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ObjectiveIn the management of patients with IBD, there is a need to identify prognostic markers and druggable biological pathways to improve mucosal repair and probe the efficacy of tumour necrosis factor alpha biologics. Vnn1 is a pantetheinase that degrades pantetheine to pantothenate (vitamin B5, a precursor of coenzyme A (CoA) biosynthesis) and cysteamine. Vnn1 is overexpressed by inflamed colonocytes. We investigated its contribution to the tolerance of the intestinal mucosa to colitis-induced injury.DesignWe performed an RNA sequencing study on colon biopsy samples from patients with IBD stratified according to clinical severity and modalities of treatment. We generated the VIVA mouse transgenic model, which specifically overexpresses Vnn1 on intestinal epithelial cells and explored its susceptibility to colitis. We developed a pharmacological mimicry of Vnn1 overexpression by administration of Vnn1 derivatives.ResultsVNN1 overexpression on colonocytes correlates with IBD severity. VIVA mice are resistant to experimentally induced colitis. The pantetheinase activity of Vnn1 is cytoprotective in colon: it enhances CoA regeneration and metabolic adaptation of colonocytes; it favours microbiota-dependent production of short chain fatty acids and mostly butyrate, shown to regulate mucosal energetics and to be reduced in patients with IBD. This prohealing phenotype is recapitulated by treating control mice with the substrate (pantethine) or the products of pantetheinase activity prior to induction of colitis. In severe IBD, the protection conferred by the high induction of VNN1 might be compromised because its enzymatic activity may be limited by lack of available substrates. In addition, we identify the elevation of indoxyl sulfate in urine as a biomarker of Vnn1 overexpression, also detected in patients with IBD.ConclusionThe induction of Vnn1/VNN1 during colitis in mouse and human is a compensatory mechanism to reinforce the mucosal barrier. Therefore, enhancement of vitamin B5-driven metabolism should improve mucosal healing and might increase the efficacy of anti-inflammatory therapy.

ObjectiveLoss of the Crohn’s disease predisposing NOD2 gene results in an intestinal microenvironment conducive for colonisation by attaching-and-effacing enteropathogens. However, it remains elusive whether it relies on the intracellular recruitment of the serine-threonine kinase RIPK2 by NOD2, a step that is required for its activation of the transcription factor NF-κB.DesignColonisation resistance was evaluated in wild type and mutant mice, as well as in ex-germ-free (ex-GF) mice which were colonised either with faeces from Ripk2-deficient mice or with bacteria with similar preferences for carbohydrates to those acquired by the pathogen. The severity of the mucosal pathology was quantified at several time points postinfection by using a previously established scoring. The community resilience in response to infection was evaluated by 16S ribosomal RNA gene sequence analysis. The control of pathogen virulence was evaluated by monitoring the secretion of Citrobacter-specific antibody response in the faeces.ResultsPrimary infection was similarly outcompeted in ex-GF Ripk2-deficient and control mice, demonstrating that the susceptibility to infection resulting from RIPK2 deficiency cannot be solely attributed to specific microbiota community structures. In contrast, delayed clearance of Citrobacter rodentium and exacerbated histopathology were preceded by a weakened propensity of intestinal macrophages to afford innate lymphoid cell activation. This tissue protection unexpectedly required the regenerating family member 3β by instigating interleukin (IL) 17A-mediated neutrophil recruitment to the intestine and subsequent phosphorylation of signal transducer and activator of transcription 3.ConclusionsThese results unveil a previously unrecognised mechanism that efficiently protects from colonisation by diarrhoeagenic bacteria early in infection.

