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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, TNF(deltaARE) mice develop a tumour necrosis factor (TNF)-driven CD-like transmural inflammation with predominant ileal involvement., Design: Heterozygous TNF(deltaARE) 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-TNF(deltaARE) mice were free of inflammation in the gut and antibiotic treatment of CONV-TNF(deltaARE) mice attenuated ileitis but not colitis, demonstrating that disease severity and location are microbiota-dependent. SPF-TNF(deltaARE) 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-TNF(deltaARE) recipients and triggered loss of lysozyme and cryptdin-2 expression. Monoassociation of GF-TNF(deltaARE) 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., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Objective: Mesenteric fat hyperplasia is a hallmark of Crohn's disease (CD), and C reactive protein (CRP) is correlated with disease activity. The authors investigated whether mesenteric adipocytes may be a source of CRP in CD and whether inflammatory and bacterial triggers may stimulate its production by adipocytes., Design: CRP expression in the mesenteric and subcutaneous fats of patients with CD and the correlation between CRP plasma concentrations and mesenteric messenger RNA (mRNA) levels were assessed. The impact of inflammatory and bacterial challenges on CRP synthesis was tested using an adipocyte cell line. Bacterial translocation to mesenteric fat was studied in experimental models of colitis and ileitis and in patients with CD., Results: CRP expression was increased in the mesenteric fat of patients with CD, with mRNA levels being 80 ± 40 (p<0.05) and 140 ± 65 (p=0.04) times higher than in the mesenteric fat of patients with ulcerative colitis and in the subcutaneous fat of the same CD subjects, respectively, and correlated with plasma levels. Escherichia coli (1230 ± 175-fold, p<0.01), lipopolysaccharide (26 ± 0.5-fold, p<0.01), tumour necrosis factor α (15 ± 0.3-fold, p<0.01) and interleukin-6 (10 ± 0.7-fold, p<0.05) increased CRP mRNA levels in adipocyte 3T3-L1 cells. Bacterial translocation to mesenteric fat occurred in 13% and 27% of healthy and CD subjects, respectively, and was increased in experimental colitis and ileitis. Human mesenteric adipocytes constitutively expressed mRNA for TLR2, TLR4, NOD1 and NOD2., Conclusion: Mesenteric fat is an important source of CRP in CD. CRP production by mesenteric adipocytes may be triggered by local inflammation and bacterial translocation to mesenteric fat, providing a mechanism whereby mesenteric fat hyperplasia may contribute to inflammatory response in CD.

Background: Iron replacement therapy is a common treatment in patients with anaemia and Crohn's disease, but oral iron supplements are less tolerated. The pathogenesis of Crohn's disease is attributed to intestinal bacteria and environmental factors that trigger disease in a genetically predisposed host. The aim of this study was to characterise the interrelationship between luminal iron sulfate, systemic iron, the gut microbiota and the development of chronic ileitis in a murine model of Crohn's disease., Methods: Wild type (WT) and heterozygous TNF(ΔARE/WT) mice were fed with an iron sulfate containing or iron sulfate free diet in combination with intraperitoneal control injections or iron injections for 11 weeks., Results: TNF(ΔARE/WT) mice develop severe inflammation of the distal ileum but remained completely healthy when transferred to an iron sulfate free diet, even if iron was systemically repleted. Absence of luminal iron sulfate reduced cellular markers of endoplasmic reticulum (ER) stress responses and pro-apoptotic mechanisms in the ileal epithelium. Phenotype or reactivity of major effector intraepithelial CD8αβ(+) T cells were not altered in the absence of luminal iron. Interestingly, ER stress mechanisms sensitised the small intestinal epithelial cell (IEC) line Mode-K to cytotoxic function of effector T cells from TNF(ARE/WT) mice. Pyrosequencing of 16S rRNA tags of the caecal microbiota revealed that depletion of luminal iron sulfate induced significant compositional alterations, while total microbial diversity (Shannon's diversity index) and number of total operational taxonomic units were not affected., Conclusion: This study showed that an iron sulfate free diet in combination with systemic iron repletion prevents the development of chronic ileitis in a murine model of Crohn's disease. Luminal iron may directly affect IEC function or generate a pathological milieu in the intestine that triggers epithelial cell stress-associated apoptosis through changes in microbial homeostasis. These results suggest that oral replacement therapy with iron sulfate may trigger inflammatory processes associated with progression of Crohn's disease-like ileitis.

