Intestinal barrier defects are common in patients with inflammatory bowel disease (IBD). To identify which components could underlie these changes, we performed an in-depth analysis of epithelial barrier genes in IBD.
A set of 128 intestinal barrier genes was selected. Polygenic risk scores were generated based on selected barrier gene variants that were associated with Crohn's disease (CD) or ulcerative colitis (UC) in our study. Gene expression was analyzed using microarray and quantitative reverse transcription polymerase chain reaction. Influence of barrier gene variants on expression was studied by cis-expression quantitative trait loci mapping and comparing patients with low- and high-risk scores.
Barrier risk scores were significantly higher in patients with IBD than controls. At single-gene level, the associated barrier single-nucleotide polymorphisms were most significantly enriched in PTGER4 for CD and HNF4A for UC. As a group, the regulating proteins were most enriched for CD and UC. Expression analysis showed that many epithelial barrier genes were significantly dysregulated in active CD and UC, with overrepresentation of mucus layer genes. In uninflamed CD ileum and IBD colon, most barrier gene levels restored to normal, except for MUC1 and MUC4 that remained persistently increased compared with controls. Expression levels did not depend on cis-regulatory variants nor combined genetic risk.
We found genetic and transcriptomic dysregulations of key epithelial barrier genes and components in IBD. Of these, we believe that mucus genes, in particular MUC1 and MUC4, play an essential role in the pathogenesis of IBD and could represent interesting targets for treatment.
Inflammatory bowel diseases (IBDs), including Crohn's disease (CD) and ulcerative colitis (UC), are a group of chronic, relapsing inflammatory disorders of the gut that affect an increasing number of individuals around the world.1 The current hypothesis on the pathogenesis of IBD is that the disease results from complex interactions among the host genome, exposome, gut microbiome, and mucosal immune system.2,3 In this regard, also a dysfunctional intestinal barrier has long been recognized as a key pathogenic factor in IBD.4 The intestinal barrier is located at the interface between the external luminal environment and the internal immune system, and has the complex task to defend against potentially harmful molecules and microorganisms, while being permeable to essential nutrients and solutes.5 It is believed that intestinal barrier defects in patients with IBD result in an increased uptake of luminal antigens across the intestinal epithelium, which in turn would trigger the immune system and the development of mucosal inflammation. However, whether mucosal barrier alterations represent a primary dysfunction in the etiology of IBD, or develop as consequence of ongoing inflammatory processes in patients with IBD, is not entirely clear.6 Observations of increased intestinal permeability in a proportion of healthy first-degree relatives of patients with IBD suggest that intestinal barrier dysfunction might be genetically determined, and not only because of the impact of inflammatory mediators.7–15 Genome-wide association studies (GWAS) have also implicated the intestinal epithelial barrier as one of the key pathways in the pathogenesis of IBD.16–18
The general structure of the intestinal barrier is based on several components contributing to its function as a physical barrier between the luminal and internal environment, together with elements from the mucosal immune system that create an immunological defense barrier.6,19 The mucus layer provides the most apical line of defense against the luminal environment and forms a sieve-like gel structure that prevents large particles and bacteria from contacting the underlying intestinal epithelium.20 Besides the predominant enterocytes, the epithelium is composed of other specialized cell types with a wide array of functions, including goblet cells that produce the gel-like mucus; Paneth cells that secrete antimicrobial peptides reinforcing the immune barrier; and microfold cells that support transport of large luminal antigens and microbiota to immune cells in the lamina propria.5,21 The intestinal epithelial cells themselves constitute by far the strongest determinants of the physical intestinal barrier through the establishment of an almost impermeable polarized monolayer along the gut wall in the absence of specific transporters. The intercellular space is furthermore sealed by junctional protein complexes, of which the tight junctions are located at the most apical pole of the epithelial cells. Tight junctions are the main gatekeepers of the paracellular space and can mediate permeability of ions and small molecules up to 20 kDa. Adherens junctions and desmosomes, by contrast, form strong adhesive bonds and are primarily responsible for maintaining tissue cohesion and integrity.22,23 Both tight junctions and adherens junctions are dependent on scaffolding proteins for their formation and may interact with the cytoskeleton and a broad range of signaling molecules for their regulation.24 At the basal side of the epithelium, hemidesmosomes take care of the firm attachment of the cells to the basement membrane and the extracellular matrix, which in turn also control intestinal functions.25 Given the complex organization and regulation of the intestinal mucosal barrier, there is a need to identify which elements are most critical for the pathophysiology of IBD.
