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Interleukin 10 (IL-10) is a pleiotropic, anti-inflammatory cytokine that has a major protective role in the intestine. Although its production by cells of the innate and adaptive immune system has been extensively studied, its intrinsic role in intestinal epithelial cells is poorly understood. In this study, we utilised both ATAC sequencing and RNA sequencing to define the transcriptional response of murine enteroids to tumour necrosis factor (TNF). We identified that the key early phase drivers of the transcriptional response to TNF within intestinal epithelium were NFκB transcription factor dependent. Using wild-type and Il10−/− enteroid cultures, we showed an intrinsic, intestinal epithelium specific effect of IL-10 deficiency on TNF-induced gene transcription, with significant downregulation of identified NFκB target genes Tnf, Ccl20, and Cxcl10, and delayed overexpression of NFκB inhibitor encoding genes, Nfkbia and Tnfaip3. IL-10 deficiency, or immunoblockade of IL-10 receptor, impacted on TNF-induced endogenous NFκB activity and downstream NFκB target gene transcription. Intestinal epithelium-derived IL-10 appears to play a crucial role as a positive regulator of the canonical NFκB pathway, contributing to maintenance of intestinal homeostasis. This is particularly important in the context of an inflammatory environment and highlights the potential for future tissue-targeted IL-10 therapeutic intervention.

Background: Inflammatory bowel disease (IBD) is a multifactorial condition influenced by the immune system, the intestinal microbiota, environmental factors, genetic and epigenetic factors. Genetic- and environment- induced dysregulation of the Nuclear Factor-kappa B (NF-κB) transcription factor pathway has been linked to IBD pathogenesis. Objective: To assess the current evidence in relation to the contribution of the classical and alternative NF-κB pathways in IBD and to discuss the epigenetic mechanisms that impact on NF-κB function. Methods: A Medline search for ‘NF-kappaB/NF-κB’, in combination with terms including ‘inflammatory bowel disease/IBD’, 'intestinal inflammation', ‘Crohn's disease’, ‘ulcerative colitis’, 'colitis'; ‘epigenetics’, ‘DNA methylation’, ‘histones’, ‘microRNAs/miRNAs’ and ‘short non-coding/long non-coding RNAs’ was performed. Results: Both NF-κB pathways contribute to the chronic inflammation underlying IBD by regulating the inflammatory immune responses and homeostasis of the intestinal epithelium (classical pathway) or regulating bowel inflammation and epithelial microfold (M) cell function (alternative pathway). DNA methylation is a common epigenetic modification in intestinal inflammation, including NFKB1 and RELA loci. Conversely, little is understood regarding epigenetic effects on genes encoding other NF-κB subunits, particularly those of the alternative pathway, and in the context of IBD. However, NF-κB interaction with chromatin modifiers is also seen to be an essential mechanism of regulation of downstream target genes relevant to NF-κB-mediated inflammatory responses. Conclusion: Further research is clearly warranted in this area, as understanding the cell-specific regulation of the NF-κB pathways will bring researchers into a position to achieve more efficient stratification of IBD patients, and more targeted and effective choice of treatment.

The etiology of Crohn’s disease (CD) is multifactorial. Bacterial and fungal microbiota are involved in the onset and/or progression of the disease. A bacterial dysbiosis in CD patients is accepted; however, less is known about the mycobiome and the relationships between the two communities. We investigated the interkingdom relationships, their metabolic consequences, and the changes in the fungal community during relapse and remission in CD. Two cohorts were evaluated: a British cohort (n = 63) comprising CD and ulcerative colitis patients, and controls. The fungal and bacterial communities of biopsy and fecal samples were analyzed, with the fecal volatiles; datasets were also integrated; and a Dutch cohort (n = 41) comprising CD patients and healthy controls was analyzed for stability of the gut mycobiome. A dysbiosis of the bacterial community was observed in biopsies and stool. Results suggest Bacteroides is likely key in CD and may modulate Candida colonization. A dysbiosis of the fungal community was observed only in the Dutch cohort; Malassezia and Candida were increased in patients taking immunosuppressants. Longitudinal analysis showed an increase in Cyberlindnera in relapse. Saccharomyces was dominant in all fecal samples, but not in biopsies, some of which did not yield fungal reads; amino acid degradation was the main metabolic change associated with CD and both bacteria and fungi might be implicated. We have shown that Bacteroides and yeasts may play a role in CD; understanding their role and relationship in the disease would shed new light on the development and treatment of CD.

Background The inflammatory bowel diseases, Crohn's disease and ulcerative colitis are related multifactorial diseases. Their pathogenesis is influenced by each individual's immune system, the environmental factors within exposome and genetic predisposition. Smoking habit is the single best-established environmental factor that influences disease phenotype, behaviour and response to therapy. Aim To assess current epidemiological, experimental and clinical evidence that may explain how smoking impacts on the pathogenesis of inflammatory bowel disease. Methods A Medline search for 'cigarette smoking', in combination with terms including 'passive', 'second-hand', 'intestinal inflammation', 'Crohn's disease', 'ulcerative colitis', 'colitis'; 'intestinal epithelium', 'immune system', 'intestinal microbiota', 'tight junctions', 'mucus', 'goblet cells', 'Paneth cells', 'autophagy'; 'epigenetics', 'genes', 'DNA methylation', 'histones', 'short noncoding/long noncoding RNAs'; 'carbon monoxide/CO' and 'nitric oxide/NO' was performed. Results Studies found evidence of direct and indirect effects of smoking on various parameters, including oxidative damage, impairment of intestinal barrier and immune cell function, epigenetic and microbiota composition changes, that contribute to the pathogenesis of inflammatory bowel disease. Conclusions Cigarette smoking promotes intestinal inflammation by affecting the function and interactions among intestinal epithelium, immune system and microbiota/microbiome.

