Philippe Seksik

Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients

A decrease in the abundance and biodiversity of intestinal bacteria within the dominant phylum Firmicutes has been observed repeatedly in Crohn disease (CD) patients. In this study, we determined the composition of the mucosa-associated microbiota of CD patients at the time of surgical resection and 6 months later using FISH analysis. We found that a reduction of a major member of Firmicutes, Faecalibacterium prausnitzii, is associated with a higher risk of postoperative recurrence of ileal CD. A lower proportion of F. prausnitzii on resected ileal Crohn mucosa also was associated with endoscopic recurrence at 6 months. To evaluate the immunomodulatory properties of F. prausnitzii we analyzed the anti-inflammatory effects of F. prausnitzii in both in vitro (cellular models) and in vivo [2,4,6-trinitrobenzenesulphonic acid (TNBS)-induced] colitis in mice. In Caco-2 cells transfected with a reporter gene for NF-κB activity, F. prausnitzii had no effect on IL-1β-induced NF-κB activity, whereas the supernatant abolished it. In vitro peripheral blood mononuclear cell stimulation by F. prausnitzii led to significantly lower IL-12 and IFN-γ production levels and higher secretion of IL-10. Oral administration of either live F. prausnitzii or its supernatant markedly reduced the severity of TNBS colitis and tended to correct the dysbiosis associated with TNBS colitis, as demonstrated by real-time quantitative PCR (qPCR) analysis. F. prausnitzii exhibits anti-inflammatory effects on cellular and TNBS colitis models, partly due to secreted metabolites able to block NF-κB activation and IL-8 production. These results suggest that counterbalancing dysbiosis using F. prausnitzii as a probiotic is a promising strategy in CD treatment.

Epidemiology and Natural History of Inflammatory Bowel Diseases

In the West, the incidence and prevalence of inflammatory bowel diseases has increased in the past 50 years, up to 8-14/100,000 and 120-200/100,000 persons, respectively, for ulcerative colitis (UC) and 6-15/100,000 and 50-200/100,000 persons, respectively, for Crohns disease (CD). Studies of migrant populations and populations of developing countries demonstrated a recent, slow increase in the incidence of UC, whereas that of CD remained low, but CD incidence eventually increased to the level of UC. CD and UC are incurable; they begin in young adulthood and continue throughout life. The anatomic evolution of CD has been determined from studies of postoperative recurrence; CD begins with aphthous ulcers that develop into strictures or fistulas. Lesions usually arise in a single digestive segment; this site tends to be stable over time. Strictures and fistulas are more frequent in patients with ileal disease, whereas Crohns colitis remains uncomplicated for many years. Among patients with CD, intestinal surgery is required for as many as 80% and a permanent stoma required in more than 10%. In patients with UC, the lesions usually remain superficial and extend proximally; colectomy is required for 10%-30% of patients. Prognosis is difficult to determine. The mortality of patients with UC is not greater than that of the population, but patients with CD have greater mortality than the population. It has been proposed that only aggressive therapeutic approaches, based on treatment of early recurrent lesions in asymptomatic individuals, have a significant impact on progression of these chronic diseases.

Ineffectiveness of Lactobacillus johnsonii LA1 for prophylaxis of postoperative recurrence in Crohn's disease: a randomised, double blind, placebo controlled GETAID trial.

Background and aims: Early endoscopic recurrence is frequent after intestinal resection for Crohn’s disease. Bacteria are involved, and probiotics may modulate immune responses to the intestinal flora. Here we tested the probiotic strain Lactobacillus johnsonii LA1 in this setting. Patients and methods: This was a randomised, double blind, placebo controlled study. Patients were eligible if they had undergone surgical resection of <1 m, removing all macroscopic lesions within the past 21 days. Patients were randomised to receive two packets per day of lyophilised LA1 (2×109 cfu) or placebo for six months; no other treatment was allowed. The primary endpoint was endoscopic recurrence at six months, with grade >1 in Rutgeerts’ classification or an adapted classification for colonic lesions. Endoscopic score was the maximal grade of ileal and colonic lesions. Analyses were performed primarily on an intent to treat basis. Results: Ninety eight patients were enrolled (48 in the LA1 group). At six months, endoscopic recurrence was observed in 30/47 patients (64%) in the placebo group and in 21/43 (49%) in the LA1 group (p = 0.15). Per protocol analysis confirmed this result. Endoscopic score distribution did not differ significantly between the LA1 and placebo groups. There were four clinical recurrences in the LA1 group and three in the placebo group. Conclusion:L johnsonii LA1 (4×109 cfu/day) did not have a sufficient effect, if any, to prevent endoscopic recurrence of Crohn’s disease.

