Atle van Beelen Granlund

Whole genome gene expression meta-analysis of inflammatory bowel disease colon mucosa demonstrates lack of major differences between Crohn's disease and ulcerative colitis.

Background In inflammatory bowel disease (IBD), genetic susceptibility together with environmental factors disturbs gut homeostasis producing chronic inflammation. The two main IBD subtypes are Ulcerative colitis (UC) and Crohn’s disease (CD). We present the to-date largest microarray gene expression study on IBD encompassing both inflamed and un-inflamed colonic tissue. A meta-analysis including all available, comparable data was used to explore important aspects of IBD inflammation, thereby validating consistent gene expression patterns. Methods Colon pinch biopsies from IBD patients were analysed using Illumina whole genome gene expression technology. Differential expression (DE) was identified using LIMMA linear model in the R statistical computing environment. Results were enriched for gene ontology (GO) categories. Sets of genes encoding antimicrobial proteins (AMP) and proteins involved in T helper (Th) cell differentiation were used in the interpretation of the results. All available data sets were analysed using the same methods, and results were compared on a global and focused level as t-scores. Results Gene expression in inflamed mucosa from UC and CD are remarkably similar. The meta-analysis confirmed this. The patterns of AMP and Th cell-related gene expression were also very similar, except for IL23A which was consistently higher expressed in UC than in CD. Un-inflamed tissue from patients demonstrated minimal differences from healthy controls. Conclusions There is no difference in the Th subgroup involvement between UC and CD. Th1/Th17 related expression, with little Th2 differentiation, dominated both diseases. The different IL23A expression between UC and CD suggests an IBD subtype specific role. AMPs, previously little studied, are strongly overexpressed in IBD. The presented meta-analysis provides a sound background for further research on IBD pathobiology.

Intestinal barrier homeostasis in inflammatory bowel disease

Abstract The single-cell thick intestinal epithelial cell (IEC) lining with its protective layer of mucus is the primary barrier protecting the organism from the harsh environment of the intestinal lumen. Today it is clear that the balancing act necessary to maintain intestinal homeostasis is dependent on the coordinated action of all cell types of the IEC, and that there are no passive bystanders to gut immunity solely acting as absorptive or regenerative cells: Mucin and antimicrobial peptides on the epithelial surface are continually being replenished by goblet and Paneth’s cells. Luminal antigens are being sensed by pattern recognition receptors on the enterocytes. The enteroendocrine cells sense the environment and coordinate the intestinal function by releasing neuropeptides acting both on IEC and inflammatory cells. All this while cells are continuously and rapidly being regenerated from a limited number of stem cells close to the intestinal crypt base. This review seeks to describe the cell types and structures of the intestinal epithelial barrier supporting intestinal homeostasis, and how disturbance in these systems might relate to inflammatory bowel disease.

Relevance of TNBS-Colitis in Rats: A Methodological Study with Endoscopic, Histologic and Transcriptomic Characterization and Correlation to IBD

Background Rectal instillation of trinitrobenzene sulphonic acid (TNBS) in ethanol is an established model for inflammatory bowel disease (IBD). We aimed to 1) set up a TNBS-colitis protocol resulting in an endoscopic and histologic picture resembling IBD, 2) study the correlation between endoscopic, histologic and gene expression alterations at different time points after colitis induction, and 3) compare rat and human IBD mucosal transcriptomic data to evaluate whether TNBS-colitis is an appropriate model of IBD. Methodology/Principal Findings Five female Sprague Daley rats received TNBS diluted in 50% ethanol (18 mg/0.6 ml) rectally. The rats underwent colonoscopy with biopsy at different time points. RNA was extracted from rat biopsies and microarray was performed. PCR and in situ hybridization (ISH) were done for validation of microarray results. Rat microarray profiles were compared to human IBD expression profiles (25 ulcerative colitis Endoscopic score demonstrated mild to moderate colitis after three and seven days, but declined after twelve days. Histologic changes corresponded with the endoscopic appearance. Over-represented Gene Ontology Biological Processes included: Cell Adhesion, Immune Response, Lipid Metabolic Process, and Tissue Regeneration. IL-1α, IL-1β, TLR2, TLR4, PRNP were all significantly up-regulated, while PPARγ was significantly down-regulated. Among genes with highest fold change (FC) were SPINK4, LBP, ADA, RETNLB and IL-1α. The highest concordance in differential expression between TNBS and IBD transcriptomes was three days after colitis induction. ISH and PCR results corresponded with the microarray data. The most concordantly expressed biologically relevant pathways included TNF signaling, Cell junction organization, and Interleukin-1 processing. Conclusions/Significance Endoscopy with biopsies in TNBS-colitis is useful to follow temporal changes of inflammation visually and histologically, and to acquire tissue for gene expression analyses. TNBS-colitis is an appropriate model to study specific biological processes in IBD.

