Identification of Restricted Subsets of Mature microRNA Abnormally Expressed in Inactive Colonic Mucosa of Patients with Inflammatory Bowel Disease

Background

Ulcerative Colitis (UC) and Crohn''s Disease (CD) are two chronic Inflammatory Bowel Diseases (IBD) affecting the intestinal mucosa. Current understanding of IBD pathogenesis points out the interplay of genetic events and environmental cues in the dysregulated immune response. We hypothesized that dysregulated microRNA (miRNA) expression may contribute to IBD pathogenesis. miRNAs are small, non-coding RNAs which prevent protein synthesis through translational suppression or mRNAs degradation, and regulate several physiological processes.

Methodology/Findings

Expression of mature miRNAs was studied by Q-PCR in inactive colonic mucosa of patients with UC (8), CD (8) and expressed relative to that observed in healthy controls (10). Only miRNAs with highly altered expression (>5 or <0.2 -fold relative to control) were considered when Q-PCR data were analyzed. Two subsets of 14 (UC) and 23 (CD) miRNAs with highly altered expression (5.2->100 -fold and 0.05–0.19 -fold for over- and under- expression, respectively; 0.001<p≤0.05) were identified in quiescent colonic mucosa, 8 being commonly dysregulated in non-inflamed UC and CD (mir-26a,-29a,-29b,-30c,-126*,-127-3p,-196a,-324-3p). Several miRNA genes with dysregulated expression co-localize with acknowledged IBD-susceptibility loci while others, (eg. clustered on 14q32.31), map on chromosomal regions not previously recognized as IBD-susceptibility loci. In addition, in silico clustering analysis identified 5 miRNAs (mir-26a,-29b,-126*,-127-3p,-324-3p) that share coordinated dysregulation of expression both in quiescent and in inflamed colonic mucosa of IBD patients. Six miRNAs displayed significantly distinct alteration of expression in non-inflamed colonic biopsies of UC and CD patients (mir-196b,-199a-3p,-199b-5p,-320a,-150,-223).

Conclusions/Significance

Our study supports miRNAs as crucial players in the onset and/or relapse of inflammation from quiescent mucosal tissues in IBD patients. It allows speculating a role for miRNAs as contributors to IBD susceptibility and suggests that some of the miRNA with altered expression in the quiescent mucosa of IBD patients may define miRNA signatures for UC and CD and help develop new diagnostic biomarkers.     

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.     

Site-Specific MicroRNA Expression May Lead to Different Subtypes in Ulcerative Colitis

Background and Aim

Ulcerative Colitis (UC) is a type of inflammatory bowel disease, considered as an important disease of gastrointestinal tract having a huge impact on the health of the patient. Prolonged inflammation of colon in UC patients increases the risk of developing colorectal cancer. MiRNA are reported as a connecting link between inflammation and cancer. Differential miRNA expression is reported in Crohn’s disease (CD) patients involving various regions of the gastrointestinal tract. The current study was performed to dissect out the site specific miRNA expression in the colon biopsy samples of UC patients from Northern India.

Methods

Biopsy samples were collected from UC patients and healthy controls from Rectosigmoid Area (RS) and Ascending Colon (AC). MiRNA expression was compared between patients with RS and AC using a microarray platform. Differential expression was further validated by Real Time PCR analysis. Demographic and pathological data of UC -associated CRC patients was collected from the hospital database and analyzed for assessing the site of cancer.

Results

Upon analysis of data generated on a microarray platform and qRT PCR revealed that the expression of six miRNAs hsa-miR-146b-5p, hsa-miR-335-3p, hsa-miR-342-3p, hsa-miR-644b-3p, hsa-miR-491-3p, hsa-miR-4732-3p were downregulated in patients where RS was involved as compared to AC. The expression of hsa-miR-141-3p was upregulated in patients where RS region was involved as compared to AC. Analysis of the registered UC patient’s database from the hospital revealed that the site of CRC was predomimnantly the rectosigmoid region of the colon in most of the cases.

Conclusion

This is the first study to show the differential expression of miRNA involving different sites of colon in UC patients. Taking our data and previous reports into consideration, we propose that differential miRNA expression during UC perhaps contribute in the development of UC-associated CRC at the rectosigmoid area.     

