Scott E. Levison

Enteroendocrine cells:neglected players in gastrointestinal disorders?

Enteroendocrine cells (EEC) form the basis of the largest endocrine system in the body. They secrete multiple regulatory molecules which control physiological and homeostatic functions, particularly postprandial secretion and motility. Their key purpose is to act as sensors of luminal contents, either in a classical endocrine fashion, or by a paracrine effect on proximate cells, notably vagal afferent fibres. They also play a pivotal role in the control of food intake, and emerging data add roles in mucosal immunity and repair. We propose that EEC are fundamental in several gastrointestinal pathologies, notably Post-infectious Irritable Bowel Syndrome, infectious enteritis, and possibly inflammatory bowel disease. Further work is needed to fully illustrate the importance, detailed biology and therapeutic potential of these frequently overlooked cells.

Colonic transcriptional profiling in resistance and susceptibility to trichuriasis: phenotyping a chronic colitis and lessons for iatrogenic helminthosis.

Background: Helminth therapy is advocated to restore and maintain control of inflammatory responses, particularly chronic colitis. However, helminths can induce chronic colitis in susceptible individuals. Susceptibility has an immunogenetic basis: defining this is essential if nematode therapy is to be successfully and safely targeted in inflammatory bowel disease (IBD). To validate a preclinical mouse model we phenotyped the response to Trichuris muris in mice. We determined colonic transcriptional activity in naïve and infected mice and linked differential gene expression to mechanistic pathways. Methods: T. muris‐infected resistant (BALB/c) and susceptible (AKR) mice were studied to a chronic colitic timepoint (day 35). Colonic genome‐wide expression was performed by microarray. Significant transcriptional changes were analyzed by cluster and gene ontology filtering and KEGG pathway mapping. Results: Day 35 infected AKR displayed chronic diarrhea, weight loss, and transmural colonic inflammation; BALB/c remained asymptomatic, cleared the infection, and demonstrated normal histology. Compared to BALB/c mice, infected AKR upregulated gene expression clusters were overrepresented by immune response, chemotaxis, and apoptosis pathways. Cellular/tissue homeostasis and tight junction pathways dominated downregulated AKR expression clusters. Infected AKR T‐helper cell development/polarization markers demonstrated predominant TH1/TH17 transcriptional activity. Colitic AKR data mirrored established murine models and human colitis. Conclusions: T. muris infection in the mouse shows striking phenotypic and transcriptional similarities to widely used models of IBD and human IBD. This preclinical mouse model presents a platform to examine biological commonalities among chronic colitides. However, these data urge caution in untargeted therapeutic helminth use until risk/benefit in susceptible individuals is more fully understood. (Inflamm Bowel Dis 2010)

Inflammatory cues enhance TGFβ activation by distinct subsets of human intestinal dendritic cells via integrin αvβ8

Regulation of intestinal T-cell responses is crucial for immune homeostasis and prevention of inflammatory bowel disease (IBD). A vital cytokine in regulating intestinal T cells is transforming growth factor-β (TGFβ), which is secreted by cells as a latent complex that requires activation to function. However, how TGFβ activation is regulated in the human intestine, and how such pathways are altered in IBD is completely unknown. Here we show that a key activator of TGFβ, integrin αvβ8, is highly expressed on human intestinal dendritic cells (DCs), specifically on the CD1c+ but not the CD141+ intestinal DC subset. Expression was significantly upregulated on intestinal DC from IBD patients, indicating that inflammatory signals may upregulate expression of this key TGFβ-activating molecule. Indeed, we found that the Toll-like receptor 4 ligand lipopolysaccharide upregulates integrin αvβ8 expression and TGFβ activation by human DC. We also show that DC expression of integrin αvβ8 enhanced induction of FOXP3 in CD4+ T cells, suggesting functional importance of integrin αvβ8 expression by human DC. These results show that microbial signals enhance the TGFβ-activating ability of human DC via regulation of integrin αvβ8 expression, and that intestinal inflammation may drive this pathway in patients with IBD.

