Nuala R. O'Shea

Disruption of macrophage pro-inflammatory cytokine release in Crohn's disease is associated with reduced optineurin expression in a subset of patients

Crohns disease (CD) is a complex and highly heterogeneous chronic inflammatory disorder, primarily affecting the gastrointestinal tract. Genetic and functional studies have highlighted a key role for innate immunity in its pathogenesis. Profound systemic defects in innate immunity and acute inflammation are understood to result in markedly delayed clearance of bacteria from the tissues, leading to local chronic granulomatous inflammation and compensatory adaptive immunological changes. Macrophages, key orchestrators of acute inflammation, are likely to play an important role in the initial impaired innate immune response. Monocyte‐derived macrophages from CD patients stimulated with Escherichia coli were shown to release attenuated levels of tumour necrosis factor and interferon‐γ with normal secretion of interleukin‐8 (IL‐8), IL‐10 and IL‐6. In controls, the secretion of these cytokines was strongly positively correlated, which was not seen with CD macrophages. The transcriptomes of CD and control macrophages were examined in an attempt to understand the molecular basis of this defect. There were no differentially expressed genes identified between the two groups, consistent with genetic heterogeneity; however, a number of molecules were found to be under‐expressed in subgroups of CD patients. The most common of these was optineurin (OPTN) which was under‐expressed in approximately 10% of the CD patients. Reduced OPTN expression coincided with lower intracellular protein levels and diminished cytokine secretion after bacterial stimulation both in the patients and with small interfering RNA knockdown in THP‐1 cells. Identifying and studying subgroups of patients with shared defective gene expression could aid our understanding of the mechanisms underlying highly heterogeneous diseases such as CD.

Optineurin deficiency in mice contributes to impaired cytokine secretion and neutrophil recruitment in bacteria-driven colitis

ABSTRACT Crohns disease (CD) is associated with delayed neutrophil recruitment and bacterial clearance at sites of acute inflammation as a result of impaired secretion of proinflammatory cytokines by macrophages. To investigate the impaired cytokine secretion and confirm our previous findings, we performed transcriptomic analysis in macrophages and identified a subgroup of individuals with CD who had low expression of the autophagy receptor optineurin (OPTN). We then clarified the role of OPTN deficiency in: macrophage cytokine secretion; mouse models of bacteria-driven colitis and peritonitis; and zebrafish Salmonella infection. OPTN-deficient bone-marrow-derived macrophages (BMDMs) stimulated with heat-killed Escherichia coli secreted less proinflammatory TNFα and IL6 cytokines despite similar gene transcription, which normalised with lysosomal and autophagy inhibitors, suggesting that TNFα is mis-trafficked to lysosomes via bafilomycin-A-dependent pathways in the absence of OPTN. OPTN-deficient mice were more susceptible to Citrobacter colitis and E. coli peritonitis, and showed reduced levels of proinflammatory TNFα in serum, diminished neutrophil recruitment to sites of acute inflammation and greater mortality, compared with wild-type mice. Optn-knockdown zebrafish infected with Salmonella also had higher mortality. OPTN plays a role in acute inflammation and neutrophil recruitment, potentially via defective macrophage proinflammatory cytokine secretion, which suggests that diminished OPTN expression in humans might increase the risk of developing CD. Summary: Optineurin plays a role in acute inflammation, proinflammatory cytokine secretion and neutrophil recruitment, which suggests that diminished optineurin expression in humans might increase the risk of developing Crohns disease.