Multiple outcome meta-analysis of gene-expression data in inflammatory bowel disease

We performed a multivariate meta-analysis of microarray data in Crohn's disease (CD) and Ulcerative colitis (UC), which are the main forms of inflammatory bowel disease (IBD). They share similar symptoms but differ in the location and extent of inflammation and in complications. We identified 249 differentially expressed genes (DEGs) in CD and 38 in UC at a false discovery rate of 1%. 20 of the DEGs were common to both diseases. A multivariate test identified 260 DEGs associated with IBD, 53 of which were not found in any of the disorders. We identified important molecular pathways implicated in the pathogenesis of IBD, such as the JAK/STAT and interferon-gamma signaling pathways, genes involved in cell adhesion, apoptosis and carcinogenesis. Among others, BCAT1 and GZMB are interesting novel DEGs that deserve further investigation in experimental models. The method could also be useful to other cases of meta-analysis of gene expression data.     

Increased expression and activation of IL-12-induced Stat4 signaling in the mucosa of ulcerative colitis patients

Inflammatory bowel diseases (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), are chronic inflammatory diseases with unsolved pathogenesis. Imbalanced Th1/Th2 may play a role in the sustained inflammation of IBD. In China, CD is rare but the incidence of UC has been rising steadily in the last two decades. We investigated the expression of IL-12 (p40) and IFN-γ, and the activational state of Stat4 signaling in mucosal tissues at the site of disease from 30 active UC patients in comparison with 30 healthy controls. RT-PCR analyses revealed increased mRNA expression of IL-12 (p40) but not IFN-γ in UC patients. Western blot analyses discovered, for the first time, increased levels of constitutive Stat4 in the cytoplasm and phosphorylated Stat4 in the nucleus of mucosal cells from UC patients. We conclude that a heightened, perhaps persistent, activational state of IL-12/Stat4, and/or IL-23/Stat4 signaling may be present in active Chinese UC patients, and possibly involved in chronic inflammation in UC.     

Genetic analysis of innate immunity in Crohn's disease and ulcerative colitis identifies two susceptibility loci harboring CARD9 and IL18RAP

The two main phenotypes of inflammatory bowel disease (IBD)--Crohn's disease (CD) and ulcerative colitis (UC)--are chronic intestinal inflammatory disorders with a complex genetic background. Using a three-stage design, we performed a functional candidate-gene analysis of innate immune pathway in IBD. In phase I, we typed 354 SNPs from 85 innate immunity genes in 520 Dutch IBD patients (284 CD, 236 UC) and 808 controls. In phase II, ten autosomal SNPs showing association at p < 0.006 in phase I were replicated in a second cohort of 545 IBD patients (326 CD, 219 UC) and 360 controls. In phase III, four SNPs with p < 0.01 in the combined phase I and phase II analysis were genotyped in an additional 786 IBD samples (452 CD, 334 UC) and 768 independent controls. Joint analysis of 1851 IBD patients (1062 CD, 789 UC) and 1936 controls demonstrated strong association to the IL18RAP rs917997 SNP for both CD and UC (p(IBD) 1.9 x 10(-8); OR 1.35). Association in CD is independently supported by the Crohn's disease dataset of the Wellcome Trust Case Control Consortium (imputed SNP rs917997, p = 9.19 x 10(-4)). In addition, an association of the CARD9 rs10870077 SNP to CD and UC was observed (p(IBD) = 3.25 x 10(-5); OR 1.21). Both genes are located in extended haplotype blocks on 2q11-2q12 and 9q34.3, respectively. Our results indicate two IBD loci and further support the importance of the innate immune system in the predisposition to both CD and UC.     

Immunolocalization and expression of kinin B1R and B2R receptors in human inflammatory bowel disease

Bradykinin is a mediator of inflammation, responsible for pain, vasodilation, and capillary permeability. Bradykinin receptor 1 (B(1)R) and bradykinin receptor 2 (B(2)R) are G protein-coupled receptors that mediate kinin effects. The latter is constitutive and rapidly desensitized; the former is induced by inflammatory cytokines and resistant to densensitization. The distribution of bradykinin receptors in human intestinal tissue was studied in patients with inflammatory bowel disease (IBD), namely ulcerative colitis (UC) and Crohn's disease (CD). Both B(2)R and B(1)R proteins are expressed in the epithelial cells of normal and IBD intestines. B(1)R protein is visualized in macrophages at the center of granulomas in CD. B(2)R protein is normally present in the apexes of enterocytes in the basal area and intracellularly in inflammatory tissue. In contrast, B(1)R protein is found in the basal area of enterocytes in normal intestine but in the apical portion of enterocytes in inflamed tissue. B(1)R protein is significantly increased in both active UC and CD intestines compared with controls. In patients with active UC, B(1)R mRNA is significantly higher than B(2)R mRNA. However, in inactive UC patients, the B(1)R and B(2)R mRNA did not differ significantly. Thus bradykinin receptors in IBD may reflect intestinal inflammation. Increased B(1)R gene and protein expression in active IBD provides a structural basis of the important role of bradykinin in chronic inflammation.     