Genetic analysis of the Trichuris muris-induced model of colitis reveals QTL overlap and a novel gene cluster for establishing colonic inflammation

BackgroundGenetic susceptibility to colonic inflammation is poorly defined at the gene level. Although Genome Wide Association studies (GWAS) have identified loci in the human genome which confer susceptibility to Inflammatory Bowel Disease (Crohn’s and Ulcerative Colitis), it is not clear if precise loci exist which confer susceptibility to inflammation at specific locations within the gut e.g. small versus large intestine. Susceptibility loci for colitis in particular have been defined in the mouse, although specific candidate genes have not been identified to date. We have previously shown that infection with Trichuris muris (T. muris) induces chronic colitis in susceptible mouse strains with clinical, histological, and immunological homology to human colonic Crohn’s disease. We performed an integrative analysis of colitis susceptibility, using an F2 inter-cross of resistant (BALB/c) and susceptible (AKR) mice following T. muris infection. Quantitative Trait Loci (QTL), polymorphic and expression data were analysed alongside in silico workflow analyses to discover novel candidate genes central to the development and biology of chronic colitis.Results7 autosomal QTL regions were associated with the establishment of chronic colitis following infection. 144 QTL genes had parental strain SNPs and significant gene expression changes in chronic colitis (expression fold-change ≥ +/-1.4). The T. muris QTL on chromosome 3 (Tm3) mapped to published QTL in 3 unrelated experimental models of colitis and contained 33 significantly transcribed polymorphic genes. Phenotypic pathway analysis, text mining and time-course qPCR replication highlighted several potential cis-QTL candidate genes in colitis susceptibility, including FcgR1, Ptpn22, RORc, and Vav3.ConclusionGenetic susceptibility to induced colonic mucosal inflammation in the mouse is conserved at Tm3 and overlays Cdcs1.1. Genes central to the maintenance of intestinal homeostasis reside within this locus, implicating several candidates in susceptibility to colonic inflammation. Combined methodology incorporating genetic, transcriptional and pathway data allowed identification of biologically relevant candidate genes, with Vav3 newly implicated as a colitis susceptibility gene of functional relevance.

Anti-inflammatory effects of infliximab in mice are independent of TNFα neutralization.

Infliximab (IFX) has been used repeatedly in mouse preclinical models with associated claims that anti‐inflammatory effects are due to inhibition of mouse tumour necrosis factor (TNF)‐α. However, the mechanism of action in mice remains unclear. In this study, the binding specificity of IFX for mouse TNF‐α was investigated ex vivo using enzyme‐linked immunosorbent assay (ELISA), flow cytometry and Western blot. Infliximab (IFX) did not bind directly to soluble or membrane‐bound mouse TNF‐α nor did it have any effect on TNF‐α‐induced nuclear factor kappa B (NF‐κB) stimulation in mouse fibroblasts. The efficacy of IFX treatment was then investigated in vivo using a TNF‐α‐independent Trichuris muris‐induced infection model of chronic colitis. Infection provoked severe transmural colonic inflammation by day 35 post‐infection. Colonic pathology, macrophage phenotype and cell death were determined. As predicted from the in‐vitro data, in‐vivo treatment of T. muris‐infected mice with IFX had no effect on clinical outcome, nor did it affect macrophage cell phenotype or number. IFX enhanced apoptosis of colonic immune cells significantly, likely to be driven by a direct effect of the humanized antibody itself. We have demonstrated that although IFX does not bind directly to TNF‐α, observed anti‐inflammatory effects in other mouse models may be through host cell apoptosis. We suggest that more careful consideration of xenogeneic responses should be made when utilizing IFX in preclinical models.