Critical Role of the Disintegrin Metalloprotease ADAM-like Decysin-1 [ADAMDEC1] for Intestinal Immunity and Inflammation

Background and Aims: ADAM [A Disintegrin And Metalloproteinase] is a family of peptidase proteins which have diverse roles in tissue homeostasis and immunity. Here, we study ADAM-like DECysin-1 [ADAMDEC1] a unique member of the ADAM family. ADAMDEC1 expression is restricted to the macrophage/dendritic cell populations of the gastrointestinal tract and secondary lymphoid tissue. The biological function of ADAMDEC1 is unknown but it has been hypothesised to play a role in immunity. The identification of reduced ADAMDEC1 expression in Crohn’s disease patients has provided evidence of a potential role in bowel inflammation. Methods: Adamdec1-/- mice were exposed to dextran sodium sulphate or infected orally with Citrobacter rodentium or Salmonella typhimurium. The clinical response was monitored. Results: The loss of Adamdec1 rendered mice more susceptible to the induction of bacterial and chemical induced colitis, as evidenced by increased neutrophil infiltration, greater IL-6 and IL-1β secretion, more weight loss and increased mortality. In the absence of Adamdec1, greater numbers of Citrobacter rodentium were found in the spleen, suggestive of a breakdown in mucosal immunity which resulted in bacteraemia. Conclusion: In summary, ADAMDEC1 protects the bowel from chemical and bacterial insults, failure of which may predispose to Crohn’s disease.

Eosinophilic oesophagitis: clinical presentation and pathogenesis

Eosinophilic oesophagitis (EoE) is an inflammatory disorder of the oesophagus which has become increasingly recognised over recent years, although it remains underdiagnosed in many centres. It is characterised histologically by a significant eosinophilic infiltration of the oesophageal mucosa (>15 eosinophils per high powered field), and clinically with features of oesophageal dysfunction such a dysphagia, food impaction, and proton pump inhibitor (PPI) resistant dyspepsia. Fibrosis and oesophageal remodelling may occur and lead to oesophageal strictures. An allergic predisposition is common in the EoE population, which appears to be primarily food antigen driven in children and aeroallergen driven in adults. Evidence suggests that the pathogenesis of EoE is due to a dysregulated immunological response to an environmental allergen, resulting in a T helper type 2 (Th2) inflammatory disease and remodelling of the oesophagus in genetically susceptible individuals. Allergen elimination and anti-inflammatory therapy with corticosteroids are currently the mainstay of treatment; however, an increasing number of studies are now focused on targeting different stages in the disease pathogenesis. A greater understanding of the underlying mechanisms resulting in EoE will allow us to improve the therapeutic options available.

DOP014 The role of optineurin in macrophage cytokine secretion and Crohn's disease

Human Scan database” (www.targetscan.org), and their impact on biological activity was searched in “GeneCodis database” (http://genecodis.cnb.csic.es). Results: 15 out of 24 tissue samples (8 responders and 7 non-responders) reached a RIN value 8 allowing miRNA sequencing. We found more than 1,300 known miRNA and about 70 new miRNA. Responders to CS had an up-regulated expression of has-miR-5701 and has-miR-625 3p, and downregulated expression of has-miR-1246 and has-miR-1291 as compared to non-responders. Bioanalysis using miRNA targets database showed up to 2,000 potential targets for the aforementioned miRNA, most of them involved in MAPK signalling pathways, cytoskeleton organization pathway, and cell differentiation endocytosis and autophagy mechanisms. Conclusions: Patients with active UC not responding to CS show a differential mucosal miRNA expression profile before starting therapy. These findings suggest that regulation of gene expression by miRNA might play a role in the response to treatment in UC patients.

Sa1791 ADAMDEC1: A Novel Molecule Linked to Crohn's Disease, Is Associated With an Increased Susceptibility to Citrobacter Rodentium Colitis in the Knock out Mouse