Expression of Toll-like receptor 2 (TLR2), TLR4, and CD14 in biopsy samples of patients with inflammatory bowel diseases: upregulated expression of TLR2 in terminal ileum of patients with ulcerative colitis.

Dysregulation of innate and adaptive intestinal immune responses to bacterial microbiota is supposed to be involved in pathogenetic mechanisms of inflammatory bowel diseases (IBDs). We investigated expression of Toll-like receptor 2 (TLR2), TLR4, and their transmembrane coreceptor CD14 in biopsy samples from patients with IBD and in non-inflamed gut mucosa from controls. Small intestine and colon samples were obtained by colonoscopy from patients with Crohn's disease (CD), ulcerative colitis (UC), and controls. Immunohistochemical analysis of cryostat sections using polyclonal and monoclonal antibodies specific for TLR2, TLR4, and CD14 showed a significant increase in TLR2 expression in the terminal ileum of patients with inactive and active UC against controls. Significant upregulation of TLR4 expression relative to controls was found in the terminal ileum and rectum of UC patients in remission and in the terminal ileum of CD patients with active disease. CD14 expression was upregulated in the terminal ileum of CD patients in remission and with active disease, in the cecum of UC patients in remission and with active disease, and in rectum of UC patients with active disease. Hence, dysregulation of TLR2, TLR4, and CD14 expression in different parts of the intestinal mucosa may be crucial in IBD pathogenesis.     

Anti-inflammatory effects of Saccharomyces boulardii mediated by myeloid dendritic cells from patients with Crohn's disease and ulcerative colitis

Saccharomyces boulardii (Sb) is a probiotic yeast that has demonstrated efficacy in pilot studies in patients with inflammatory bowel disease (IBD). Microbial antigen handling by dendritic cells (DC) is believed to be of critical importance for immunity and tolerance in IBD. The aim was to characterize the effects of Sb on DC from IBD patients. Highly purified (>95%), lipopolysaccharide-stimulated CD1c(+)CD11c(+)CD123(-) myeloid DC (mDC) from patients with ulcerative colitis (UC; n = 36), Crohn's disease (CD; n = 26), or infectious controls (IC; n = 4) were cultured in the presence or absence of fungal supernatant from Sb (SbS). Phenotype and cytokine production and/or secretion of IBD mDC were measured by flow cytometry and cytometric bead arrays, respectively. T cell phenotype and proliferation were assessed in a mixed lymphocyte reaction (MLR) with allogenic CD4(+)CD45RA(+) na?ve T cells from healthy donors. Mucosal healing was investigated in epithelial wounding and migration assays with IEC-6 cells. SbS significantly decreased the frequency of CD40-, CD80-, and CD197 (CCR7; chemokine receptor-7)-expressing IBD mDC and reduced their secretion of tumor necrosis factor (TNF)-α and interleukin (IL)-6 while increasing IL-8. In the MLR, SbS significantly inhibited T cell proliferation induced by IBD mDC. Moreover, SbS inhibited T(H)1 (TNF-α and interferon-γ) polarization induced by UC mDC and promoted IL-8 and transforming growth factor-β-dependent mucosal healing. In summary, we provide novel evidence of synergistic mechanisms how Sb controls inflammation (inhibition of T cell costimulation and inflammation-associated migration and mobilization of DC) and promotes epithelial restitution relevant in IBD.     