Objective: Melanin-concentrating hormone (MCH) is a hypothalamic orexigenic neuropeptide that regulates energy balance. However, the distribution of MCH and its receptor MCHR1 in tissues other than brain suggested additional, as yet unappreciated, roles for this neuropeptide. Based on previous paradigms and the presence of MCH in the intestine as well as in immune cells, its potential role in gut innate immune responses was examined., Methods: In human intestinal xenografts grown in mice, changes in the expression of MCH and its receptors following treatment with Clostridium difficile toxin A, the causative agent of antibiotic-associated diarrhoea in hospitalised patients, were examined. In colonocytes, the effect of C difficile toxin A treatment on MCHR1 expression, and of MCH on interleukin 8 (IL8) expression was examined. MCH-deficient mice and immunoneutralisation approaches were used to examine the role of MCH in the pathogenesis of C difficile toxin A-mediated acute enteritis., Results: Upregulation of MCH and MCHR1 expression was found in the human intestinal xenograft model, and of MCHR1 in colonocytes following exposure to toxin A. Treatment of colonocytes with MCH resulted in IL8 transcriptional upregulation, implying a link between MCH and inflammatory pathways. In further support of this view, MCH-deficient mice developed attenuated toxin A-mediated intestinal inflammation and secretion, as did wild-type mice treated with an antibody against MCH or MCHR1., Conclusion: These findings signify MCH as a mediator of C difficile-associated enteritis and possibly of additional gut pathogens. MCH may mediate its proinflammatory effects at least in part by acting on epithelial cells in the intestine.

Background: In the course of inflammatory bowel diseases (IBD) and acute murine ileitis following peroral Toxoplasma gondii infection, commensal Escherichia coli accumulate at inflamed mucosal sites and aggravate small intestinal immunopathology., Aim: To unravel the molecular mechanisms by which commensal E coli exacerbate ileitis., Methods: Ileitis was investigated in mice that lack Toll-like receptors (TLR) 2 or 4, specific for bacterial lipoproteins (LP) or lipopolysaccharide (LPS), respectively. Gnotobiotic mice, in which any cultivable gut bacteria were eradicated by antibiotic treatment, were used to study the role of LPS in ileitis., Results: Microbiological analyses revealed that E coli increase in the inflamed ileum. TLR4(-/-), but not TLR2(-/-), mice displayed reduced mortality and small intestinal immunopathology. Decreased interferon (IFN)-gamma and nitric oxide (NO) levels in the inflamed terminal ileum of TLR4(-/-) mice indicated that TLR4 signalling aggravates ileitis via local mediator release from immune cells. E coli strains isolated from the inflamed ileum activated cultured mouse macrophages and induced TLR4-dependent nuclear factor kappaB activation and NO production in human embryonic kidney 293 cells and in peritoneal macrophages, respectively. Most strikingly, in contrast with wild-type mice, gnotobiotic TLR4(-/-) mice were protected from induction of ileitis by treatment with purified E coli lipid A or colonisation with live E coli. Finally, prophylactic treatment with the LPS scavenger polymyxin B ameliorated T gondii-induced ileitis., Conclusion: These findings highlight the innate immune system as a key player in T gondii-induced ileal immunopathology. Treatment with LPS or TLR4 antagonists may represent a novel strategy for prophylaxis and/or therapy of small intestinal inflammation in IBD.