Introduction Innate immunity is attenuated in patients with Crohn’s disease (CD), with impaired neutrophil recruitment, delayed clearance of E. coli , and defective secretion of pro-inflammatory cytokines from macrophages 1,2 . This primary macrophage defect may result in failure to eradicate bacterial flora entering the tissues and lead to the chronic granulomatous inflammation characteristic of CD. To discover the molecules responsible, transcriptomic profiles were obtained from cultured human macrophages from CD patients and controls. ADAMDEC1 a Disintegrin and Metalloprotease was under-expressed in ~10% of CD patients. This protein is almost exclusively found in macrophages and dendritic cells in the small and large bowel lamina propria. Here we describe the response of Adamdec1 -/- mice to an enteric bacterial infection with Citrobacter rodentium . Methods Adamdec1 -/- and wild type mice were administered ~108 or 109 C. rodentium by oral gavage and body weight monitored for three weeks. At intervals mice were sacrificed and samples of serum, stool, colon and spleen were collected. Serum cytokine levels were measured and bacteria counted, in stool and spleen. Bowel inflammation was assessed histologically. Neutrophil and immune cell recruitment to the colon were measured by MPO assay and qPCR respectively. Results During infection, control mice experienced a mild self-limiting colitis, with minimal weight loss. Expression of Adamdec1 was up-regulated in the colon and this normalised with resolution. Adamdec1 -/- mice were more susceptible to C. rodentium infection: they demonstrated dramatic weight loss (p Conclusion By analysing the transcriptome of macrophages from CD patients we have identified a novel molecule involved in mucosal immunity. Further work is underway to elucidate the precise role of ADAMDEC1 in the immune response. Individuals with grossly attenuated expression levels may be at an increased risk of developing CD as a consequence of an impaired ability to handle enteric bacterial pathogens. Disclosure of Interest None Declared References Segal & Loewi, Lancet 1976 Jul 31; 2(7979):219–21. Smith AM et al. JEM 2009; 206:1883.

Su1770 Fam5c, a Possible Causal Molecule in Ulcerative Colitis Revealed Through a Transcriptomic Analysis of the Bowel Mucosa

Introduction Abnormalities of the colonic mucosa have been implicated in the pathogenesis of ulcerative colitis (UC). We investigated mRNA profiles of macroscopically non-inflamed mucosal biopsies from the colon in patients with UC, Crohn’s disease (CD) and control subjects without gastrointestinal disease (HC), to identify genes that might be involved in the aetiology of the disease. Methods Paired biopsies were taken for histology and mRNA extraction from macroscopically non-inflamed mucosa in the ascending and descending colon, and the rectum, from 24 patients with UC, 14 with CD and 27 HCs undergoing routine colonoscopy. Patients were in complete clinical remission and were either on no treatment or on 5-aminosalicylates ± azathioprine. cRNA was hybridised to Illumina HumanHT-12 v4 Expression Beadchips. Array expression data were log transformed and normalised. Only probes with a detection p-value Results In group comparisons, of the 26,261 expressed probes, Family with Sequence Similarity 5, member C ( FAM5C ) was the only gene to be significantly under-expressed in UC, both in the rectum (FC = –1.58, p = 0.0008) and the descending colon (FC = –1.64, p = 0.0011). Outlier analysis showed that FAM5C was also grossly under-expressed in the ascending colon in 37.5% of UC patients, demonstrating that its expression is abnormal throughout the colon in a significant proportion of individuals. Expression levels were not abnormal in CD. Expression of FAM5C in UC did not correlate with the known markers of inflammation, IL-8 , S100A8 , DEFA5 and DEFA6 , or with treatment. The under-expression of FAM5C in UC was confirmed in biopsies of non-inflamed rectal mucosa from an independent cohort of patients (FC = –1.68, p = 0.0073) and by qPCR (p Conclusion This is the first description of the under-expression of FAM5C in UC. As these observations were made in non-inflamed mucosa, low levels of this protein might be involved in the pathogenesis of the disease. Indications that FAM5C may function as tumour suppressor [1], could link to the observed predisposition to colonic malignancy in UC. Disclosure of Interest None Declared. Reference Kuriowa T et al. (2009) Oncol Rep, 1005–11.