Association of genetic variation in the NR1H4 gene, encoding the nuclear bile acid receptor FXR, with inflammatory bowel disease

ABSTRACT: BACKGROUND: Pathogenesis of inflammatory bowel diseases (IBD), ulcerative colitis (UC) and Crohn's disease (CD), involves interaction between environmental factors and inappropriate immune responses in the intestine of genetically predisposed individuals. Bile acids and their nuclear receptor, FXR, regulate inflammatory responses and barrier function in the intestinal tract. METHODS: We studied the association of five variants (rs3863377, rs7138843, rs56163822, rs35724, rs10860603) of the NR1H4 gene encoding FXR with IBD. 1138 individuals (591 non-IBD, 203 UC, 344 CD) were genotyped for five NR1H4 genetic variants with TaqMan SNP Genotyping Assays. RESULTS: We observed that the NR1H4 SNP rs3863377 is significantly less frequent in IBD cases than in non-IBD controls (allele frequencies: P = 0.004; wild-type vs. SNP carrier genotype frequencies: P = 0.008), whereas the variant rs56163822 is less prevalent in non-IBD controls (allele frequencies: P = 0.027; wild-type vs. SNP carrier genotype frequencies: P = 0.035). The global haplotype distribution between IBD and control patients was significantly different (P = 0.003). This also held true for the comparison between non-IBD and UC groups (P = 0.004), but not for the comparison between non-IBD and CD groups (P = 0.079). CONCLUSIONS: We show that genetic variation in FXR is associated with IBD, further emphasizing the link between bile acid signaling and intestinal inflammation.     

Loss of Setd2 associates with aberrant microRNA expression and contributes to inflammatory bowel disease progression in mice

Both SETD2-mediated H3K36me3 and miRNAs play critical epigenetic roles in inflammatory bowel disease (IBD) and involve in the dysfunctional intestinal barrier. However, little is known about cross-talk between these two types of regulators in IBD progression. We performed small RNA sequencing of Setd2 epithelium-specific knockout mice (Setd2^Vil-KO) and wild-type controls, both with DSS-induced colitis, and designed a framework for integrative analysis. Firstly, we integrated the downloaded ChIP-seq data with miRNA expression profiles and identified a significant intersection of pre-miRNA expression and H3K36me3 modification. A significant inverse correlation was detected between changes of H3K36me3 modification and expression of the 171 peak-covered miRNAs. We further integrated RNA-seq data with predicted miRNA targets to screen negatively regulated miRNA-mRNA pairs and found the H3K36me3-associated differentially expressed microRNAs significantly enriched in cell-cell junction and signaling pathways. Using network analysis, we identified ten hub miRNAs, among which six are H3K36me3-associated, suggesting therapeutic targets for IBD patients with SETD2-deficiency.     

Resistin,Elastase, and Lactoferrin as Potential Plasma Biomarkers of Pediatric Inflammatory Bowel Disease Based on Comprehensive Proteomic Screens

Inflammatory bowel disease (IBD) is an immune-mediated chronic inflammation of the intestine, which can present in the form of ulcerative colitis (UC) or as Crohn’s disease (CD). Biomarkers are needed for reliable diagnosis and disease monitoring in IBD, especially in pediatric patients. Plasma samples from a pediatric IBD cohort were interrogated using an aptamer-based screen of 1322 proteins. The elevated biomarkers identified using the aptamer screen were further validated by ELISA using an independent cohort of 76 pediatric plasma samples, drawn from 30 CD, 30 UC, and 16 healthy controls. Of the 1322 proteins screened in plasma from IBD patients, 129 proteins were significantly elevated when compared with healthy controls. Of these 15 proteins had a fold change greater than 2 and 28 proteins had a fold change >1.5. Neutrophil and extracellular vesicle signatures were detected among the elevated plasma biomarkers. When seven of these proteins were validated by ELISA, resistin was the only protein that was significantly higher in both UC and CD (p < 0.01), with receiver operating characteristic area under the curve value of 0.82 and 0.77, respectively, and the only protein that exhibited high sensitivity and specificity for both CD and UC. The next most discriminatory plasma proteins were elastase and lactoferrin, particularly for UC, with receiver operating characteristic area under the curve values of 0.74 and 0.69, respectively. We have identified circulating resistin, elastase, and lactoferrin as potential plasma biomarkers of IBD in pediatric patients using two independent diagnostic platforms and two independent patient cohorts.     

The Involvement of Immune Semaphorins in the Pathogenesis of Inflammatory Bowel Diseases (IBDs)

ResultsThe percentage (%) of T regulatory cells (Tregs) expressing sema3A in patients with active CD (64.5% ±14.49%) and active UC (49.8% ±16.45%) was significantly lower when compared to that of healthy controls (88.7% ±3.6%, p< 0.001 and p< 0.0001, respectively). This expression was seen to be in negative correlation with CD activity. Serum levels of Sema4A were significantly lower in patients with CD and UC when compared to that of controls (5.69±1.48ng\ml for CD, 5.26±1.23 ng/ml for UC patients vs 9.74±2.73ng/ml for normal controls, P<0.001). Sema4A was highly expressed in lymphocytes of the lamina propria of CD and UC patients but absent in patients with diverticulitis or in normal individuals.ConclusionsAltered % of Tregs expressing sema3A in patients with inflammatory bowel diseases (IBD) is partially responsible for their failure in preventing CD4+ effector T cell induced inflammation in IBD in peripheral blood. The increased expression of sema4A in bowel biopsies from CD and UC patients is suggestive of its central role in regulating local tissue inflammation in the bowel.     