Specificities of the fecal microbiota in inflammatory bowel disease.

Background: Abnormalities have been described in the fecal microbiota of patients with IBD, but it is not known whether they are specific for inflammatory bowel disease (IBD) or to some extent common to other forms of colitis. The aim of this study was to compare the bacterial composition of the dominant fecal microbiota in patients with Crohns disease (CD), ulcerative colitis (UC), infectious colitis (IC), and in healthy subjects (HS). Methods: Fluorescent in situ hybridization adapted to flow cytometry was used to analyze the bacterial composition of fecal samples from 13 patients with active CD, 13 patients with active UC, 5 patients with IC, and 13 HS. We used 6 group‐specific probes targeting 16S rRNA and spanning the main phylogenetic groups of the fecal microbiota. Results: A significantly higher proportion of the total fecal bacteria were recognized by the 6 probes in HS (86.6% ± 12.7) and in IC (84.0% ± 11.7) than in patients with IBD (70.9% ± 15 in CD and 60.1% ± 25.7 in UC). The Clostridium coccoides group was reduced in UC (20.0% ± 13.3 versus 42.0% ± 12.0 in HS; P < .001), whereas the C leptum group was reduced in CD (13.1% ± 11.9 versus 25.2% ± 14.2 in HS; P = .002). The Bacteroides group was more abundant in IC (36.4% ± 22.9) than in the other 3 groups (13.8% ± 11.8 in CD, 11.7% ± 11.7 in UC, 12.1% ± 7.0 in HS; P < .001 for all 3 comparisons). Conclusions: In IBD the dominant fecal microbiota comprises unusual bacterial species. Moreover, CD and UC fecal microbiota harbor specific discrepancies and differ from that of IC and healthy subjects.

Biodiversity of the mucosa-associated microbiota is stable along the distal digestive tract in healthy individuals and patients with IBD

Background: The mucosa‐associated microbiota, being very close to the inflammatory process associated with inflammatory bowel disease (IBD), may have a pathogenic role. We used a culture‐independent method to analyze the mucosa‐associated microbiota in IBD patients at various points of the distal digestive tract. Methods: Thirty‐five patients (20 with Crohns disease, 11 with ulcerative colitis, and 4 controls) underwent colonoscopy. Biopsies (n = 126) were taken from 4 sites: the ileum, right colon, left colon, and rectum. Fecal samples were also obtained from 7 individuals. Temporal temperature gradient gel electrophoresis (TTGE) of 16S rDNA was used to evaluate dominant species diversity. TTGE profiles were compared using software that measures the degree of similarity. Results: In a given individual, the overall similarity percentage between the 4 segments of the distal digestive tract was 94.7 ± 4.0%, regardless of clinical status. The average similarity of all profiles for a given segment was 59.3 ± 18.3% in the overall population. Dendrogram analysis showed that TTGE profiles did not cluster with clinical status. Differences were observed between the dominant fecal microbiota and the mucosa‐associated microbiota of all 4 sites, with similarity percentages less than 92%. Conclusions: These results confirm that the dominant species differ between the mucosa‐associated and fecal microbiota. They also show that, in a given individual, the microbiota is relatively stable along the distal digestive tract, showing a slight evolution in dominant species diversity from the ileum to the rectum, in both healthy subjects and patients with IBD.

Connecting dysbiosis, bile-acid dysmetabolism and gut inflammation in inflammatory bowel diseases