PWE-246 Infliximab treatment significantly reduces inflammatory macrophage numbers while preserving regulatory macrophages in a mouse model of chronic Crohn's colitis

Introduction Inappropriate inflammatory responses to intestinal flora, augmented by host susceptibility genetics, contribute to the pathogenesis of Crohns disease (CD). Transmural intestinal inflammation results from innate and adaptive immune cell infiltration, and pro-inflammatory cytokine accumulation. Activated macrophages represent a major source of TNFα production. The treatment of CD with anti-TNFα antibody (Ab) therapy has proved clinically beneficial, yet over 30% of patients fail to respond. We characterised the biological and immunological effects of Infliximab therapy in a model of experimental colitis. Methods Genetically identical mice (AKR), susceptible to chronic Trichuris muris-induced colitis, were infected with 300 T muris eggs. A single 5 mg/kg dose of Infliximab was administered intra-peritoneally once chronic colitis had established (from day 35 post-infection, p.i.). Systemic, mesenteric lymph node (MLN) and colonic effects were analysed at day 45 p.i. MLN cell cytokine bead-array and colonic gene expression (RT-qPCR) analysis were performed. Colonic histopathology, tissue Foxp3+, and macrophage recruitment and phenotype were determined. The treatment group was compared to untreated-infected, naive, and naïve AKR administered Infliximab (n=5 per group). Results Treatment did not alter worm expulsion. Anti–TNFα Ab therapy preserved colonic length compared to untreated disease (p=0.049). Colonic inflammation was less severe with Infliximab treatment (p=0.07). Reduced TNFα, CCL2, and GMCSF proteins were measured in the MLN of Infliximab treated infected AKR. Reduced colonic expression of TNFα, IL-1β, IFNγ and IL-12p40, and increased IL-13 was observed following Infliximab treated disease. Colonic Foxp3+ cell numbers increased with disease but were unaltered by treatment. Infected mice treated with Infliximab demonstrated a 50% reduction in colonic F4/80+ macrophages (p=0.036). A relative increase of the proportion of colonic Arg+ alternatively activated macrophages (AAMФ) was observed with Infliximab treatment compared to untreated disease (29% vs 14%). Conclusion Infliximab therapy suppresses TH1-driven experimental colitis. Anti–TNFα Ab treatment reduced pro-inflammatory macrophages recruitment, and for the first time in vivo has been shown to preserve colonic tissue regulatory AAMФ. Whether a result of a fundamental alteration to macrophage recruitment, or the differentiation of a specific macrophage phenotype, requires further study. The presence of AAMФ at index biopsy, or an increase in AAMФ numbers following treatment initiation, may help to identify patient responders to Anti-TNFα Ab therapy. Competing interests None declared.

PWE-245 Anti-TNFα antibody therapy and parenteral corticosteroids demonstrate distinct effects in the treatment of experimental Trichuris muris-induced chronic colitis