Objective Epidemiological and clinical data indicate that patients suffering from IBD with long-standing colitis display a higher risk to develop colorectal high-grade dysplasia. Whereas carcinoma invasion and metastasis rely on basement membrane (BM) disruption, experimental evidence is lacking regarding the potential contribution of epithelial cell/BM anchorage on inflammation onset and subsequent neoplastic transformation of inflammatory lesions. Herein, we analyse the role of the alpha 6 beta 4 integrin receptor found in hemidesmosomes that attach intestinal epithelial cells (IECs) to the laminin-containing BM. Design We developed new mouse models inducing IEC-specific ablation of alpha 6 integrin either during development (alpha 6(Delta IEC)) or in adults (alpha 6(Delta IEC-TAM)). Results Strikingly, all alpha 6(Delta IEC) mutant mice spontaneously developed long-standing colitis, which degenerated overtime into infiltrating adenocarcinoma. The sequence of events leading to disease onset entails hemidesmosome disruption, BM detachment, IL-18 overproduction by IECs, hyperplasia and enhanced intestinal permeability. Likewise, IEC-specific ablation of alpha 6 integrin induced in adult mice (alpha 6(Delta IEC-TAM)) resulted in fully penetrant colitis and tumour progression. Whereas broad-spectrum antibiotic treatment lowered tissue pathology and IL-1 beta secretion from infiltrating myeloid cells, it failed to reduce Th1 and Th17 response. Interestingly, while the initial intestinal inflammation occurred independently of the adaptive immune system, tumourigenesis required B and T lymphocyte activation. Conclusions We provide for the first time evidence that loss of IECs/BM interactions triggered by hemidesmosome disruption initiates the development of inflammatory lesions that progress into high-grade dysplasia and carcinoma. Colorectal neoplasia in our mouse models resemble that seen in patients with IBD, making them highly attractive for discovering more efficient therapies.

Background: Crohn’s disease is one of the principal human chronic inflammatory bowel diseases. Although its aetiology is still unknown, its complex pathogenesis has environmental, immunological, and genetic determinants. CARD15 is the first susceptibility gene implicated in the predisposition to Crohn’s disease and is known to be expressed only in monocytes. However, its expression in situ has not yet been studied. Aims: To analyse the tissue distribution of CARD15 and identify cells producing CARD15 in samples of colon from patients with Crohn’s disease and control subjects. Patients and methods: We analysed CARD15 gene expression in surgical specimens of colon from eight children with Crohn’s disease and nine controls by immunohistochemistry, in situ hybridisation, and reverse transcription-polymerase chain reaction (RT-PCR). Results: We showed that CARD15 was present only in the cytoplasm of macrophages in the normal colon. Increased CARD15 expression was detected in Crohn’s disease lesions. There were more CARD15 positive cells in Crohn’s disease lesions than in uninvolved areas. Both intestinal epithelial cells, macrophages, and their derivatives overproduced CARD15 in Crohn’s disease. To further assess CARD15 expression by intestinal epithelial cells, we performed RT-PCR on freshly isolated intestinal epithelial cells, and showed that these cells isolated from Crohn’s disease samples contained more CARD15 mRNA than intestinal epithelial cells from controls. Conclusions: We have demonstrated that colonic involvement in active Crohn’s disease is associated with increased CARD15 gene expression in both macrophages and intestinal epithelial cells. Therefore, this deregulation can affect the host-environment interaction and thus contribute to the pathogenesis of this disease.

It is becoming increasingly clear that the ‘one microbe-one disease’ paradigm must be reconsidered, because it fails to account for the pathogenesis of several common human illnesses with ever-increasing socioeconomic impacts (such as IBD). Notably, instability within the gut's microbial communities (referred to as dysbiosis) has been linked to loss of gut barrier function in IBD. In particular, IBDs are associated with a greater preponderance of certain Bacteroidetes and a lower abundance of Firmicutes. 1 ,2 Likewise, several targeted approaches in mice have revealed that the major Crohn's disease-predisposing NOD2 gene has a role in protecting against intestinal inflammation afforded by some Bacteroides spp (including B vulgatus ).3 ,4 Remarkably, reciprocal transplantation of faecal microbiota from Nod2 -deficient mice enhanced the vulnerability of control animals to colonic injury.3 In line with reverse genetic studies in mice, subclinical dysbiosis has been observed in healthy, first-degree relatives of patients with Crohn's disease.5 One can therefore reasonably presume that familial similarities in the microbiome are related to similar host genetic compositions.3 ,6 Consistently, twin studies have demonstrated that variations in the host genome may influence the structure and composition of the …