Rapid and noninvasive metabonomic characterization of inflammatory bowel disease

Inflammatory bowel diseases (IBD) including Crohn's disease (CD) and ulcerative colitis (UC) have a major impact on the health of individuals and populations. Accurate diagnosis of inflammatory bowel disease (IBD) at an early stage, and correct differentiation between Crohn's disease (CD) and ulcerative colitis (UC), is important for optimum treatment and prognosis. We present here the first characterization of fecal extracts obtained from patients with CD and UC by employing a noninvasive metabonomics approach, which combines high resolution 1H NMR spectroscopy and multivariate pattern recognition techniques. The fecal extracts of both CD and UC patients were characterized by reduced levels of butyrate, acetate, methylamine, and trimethylamine in comparison with a control population, suggesting changes in the gut microbial community. Also, elevated quantities of amino acids were present in the feces from both disease groups, implying malabsorption caused by the inflammatory disease or an element of protein losing enteropathy. Metabolic differences in fecal profiles were more marked in the CD group in comparison with the control group, indicating that the inflammation caused by CD is more extensive in comparison with UC and involves the whole intestine. Furthermore, glycerol resonances were a dominant feature of fecal spectra from patients with CD but were present in much lower intensity in the control and UC groups. This work illustrates the potential of metabonomics to generate novel noninvasive diagnostics for gastrointestinal diseases and may further our understanding of disease mechanisms.     

A microRNA panel that regulates proinflammatory cytokines as diagnostic and prognosis biomarkers in colon cancer

Colon cancer (CC) is the third most common neoplasm and the fourth cause of cancer-related death worldwide in both sexes. It has been established that inflammation plays a critical role in tumorigenesis and progression of CC. Immune, stromal and tumor cells supply the tumor microenvironment with pro-inflammatory cytokines such as interleukin 1β, TNFα, IL-6 and IL-11, to hyperactivate signaling pathways linked to cancerous processes. Recent findings suggest a putative role of microRNAs (miRNAs) in the progression and management of the inflammatory response in intestinal diseases. Moreover, miRNAs are able to regulate expression of molecular mediators that are linking inflammation and cancer. In this work a miRNA panel differentially expressed between healthy, inflammatory bowel disease (IBD) and CC tissue was established. Identified miRNAs regulate signaling pathways related to inflammation and cancer progression. An inflammation associated-miRNA panel composed of 11-miRNAs was found to be overexpressed in CC but not in inflamed or normal tissues (miR-21-5p, miR-304-5p, miR-577, miR-335-5p, miR-21-3p, miR-27b-5p, miR-335-3p, miR-215-5p, miR-30b-5p, miR-192-5p, miR-3065-5p). The association of top hit miRNAs, miR-3065-5p and miR-30b-5p expression with overall survival of CC patients was demonstrated using Kaplan-Meier tests. Finally, differential miRNA expression was validated using an inflammation-associated CC model induced by Azoxymethane/Dextran Sodium Sulfate (AOM/DSS) to compare miRNA expression in normal and inflamed tissue versus CC tissues. Based on these findings we propose the identified inflammatory miRNA panel as a potent diagnostic tool for CC determination.     

Integrated miRNA and mRNA Expression Profiling in Inflamed Colon of Patients with Ulcerative Colitis

Background

Ulcerative colitis (UC) is associated with differential colonic expression of genes involved in immune response (e.g. IL8) and barrier integrity (e.g. cadherins). MicroRNAs (miRNAs) are regulators of gene expression and are involved in various immune-related diseases. In this study, we investigated (1) if miRNA expression in UC mucosa is altered and (2) if any of these changes correlate with mucosal mRNA expression. Integration of mRNA and miRNA expression profiling may allow the identification of functional links between dysregulated miRNAs and their target mRNA.