In this study, we performed an in-depth characterization of intestinal epithelial barrier genes in patients with IBD and combined genetic and transcriptomic approaches to get a better view on disease-relevant genes and components of the intestinal epithelial barrier. We first evaluated genetic risk scores based on variants in barrier genes and searched for genes and barrier components that were most enriched at genetic level. Second, we investigated expression levels of barrier genes using intestinal mucosal tissue from patients with IBD. Finally, we also analyzed whether the barrier gene variants regulated the mucosal gene expression levels in our study cohort.
Materials and Methods
Ethical Statement
Subjects were recruited at the outpatient IBD clinic of the University Hospitals Leuven, Belgium. The study was approved by the ethics committee of the UZ/KU Leuven (S53684/B322201213950), with written informed consent from all individuals before sample collection.
Selection of Intestinal Barrier Genes
A literature search was performed in PubMed to select genes involved in intestinal epithelial barrier function. Different combinations of the following search terms were used: “inflammatory bowel disease,” “Crohn's disease,” “ulcerative colitis,” “intestinal barrier function,” “intestinal integrity,” “intestinal epithelium,” “gut barrier,” “mucosal permeability,” and “barrier genes.” Importantly, also genes without previous evidence for their significance in IBD but essential for the structure of the intestinal barrier were included. For gene selection, we focused on the intestinal epithelium as physical barrier, and excluded genes involved in immunological barrier function. Subdivision of the genes into barrier components/categories was performed at the end of the selection.
Genetic Risk Study
Genotyping of 1696 patients with CD, 884 patients with UC, and 849 unrelated controls from our center was performed before using Immunochip (Table 1).17,18 For this study, we first extracted all single-nucleotide polymorphisms (SNPs) in the selected barrier genes, including markers located within 50 kb up- or downstream of the transcription start/end site of the genes (n = 3220). All these SNPs passed quality control according to the criteria as described before.17,18 Highly correlated SNPs (SNPs in high linkage disequilibrium, r2 > 0.7) were subsequently excluded, leaving 1317 barrier SNPs for association. Comparative analysis between cases (CD or UC) and controls was performed using logistic regression in PLINK (v1.07). A CD or UC polygenic barrier risk score was defined for each individual by counting the total number of risk alleles for the nominally significant disease-associated SNPs (defined as uncorrected P < 0.05) in the CD or UC versus controls analyses. Comparison of the combined barrier risk scores between cases (CD or UC) and controls was performed using Mann–Whitney U tests. Quartile analysis was performed using the Chi-squared test in R 3.2.5.
To evaluate if specific genes or barrier components were enriched in independently associated SNPs, we compared the number of (non)associated variants in a given gene with the number of (non)associated variants in the other genes for gene-level enrichment and the number of (non)significant genes (significant defined as having at least 1 associated SNP) in a given barrier component with those in the other components for component-level enrichment. Comparisons were performed using the Fisher's exact test in R 3.2.5 for 2 × 2 contingency tables, taking into account the total number of variants in each gene or barrier component. P < 0.05 was considered as enriched.
Mucosal Gene Expression Study
Patients and Biopsies
Endoscopic mucosal biopsies were obtained from 198 patients with IBD and 22 controls for microarray and/or quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. The biopsy specimens included colon from 97 patients with UC (74 with active disease and 23 with inactive disease), 34 patients with CD (8 with active colonic disease and 26 with inactive disease), and 11 controls; and terminal ileum from 67 patients with CD (51 with active ileal disease and 16 with inactive disease) and 11 controls (Table 1). The uninflamed colon biopsies from patients with CD were solely used for qRT-PCR analysis. Baseline characteristics from the individuals are presented in Table 1, Supplemental Digital Content 1, http://links.lww.com/IBD/B619. All biopsies were taken from different patients (no paired samples). Disease activity of the patients was based on endoscopic findings, with active disease defined as Mayo endoscopic subscore ≥2 for UC, and the presence of ulcers for patients with CD. The control group, who underwent endoscopy for polyp screening, had normal mucosa at endoscopic level. The biopsies were immediately snap-frozen in liquid nitrogen on extraction and stored at −80°C until RNA isolation.