Objective Gut microbiota metabolises bile acids (BA). As dysbiosis has been reported in inflammatory bowel diseases (IBD), we aim to investigate the impact of IBD-associated dysbiosis on BA metabolism and its influence on the epithelial cell inflammation response. Design Faecal and serum BA rates, expressed as a proportion of total BA, were assessed by high-performance liquid chromatography tandem mass spectrometry in colonic IBD patients (42) and healthy subjects (29). The faecal microbiota composition was assessed by quantitative real-time PCR. Using BA profiles and microbiota composition, cluster formation between groups was generated by ranking models. The faecal BA profiles in germ-free and conventional mice were compared. Direct enzymatic activities of BA biotransformation were measured in faeces. The impact of BA on the inflammatory response was investigated in vitro using Caco-2 cells stimulated by IL-1β. Results IBD-associated dysbiosis was characterised by a decrease in the ratio between Faecalibacterium prausntizii and Escherichia coli. Faecal-conjugated BA rates were significantly higher in active IBD, whereas, secondary BA rates were significantly lower. Interestingly, active IBD patients exhibited higher levels of faecal 3-OH-sulphated BA. The deconjugation, transformation and desulphation activities of the microbiota were impaired in IBD patients. In vitro, secondary BA exerted anti-inflammatory effects, but sulphation of secondary BAs abolished their anti-inflammatory properties. Conclusions Impaired microbiota enzymatic activity observed in IBD-associated dysbiosis leads to modifications in the luminal BA pool composition. Altered BA transformation in the gut lumen can erase the anti-inflammatory effects of some BA species on gut epithelial cells and could participate in the chronic inflammation loop of IBD.

CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands

Complex interactions between the host and the gut microbiota govern intestinal homeostasis but remain poorly understood. Here we reveal a relationship between gut microbiota and caspase recruitment domain family member 9 (CARD9), a susceptibility gene for inflammatory bowel disease (IBD) that functions in the immune response against microorganisms. CARD9 promotes recovery from colitis by promoting interleukin (IL)-22 production, and Card9−/− mice are more susceptible to colitis. The microbiota is altered in Card9−/− mice, and transfer of the microbiota from Card9−/− to wild-type, germ-free recipients increases their susceptibility to colitis. The microbiota from Card9−/− mice fails to metabolize tryptophan into metabolites that act as aryl hydrocarbon receptor (AHR) ligands. Intestinal inflammation is attenuated after inoculation of mice with three Lactobacillus strains capable of metabolizing tryptophan or by treatment with an AHR agonist. Reduced production of AHR ligands is also observed in the microbiota from individuals with IBD, particularly in those with CARD9 risk alleles associated with IBD. Our findings reveal that host genes affect the composition and function of the gut microbiota, altering the production of microbial metabolites and intestinal inflammation.

Analysis of bacterial bowel communities of IBD patients: what has it revealed?

The bacterial community, in whole or in part, resident in the bowel of humans is considered to fuel the chronic immune inflammatory conditions characteristic of Crohns disease and ulcerative colitis. Chronic or recurrent pouchitis in ulcerative colitis patients is responsive to antibiotic therapy, indicating that bacteria are the etiological agents. Microbiological investigations of the bacterial communities in stool or of biopsy‐associated bacteria have so far failed to reveal conclusively the existence of pathogens or bacterial communities of consistently altered composition in IBD patients relative to control subjects. Confounding factors need to be eliminated from future studies by using better‐defined patient populations of newly diagnosed and untreated individuals and by improved sampling procedures.

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.

Usefulness of co-treatment with immunomodulators in patients with inflammatory bowel disease treated with scheduled infliximab maintenance therapy

Background and aims Concomitant use of immunosuppressants (IS) with scheduled infliximab (IFX) maintenance therapy for Crohns disease (CD) or ulcerative colitis (UC) is debated. The aim of this study was to assess whether IS co-treatment is useful in patients with inflammatory bowel disease (IBD) on scheduled IFX infusions. Methods 121 consecutive patients with IBD (23 UC, 98 CD) treated by IFX and who received at least 6 months of IS co-treatment (azathioprine (AZA) or methotrexate (MTX)) were studied. In each patient, the IFX treatment duration was divided into semesters which were independently analysed regarding IBD activity. Results Semesters with IS (n=265) and without IS (n=319) were analysed. IBD flares, perianal complications and switch to adalimumab were less frequently observed in semesters with IS than in those without IS (respectively: 19.3% vs 32.0%, p=0.003; 4.1% vs 11.8%, p=0.03; 1.1% vs 5.3%, p=0.006). Maximal C-reactive protein (CRP) level and IFX dose/kg observed during the semesters were lower in semesters with IS. Within semesters with IS, IBD flares and perianal complications were less frequently observed in semesters with AZA than in those with MTX. In multivariate analysis, IS co-treatment was associated with a decreased risk of IBD flare (OR 0.52; 95% CI 0.35 to 0.79) Conclusion In patients with IBD receiving IFX maintenance therapy, IS co-treatment is associated with reduced IBD activity, IFX dose and switch to adalimumab. In this setting, co-treatment with AZA seems to be more effective than co-treatment with MTX. Benefit of such a combination treatment has to be balanced with potential risks, notably infections and cancers.