Alternative pathway NF-κB signalling regulates susceptibility towards developing inflammatory bowel disease (IBD), colitis-associated cancer and sepsis-associated intestinal epithelial cell apoptosis and shedding. However, the cell populations responsible for the perturbed alternative pathway NF-κB signalling in intestinal mucosal pathology remain unclear. In order to investigate the contribution of the epithelial compartment, we have tested whether NF-κB2 regulated transcription in intestinal epithelial cells controls the intestinal epithelial response to cytokines that are known to disrupt intestinal barrier permeability. Enteroids were generated from the proximal, middle and distal regions of small intestine (SI) from C57BL/6J wild-type mice and displayed region-specific morphology that was maintained during sub-culture. Enteroids treated with 100 ng/mL TNF were compared with corresponding regions of SI from C57BL/6J mice treated systemically with 0.33 mg/kg TNF for 1.5 h. TNF-induced apoptosis in all regions of the intestine in vitro and in vivo but resulted in Paneth cell degranulation only in proximal tissue-derived SI and enteroids. TNF also resulted in increased enteroid sphericity (quantified as circularity from two-dimensional bright field images). This response was dose and time-dependent and correlated with active caspase-3 immunopositivity. Proximal tissue-derived enteroids generated from Nfκb2−/− mice showed a significantly blunted circularity response following the addition of TNF, IFNγ, lipopolysaccharide (LPS) activated C57BL/6J-derived bone marrow-derived dendritic cells (BMDC) and secreted factors from LPS-activated BMDCs. However, Nfκb1−/− mouse-derived enteroids showed no significant changes in response to these stimuli. In conclusion, the selection of SI region is important when designing enteroid studies as region-specific identity and response to stimuli such as TNF are maintained in culture. Intestinal epithelial cells are at least partially responsible for regulating their own fate by modulating NF-κB2 signalling in response to stimuli known to be involved in multiple intestinal and systemic diseases. Future studies are warranted to investigate the therapeutic potential of intestinal epithelial NF-κB2 inhibition.

The heterogeneous nature of inflammatory bowel disease (IBD) presents challenges, particularly when choosing therapy. Activation of the NF-κB transcription factor is a highly-regulated, dynamic event in IBD pathogenesis. We expressed the human NF-κB/p65 subunit in blood-derived macrophages, using lentivirus. Confocal imaging of p65 activation revealed that a higher proportion of macrophages from Crohn’s patients responded to lipid-A compared to controls. In contrast, cells from ulcerative colitis (UC) patients exhibited a shorter duration of p65 nuclear localisation compared to healthy controls and Crohn’s donors. Using a similar lentivirus approach, NF-κB-regulated luciferase was expressed in patient macrophages, isolated from frozen peripheral blood mononuclear cell samples. Following activation, samples could be segregated into three clusters based on the NF-κB-regulated luciferase response. The majority of UC samples appeared in hypo-responsive cluster 1, with Crohn’s patients representing the majority of hyper-responsive cluster 3. A positive correlation was seen between NF-κB-induced luciferase activity and cytokine levels released to medium from stimulated macrophages, but not in serum or biopsy. Analysis of macrophage cytokine responses and patient metadata revealed a strong correlation between Crohn’s patients who smoked and hyper-activation of p65. Thesein vitrodynamic assays of NF-κB activation in blood-derived macrophages segregate IBD patients into groups with different phenotypes and therefore may help determine response to therapy.Significance statementThis manuscript describes two dynamic assays of NF-κB activation in blood-derived macrophages that can segregate IBD patients into groups with different phenotypes. For the first time we introduce the use of dynamic measurements of a transcription factor activation as a method to stratify patients and we are confident that our approach will lead in future to early patient stratification and prediction of treatment outcome.

When transmitted through the oral route, Toxoplasma gondii first interacts with its host at the small intestinal epithelium. This interaction is crucial to controlling initial invasion and replication, as well as shaping the quality of the systemic immune response. It is therefore an attractive target for the design of novel vaccines and adjuvants. However, due to a lack of tractable infection models, we understand surprisingly little about the molecular pathways that govern this interaction. The in vitro culture of small intestinal epithelium as 3D enteroids shows great promise for modeling the epithelial response to infection. However, the enclosed luminal space makes the application of infectious agents to the apical epithelial surface challenging. Here, we have developed three novel enteroid-based techniques for modeling T. gondii infection. In particular, we have adapted enteroid culture protocols to generate collagen-supported epithelial sheets with an exposed apical surface. These cultures retain epithelial polarization, and the presence of fully differentiated epithelial cell populations. They are susceptible to infection with, and support replication of, T. gondii. Using quantitative label-free mass spectrometry, we show that T. gondii infection of the enteroid epithelium is associated with up-regulation of proteins associated with cholesterol metabolism, extracellular exosomes, intermicrovillar adhesion, and cell junctions. Inhibition of host cholesterol and isoprenoid biosynthesis with Atorvastatin resulted in a reduction in parasite load only at higher doses, indicating that de novo synthesis may support, but is not required for, parasite replication. These novel models therefore offer tractable tools for investigating how interactions between T. gondii and the host intestinal epithelium influence the course of infection.

