Bénédicte Pigneur

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.

Defective IL10 signaling defining a subgroup of patients with inflammatory bowel disease.

OBJECTIVES:Early onset inflammatory bowel diseases (EO-IBD) developing during the first year of life are likely to reflect inherited defects in key mechanism(s) controlling intestinal homeostasis, as recently suggested for interleukin 10 (IL10). Thus, we aimed to further elaborate the hypothesis of defective anti-inflammatory responses in patients with IBD.METHODS:The capacities of transforming growth factor β (TGFβ) and IL10 to inhibit proinflammatory cytokine production by monocyte-derived dendritic cells (MoDC) or peripheral blood cells (PBMC) was analyzed in 75 children with IBD, including 13 infants with EO-IBD (in whom autoimmune diseases or classical immunodeficiencies were ruled out). IL10 receptor-A/-B expression, STAT3 activation in response to IL6, IL10, IL21, IL22 were analyzed by FACS and western blotting. IL10RA and B genes were sequenced. The response to IL22 was tested in ileal/colonic tissue cultures. Tissue gene expression was analyzed by Taqman real-time polymerase chain reaction.RESULTS:Production of IL10 in response to bacterial motifs was normal in all IBD patients. In contrast to our original hypothesis, no defect of the anti-inflammatory potential of TGFβ and IL10 was observed in children with IBD or EO-IBD except two infants who presented with granuloma-positive colitis at 3 months of life: no response to IL10 was observed secondary to mutations in the α (p.R262C) or β (p.E141X) chain of IL10R, respectively, although a fully functional Jak-STAT3 pathway was present in both patients. When analyzing the regulation of intestinal bacterial clearance, we detected a defect in the patient with absent IL10 RB to upregulate protective transcripts in response to IL22, whereas all other EO-IBD patients, including the patient with an abnormal α chain, responded normally.CONCLUSIONS:Impaired IL10 signaling characterizes a subgroup of IBD patients, whereas the majority of children with severe IBD including EO forms normally produces and responds to IL10. Defective IL22 signaling may additionally impair intestinal epithelial clearance. Our data point out the complexity of IBD, which represent a group of distinct diseases with several pathogenetic abnormalities.

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.

Natural history of Crohn's disease: Comparison between childhood- and adult-onset disease

Background: Childhood‐onset Crohns disease (CD) might reflect a more severe form of disease. To test this hypothesis we analyzed the long‐term natural history of CD in an adult cohort of patients with childhood‐onset compared to adult‐onset CD. Methods: We selected 206 childhood‐onset CD patients among 2992 adult patients with a diagnosis of CD established before December 31, 2000. Disease characteristics were prospectively assessed during follow‐up until December 2007 and compared to adult‐onset CD patients matched 2 to 1 on gender, year of CD diagnosis, and disease location. Results: Compared to adult‐onset CD, patients with childhood‐onset CD were more likely to have a severe disease, with an increased year‐by‐year disease activity index (37% of patient‐years in childhood‐onset group versus 31% in the adult‐onset group, P < 0.001). Immunosuppressant requirement was also increased with a 10‐year cumulative risk of 54 ± 3% in childhood‐onset CD group versus 45 ± 2%, in the adult‐onset CD group (P < 0.001). Cumulative risks of stricturing and penetrating complications and surgical resections were not statistically different between groups. Accordingly, these events occurred at a younger age in the childhood‐onset CD group. At the age of 30 years the actuarial risk of having undergone an extensive intestinal resection was 48 ± 5% in the childhood‐onset group versus 14 ± 2% in the adult‐onset group (P < 0.001). Conclusions: Patients with childhood‐onset CD exhibit a more active disease and require more immunosuppressive therapy. This feature is observed irrespective of the disease location, suggesting an intrinsic more severe phenotype. Inflamm Bowel Dis 2009

The efficacy of exclusive nutritional therapy in paediatric Crohn’s disease, comparing fractionated oral vs. continuous enteral feeding

Background  Nutritional therapy has an established role as induction therapy in paediatric Crohn’s disease. However, compliance is the main difficulty and may be greatly influenced by the administration route.