Introduction Anti-tumour necrosis factor (TNF) α antibody treatment and corticosteroid therapy represent central strategies in the management of Crohns disease. Yet, over 30% of patients fail to respond. Understanding the mechanism of effect for each therapy is complicated by disease heterogeneity, and the complexities of effector and regulatory immune cell responses. We characterised the biological and immunological effects of infliximab and hydrocortisone therapy in a genetically identical murine model of experimental colitis. Methods Mice (AKR) susceptible to chronic Trichuris muris-induced colitis were infected with 300 T muris eggs. A single 5 mg/kg dose of Infliximab, or daily hydrocortisone treatment (2 mg/kg, QDS) were administered intraperitoneally once chronic colitis had established (from day 35 post-infection, p.i.). Systemic, mesenteric lymph node (MLN) and colonic effects were analysed at day 45 p.i. MLN cell cytokine bead-array and colonic gene expression (RT-qPCR) analysis were performed. Colonic histopathology, tissue Foxp3+ and macrophage recruitment were determined. Treated groups were compared to naïve and untreated-infected AKR (n=5 per group). Results Neither treatment altered worm expulsion. Anti–TNFα Ab and corticosteroid therapy preserved colonic length, compared to untreated disease. Colonic inflammation was less severe with steroid treatment (p=0.005) and infliximab (p=0.07). An increase in MLN TH2 cytokines was suggested with both treatments. Reduced colonic TNFα, IL-1β, IFNγ and IL-12p40, and increased IL-13 expression were observed following Infliximab. Down-regulated TH1 cytokines, elevated TH2 cytokines (IL-4, IL-5, IL-13), and up-regulated colonic IL-10 expression were detected following corticosteroid treatment. Colonic Foxp3+ cell numbers increased with disease but were unaltered by either treatment. A significant reduction in tissue F4/80+ macrophages was observed with infliximab treatment alone. Conclusion Anti–TNFα Ab and corticosteroid therapy suppress TH1-driven experimental colitis. Up-regulated transcription of TH2 and regulatory (IL-10, TGFb, Foxp3) pathway molecules was seen with corticosteroid treatment. This was not accompanied by an increased influx of Foxp3+ T-cell, suggestive that corticosteroids may alter regulatory-cell function more significantly than recruitment, in the reduction of pathology and disease activity. Anti–TNFα Ab treatment reduced colonic pro-inflammatory macrophage recruitment. With differing modalities of immunosuppression demonstrated, this model may increase understanding of why either mode of therapy can induce benefit in man even if the other has failed. Competing interests None declared.

Genetic susceptibility to chronic trichuris muris induced experimental colitis: translation and relevance to human Crohn's disease

Introduction Unraveling the genetic architecture of complex traits presents a true challenge. The analysis of experimental models with strict phenotypic documentation and homology to human traits, alongside sensitive scientific methodologies, will facilitate future discoveries. Trichuris muris (T muris) induces chronic colitis in susceptible mouse strains, with previously described clinical, histological, and immunological commonalities to human Crohns disease (CD). Conversely, resistant mouse strains exhibit acute infection, with quick and full recovery. Cross-breeding susceptible and resistant mice has permitted Quantitative Trait Loci (QTL) mapping, pertaining to chronic T muris inflammation. Aims (1.) Characterize the genetic basis of chronic Trichuris induced colitis, and identify biologically relevant susceptibility genes. (2) Correlate initial experimental findings to human CD genome sequence variation data. Methods An F2 inter-cross of resistant (BALB/c) and susceptible (AKR) mice were generated and infected with T muris (300 ova). Colonic and systemic phenotype was established (worm burden, histology, immune response), and QTL mapping performed. Genome-wide colonic transcriptional activity of parental strains was also determined (Affymetrix). Hypothesis-free in silico work-flow analysis identified candidate genes from the QTL and expression data. Genes lacking parental strain SNPs were excluded. Polymorphic QTL genes, with differential transcription were subsequently compared to experimental and CD data for potential homology. Results QTL associated with chronic T muris colitis susceptibility were identified on 7 murine chromosomes (Chr). 144 QTL-genes displayed parental strain SNPs and significant gene expression changes in chronic colitis (ANOVA, p<0.05, expression fold-change ≤±1.4). QTL-Chr3 demonstrated overlap with published QTL of three unrelated experimental models of colitis. Of 33 potential QTL-Chr3 gene candidates, Ptpn22 and Fcgr1 display human and experimental homology. Additional transcribed polymorphic genes, central to biological (KEGG) pathways, were highlighted at this locus. Conclusion 7 QTL indicate susceptibility to chronic T muris colitis. Unbiased, systematic, reductionist analysis prioritised cis-QTL gene candidates. With experimental and human trait overlap, this model and methodology can inform the biology and outcome of transmural colonic inflammation. Positional, mechanistic pathway genes may thus be identified for translational studies.