Chronic inflammation is a common factor in the development of many gastrointestinal malignancies. Examples include inflammatory bowel disease predisposing to colorectal cancer, Barrett's esophagus as a precursor of esophageal adenocarcinoma, and Helicobacter pylori-induced gastric cancer. The classical activation pathway of NF-κB signaling has been identified as regulating several sporadic and inflammation-associated gastrointestinal tract malignancies. Emerging evidence suggests that the alternative NF-κB signaling pathway also exerts a distinct influence on these processes. This review brings together current knowledge of the role of the alternative NF-κB signaling pathway in the gastrointestinal tract, with a particular emphasis on inflammation-associated cancer development.\ud \ud \ud members of the nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) family were initially described as transcription factors in B lymphocytes in 1986 (68). Since then, they have been shown to be widely expressed and are conserved across both vertebrates and invertebrates (5, 27).\ud \ud The conventional model of NF-κB signaling proposes two main arms of the pathway. These share similar features but are triggered independently and activate different target genes (76). The classical (canonical) NF-κB activation pathway is triggered by Th1 cytokines and is typified by the action of reticuloendotheliosis viral oncogene homolog A (RelA) (p65)-NF-κB1(p50) heterodimers, whereas the alternative (noncanonical) activation pathway signals through the adaptor protein NF-κB-inducing kinase (NIK). Activation of this mechanism leads to nuclear translocation of transcriptionally active v-rel avian reticuloendotheliosis viral oncogene homolog B (RelB)-NF-κB2(p52) heterodimers.\ud \ud Signaling through either pathway can influence multiple different cellular functions and can exert effects that may appear contradictory. For example, both pro- and anti-apoptotic effects, as well as proliferation (18) and senescence (70) signals, have been attributed to the classical activation pathway of NF-κB signaling. Because of the wide variation in outcomes following pathway activation, it is difficult to extrapolate the effects of NF-κB signaling from one context to another. Classical pathway NF-κB signaling has been identified as a key regulator of inflammation-associated carcinogenesis in several tissues since the early 2000s when Greten et al. demonstrated increased sensitivity to colitis-associated carcinogenesis in mice lacking IKK-β in intestinal epithelial cells (31), and, almost simultaneously Pikarsky et al. identified a similar increase in tumor burden in Mdr2 mice lacking IKK-β in hepatocytes (60). More recent evidence has established that alternative activation pathway NF-κB signaling is also important during the development of several gastrointestinal pathologies in mouse and humans. This article seeks to review this evidence and to establish questions for future research.

Infliximab is important in the therapeutic arsenal of Crohn's disease (CD). However, its effect on mucosal barrier function is not fully understood. Adherent-invasive Escherichia coli (AIEC) are important in CD pathophysiology, but the transmucosal uptake routes are partly unknown. We investigated effects of infliximab on uptake of colon-specific AIEC HM427 across CD colonic mucosa.Endoscopic biopsies from non-inflamed colon of seven patients with CD, before and after two infliximab infusions, and eight non-inflammation controls, were mounted in Ussing chambers. Paracellular permeability (Before infliximab treatment, colonic passage of HM427 [CD: 2475 CFU (450-3000); controls 1163(225-1950)] andInfliximab restored the colonic barrier to AIEC in CD; an effect partially mediated by blocking lipid rafts in epithelial cells. This ability likely contributes to infliximab's clinical efficacy in colonic CD.

Inflammatory bowel disease (IBD) is associated with anaemia and oral iron replacement to correct this can be problematic, intensifying inflammation and tissue damage. The intestinal microbiota also plays a key role in the pathogenesis of IBD, and iron supplementation likely influences gut bacterial diversity in patients with IBD. Here, we assessed the impact of dietary iron, using chow diets containing either 100, 200 or 400 ppm, fed ad libitum to adult female C57BL/6 mice in the presence or absence of colitis induced using dextran sulfate sodium (DSS), on (i) clinical and histological severity of acute DSS-induced colitis, and (ii) faecal microbial diversity, as assessed by sequencing the V4 region of 16S rRNA. Increasing or decreasing dietary iron concentration from the standard 200 ppm exacerbated both clinical and histological severity of DSS-induced colitis. DSS-treated mice provided only half the standard levels of iron ad libitum (i.e. chow containing 100 ppm iron) lost more body weight than those receiving double the amount of standard iron (i.e. 400 ppm); p

Background/Aims: Crohn's disease (CD) and ulcerative colitis (UC) are both typified by an altered intestinal microbiota, and gene associations imply various defects in the mucosal barrier and in the innate immune response to bacteria. This review aims to assess how alterations in diet or use of modified bacteria could have therapeutic effects in CD or UC. Methods: A MEDLINE search using the terms ‘prebiotic', ‘genetically modified bacteria', ‘mucosal barrier in association with ulcerative colitis', ‘Crohn's disease‘ or ‘microbiota'. Results: A large body of data from in vitro and animal studies shows promise for therapeutic approaches that target the microbiota. Approaches include dietary supplementation with fermentable fibres (prebiotics) and soluble fibres that block bacterial-epithelial adherence (contrabiotics), enhancement of the mucosal barrier with phosphatidylcholine, and use of genetically modified bacteria that express IL-10 or protease inhibitors. Vitamin D supplementation also shows promise, acting via enhancement of innate immunity. Clinical trials have shown benefit with enterically delivered phosphatidylcholine supplementation in UC and near-significant benefit with vitamin D supplementation in CD. Conclusion: Strategies that target the microbiota or the host defence against it appear to be good prospects for therapy and deserve greater investment.

Inflammatory bowel disease (IBD), a term encompassing the conditions ulcerative colitis (UC) and Crohn's disease, affects around 1 in 300 people in the UK and causes significant morbidity, although, thankfully, little mortality1. Although UC is limited to the colon and causes mucosal ulceration, Crohn's disease can affect any part of the intestine and causes ulceration, transmural inflammation, stricturing, fistula and abscess formation. Both are relapsing and remitting diseases and, despite advances in medical treatments, including immunosuppressants and drugs which specifically block pro-inflammatory molecules (tissue necrosis factor; TNF), about 25% with UC and 50–80% with Crohn's disease will require major surgery at least once in their lives.