BACKGROUND AND AIMS—Inflammatory bowel disease (IBD) includes ulcerative colitis and Crohn's disease, both of which are multifactorial diseases involving the interaction of genetic and environmental factors. A region on chromosome 12 centred around the marker locus D12S83 has previously been associated with IBD predisposition. The aim of the study was to investigate this genetic region in an independent panel of European families affected by Crohn's disease. METHODS—A sample of 95 families with two or more affected relatives and 75 simplex nuclear families were genotyped for 19 microsatellite loci located on chromosome 12. A search for linkage and linkage disequilibrium was performed using non-parametric two point and multipoint analyses with the Analyze and Genehunter packages. RESULTS—No evidence of linkage or linkage disequilibrium was observed for any of the marker loci, including D12S83 (p=0.35 for the two point linkage test). Multipoint linkage analysis also failed to reveal positive linkage on chromosome 12. Power calculations allowed us to reject the hypothesis that the genetic region of chromosome 12 centred on D12S83 contains a susceptibility locus with a relative risk (λs) equal to or greater than 2.0 in these families. CONCLUSION—Failure to detect linkage or linkage disequilibrium in these families suggests that the chromosome 12 locus previously reported to be associated with genetic predisposition to IBD does not play a role in all European family samples. This observation is compatible with heterogeneity in the genetic basis of susceptibility to the disease and/or exposure to various environmental factors among Caucasian families. Keywords: chromosome 12; inflammatory bowel disease; Crohn's disease; linkage analyses; replication study

Objectives Dysbiosis of the intestinal microbiota is associated with Crohn's disease (CD). Functional evidence for a causal role of bacteria in the development of chronic small intestinal inflammation is lacking. Similar to human pathology, TNFdeltaARE mice develop a tumour necrosis factor (TNF)-driven CD-like transmural inflammation with predominant ileal involvement. Design Heterozygous TNFdeltaARE mice and wildtype (WT) littermates were housed under conventional (CONV), specific pathogen-free (SPF) and germ-free (GF) conditions. Microbial communities were analysed by high-throughput 16S ribosomal RNA gene sequencing. Metaproteomes were measured using LC-MS. Temporal and spatial resolution of disease development was followed after antibiotic treatment and transfer of microbial communities into GF mice. Granulocyte infiltration and Paneth cell function was assessed by immunofluorescence and gene expression analysis. Results GF-TNFdeltaARE mice were free of inflammation in the gut and antibiotic treatment of CONV-TNFdeltaARE mice attenuated ileitis but not colitis, demonstrating that disease severity and location are microbiota-dependent. SPF-TNFdeltaARE mice developed distinct ileitis-phenotypes associated with gradual loss of antimicrobial defence. 16S analysis and metaproteomics revealed specific compositional and functional alterations of bacterial communities in inflamed mice. Transplantation of disease-associated but not healthy microbiota transmitted CD-like ileitis to GF-TNFdeltaARE recipients and triggered loss of lysozyme and cryptdin-2 expression. Monoassociation of GF-TNFdeltaARE mice with the human CD-related Escherichia coli LF82 did not induce ileitis. Conclusions We provide clear experimental evidence for the causal role of gut bacterial dysbiosis in the development of chronic ileal inflammation with subsequent failure of Paneth cell function.

The peroxisome proliferator activated receptor γ (PPARγ) is a nuclear receptor highly expressed in the colon and playing a key role in bacterial induced inflammation. Regulation of colon inflammation by this receptor has been well demonstrated in many experimental models of colitis but also in patients with ulcerative colitis, characterised by impaired expression of PPARγ confined to their colon epithelial cells. Recent data showing that PPARγ was the major functional receptor mediating the common aminosalicylate activities in inflammatory bowel diseases (IBD) have also reinforced the roles of this receptor in the control of intestinal inflammation. The aims of this review are to discuss the potential roles of PPARγ in the physiopathology of IBD, as well as the emerging therapeutic strategies targeting this receptor. Current evidence suggests that Crohn’s disease (CD) and ulcerative colitis (UC) result from a complex interplay between genetic and environmental factors, leading to an abnormal innate and adaptive immune response of the gut directed against luminal constituents in genetically determined patients. Identification of cytoplasmic receptors of bacterial peptidoglycan, namely nucleotide oligomerisation domain (NOD)2/caspase recruitment domain (CARD)15 and NOD1/CARD4, as CD susceptibility genes reinforced the pivotal role of the interactions between enteric microbes and the intestinal immune system in the physiopathology of IBD.1–3 Furthermore, recent advances in our laboratory and others also indicate the involvement of another key receptor, PPARγ, which regulates colon inflammation. This represents a new target in the development of therapeutic molecules in IBD. PPARγ is a nuclear receptor discovered in mammals in 1993 as an orphan receptor.4 Until recently, PPARγ was known as a receptor mainly expressed by adipose tissue and involved in the regulation of insulin resistance. PPARγ is activated by antidiabetic thiazolidinedione drugs.5 In 1998, the first studies were published reporting a potential link between this receptor and …