Tu1280 Screening Using the European Crohn's and Colitis Organisation (ECCO) Guidelines Demonstrates High Strongyloides Sero-Prevalence in Migrants With Inflammatory Bowel Disease

Introduction Strongyloidiasis can persist and cause hyperinfection years after acquisition when host immunity is impaired. European Crohn9s and Colitis Organisation guidelines 1 on opportunisitc infections recommend that Inflammatory Bowel Disease (IBD) patients returning from endemic areas be screened. However, prevalence of intestinal helminths in migrant IBD patients is unknown. We investigated the sero-prevalence of Strongyloidiasis and factors associated with infection. Methods Migrant patients attending IBD clinic over a 10-month period, with a diagnosis of Crohn9s disease (CD) or Ulcerative colitis (UC), were tested for Strongyloides serology. Eosinophil count and inflammatory markers were measured. Ethnicity was used as a proxy for migrant status. Sero-positive patients were followed-up with a Strongyloides charcoal culture before treatment with Ivermectin. Repeat eosinophil count and inflammatory markers were performed 3 months later. T test and χ 2 analysis (p Results 97 migrant patients (54 CD vs 43 UC) were tested. 13/97 patients were sero-positive. In both groups, over 70% of patients were from Asia. Mean eosinophil counts (×10 9 /l) were not different between the two groups (0.29 vs 0.22, p>0.05). No significant change was seen in eosinophil count or in inflammatory markers post treatment. In the sero-positive group 23% had past and current eosinophilia, but this was not statistically different from sero-negative patients. 9/13 reportable charcoal stool cultures were negative. No patients with Strongyloides were taking steroids, compared to 23% of sero-negative patients. In both groups, >40% were on two or more immunosuppressants. Conclusion There is a high sero-prevalence of Strongyloides in migrant IBD patients. Patients from Asia demonstrated the highest prevalence. Eosinophilia and raised inflammatory markers were not predictive of positive serology, most likely due to the high rate of immunosuppression. We cannot confirm all sero-positive patients were infected; published data 2 supports the specificity of Strongyloides serology for current infection. We recommend ECCO guidelines and current British Society of Gastroenterology guidelines are adapted to include targetting IBD patients who originate from endemic areas and serological testing be first line. Follow-up of patients is required to assess the impact of treatment on IBD activity. Competing interests None declared. References 1. Rahier JF , et al. J Crohn9s Colitis 2009; 3 :47–91. 2. Loutfy MR , et al. Am J Trop Med Hyg 2002; 66 :749–52.

Tu1353 Mucosal mRNA Expression Profiling From the Terminal Ileum and Colon Reveals Under Expression of Claudin 8, a Tight Junction Molecule, as Potentially Causal in Ulcerative Colitis

Introduction Intestinal barrier dysfunction plays an important role in the pathogenesis of ulcerative colitis (UC). We investigated mRNA profiles of mucosa from the colon and terminal ileum, in patients with UC and controls (HC) to identify genes that might be implicated in the pathogenesis of the disease. Methods Mucosal biopsies were taken from 24 quiescent UC patients (Mayo score <3) and 33 HCs undergoing colonoscopy. Patients were on no treatment or on 5-aminosalicylates ± azathioprine. HCs were patients without organic disease. Parallel biopsies were taken for RNA extraction and histology from macroscopically non-inflamed mucosa in the terminal ileum (TI), ascending, descending and sigmoid colon, and rectum. cRNA was hybridised to Illumina HumanHT v12.0 Expression Beadchips. Expression data were log transformed and normalised. Probes with a detection p value <0.01 were analysed. Comparing 85 biopsies from HCs and 68 biopsies from UC patients across the colon, the data for each bowel location were adjusted to the mean HC rectal expression level. Where multiple biopsies were taken from the same individual, the adjusted data across all biopsies for that individual were averaged. T tests between groups and outlier analysis (p<0.005, fold change (FC) ≥1.5) were performed using proprietary software. Results Of the ∼30K probes analysed, the two most significantly under-expressed in UC in the colon were those of claudin 8 (CLDN8) with FCs 2.94 (p=1.29×10−5) and 3.45 (p=3.92×10−5). The expression of claudin 8 increased distally in the colon, whereas claudins 3, 7 and 23 were highly, and uniformly, expressed throughout the colon and were normal in UC. Outlier analysis between HC and UC showed CLDN8 to be significantly under-expressed in 25%>40% of UC patients at all 4 colonic sites. There were no CLDN8 UC outliers in the TI. Most of these outlier patients demonstrated consistent levels of under-expression throughout the colon, and their histology revealed microscopic inflammation. Other patients with histologically active disease had normal CLDN8 expression. Conclusion CLDN8 is significantly under-expressed in the UC colon. Outlier analysis has also identified a group of patients in whom CLDN8 is grossly under-expressed. Low expression of CLDN8 in UC could be secondary to inflammation, although the evidence presented here is against this. Reduced levels of CLDN8 could lead to a weak and permeable mucosa predisposing to UC by reducing barrier resistance and allowing penetration by microbes. Competing interests None declared.