Purpose of review Inflammatory bowel disease is thought to result from an abnormal response to the gut microbiota. This review discusses advances in knowledge of the changes in gut microbiota and host response in inflammatory bowel disease. Recent findings Approximately 15% of Crohn's disease cases in western populations result from mutations in NOD2/CARD15. This disease leads to defective intestinal defensin production and defective monocyte interleukin-8 response to bacterial peptidoglycan. A similar defective interleukin-8 response and consequent delayed neutrophil recruitment have also been shown in patients with Crohn's disease who do not have the NOD2 mutation. A consequence seems to be the accumulation in tissue of macrophages containing various bacteria, perhaps particularly Escherichia coli. In keeping with this patients with Crohn's disease have circulating antibodies against bacterial flagellar proteins of enterobacteria and clostridia. In ulcerative colitis, there is less evidence for invasion by or immune response to bacteria but changes in gut microbiota include a relative deficiency of bifidobacteria. There is considerable interest in probiotic or prebiotic therapies although so far little evidence for their efficacy. Summary Molecular techniques are giving us better insight into the gut microbiota in inflammatory bowel disease that should translate into improved therapies.

Crohn's disease (CD) and ulcerative colitis (UC) have features that suggest bacterial involvement, and all genetic models of inflammatory bowel disease (IBD) require the presence of commensal bacteria. CD is associated with innate immune response genes such as NOD2/CARD15 and the autophagy genes ATG16L1 and IRGM. However, IBD responds to immunosuppression, suggesting that any bacteria involved are not acting as conventional pathogens. Molecular techniques are rapidly advancing our knowledge of the gut microbiota. In CD there is reduced diversity, and notably a reduction in the probiotic Faecalibacterium prausnitzii, the presence of which in the terminal ileum is associated with a reduced risk of recurrence following surgery. There is also a consistent increase in mucosa-associated Escherichia coli with an "adherent and invasive" phenotype, which allows them to replicate inside macrophages and induce granulomas. Speculation that CD could be caused by the Mycobacterium avium subspecies paratuberculosis (MAP) continues. The response to antitumor necrosis factor treatments suggests that, if relevant at all, MAP is not acting as a conventional pathogen. However, there is increased colonization by MAP in CD, and there is evidence that it could have an indirect effect mediated by the suppression of macrophage function. UC relapse is frequently associated with infection by pathogens, but there is less evidence for involvement of a specific bacterial species. Poor barrier integrity followed by an inflammatory reaction to bacterial components, with chronicity maintained by an autoimmune process, seems a plausible pathogenic model. Bacterial theories of pathogenesis are now becoming testable by targeted therapeutic interventions.

Nucleotide-binding oligomerization domain-containing-2 (NOD2) acts as a bacterial sensor in dendritic cells (DCs), but it is not clear how bacterial recognition links with antigen presentation after NOD2 stimulation. NOD2 variants are associated with Crohn's disease, where breakdown in self-recognition of commensal bacteria leads to gastrointestinal inflammation. Here we show NOD2 triggering by muramyldipeptide induces autophagy in DCs. This effect requires receptor-interacting serine-threonine kinase-2 (RIPK-2), autophagy-related protein-5 (ATG5), ATG7 and ATG16L1 but not NLR family, pyrin domain containing-3 (NALP3).We show that NOD2-mediated autophagy is required for both bacterial handling and generation of major histocompatibility complex (MHC) class II antigen-specific CD4(+) T cell responses in DCs. DCs from individuals with Crohn's disease expressing Crohn's disease-associated NOD2 or ATG16L1 risk variants are defective in autophagy induction, bacterial trafficking and antigen presentation. Our findings link two Crohn's disease-associated susceptibility genes in a single functional pathway and reveal defects in this pathway in Crohn's disease DCs that could lead to bacterial persistence via impaired lysosomal destruction and immune mediated clearance.

Introduction/background Inflammatory bowel disease (IBD) results from complex interactions between: host genome, immune system, mucosa, bacteria, and environment. Sources of data Review of PubMed database using search terms 'bacteria and inflammatory bowel disease' and 'genetics and inflammatory bowel disease'. PubMed 'related reference' feature and references from retrieved articles were examined. Areas of agreement IBD results from interaction between the microbiota of the gut and the immune system. Key gene defects associated with IBD are involved in bacterial recognition and processing. The environment at least modifies and may determine pathogenesis. Areas of controversy It has been disputed whether the primary defect in IBD is immunological or bacterial, and which bacteria are key. GROWING POINTS/AREAS FOR RESEARCH: 'M cells', the specialized epithelial cells that overlie Peyer's patches, are a major interface between gut bacteria and the immune system. Improved understanding is needed of the bacteria involved in IBD pathogenesis, their genotypes and phenotypes, their portal of entry and their mechanism for escaping attack from the immune system. Bacterial ligands involved in bacteria-epithelial adhesion are emerging, and molecular techniques are rapidly increasing our knowledge of the human intestinal microbiota.

Introduction Mucosa-associated E.coli are commonly found in patients with Crohn’s disease (CD), colorectal cancer (CRC) and to a lesser extent in ulcerative colitis (UC)1,2. They are able to replicate within macrophage phagolysosomes and are found inside CD tissue macrophages3,4. Our aim here was to assess these mucosal E.coli isolates for ability to tolerate stress conditions characteristic of that found within the phagolysosome. Methods E.coli isolates from patients with CD (n = 16), UC (6), CRC (7), and those obtained from other patients (n = 7; IBS (2), sporadic polyps (3), piles (1), healthy individuals (3)) and laboratory strains (6) were grown in LB medium (OD600nm 0.1). Ten-fold dilutions were plated under stress conditions; 100 mM MES pH5 with or without 1 mM NaNO2 (low pH and nitrosative stress), 1 mM H2O2 pH7 (peroxidative stress), 1 mM methyl viologen pH7 (superoxidative stress) and compared to growth on LB agar pH7. Data was correlated with ability of isolates to replicate within J774 macrophages.4 Host oxidative stress response of macrophages infected with either CD isolate HM605 or E.coli K12EPI300 (susceptible to macrophage killing) were studied. cDNA synthesised from isolated RNA, was subjected to Qiagen RT2 PCR Oxidative stress arrays (n = 3 replicates/arrays performed for condition). Results CD isolates showed greater tolerance to superoxidative stress than isolates from UC, CRC and other patients/laboratory E.coli strains (p 2-fold) in both E.coli HM605- and K12EPI300-infected macrophages compared to uninfected controls (p Conclusion CD mucosa-associated E.coli are tolerant of superoxidative stress to support their survival and replication within host macrophages. Adaptation to the phagolysosome niche appears not to be through ability to alter host oxidative stress response to infection. References 1 Thomazini, et al. Int J Med Microbiol 2011;301:475–79. 2 Martin, et al. Gastroenterology 2004;127: 80–93. 3 Bringer, et al. Cell. Microbiol 2006;8:471–84. 4 Subramanian, et al. AAC 2008;52:427–34. Disclosure of Interest A. Tawfik: None Declared, J. Rhodes Consultant for: is/has been a member of advisory boards for Atlantic, Procter & Gamble and Falk, Conflict with: has received honoraria from Abbott, Falk, Ferring, Glaxo Smith Kline, Procter & Gamble and Schering Plough, B. Campbell Conflict with: has received honoraria from Amgen, Falk and Enterome