RNA Isolation and Microarray Analysis
Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Venlo, the Netherlands). Assessment of RNA integrity and quantity was performed by the 2100 Bioanalyzer (Agilent, Waldbronn, Germany) and the Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE), respectively. The isolated RNA was analyzed with Affymetrix GeneChip Human Gene 1.0 ST Arrays (Affymetrix, Santa Clara, CA) (GSE75214) and as previously described by Vanhove et al.26 Comparative analyses between the studied groups were performed using the R/Bioconductor LIMMA (linear models for microarray data) package.27 Differential expression was calculated based on moderated t-statistics with correction for multiple testing according to the Benjamini–Hochberg false discovery rate (FDR).28 Because the main focus of this study was to evaluate the intestinal epithelial barrier, we filtered the results from the genome-wide comparative analyses for the gene probe sets representing the selected barrier genes. Gene probe sets with a >2-fold change and FDR <0.05, showing multiple testing correction for the entire array, were considered biologically significant. The gene probe set of IL8 (inflammatory marker) was included to evaluate inflammation. Enrichment of genes in the specified categories of the intestinal barrier was evaluated using the Fisher's exact test in R 3.2.5. Correlations with IL8 were studied with the Spearman's rank correlation test in IBM SPSS statistics 22. The microarray log2 expression values were used for the correlation analyses, and the colon (n = 116) and ileum (n = 78) samples were studied separately. P < 0.05 was considered significant.
To evaluate the relation between the barrier gene expression levels and genetic barrier risk, pairwise comparisons in LIMMA (as above) were performed for patients with low and high genetic barrier risk. Low and high risk was defined as a genetic barrier risk score below the 25th percentile value (quartile [Q] 1 = Q1) or above the 75th percentile value (Q4), respectively (Table 1).
qRT-PCR
Based on the significance levels in the comparisons and/or their relevance for both tissue types (colon and ileum), the following genes were selected for validation by qRT-PCR: MUC1, MUC4, TFF1, CLDN1, CLDN8, OCLN, DSG3, and MAGI1. Beta-actin was used as endogenous reference gene. The primer and probe sequences (Sigma-Aldrich, Diegem, Belgium) for the genes were custom designed using OligoAnalyzer 3.1 software (see Table 2, Supplemental Digital Content 1, http://links.lww.com/IBD/B619). The RevertAid H Minus First Strand cDNA synthesis kit (Fermentas, St. Leon-Rot, Germany) was used to synthesize cDNA from 0.5 μg total RNA, according to the manufacturer's protocol. Five samples were excluded for qRT-PCR analysis because insufficient material was available. The qRT-PCR experiments were performed in duplicate using the SensiFast Probe No-ROX Kit (GC Biotech, Alphen aan den Rijn, the Netherlands) in a final reaction volume of 20 μL, on a Rotor-Gene 3000 instrument (Corbett Research, Mortlake, Australia). Cycle threshold values for each gene were determined by the Rotor-Gene 6 software package. The relative messenger RNA (mRNA) expression values of the barrier genes were calculated as ratio relative to the endogenous reference gene beta-actin (Pfaffl method).29 Statistical analysis of the results was performed using 2-tailed Mann–Whitney U tests for unpaired samples (IBM SPSS statistics 22), and a significance level of 0.05 was used.
Expression Quantitative Trait Loci (eQTL) Mapping
The genotype profiles and gene expression data were combined by performing cis-eQTL mapping on the available set of samples in our cohort with both genetic marker and microarray expression information: inflamed (n = 56) and normal (n = 20) colon from patients with UC, inflamed colon (n = 3) from patients with CD, and inflamed (n = 34) and normal (n = 12) ileum from patients with CD (Table 1). The maximal distance between each gene and SNP was limited to 1 mega-base (Mb). Only SNPs with a minor allele frequency > 0.05 and low linkage disequilibrium (r2 < 0.1) were selected for analysis (n = 17.108, of which 2329 in cis of the barrier genes). Direct pairwise regressions were performed using the Matrix eQTL package in R 3.2.5.30 Each location (colon and ileum) and disease activity status (active and inactive) was analyzed separately because the microarray results pointed toward distinct profiles for these groups. Within each subgroup, we again filtered for minor allele frequency <0.05 during the eQTL analysis to avoid false-positive results. Correction for multiple testing was performed using the Benjamini–Hochberg procedure implemented in Matrix eQTL.
