Thean Soon Chew

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.

PWE-232 ADAMDEC1: a novel molecule in inflammation and bowel disease

Introduction Innate immunity is attenuated in patients with Crohns disease (CD) with impaired neutrophil recruitment to skin and bowel, delayed clearance of Escherichia 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 peripheral blood 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 the Adamdec1 knockout (KO) mouse. Methods 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, 5×108 heat killed E coli (HkEc) were injected subcutaneously (SC) into two sites on the backs of KO and WT mice (n=8 per group). Mice were weighed, injection sites inspected for ulceration and subcutaneous inflammatory nodules measured, daily. Injection sites were excised at different time points for histology and identification of infiltrating cells by FACS. Results Adamdec1 KO mice 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 KO mice had significantly smaller inflammatory nodules and less ulceration at the injection sites after 48–72 h, compared with 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 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. Competing interests None declared.

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.

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.