The mucosal barrier has three major components, the mucus layer, the epithelial glycocalyx and the surface epithelium itself, whose integrity largely depends on tight junction function. In health, there is relatively little direct interaction between the luminal microbiota and the epithelium - the continuous mucus layer in the colon keeps the surface epithelium out of contact with bacteria and the ileo-caecal valve ensures that the distal small intestine is relatively microbe free. Most interaction takes place at the Peyer's patches in the distal ileum and their smaller colonic equivalents, the lymphoid follicles. Peyer's patches are overlain by a ‘dome' epithelium, 5% of whose cells are specialised M (microfold) epithelial cells, which act as the major portal of entry for bacteria. There are no goblet cells in the dome epithelium and M cells have a very sparse glycocalyx allowing easy microbial interaction. It is intriguing that the typical age range for the onset of Crohn's disease (CD) is similar to the age at which the number of Peyer's patches is greatest. Peyer's patches are commonly the sites of the initial lesions in CD and the ‘anti-pancreatic' antibody associated with CD has been shown to have as its epitope the glycoprotein 2 that is the receptor for type-1 bacterial fimbrial protein (fimH) on M cells. There are many reasons to believe that the mucosal barrier is critically important in the pathogenesis of inflammatory bowel disease (IBD). These include (i) associations between both CD and ulcerative colitis (UC) with genes that are relevant to the mucosal barrier; (ii) increased intestinal permeability in unaffected relatives of CD patients; (iii) increased immune reactivity against bacterial antigens, and (iv) animal models in which altered mucosal barrier, e.g. denudation of the mucus layer associated with oral dextran sulphate in rodents, induces colitis. Whilst some IBD patients may have genetic factors leading to weakening of the mucosal barrier, it is likely that environmental factors may be even more important. Some may be subtle and indirect, e.g. the effects of stress on the mucosa barrier, whilst others may be more obvious, e.g. the effect of pathogen-related gastroenteritis, known often to act as trigger for IBD relapse. We have also been very interested in the potentially harmful effects of ingested detergents - either by contamination of cutlery by inadequate rinsing or via ingestion of processed foods containing permitted emulsifiers. In vitro and ex vivo studies show that even very small trace amounts of these surfactants can greatly increase bacterial translocation. Implications for therapy are not yet so obvious. We advise our IBD patients to avoid processed foods containing emulsifiers and to rinse their dishes well - whilst accepting that there is no direct evidence yet to support this. Therapies that aim to enhance the mucosal barrier have yet to come to market, but trials of enteric-delivered phosphatidylcholine in UC are promising. The faecal concentration of mucus-degrading bacterial enzymes (particularly proteases, sulphatases and sialidases) correlates with disease activity in UC, and these represent good targets for therapy.

Ulcerative colitis and Crohn's disease (together known as Inflammatory Bowel Disease or IBD) are both associated with increased risk for colorectal cancer. Although it is conventional to emphasise differences between IBD-associated and sporadic colon cancer, such as a lower rate of Adenomatosis Polyposis Coli mutations and earlier p53 mutations, IBD-associated cancer has a similar dysplasia-cancer sequence to sporadic colon cancer, similar frequencies of major chromosomal abnormalities and of microsatellite instability and similar glycosylation changes. This suggests that IBD-associated colon cancer and sporadic colon cancer might have similar pathogenic mechanisms. Because the normal colon is arguably in a continual state of low-grade inflammation in response to its microbial flora, it is reasonable to suggest that both IBD-associated and sporadic colon cancer may be the consequence of bacteria-induced inflammation. We have speculated that the glycosylation changes might result in recruitment to the mucosa of bacterial and dietary lectins that might otherwise pass harmlessly though the gut lumen. These could then lead to increased inflammation and/or proliferation and thence to ulceration or cancer. The glycosylation changes include increased expression of onco-fetal carbohydrates, such as the galactose-terminated Thomsen-Friedenreich antigen (Gal beta1,3GalNAc alpha-), increased sialylation of terminal structures and reduced sulphation. These changes cannot readily be explained by alterations in glycosyltransferase activity but similar changes can be induced in vitro by alkalinisation of the Golgi lumen. Consequences of these changes may be relevant not only for cell-surface glycoconjugates but also for intracellular glycoconjugates.