Results
Epithelial Barrier Gene Selection and Classification
We selected 128 genes related to physical intestinal barrier function. Of these, 25 were classified as part of the mucus layer, 34 as tight junctions, 5 as adherens junctions, 14 as desmosomes, 4 as hemidesmosomes, 17 as cytoskeleton, 9 as extracellular matrix, and 20 as regulating proteins (see Table 3, Supplemental Digital Content 1, http://links.lww.com/IBD/B619).
Genetics of Epithelial Barrier Genes
Eighty-two SNPs were nominally significant for association with CD and 69 SNPs with UC (see Table 4, Supplemental Digital Content 1, http://links.lww.com/IBD/B619). None of these remained significant after correction for multiple testing (<3.8 × 10−5 for 1317 SNPs). When considering the total number of risk alleles for the nominally significant SNPs per individual, patients with CD had significantly higher CD barrier risk scores compared with controls (median 82 [interquartile range (IQR): 77–87] versus 78 [IQR: 73–83], P < 2.2 × 10−16) (see Fig. 1A [Supplemental Digital Content 1, http://links.lww.com/IBD/B619] for the distribution of the CD barrier risk scores). The median number of UC barrier risk alleles also was significantly higher in patients with UC than in controls (68 [IQR: 64–73] versus 64 [IQR: 60–69], P < 2.2 × 10−16) (see Fig. 1B [Supplemental Digital Content 1, http://links.lww.com/IBD/B619] for the distribution of the UC barrier risk scores). Quartile analysis of the barrier risk scores showed that a higher proportion of patients with CD had CD barrier risk scores in Q4 versus controls (32.8% versus 17.1%), with proportionally less patients in Q1 versus controls (16.5% versus 32.4%) (P < 2.2 × 10−16) (Fig. 1A). Similar findings were seen for the UC barrier risk scores: more patients with UC in Q4 (40.2% versus 19.8%), whereas fewer patients were found in Q1 compared with controls (15.4% versus 31.3%) (P < 2.2 × 10−16) (Fig. 1B).
Quartile analysis of the barrier risk scores in patients and controls. The percentage of individuals in the quartiles (Q1–Q4) of the CD barrier risk scores (A) and UC barrier risk scores (B). Comparisons of the number of individuals in Q1 and Q4 were performed with Chi-squared testing. *Statistically significant (P < 2.2 × 10−16 for A and B).
In addition to the combined risk of the genetic barrier variants, we evaluated if the nominally associated SNPs were overrepresented in specific genes or components of the intestinal barrier. Comparison of the numbers of associated variants in the selected barrier genes showed enrichment in MUC19, MUC22 and TFF1 (mucus layer), and PTGER4 (regulating proteins) for CD (P = 4.30 × 10−2, 9.41 × 10−3, 1.12 × 10−2, and 8.94 × 10−4, respectively), whereas for UC most enrichment was seen in MUC21 and MUC22 (mucus layer) and GNA12 and HNF4A (regulating proteins) (P = 4.87 × 10−2, 2.47 × 10−2, 7.85 × 10−3, and 5.67 × 10−3, respectively) (see Table 5, Supplemental Digital Content 1, http://links.lww.com/IBD/B619). The barrier component with most genes associated with CD and UC was the group of regulating proteins, although enrichment of this component was only significant for UC (P = 2.18 × 10−3) (see Table 6 [Supplemental Digital Content 1, http://links.lww.com/IBD/B619] and Fig. 2).
Enrichment analysis of associated barrier genes (≥1 SNP) per component. Bar plots representing the percentage of significant genes in each of the barrier components for CD (left) and UC (right). Only the set of regulating proteins was significantly enriched for UC in associated barrier genes compared with the other barrier components using Fisher's exact testing (*P < 0.05).
Mucosal Barrier Gene Expression
Microarray Analysis
Of the 128 selected genes, 125 were represented on the Human Gene 1.0 ST arrays by 132 different gene probe sets. To correlate the barrier gene mRNA expression levels with inflammation, we included the expression profile of IL8, represented by 1 extra gene probe set. In agreement with endoscopic disease activity, IL8 expression was significantly higher in active IBD (UC and/or CD) compared with controls, whereas no differences were detected for IL8 in uninflamed biopsies of patients with IBD versus controls. Results of all comparisons are given in Table 7, Supplemental Digital Content 1, http://links.lww.com/IBD/B619. A heat map of the colonic and ileal expression values per gene probe set and individual is provided as Figs. 2 and 3, Supplemental Digital Content 1, http://links.lww.com/IBD/B619.