The role of mechanical forces and adenosine in the regulation of intestinal enterochromaffin cell serotonin secretion

Enterochromaffin (EC) cells of the diffuse neuroendocrine cell system secrete serotonin (5-HT) with activation of gut motility, secretion, and pain. These cells express adenosine (ADORA) receptors and are considered to function as mechanosensors. Physiological pathways mediating mechanosensitivity and adenosine responsiveness remain to be fully elucidated, as do their roles in inflammatory bowel disease (IBD) and neoplasia. Pure (98-99%) FACS-sorted normal and IBD human EC cells and neoplastic EC cells (KRJ-I) were studied. IBD-EC cells and KRJ-I overexpressed ADORA2B. NECA, a general ADORA receptor agonist, stimulated, whereas the A2B receptor antagonist MRS1754 inhibited, 5-HT release (EC50 = 1.8 × 10-6 M; IC50 = 3.7 × 10-8 M), which was associated with corresponding alterations in intracellular cAMP levels and pCREB (Ser133). Mechanical stimulation using a rhythmic flex model induced transcription and activation of Tph1 (tryptophan hydroxylase) and VMAT₁ (vesicular monoamine transporter 1) and the release of 5-HT, which could be inhibited by MRS1754 and amplified by NECA. Secretion was also inhibited by H-89 (PKA inhibitor) while Tph1 and VMAT₁ transcription was regulated by PKA/MAPK and PI₃K-mediated signaling. Normal and IBD-EC cells also responded to NECA and mechanical stimulation with PKA activation, cAMP production, and 5-HT release, effects reversible by MRS1754. EC cells express stimulatory ADORA2B, and rhythmic stretch induces A2B activation, PKA/MAPK/IP3-dependent transcription, and PKA-dependent secretion of 5-HT synthesis and secretion. Receptor expression is amplified in IBD and neoplasia, and 5-HT release is increased. Determination of factors that regulate EC cell function are necessary for understanding its role as a mechanosensory cell and to facilitate the development of agents that can selectively target cell function in EC cell-associated disease.

Enhanced expression of CXCL10 in inflammatory bowel disease: potential role of mucosal Toll-like receptor 3 stimulation.

Background:We explored the gene expression in colonic biopsies of active and inactive inflammatory bowel disease (IBD) in an extensive material of ulcerative colitis (UC) and Crohn’s disease (CD). The chemokine CXCL10 and its receptor CXCR3 were among the upregulated genes. This study examined the expression of CXCL10 and the mechanisms for its release in patients with UC or CD and in intestinal epithelial cell (IEC) lines. Methods:A microarray gene expression analysis was done on colonic biopsies (n = 133) from patients with IBD. Biopsies were studied with immunohistochemistry for CXCL10 and CXCR3 expression. Mechanisms for CXCL10 release in peripheral blood mononuclear cells (PBMCs) and in the colonic epithelial cell lines HT-29 and SW620 were studied upon pattern recognition receptor (PRR) stimulation. Results:CXCL10 and CXCR3 mRNA abundances were increased in biopsies from active UC and CD compared to inactive disease and controls. CXCL10 was mainly localized to mucosal epithelial cells, with increased immunostaining in active IBD. CXCR3-positive cells were scattered in the lamina propria. CXCL10 was secreted from the colonic epithelial cell lines in response to the Toll-like receptor 3 (TLR3) ligand polyinosinic: polycytidylic acid (poly(I:C)). This ligand also induced a marked release of CXCL10 in PBMCs from IBD patients and controls. Conclusions:We identified CXCL10 and CXCR3 as upregulated genes in colonic mucosa in active IBD. The TLR3-ligand poly(I:C) markedly increased release of CXCL10 in colonic epithelial cell lines, suggesting a TLR3-mediated CXCL10 release from mucosal epithelial cells in IBD patients.