SummaryThe gut barrier, composed of a single layer of intestinal epithelial cells (IECs) held together by tight junctions, prevents the entrance of harmful microorganisms, antigens and toxins from the gut lumen into the blood. Small intestinal homeostasis is normally maintained by the rate of shedding of senescent enterocytes from the villus tip exactly matching the rate of generation of new cells in the crypt. However, in various localized and systemic inflammatory conditions, intestinal homeostasis can be disturbed as a result of increased IEC shedding. Such pathological IEC shedding can cause transient gaps to develop in the epithelial barrier and result in increased intestinal permeability. Although pathological IEC shedding has been implicated in the pathogenesis of conditions such as inflammatory bowel disease, our understanding of the underlying mechanisms remains limited. We have therefore developed a murine model to study this phenomenon, because IEC shedding in this species is morphologically analogous to humans. IEC shedding was induced by systemic lipopolysaccharide (LPS) administration in wild-type C57BL/6 mice, and in mice deficient in TNF-receptor 1 (Tnfr1-/-), Tnfr2 (Tnfr2-/-), nuclear factor kappa B1 (Nfκb1-/-) or Nfκb2 (Nfκb2-/-). Apoptosis and cell shedding was quantified using immunohistochemistry for active caspase-3, and gut-to-circulation permeability was assessed by measuring plasma fluorescence following fluorescein-isothiocyanate–dextran gavage. LPS, at doses ≥0.125 mg/kg body weight, induced rapid villus IEC apoptosis, with peak cell shedding occurring at 1.5 hours after treatment. This coincided with significant villus shortening, fluid exudation into the gut lumen and diarrhea. A significant increase in gut-to-circulation permeability was observed at 5 hours. TNFR1 was essential for LPS-induced IEC apoptosis and shedding, and the fate of the IECs was also dependent on NFκB, with signaling via NFκB1 favoring cell survival and via NFκB2 favoring apoptosis. This model will enable investigation of the importance and regulation of pathological IEC apoptosis and cell shedding in various diseases.

Introduction Increased numbers of adherent, invasive E.coli (AIEC) have been reported within intestinal epithelium of patients with Crohn’s disease (CD) and colorectal cancer (CRC). 1 Genotoxicity and angiogenic activity of AIEC have been described by our group and others. 2–4 We hypothesised that a key contribution of cancer-promoting activity of AIEC may also be through their ability to activate Wnt/b-catenin signalling, and reported that Wnt target-genes were up-regulated in colonocytes at mRNA and protein level, including cyclooxygenase-2 (COX-2). 5 Here, we further investigated the ability of AIEC to activate Wnt transcription and nuclear translocation of b-catenin. We sought also to confirm our findings in vivo using an AIEC mono-association mouse model. Methods Activation of Wnt transcription activity in response to E.coli isolates 1 (MOI: 10; for 4 h) was assessed using a TCF/LEF HeLa cell luciferase reporter assay. Infected cells were also pre-treated with and without COX inhibitors. Nuclear translocation of b-catenin was assessed by immunofluorescence in CRC cell-lines SW480 and DLD1. Following 6 week mono-association of Il10 -/- 129 SvEv mice with CRC AIEC isolate HM44, intestinal tissue was fixed, Cox-2 and β-catenin expression assessed by immunohistochemistry and compared to germ-free controls. Results Mucosa-associated E.coli isolated from CRC (HM44, HM358, HM545), Crohn’s disease (HM95, HM605), and ulcerative colitis (HM250, HM374) patients significantly increased Wnt TCF/LEF signalling in HeLa cells, ranging from 1.56±0.11 to 2.60±0.06 fold above uninfected controls (1.0±0.03); all p b -catenin nuclear translocation as per prostaglandin E2 treatment (1–10 μM). These responses were blocked using COX inhibitors (diclofenac>indomethecin>aspirin; 1–100 μM). Increased intestinal Cox-2 expression and Wnt signalling was observed in vivo in Il10 -/- mice infected with E. coli HM44 (n = 15) compared to germ-free mice (n = 5), with Cox-2 elevated 2.04±0.10 fold, and nuclear localisation of β-catenin elevated 1.98±0.13 fold; both p Conclusion IBD and CRC mucosa-associated E.coli activate intestinal Wnt-signalling in vitro and in vivo. The specific bacterial factors triggering early cancer-promoting signals such as elevated COX-2 and Wnt pathway activation are currently being investigated using a validated CRC E.coli fosmid-library screening approach, 3 with 12 confirmed positive clones currently undergoing sequence analysis. References 1 Martin HM, et al . Gastroenterology 2004; 127 :80–93 2 Arthur JC, et al . Science 2012; 338 :120–23 3 Prorok-Hamon M, et al . Gut 2014; 63 :761–70 4 Buc E, et al . PLoS One 2013; 8 (2):e56964 5 Meehan B, et al . Gut 2015; 64 (Supp 1):A362–A363 (abstract) Disclosure of Interest B. Meehan: None Declared, B. Campbell Conflict with: Has received honoraria from Amgen, Falk and Enterome, J. Rhodes Conflict with: Is/has been a member of advisory boards for Atlantic, Procter & Gamble and Falk, has received honoraria from Abbott, Falk, Ferring, Glaxo Smith Kline, Procter & Gamble and Schering Plough.