Background and Aims: The colonic microflora is involved in the pathogenesis of Crohn's disease (CD) but less than 30% of the microflora can be cultured. We investigated potential differences in the faecal microflora between patients with colonic CD in remission (n=9), patients with active colonic CD (n=8), and healthy volunteers (n=16) using culture independent techniques., Methods: Quantitative dot blot hybridisation with six radiolabelled 16S ribosomal ribonucleic acid (rRNA) targeting oligonucleotide probes was used to measure the proportions of rRNA corresponding to each phylogenetic group. Temporal temperature gradient gel electrophoresis (TTGE) of 16S rDNA was used to evaluate dominant species diversity., Results: Enterobacteria were significantly increased in active and quiescent CD. Probe additivity was significantly lower in patients (65 (11)% and 69 (6)% in active CD and quiescent CD) than in healthy controls (99 (7)%). TTGE profiles varied markedly between active and quiescent CD but were stable in healthy conditions., Conclusion: The biodiversity of the microflora remains high in patients with CD. Enterobacteria were observed significantly more frequently in CD than in health, and more than 30% of the dominant flora belonged to yet undefined phylogenetic groups.

Background: A new subline of the senescence accelerated mouse (SAM) P1/Yit strain has been established which shows spontaneous enteric inflammation under specific pathogen free (SPF) conditions., Aims: To elucidate the pathogenesis of enteric inflammation in this new subline., Methods: The SPF and germ free (GF) SAMP1/Yit strains were used. Histological, immunological, and microbiological characterisation of the mice with enteric inflammation was performed., Results: Histologically, enteritic inflammation developed as a discontinuous lesion in the terminal ileum and caecum with the infiltration of many inflammatory cells after 10 weeks of age. the activity of myeloperoxidase, and both immunolocalisation and mRNA expression of inducible nitric oxide synthase increased in the lesion. CD3-epsilon positive T cells, neutrophils, and macrophages were more numerous in the inflamed mucosa of the SAMP1/Yit strain. The GF SAMP1/Yit strain did not show any inflammation in the intestinal wall, by the age of 30 weeks, and the enteritis and caecitis developed 10 weeks after the conventionalisation of the GF SAMP1/Yit strain., Conclusion: Enteric inflammation in the ileum and caecum developed in the SAMP1/Yit strain. The pathophysiological characteristics of the disease in this mouse have some similarities to those of human inflammatory bowel disease (IBD). This mouse strain should be a useful model system for elucidating the interaction between the pathogenesis of IBD and the gut microflora.

To elucidate the role of microbiological factors in pouchitis, this study investigated the composition of ileal reservoir microflora, the mucus degrading capacity of bacterial enzymes as well as the pH and the proteolytic activity of pouch effluent. Stool samples were collected from five patients with pouchitis and nine patients without pouchitis. The flora of patients with pouchitis had an increased number of aerobes, a decreased ratio anaerobes to aerobes, less bifidobacteria and anaerobic lactobacilli, more Clostridium perfringens, and several species that were not found in control patients (for example, fungi). Furthermore the pH was significantly higher in patients with pouchitis (median value 6.5) than in control patients (5.4). To find out if the pH might influence the breakdown of intestinal mucus glycoproteins, the activity of glycosidases and proteases, and the degradation of hog gastric mucin by the pouch flora was tested at pH 5.2-7.6. Some glycosidases were inhibited, others were stimulated by a low pH, however, in each sample the proteolytic activity was inhibited for 75% at pH 5.2 compared with pH 6.8 and 7.6. Degradation of hog gastric mucin by the pouch flora was an active process at pH 7.2: within two to four hours of incubation more than half of the mucin was degraded. At pH 5.2 it took twice as long. It is concluded that pouchitis possibly results from instability of the flora in the pouch, which causes homeostasis to disappear (dysbiosis), and the protection of the pouch epithelium by the mucus layer becomes affected by increased activity of bacterial and host derived enzymes.