Mo1786 ADAMDEC1: A Novel Molecule in Inflammation and Bowel Disease

Background: Innate immunity is attenuated in patients with Crohns disease (CD) with impaired neutrophil recruitment to skin and bowel, delayed clearance of E. coli from the skin, and impaired secretion of pro-inflammatory cytokines from macrophages (Marks et al, 2006; Smith et al, 2009). The primary defect of acute inflammation results in failure to eradicate bacterial flora entering the bowel wall leading to the chronic granulomatous inflammation characteristic of CD. Microarray analysis of monocyte derived macrophage mRNA expression, confirmed by qPCR, revealed that ADAMDEC1 (ADAM like Decysin1) was under-expressed in 10% (6/60) of CD patients. ADAMDEC1, a Metalloprotease and Decysin, is part of a family of proteins involved in wound healing and tissue repair, and is almost exclusively expressed in macrophages, dendritic cells and the gastrointestinal tract. To determine the role of this protein we examined E. coli induced inflammation and Dextran Sodium Sulphate (DSS) colitis in Adamdec1 knockout (KO) mice. Method: In an acute colitis model, Adamdec1 KO mice were exposed to 2% DSS for 7 days. Controls, wild type (WT) litter mates, were age, weight and sex matched (n=11 per group). Clinical colitis scores (weight loss, PR blood, loose stool) were recorded daily. Histology was obtained from small and large bowels. For bacterial inflammation, 5x108 heat killed E. coli (HkEc) were injected subcutaneously (SC) into two sites on the backs of KO andWTmice (n=8 per group). Mice were weighed, injection sites inspected for ulceration and subcutaneous inflammatory nodules measured daily. Injection sites were excised at different times for histology and identification of infiltrating cells by FACS. Results: Adamdec1 KOmice were more susceptible to DSS colitis. They demonstrated higher clinical colitis scores with an earlier and more dramatic weight loss (p<0.001). A more florid inflammatory response was seen on histology. In response to a subcutaneous injection of E. coli, Adamdec1 KOmice had smaller inflammatory nodules and less ulceration at the injection sites after 48-72 hours, than WT mice (p<0.001). Conclusion: Mice lacking Adamdec1 develop a phenotype that closely mirrors that observed in patients with CD, an attenuated and delayed E.coli induced acute inflammatory response and an increased susceptibility to bowel inflammation. These results suggest ADAMDEC1 plays an important role in the acute inflammatory response to bacteria and has a protective role within the intestine; reduced levels may have a pivotal role in the development and persistence of CD. Studies are currently underway to further investigate the impaired cellular recruitment and potentially defective bacterial clearance at these early stages of inflammation in our model which could predispose to a more exuberant secondary response and chronic inflammation.