Expression of Toll-like receptor-3 is enhanced in active inflammatory bowel disease and mediates the excessive release of lipocalin 2

Anti‐microbial peptides might influence the pathogenesis and course of inflammatory bowel disease (IBD). We sought to clarify the role of the anti‐microbial glycoprotein lipocalin 2 (LCN2) in the colon by determining its localization and regulation in IBD. Following a microarray gene expression study of colonic biopsies from a large IBD population (n = 133), LCN2 was localized using immunohistochemistry and in‐situ hybridization. Moreover, we examined the regulation of LCN2 in HT‐29 cells with a panel of pattern recognition receptors (PRRs) and sought evidence by immunohistochemistry that the most relevant PRR, the Toll‐like receptor (TLR)‐3, was indeed expressed in colonic epithelium in IBD. LCN2 was among the 10 most up‐regulated genes in both active ulcerative colitis (UCa) and active Crohns disease (CDa) versus healthy controls. LCN2 protein was found in both epithelial cells and infiltrating neutrophils, while mRNA synthesis was located solely to epithelial cells, indicating that de‐novo synthesis and thus regulation of LCN2 as measured in the gene expression analysis takes place in the mucosal epithelial cells. LCN2 is a putative biomarker in faeces for intestinal inflammation, different from calprotectin due to its epithelial site of synthesis. LCN2 release from the colonic epithelial cell line HT‐29 was enhanced by both interleukin (IL)‐1β and the TLR‐3 ligand poly(I:C), and TLR‐3 was shown to be expressed constitutively in colonic epithelial cells and markedly increased during inflammation.

REG gene expression in inflamed and healthy colon mucosa explored by in situ hybridisation

The regenerating islet-derived (REG) gene family encodes a group of proteins highly expressed in several human pathologies, many of which are associated with epithelial inflammation. All human family members, namely REG1A, REG1B, REG3A and REG4, are closely related in genomic sequence and all are part of the c-type lectin superfamily. REGs are highly expressed during inflammatory bowel disease (IBD)-related colonic inflammation and the in vivo expression pattern of REG1A and REG4 has been localised by using immunohistochemistry. However, the function of the encoded proteins is largely unknown and the cellular localisation of REG expression during colonic inflammation has not been described. Therefore, we have used in situ hybridisation to demonstrate the cellular localisation of REG expression in healthy and diseased colonic mucosa. Samples drawn from an IBD cohort including both inflamed and un-inflamed colonic mucosa are described, as are samples from non-IBD inflammation and healthy controls. Immunohistochemistry against known cell-type markers on serial sections has localised the expression of REGs to metaplastic Paneth cells (REG1A, REG1B and REG3A) and enteroendocrine cells (REG4), with a marked expansion of expression during inflammation. The group of REGs can, based on gene expression patterns, be divided into at least two groups; REG1A, REG1B and REG3A with their expression focused in the crypt base spreading from Paneth cells and REG4 being more highly expressed towards the luminal face. This exploration of expression pattern forms provides the background for further exploration of REG function in the intestine.

The guanylate cyclase-C signaling pathway is down-regulated in inflammatory bowel disease

Abstract Objective. Activation of membrane receptor guanylate cyclase-C (GC-C) is implicated in gastrointestinal fluid and electrolyte balance, preservation of intestinal barrier integrity, anti-trophic effects and inhibition of pain sensation. To evaluate GC-C signaling, we examined the regulation of GC-C (GUCY2C/Gucy2c) and its endogenous ligands guanylin (GN/GUCA2A/Guca2a) and uroguanylin (UGN/GUCA2B/Guca2b) in colonic Crohn’s disease (CD), ulcerative colitis (UC) and in rats with 2,4,6-Trinitrobenzene sulphonic acid (TNBS) colitis. Correlation analyses between expression of GUCA2A and GUCY2C and expression of inflammatory cytokines (IL1A, IL1B, TNFA and IFNG) were conducted. Additionally, expression of transcription factors for GUCA2A and GUCY2C, and the GC-C signaling pathway, were examined. Material and methods. Biopsies from active UC/CD, un-inflamed UC/CD and healthy controls, and inflamed and healthy rat colon were investigated with gene expression microarray, immunohistochemistry (IHC) and in situ hybridization (ISH). Results. GUCA2A/Guca2a, GUCA2B, GUCY2C/Gucy2c, transcription factors, as well as several cyclic guanosine-3′,5′-monophosphate downstream mediators were all significantly down-regulated in both inflamed colonic inflammatory bowel disease (IBD) mucosa and TNBS colitis. Expression of GUCA2A and GUCY2C negatively correlated to expression of inflammatory cytokines. IHC and ISH confirmed microarray results for GUCA2A/Guca2a and GUCY2C/Gucy2c in inflamed samples. We identified a highly significant positive correlation between the expression of the transcription factor caudal type homeobox 2 (CDX2) and the expression of the downstream target gene GUCY2C. Conclusions. GUCA2A, GUCA2B and GUCY2C as well as several steps of the GC-C signaling pathway are down-regulated in IBD. This may have implications in IBD pathogenesis.

