490976.2014.990784. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 44.Heinken A, Khan MT, Paglia G, Rodionov DA, Harmsen HJ, Thiele I. 2014. Functional metabolic map ofFaecalibacterium prausnitzii, a beneficial human gut microbe. J Bacteriol196:3289–3302. doi: 10.1128/JB.01780-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 45.Perła-Kaján J, Twardowski T, Jakubowski H. 2007. Mechanisms of homocysteine toxicity in humans. Amino Acids32:561–572. doi: 10.1007/s00726-006-0432-9. [DOI] [PubMed] [Google Scholar]
• 46.Pérez-Cano FJ, Franch A, Castellote C, Castell M. 2003. Immunomodulatory action of spermine and spermidine on NR8383 macrophage line in various culture conditions. Cell Immunol226:86–94. doi: 10.1016/j.cellimm.2003.09.009. [DOI] [PubMed] [Google Scholar]
• 47.Wikoff WR, Anfora AT, Liu J, Schultz PG, Lesley SA, Peters EC, Siuzdak G. 2009. Metabolomics analysis reveals large effects of gut microflora on mammalian blood metabolites. Proc Natl Acad Sci U S A106:3698–3703. doi: 10.1073/pnas.0812874106. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 48.Waddell TG, Henderson BS, Morris RT, Lewis CM, Zimmermann AG. 1987. Chemical evolution of the citric acid cycle: sunlight photolysis of alpha-ketoglutaric acid. Orig Life Evol Biosph17:149–153. doi: 10.1007/BF01808242. [DOI] [PubMed] [Google Scholar]
• 49.Duncan SH, Hold GL, Harmsen HJ, Stewart CS, Flint HJ. 2002. Growth requirements and fermentation products ofFusobacterium prausnitzii, and a proposal to reclassify it asFaecalibacterium prausnitzii gen. nov., comb. nov. Int J Syst Evol Microbiol52:2141–2146. doi: 10.1099/ijs.0.02241-0. [DOI] [PubMed] [Google Scholar]
• 50.Joly F, Mayeur C, Bruneau A, Noordine ML, Meylheuc T, Langella P, Messing B, Duée PH, Cherbuy C, Thomas M. 2010. Drastic changes in fecal and mucosa-associated microbiota in adult patients with short bowel syndrome. Biochimie92:753–761. doi: 10.1016/j.biochi.2010.02.015. [DOI] [PubMed] [Google Scholar]
• 51.Rochat T, Bermúdez-Humarán L, Gratadoux JJ, Fourage C, Hoebler C, Corthier G, Langella P. 2007. Anti-inflammatory effects ofLactobacillus casei BL23 producing or not a manganese-dependent catalase on DSS-induced colitis in mice. Microb Cell Fact6:22. doi: 10.1186/1475-2859-6-22. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 52.Ameho CK, Adjei AA, Harrison EK, Takeshita K, Morioka T, Arakaki Y, Ito E, Suzuki I, Kulkarni AD, Kawajiri A, Yamamoto S. 1997. Prophylactic effect of dietary glutamine supplementation on interleukin 8 and tumour necrosis factor alpha production in trinitrobenzene sulphonic acid induced colitis. Gut41:487–493. doi: 10.1136/gut.41.4.487. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 53.Wallace JL, MacNaughton WK, Morris GP, Beck PL. 1989. Inhibition of leukotriene synthesis markedly accelerates healing in a rat model of inflammatory bowel disease. Gastroenterology96:29–36. [DOI] [PubMed] [Google Scholar]
• 54.Bradley PP, Priebat DA, Christensen RD, Rothstein G. 1982. Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker. J Invest Dermatol78:206–209. doi: 10.1111/1523-1747.ep12506462. [DOI] [PubMed] [Google Scholar]
• 55.Fiehn O, Kopka J, Dörmann P, Altmann T, Trethewey RN, Willmitzer L. 2000. Metabolite profiling for plant functional genomics. Nat Biotechnol18:1157–1161. doi: 10.1038/81137. [DOI] [PubMed] [Google Scholar]
• 56.Kechaou N, Chain F, Gratadoux JJ, Blugeon S, Bertho N, Chevalier C, Le Goffic R, Courau S, Molimard P, Chatel JM, Langella P, Bermúdez-Humarán LG. 2013. Identification of one novel candidate probioticLactobacillus plantarum strain active against influenza virus infection in mice by a large-scale screening. Appl Environ Microbiol79:1491–1499. doi: 10.1128/AEM.03075-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
• 57.Oliveros JC.2007. An interactive tool for comparing lists with Venn diagrams.http://bioinfogp.cnb.csic.es/tools/venny/index.html.

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