Introduction Iron deficiency anaemia is common in IBD. Iron supplementation may induce or exacerbate colitis in rats (APT 2001;15:1989–99).Dysbiosis of the microbiota is common in IBD and iron contributes to this as it is a growth factor for pathogenic bacteria.We investigated the effect of dietary iron supplementation and/or reduction on the severity of chronic colitis in a murine model using clinical, histological and biochemical parameters. Methods Studies were performed on 6 groups of 8 wild type (WT) C57BL/6 mice.Chronic colitis was induced with 1.25% Dextran Sodium Sulphate (DSS) for 5 days,followed by16 further days on water [for 3 consecutive cycles]. DSS-treated mice were fed one of three diets:low iron [LI] (100 ppm), normal iron [NI] (200 ppm) and high iron[HI] (400 ppm) supplemented chow. Also, 3 non-DSS-treated groups were studied and fed similarly. Half of the mice in each control group were treated with 1 cycle of acute 2% DSS for 5 days at day 53, followed by 5 further days on water. All mice were sacrificed at day 63. Daily weights and clinical features were recorded. Histological colitis was scored using the Bauer score(Gut 2010; 59:1192–99).Faecal calprotectin was measured by ELISA and faecal iron by immunoassay at various time points [day (d) 1, 21, 42 & 63]. Statistical analyses used the Kruskal–Wallis test with post-hoc analysis. Results Oral DSS administration induced colitis in all treated mice. While chronic DSS colitis was not associated with weight loss, there was severe weight loss in acute DSS mice which was greatest in the low iron diet group (p = 0.001 LI vs. HI; p = 0.01 for LI vs. NI). Histologically, the colitis features were more prominent in acute DSS-treated mice ingesting low and high iron diets, with median colitis scores 6 & 5.5 respectively.Cyclic administration of DSS in drinking water resulted in a significant rise in faecal calprotectin, from baseline to d63 in LI (p = 0.05) and for HI (p = 0.01), but this was not significant in the NI group. In acute DSS, the rise was greater in LI and HI (p = 0.001) and less in NI (p = 0.01).Total faecal iron was increased in a dose-dependent manner within 9 weeks in all non-DSS groups. Nevertheless, in chronic DSS groups, p = 0.001 at d1 vs. d63 for all groups [382% change for LI, 331% for NI and 355% for HI].However, in acute DSS p = 0.05 for LI, p = 0.001 NI & HI (d1 vs. d10). Conclusion Changes in nutritional luminal iron exacerbate colitis.Oral administration of DSS causes a reproducible acute colitis, followed by a slow recovery phase with a concomitant chronic inflammation. Chronic colitis was worse in mice fed low or high iron diets, as shown by elevated calprotectin. Faecal iron rose equally in all 3 groups: with iron increases likely arising from diet and bleeding during colitis. Dysbiosis may be a consequence of this change in luminal iron. Disclosure of Interest None Declared

Introduction Iron deficiency anaemia is a common complication in inflammatory bowel disease (IBD). There is evidence that iron supplementation induces inflammation both in normal rats and in rodent models of IBD (Carrier J et al , Aliment Pharmacol Ther. 2001 Dec; 15(12): 1989–99.). Changes in the gut microbiota [dysbiosis] are common in IBD patients, but the mechanism is unclear. Iron may be a factor, as it is a growth factor for some bacteria. We have therefore investigated the effect of dietary iron intake upon a murine model of colitis. Method Animal studies were performed on 6 groups of female C57BL/6 mice. Acute colitis was induced with 2% Dextran Sodium Sulphate (DSS) for 5 days in drinking water before go back on a normal water for a another 5 days. Two modifications of diet (normal chow 200 ppm iron) were used: low iron (100 ppm) and high iron (400 ppm). The groups were 1 controls; 2 acute DSS normal diet; 3 acute DSS with low iron diet for 10 days; 4 acute DSS with high iron diet for 10 days; 5 low iron diet for 4 weeks; 6 high iron diet for 4 weeks. Daily weight and clinical features were logged. Histology was performed at day 10 for groups 1–4 and day 28 for the rest. Colitis was scored using the Bauer score (Bauer et al , Gut 2010; 59:9 1192–1199). Faecal Calprotectin measured by ELISA (Biohit Healthcare Ltd.), and faecal iron by immunoassay. One ANOVA and t tests were performed to compare groups or pairs of samples. Results Weight loss: DSS induced colitis in groups 2–4; it was most severe in the low iron group. Mean changes in weight were +4% in control, -5.3% in group 2, -9.2% in group 3, and -1% in group 4. Post-hoc testing showed p values of 0.0005, 0.16 and 0.01 for low iron diet vs groups 1, 2 and 4, respectively. Histology: mean colitis scores were; 0, 1.4, 1.9, 1.4, 0, 0 for groups 1–6, respectively. The score to low iron DSS mice was significantly worse than that in untreated mice and marginally worse than those on normal or high iron diets. Calprotectin: calprotectin concentrations were greatest in DSS mice on the low iron diet. Faecal iron: this was increased in a dose-dependent manner within 4 weeks in the groups of mice that did not receive DSS. In DSS treated mice, the low iron diet mice resulted in the highest faecal iron results p = 0.010, 0.0024, and 0.0019 for groups 1, 2 and 4 respectively. Conclusion Clinical observation and histological scoring showed that a low iron diet exacerbated DSS colitis. Mice consuming 50% of the normal amount of iron lost more weight and developed more severe colitis than other mice given DSS. The high iron diet had no measureable effect on the severity of DSS colitis. We will now investigate the effects of iron on the intestinal microbiota in the 6 groups. Disclosure of interest None Declared.

Introduction Increased numbers of adherent, invasive E.coli (AIEC) have been reported on and within intestinal epithelial cells of patients with colorectal cancer (CRC). 1 Cancer-inducing activity of AIEC have recently been described by our group and others, including potential for genotoxicity and promotion of angiogenesis. 2,3,4 We hypothesise that CRC-associated AIEC may also trigger key cancer signalling pathways that support development and progression of early carcinogenesis, including (Wingless) Wnt/b-catenin cell signalling pathway. 5 Method Two human CRC cell-lines SW-480 and DLD1 were infected with CRC mucosa-associated AIEC isolates HM44 and HM358 1 for 4h at multiplicity of infection of 10. RNA was isolated, and synthesised cDNA subjected to RT 2 PCR Wnt Target Array (84 Wnt-relevant genes). Gene expression changes were confirmed by Roche Lightcycler qPCR and protein expression by immunoblot (0.5h to 4h infections). Results In response to 4h infection with CRC-associated AIEC isolates HM44 and HM358, 11 Wnt target-genes were significantly up-regulated and 7 significantly down-regulated, many regulating cell cycle, cell proliferation, migration and angiogenesis. Key genes up-regulated in SW480 cells included cyclooxygenase-2 ( PTGS-2/COX2 ), increased 8.1 ± 0.9 and 9.2 ± 1.0 fold (both P = 0.0002), Fos-related antigen 1 ( FOSL1 ) increased 6.1 ± 0.6 and 7.9 ± 1.9 fold (P VEGFA ) upregulated 4.6 ± 0.3 and 4.2 ± 0.2 fold respectively (P 2 fold change) included a known CRC tumour suppressor, Wnt-1 inducible signalling pathway 2 ( WISP2 ); P Conclusion Early indications suggest a key contribution of cancer-promoting activity of CRC-mucosa-associated E.coli events may be through ability to activate the Wnt signalling pathway. Confirmation of the importance of these findings is being investigated in vivo using a mono-association mouse model, 2 along with a CRC E.coli fosmid-library screening approach 3 to identify specific bacterial factors triggering these early cancer-promoting signals. Disclosure of interest B. Meehan: None Declared, B. Campbell Conflict with: Received honoraria from Amgen and Falk., J. Rhodes Conflict with: Is/has been a member of advisory boards for Atlantic, Procter and Gamble and Falk, has received honoraria from Abbott, Falk, Ferring, Glaxo Smith Kline, Procter and Gamble and Schering Plough. References Martin, et al . Gastroenterology 2004;127:80–93 Arthur JC, et al . Science 2012;338:120–23 Prorok-Hamon M, et al . Gut 2014;63:761–70 Buc, et al . PLoS One 2013;8(2):e56964 Schneikert J, Behrens J. Gut 2007;56:417–25