Expression of CCL20 and Its Corresponding Receptor CCR6 Is Enhanced in Active Inflammatory Bowel Disease, and TLR3 Mediates CCL20 Expression in Colonic Epithelial Cells

Background The chemokine CCL20 and its receptor CCR6 are putative drug targets in inflammatory bowel disease, and CCL20 is a novel IBD predilection gene. Previous findings on the CCL20 response in these diseases are divergent. This study was undertaken to examine CCL20 and CCR6 during active and inactive disease, and mechanisms for CCL20 regulation by the innate immune system. As TLR3 has recently emerged as a possible mediator of CCL20 production, we hypothesised that this TLR plays an important role in enterocytic CCL20 production. Methods A large microarray study on colonic pinch biopsies from active and inactive ulcerative colitis and Crohn’s disease provided background information. CCL20 and CCR6 were localized and their expression levels assessed in biopsies using in situ hybridization and immunohistochemistry. Regulation of CCL20 was studied in the HT29 cell line using a panel of pattern recognition receptor ligands followed by a TLR3 siRNA assay. Results CCL20 and CCR6 mRNA abundances were increased during active inflammation (CCL20 5.4-fold in ulcerative colitis and 4.2-fold in Crohn’s disease; CCR6 1.8 and 2.0, respectively). CCL20 and CCR6 mRNA positive immune cells in lamina propria were more numerous, and CCL20 immunoreactivity increased massively in the epithelial cells during active inflammation for both diseases. TLR3 stimulation potently induced upregulation and release of CCL20 from HT29 cells, and TLR3 silencing reduced CCL20 mRNA and protein levels. Conclusions The CCL20-CCR6 axis is involved during active inflammation in both ulcerative colitis and Crohn’s disease. The epithelial cells seem particularly involved in the CCL20 response, and results from this study strongly suggest that the innate immune system is important for activation of the epithelium, especially through TLR3.

Mucosal toll-like receptor 3-dependent synthesis of complement factor B and systemic complement activation in inflammatory bowel disease

Background:Recent studies link Toll-like receptor 3 (TLR3) to the pathogenesis of inflammatory bowel disease (IBD). Screening TLR3-agonist response in an intestinal epithelial cell line, we found complement factor B mRNA (CFB) potently upregulated and went on to further study localization of complement factor B synthesis and systemic activation of complement in ulcerative colitis and Crohns disease. Methods:In a transcriptome analysis of poly (I:C) stimulated HT-29 cells, we found CFB highly upregulated downstream of TLR3. We sought to confirm CFB upregulation in a microarray gene expression analysis on colonic biopsies from an IBD population (n = 133). Immunohistochemical staining and in situ hybridization were done to identify cellular sources of factor B and CFB. Systemic complement activation was assessed in plasma (n = 18) using neoepitope-based enzyme linked immunosorbent assay. Results:CFB mRNA and protein were abundantly expressed in the colonic epithelial cell line, and synthesis enhanced by the poly (I:C) TLR3 ligand. In inflamed versus normal colonic mucosa of ulcerative colitis and Crohns disease, CFB mRNA was the most significantly overexpressed gene and the mRNA abundance ratio was among the 50 highest. Epithelial cells were the dominating site of factor B expression. Systemic complement activation was significantly higher in active than in nonactive IBD. Conclusions:This study is the first to link TLR3 to activation of the alternative complement pathway. Complement factor B is potently upregulated locally in IBD in addition to having a possible central role in systemic complement activation. This suggests a prominent role for complement in IBD pathogenesis.