Phenotypic Characterization of Very Early-onset IBD Due to Mutations in the IL10, IL10 Receptor Alpha or Beta Gene: A Survey of the GENIUS Working Group.

Objective:Early-onset inflammatory bowel disease starting within the first months of life could be due to a particular genetic defect. We set up the GENetically determined ImmUne-mediated enteropathieS (GENIUS) network and collected infants with a proven defect of the IL10 axis for accurate phenotyping of disease presentation and evolution. Design:Ten patients with early-onset inflammatory bowel disease with confirmed mutations in IL10, IL10RA, or IL10RB genes were characterized on clinical, endoscopic–histological, immunobiological, and radiological findings. Functional assays to confirm defective responses to IL10 were performed on peripheral blood mononuclear cells. Results:A functional defect in IL10 signaling was confirmed in all IL10R patients tested. Disease started with severe diarrhea within the first 12 weeks in all patients. All infants showed Crohns disease–like ulcerations limited to the colon with marked perianal inflammation (fissures, abscess, and fistula); disease progression to the small bowel occurred in only 1 patient. Four of the 10 patients had granulomata on histology, and all patients showed Crohns disease–like mesenteric infiltration on imaging. Disease pattern was indistinguishable between IL10R alpha or beta chain or IL10 defects; autoimmunity was not observed. Mutations in IL10 were more frequently associated with bacterial and viral infections. Patients responded partially to treatment with steroids or anti–tumor necrosis factor drugs, whereas hematopoietic stem cell transplantation proved efficacious. Conclusion:The importance of the IL10 pathway within the colonic mucosa is highlighted by the development of severe colitis within a few weeks in infants with mutations in IL10, IL10RA, or IL10RB. Immunosuppression failed to correct the defect in this pathway, which seems to be a key to controlling inflammation in the colon.

Current smoking differentially affects blood mononuclear cells from patients with crohn's disease and ulcerative colitis: Relevance to its adverse role in the disease

Background: Epidemiologic data suggest that smoking increases the risk and the severity of Crohns disease (CD), although it may protect patients with ulcerative colitis (UC). To investigate this paradox, we evaluated the effect of cigarette smoke in the function of blood mononuclear cells from healthy subjects and patients with CD or UC in flare up. Methods: The production of mediators associated with inflammation but also with protective functions was evaluated by enzyme‐linked immunosorbent assay (ELISA) and enzyme immunoassay (EIA), following either in vivo or in vitro exposure to cigarette smoke. Results: We found that mononuclear cells from smokers with CD were functionally impaired. These cells secreted lower levels of chemokines and cytokines as compared with nonsmoker counterparts, whereas healthy smokers or smokers with UC were not affected. Similar findings were noted after in vitro exposure to cigarette smoke extract. In addition, cells from patients with CD who smoke presented a defective sensitivity to antiinflammatory or antioxidant protection, and particularly synthesized lower levels of cytoprotective Hsp70. The effects observed were not due to diminished cell viability. Our experiments suggest that cigarette smoke‐related responses were largely dependent on oxidative stress generated, and not on the nicotine component. Conclusions: Overall, our data point out the presence of biological differences between blood mononuclear cells from patients with CD and UC toward cigarette smoke that might support its opposite role in both diseases. (Inflamm Bowel Dis 2012;)