Methodology

Colonic mucosal biopsies were obtained from 17 UC (10 active and 7 inactive) patients and 10 normal controls. Total RNA was used to analyze miRNA and mRNA expression via Affymetrix miRNA 2.0 and Affymetrix Human Gene 1.0ST arrays, respectively. Both miRNA and gene expression profiles were integrated by correlation analysis to identify dysregulated miRNAs with their corresponding predicted target mRNA. Microarray data were validated with qRT-PCR. Regulation of IL8 and CDH11 expression by hsa-miR-200c-3p was determined by luciferase reporter assays.

Results

When comparing active UC patients vs. controls, 51 miRNAs and 1543 gene probe sets gave significantly different signals. In contrast, in inactive UC vs. controls, no significant miRNA expression differences were found while 155 gene probe sets had significantly different signals. We then identified potential target genes of the significantly dysregulated miRNAs and genes in active UC vs. controls and found a highly significant inverse correlation between hsa-miR-200c-3p and IL8, an inflammatory marker, and between hsa-miR-200c-3p and CDH11, a gene related to intestinal epithelial barrier function. We could demonstrate that hsa-miR-200c-3p directly regulates IL8 and CDH11 expression.

Conclusion

Differential expression of immune- and barrier-related genes in inflamed UC mucosa may be influenced by altered expression of miRNAs. Integrated analysis of miRNA and mRNA expression profiles revealed hsa-miR-200c-3p for use of miRNA mimics as therapeutics.     

Farnesoid X receptor (FXR) activation and FXR genetic variation in inflammatory bowel disease

Background

We previously showed that activation of the bile salt nuclear receptor Farnesoid X Receptor (FXR) protects against intestinal inflammation in mice. Reciprocally, these inflammatory mediators may decrease FXR activation. We investigated whether FXR activation is repressed in the ileum and colon of inflammatory bowel disease (IBD) patients in remission. Additionally, we evaluated whether genetic variation in FXR is associated with IBD.

Methods

mRNA expression of FXR and FXR target gene SHP was determined in ileal and colonic biopsies of patients with Crohn''s colitis (n = 15) and ulcerative colitis (UC; n = 12), all in clinical remission, and healthy controls (n = 17). Seven common tagging SNPs and two functional SNPs in FXR were genotyped in 2355 Dutch IBD patients (1162 Crohn''s disease (CD) and 1193 UC) and in 853 healthy controls.

Results

mRNA expression of SHP in the ileum is reduced in patients with Crohn''s colitis but not in patients with UC compared to controls. mRNA expression of villus marker Villin was correlated with FXR and SHP in healthy controls, a correlation that was weaker in UC patients and absent in CD patients. None of the SNPs was associated with IBD, UC or CD, nor with clinical subgroups of CD.

Conclusions

FXR activation in the ileum is decreased in patients with Crohn''s colitis. This may be secondary to altered enterohepatic circulation of bile salts or transrepression by inflammatory signals but does not seem to be caused by the studied SNPs in FXR. Increasing FXR activity by synthetic FXR agonists may have benefit in CD patients.     

An ileal Crohn's disease gene signature based on whole human genome expression profiles of disease unaffected ileal mucosal biopsies

Previous genome-wide expression studies have highlighted distinct gene expression patterns in inflammatory bowel disease (IBD) compared to control samples, but the interpretation of these studies has been limited by sample heterogeneity with respect to disease phenotype, disease activity, and anatomic sites. To further improve molecular classification of inflammatory bowel disease phenotypes we focused on a single anatomic site, the disease unaffected proximal ileal margin of resected ileum, and three phenotypes that were unlikely to overlap: ileal Crohn's disease (ileal CD), ulcerative colitis (UC), and control patients without IBD. Whole human genome (Agilent) expression profiling was conducted on two independent sets of disease-unaffected ileal samples collected from the proximal margin of resected ileum. Set 1 (47 ileal CD, 27 UC, and 25 Control non-IBD patients) was used as the training set and Set 2 was subsequently collected as an independent test set (10 ileal CD, 10 UC, and 10 control non-IBD patients). We compared the 17 gene signatures selected by four different feature-selection methods to distinguish ileal CD phenotype with non-CD phenotype. The four methods yielded different but overlapping solutions that were highly discriminating. All four of these methods selected FOLH1 as a common feature. This gene is an established biomarker for prostate cancer, but has not previously been associated with Crohn's disease. Immunohistochemical staining confirmed increased expression of FOLH1 in the ileal epithelium. These results provide evidence for convergent molecular abnormalities in the macroscopically disease unaffected proximal margin of resected ileum from ileal CD subjects.