Introduction Patients with Crohn’s disease exhibit an attenuated inflammatory response after trauma or Escherichia coli infection and have delayed clearance of subcutaneous bacteria compared to healthy controls. Adherent, invasive E. coli , increased in Crohn’s disease, replicate within macrophages and may have a primary pathogenic role. Crohn’s disease patients’ macrophages may have a primary defect in bacterial killing, allowing survival of AIEC. Methods We aimed to assess the relative ability of monocyte-derived macrophages from Crohn’s patients to kill intracellular E. coli and Staphylococcus aureus compared to healthy controls. Peripheral blood monocytes were obtained from consenting adults by centrifugation over Lymphoprep™ followed by 2h adherence to plastic Nunco tissue culture dishes and subsequent differentiation into macrophages by 5d culture (as per Smith et al , J. Exp. Med. 2009, 206: 1883–97). Macrophages were infected with an adherent, invasive E. coli HM605, E. coli K12 or Staph. aureus Oxford strain. Intramacrophage killing was assessed using the gentamicin protection assay. Cytokine release to the culture medium was also determined by sandwich ELISA. Macrophage-mediated chemotaxis of human neutrophils, obtained from healthy controls by centrifugation over Polymorphoprep™, was quantified in Boyden chambers. Results No significant difference in the relative killing of E.coli K12 (-14 ± 11% vs. -45 ± 9%) and Staph. aureus (-52 ± 4% vs. -63 ± 5%) nor in the relative survival of AIEC HM605 (+50 ± 26% vs. +8 ± 22%) within monocyte-derived macrophages was seen (healthy controls vs. Crohn’s disease respectively; n = 10 each group, ANOVA). TNFα, IL-6 and IL-8 production were not significantly different between the two groups and macrophage mediated neutrophil chemotaxis was equivalent. Smoking status did not affect bacterial survival, with no differences observed in killing between current smokers, ex-smokers and non-smokers. Conclusion AIEC are ineffectively killed by both Crohn’s disease and healthy macrophages. Macrophages from patients with Crohn’s disease do not appear to have an inherent defect in killing and exhibit equivalent ability to induce neutrophil chemotaxis relative to controls. These data suggest circulating inhibitors of Neutrophil chemotaxis may explain the previously observed defective neutrophil chemotaxis and bacterial clearance in vivo . Disclosure of Interest P. Flanagan Grant/research support from: Awarded a Shire innovation fund for SpRs, S. Subramanian: None Declared, B. Campbell: None Declared, J. Rhodes Consultant for: A member of advisory boards for Atlantic, Procter and Gamble and Falk, Speaker bureau with: Has received speaking honoraria from Abbott, Falk, Ferring, Glaxo Smith Kline, Procter and Gamble, Schering Plough, Shire and Wyeth, Conflict with: With the University of Liverpool and Provexis UK, holds a patent for use of a soluble fibre preparation as maintenance therapy for Crohn’s disease plus a patent pending for its use in antibiotic-associated diarrhoea.

Increased mucosal expression of TF, the Thomsen-Friedenreich oncofetal blood group antigen (galactose beta1-3 N-acetylgalactosamine alpha-) occurs in colon cancer and colitis. This allows binding of TF-specific lectins, such as peanut agglutinin (PNA), which is mitogenic to the colorectal epithelium. To identify the cell surface TF-expressing glycoprotein(s), HT29 and Caco2 colon cancer cells were surface-labeled with Na[(125)I] and subjected to PNA-agarose affinity purification and electrophoresis. Proteins, approximately 110-180 kDa, present in HT29 but not Caco2 were identified by Western blotting as high molecular weight splice variants of CD44 (CD44v). Selective removal of TF antigen by Streptococcus pneumoniae endo-alpha-N-acetylgalactosaminidase substantially reduced PNA binding to CD44v. Immunoprecipitated CD44v from HT29 cell extracts also expressed sialyl-Tn (sialyl 2-6 N-acetylgalactosaminealpha-). Incubation of PNA 15 microg/ml with HT29 cells caused no additional proliferative effect in the presence of anti-CD44v6 mAb. In colon cancer tissue extracts (N = 3) PNA bound to CD44v but not to standard CD44. These data show that CD44v is a major PNA-binding glycoprotein in colon cancer cells. Because CD44 high molecular weight splice variants are present in colon cancer and inflammatory bowel disease tissue but are absent from normal mucosa, these results may also explain the increased PNA reactivity in colon cancer and inflammatory bowel disease. The coexpression of oncofetal carbohydrate antigens TF and sialyl-Tn on CD44 splice variants provides a link between cancer-associated changes in glycosylation and CD44 splicing, both of which correlate with increased metastatic potential.