Tolerance and efficacy of azathioprine in pediatric Crohn's disease

Background: Thiopurines are considered first‐line immunomodulators for the prevention of relapse in moderate to severe pediatric Crohns disease (CD). Early introduction of thiopurines was shown in a pediatric trial to maintain steroid‐free remission in 90% of patients for 18 months. In the present study we analyzed the tolerance and efficacy of azathioprine (AZA) to maintain remission in a homogenous single‐center observational cohort of children with CD. Methods: In all, 105 pediatric CD patients (male/female 68/37) were retrospectively evaluated for the efficacy of AZA (doses 1.4–4 mg/kg) to maintain remission at 6, 12, 18, and 24 months of follow‐up. Overall, 93 children were included with active disease (pediatric Crohns disease activity index [PCDAI] >30), steroid/enteral‐nutrition dependency, or postileocecal resection. Remission was defined as PCDAI ≤10 without steroids. Patients requiring antitumor necrosis factor (TNF) medication, other immunomodulators, or surgery were considered to experience a relapse. Results: Based on PCDAI, steroid‐free remission was achieved in 56/93 (60.2%), 37/93 (39.8%), 31/93 (33.3%), and 29/93 (31.2%) at visits month (M)6, M12, M18, and M24, respectively. Within the first 4 weeks, AZA was stopped in 10/93 patients due to adverse reactions (pancreatitis, nausea, vomiting, skin reactions, general weakness), or not introduced due to low thiopurine methyl transferase (TPMT) activity (n = 3). No neutropenia occurred in patients with normal TPMT activity. Three infectious episodes were documented requiring temporary AZA suspension. Conclusions: AZA is efficacious in maintaining remission in pediatric CD patients, but to a lesser extent than previously suggested. The majority of patients who are in steroid‐free remission at 12 months remained in prolonged remission. Overall tolerance of AZA was excellent. Inflamm Bowel Dis 2011

Fecal microbiota transplantation in inflammatory bowel disease: the quest for the holy grail

Inflammatory bowel disease (IBD) is due to an aberrant immune response toward luminal antigens, probably commensal bacteria, in genetically susceptible subjects and is also influenced by environmental factors. An imbalanced intestinal microbiota known as “dysbiosis,” characterized by an increased proportion of pro-inflammatory microorganisms and a decreased proportion of anti-inflammatory microorganisms, has been repeatedly observed in IBD and is now recognized as a key factor in the gut inflammatory process. Fecal microbiota transplantation (FMT) has gained interest as a novel treatment option in IBD. The goal of FMT in IBD is not only to correct the dysbiosis, but also to restore a normal dialog between the host immune system and the microbiota. Data are still scarce, but the results of the first studies suggest that FMT could be a promising therapy in IBD. More studies are needed to define the best indications, optimal timing, frequency, mode of delivery, and the optimal donor for each patient.

Outcome of home parenteral nutrition in 251 children over a 14-y period: report of a single center.

BACKGROUND Parenteral nutrition (PN) is the main treatment for intestinal failure. OBJECTIVE We aimed to review the indications for home parenteral nutrition (HPN) in children and describe the outcome over a 14-y period from a single center. DESIGN We conducted a retrospective study that included all children who were referred to our institution and discharged while receiving HPN between 1 January 2000 and 31 December 2013. The indications for HPN were divided into primary digestive diseases (PDDs) and primary nondigestive diseases (PNDDs). We compared our results to a previous study that was performed in our unit from 1980 to 2000 and included 302 patients. RESULTS A total of 251 patients were included: 217 (86%) had a PDD. The mean ± SD age at HPN onset was 0.7 ± 0.3 y, with a mean duration of 1.9 ± 0.4 y. The indications for HPN were short bowel syndrome (SBS) (59%), PNDD (14%), congenital enteropathies (10%), chronic intestinal pseudo-obstruction syndromes (9%), inflammatory bowel diseases (5%), and other digestive diseases (3%). By 31 December 2013, 52% of children were weaned off of HPN, 9% of the PDD subgroup had intestinal transplantation, and 10% died mostly because of immune deficiency. The major complications of HPN were catheter-related bloodstream infections (CRBSIs) (1.7/1000 d of PN) and intestinal failure-associated liver disease (IFALD) (51 children; 20% of cohort). An increased rate of CRBSIs was observed compared with our previous study, but we saw a decreasing trend since 2012. No noteworthy deceleration of growth was observed in SBS children 6 mo after weaning off HPN. CONCLUSIONS SBS was the major indication for HPN in our cohort. IFALD and CRBSIs were potentially life-threatening problems. Nevertheless, complication rates were low, and deaths resulted mostly from the underlying disease.