Colonic expression of the epithelial barrier genes did not differ between UC and CD patients with active disease. As compared to controls, however, the expression of many barrier genes was dysregulated in the colon of patients with active IBD (UC and/or CD). The mRNA expression levels of MUC1, MUC5B, EMCN, MCAM and TFF1 (mucus layer), CLDN1 and JAM2 (tight junctions), DSG3 (desmosomes), LAMA4 and LAMC1 (extracellular matrix), and TCF4 and F2RL2 (regulating proteins) were >2-fold significantly upregulated in the inflamed colon of patients with IBD, whereas the mRNA expression levels of RETNLB (mucus layer), CLDN8 and OCLN (tight junctions), and MAGI1 and MEP1A (regulating proteins) were >2-fold significantly downregulated in active patients with IBD when compared with the colon of controls (Table 2). Of the different barrier components, the mucus layer was most enriched in differentially expressed genes (P = 4.97 × 10−2) (see Table 8, Supplemental Digital Content 1, http://links.lww.com/IBD/B619). None of the barrier genes remained significantly dysregulated in the colon of UC patients with inactive disease as compared to their expression levels in controls (Table 2). All colonic dysregulated genes showed a highly significant correlation with IL8, confirming the direct impact of inflammation on epithelial barrier gene expression (see Table 9 and Fig. 4 [Supplemental Digital Content 1, http://links.lww.com/IBD/B619] for the highest correlated ones).
Significant Barrier Genes in the Colon of Patients with UC and Patients with CD Versus Controls
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Significant Barrier Genes in the Colon of Patients with UC and Patients with CD Versus Controls
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In addition to the colonic mRNA expression levels, differences in barrier gene expression in the terminal ileum of CD patients with active and inactive disease, and controls were evaluated. Eight genes (MUC1, MUC4, MUC5B, MUC6 and TFF1 [mucus layer], CLDN1 and CLDN18 [tight junctions], and F2RL2 [regulating proteins]) showed a >2-fold significantly increased expression in the inflamed ileal mucosa of patients with CD compared with uninflamed tissue of controls, whereas the expression of CLDN8 (tight junctions) was significantly downregulated (Table 3). The barrier component most enriched in genes with differential expression in the ileum of CD patients with active disease versus controls also was the mucus layer (P = 8.54 × 10−3) (see Table 8, Supplemental Digital Content 1, http://links.lww.com/IBD/B619). Interestingly, the ileal expression of MUC1, MUC4 (mucus layer), and CLDN8 (tight junctions) remained dysregulated in the ileum of CD patients with inactive disease in comparison with controls. The mRNA expression of MUC1 and MUC4 was >2-fold significantly upregulated in inactive patients with CD, whereas CLDN8 was >2-fold significantly downregulated in patients compared with controls (Table 3). Again, significant correlations were found between the ileal mRNA levels of the dysregulated genes and IL8 (see Table 9 and Fig. 4 [Supplemental Digital Content 1, http://links.lww.com/IBD/B619] for the highest correlated ones).
Validation by qRT-PCR
The differential barrier gene expression levels of MUC1, MUC4 and TFF1 (mucus layer), CLDN1, CLDN8 and OCLN (tight junctions), DSG3 (desmosomes), and MAGI1 (regulating proteins) from the microarray analysis were confirmed by qRT-PCR (Fig. 3).
Relative expression levels of 8 barrier genes with qRT-PCR. Bar plots representing the relative expression levels of 8 barrier genes (A–H) measured by qRT-PCR in colon (white bars) from controls (n = 11), active UC (n = 72), inactive UC (n = 22), active patients with CD (n = 8), and inactive patients with CD (n = 26); and ileum (black bars) from controls (n = 11), active CD (n = 51), and inactive patients with CD (n = 14). The expression levels are normalized to beta-actin. Data are expressed as medians with IQR. Comparisons between the subgroups were performed with Mann–Whitney U testing. Significant differences as described in the main text are indicated (*P < 0.05, **P < 0.01). CDc, colon of patients with Crohn's disease; CDi, ileum of patients with CD.