Genotype-Phenotype Correlation, Colonic Transcription and Functional Analysis Implicate Vav3 in Colitis

203025(50mg/kg, BID) or PBS were transanally administered for 14 days. After treatment, mice were killed to assess the colon macroand microscopically. Mucosal mRNA expression of pro-inflammatory cytokines and chemokines were examined using RT-PCR. To address differences in action of the two compounds, immunohistochemical staining of ACE and immunofluorescent staining of AT1aR was performed Results: In the DSS model, weight loss and histology score for CCG-203025(-3.6±2.9, 3.9±1.2) were superior(P<0.05) to ACEI(11.0±3.2, 6.3±2.6) vs. placebo(-6.8±3.3, 10.7±1.2 ). In the IL10-/-model, ACEI suppressed histologic damage better than CCG-203025(Table). Both enalaprilat and CCG-203025 reduced pro-inflammatory cytokines, and chemokines, but ACEI was superior to CCG203025(Table). Immunological staining showed dense distribution of ACE on the colonic mucosal surface, and AT1aR within the lamina propria. Conclusions: This study demonstrates the therapeutic efficacy of both ACEI and AT1aR-A in preventing the development of both acute and immunologically-relevant colitis models. The differential expression of ACE and AT1aR may account for the different action of the two compounds in the two models. RAS blockade may be a promising target for a new therapeutic agent for inflammatory bowel disease.

OC-010 PhenotypingTrichuriscolitis reveals innate and adaptive immunological commonalities to human Crohn's disease

Introduction Trichuris muris, an intestinal dwelling nematode, induces chronic colitis in susceptible mouse strains. Pathology is characterised by patchy, full thickness colonic inflammation, similar to Crohns disease (CD). Conversely, resistant mouse strains exhibit transient acute infection (<14 days) with quick and full resolution. Although not an aetiological trigger for CD, the similar histology and strain-dependent response to Trichuris may permit identification of immuno-genetic mechanisms which initiate or perpetuate chronic colitis/resolution. Accurate mechanistic and clinical phenotyping relevant to outcome is essential, and permits biological comparison between T muris colitis and human colitides. Aims (1) Characterise colonic mucosal gene expression in resistant and susceptible mouse strains following T muris infection. (2) Phenotype and compare the clinical and immunological profiles of T muris chronic colitis with available CD data. Method Susceptible (AKR) and resistant (BALB/c) mice were administered 300 T muris ova by oral gavage. Naïve, resistant and susceptible mice were compared at day 35 post-infection. Wellbeing, body weight and stools were assessed throughout. Worm count, serum IgG, macroscopic and microscopic colonic changes were determined. Pooled colonic mRNA (n=5) was applied to Affymetrix GeneChip Mouse Exon 1.0 ST arrays. 3 replicate GeneChips were processed per study group. Bio-informatic and ANOVA statistical analysis, qPCR replication, and in silico data mining was performed. Results Resistant BALB/c exhibited “normal” macroscopic and microscopic appearances and a strong Th2 immune response post-infection. Diarrhoea, weight loss, and colonic shortening were seen in susceptible infected AKR, with moderate-to-severe focal, transmural colonic inflammation and fat hypertrophy. Consistent with clinical and histological findings, infected AKR demonstrated differentially upregulated innate pattern-recognition and autophagy transcripts (eg, TLR4, MyD88, Ripk2, NFkB, NOD2, IRGM) (>×1.2-fold upregulation cf. naïve, p<0.05). Similarly significant upregulated expression of adaptive Th1 and Th17/IL-23R pathway cytokines, receptors and transcription factors was found in colitic AKRs post-infection (eg, IFNg, TNFa, IL23a, IL-12b, IL-12Rb1, TGFb, Stat3, IL-17F, IL-6, IL-21). Mechanistic gene expression patterns with parallels to CD were demonstrated. Conclusion With clinical, histological, and immunological commonalities with human Crohns disease, chronic Trichuriasis offers a validated, immuno-competent and reproducible tool for colitis research. The exploration and translation of resistance and susceptibility phenotypes/genotypes in this model of colonic inflammation may be far reaching.