As compared to the normal colon of controls, we found that the mRNA levels of MUC1, TFF1, CLDN1, and DSG3 were significantly upregulated in the inflamed colon of patients with UC and/or patients with CD, whereas the colonic expression levels of CLDN8, OCLN, and MAGI1 were significantly decreased in active patients with IBD compared with controls. The more sensitive qRT-PCR results also showed increased mRNA expression levels of MUC4 for these comparisons, whereas OCLN and MAGI1 levels were significantly decreased in active patients with CD. In addition, although no significant alterations were previously found for the colonic expression of the barrier genes between UC and CD patients with active disease, qRT-PCR analysis did identify significantly different levels of CLDN8, DSG3, TFF1, and MAGI1 in the colon of active patients with UC when compared with active patients with CD. Finally, qRT-PCR showed significantly increased expression levels of MUC1, MUC4, and DSG3 in the colon of UC patients with inactive disease versus controls. Evaluation of the genes in an additional cohort of 26 inactive patients with CD demonstrated that MUC1 and MUC4 also were significantly upregulated in uninflamed colon samples from patients with CD compared with healthy controls (P = 0.043 and 0.009, respectively) (Fig. 3).
In the ileum, we confirmed the differential expression of MUC1, MUC4, TFF1, CLDN1, and CLDN8 in active patients with CD when compared with controls. The ileal expression of MUC1, MUC4, and CLDN8 also remained dysregulated in CD patients with inactive disease as seen in the microarray analysis. Additional differences were observed for OCLN and MAGI1, having significantly decreased levels in the inflamed ileum of patients with CD when compared with the ileum of controls.
Influence of Genetics on Mucosal Barrier Gene Expression
To evaluate if there were cis-acting genetic variants affecting the barrier gene expression levels, we performed cis-eQTL mapping in each of the patient sample groups (inflamed colon, normal colon, inflamed ileum, and normal ileum). No significant cis-eQTL signals were found after correction for multiple testing in any of the groups. We also did not find significant differences in the barrier gene expression levels between CD and UC patients with the lowest (<75 for CD, <62 for UC) and highest genetic barrier risk scores (>86 for CD, >70 for UC).
Discussion
This study represents a comprehensive report in which the different components of the intestinal epithelial barrier were analyzed at genetic and transcriptomic levels in the context of IBD, taking into account disease type (CD and UC), biopsy location (colon and ileum), and activity status (inflamed and uninflamed).
We found that the total number of risk alleles in epithelial barrier genes was significantly higher in patients with CD and patients with UC compared with controls, validating the known impact of the intestinal barrier for the pathogenesis of IBD. Further analysis of barrier gene variants highlighted the potential role of MUC19, MUC22, TFF1, and PTGER4 for CD and MUC21, MUC22, GNA12, and HNF4A for UC. At component level, genes with associated variants were most enriched in the group of regulating proteins for both CD and UC. The mucosal gene expression study showed that the mRNA expression of many epithelial barrier genes was dysregulated in the inflamed colon and ileum of patients with IBD, with a significant over representation of mucus layer genes in both. During inactive disease, the expression of MUC1 and MUC4 remained commonly disturbed in intestinal samples of patients with CD and patients with UC, suggesting that these genes act as crucial players in IBD. In CD ileum, CLDN8 also remained significantly lower expressed compared with controls as evaluated by both microarray and qRT-PCR. Analysis of the link between the genetic variants in the barrier genes and their expression alterations, however, did not show significant findings, which might indicate that both levels are not necessarily directly related and influenced by many other disease-specific factors. A schematic overview of the most interesting results is shown in Fig. 4.
Schematic overview of the main results. The genetic and transcriptomic approaches in this study identified the potential role of particular epithelial barrier genes and components in the context of IBD. We found that disease-associated variants were significantly enriched in MUC19 (secreted mucin), MUC22 (membrane-bound mucin), TFF1 (stabilizing mucus layer protein), and PTGER4 (regulating protein) for CD, and MUC21 (membrane-bound mucin), MUC22 (membrane-bound mucin), GNA12 (regulating protein), and HNF4A (regulating protein) for UC. The most enriched barrier component was the set of regulating proteins, although only significant for UC. At mRNA level, persistent changes in ileal and colonic MUC1 (membrane-bound mucin) and MUC4 (membrane-bound mucin) expression were found during inactive disease for CD and UC, pinpointing to a possible central role of these genes in IBD onset/relapse. In uninflamed CD ileum, also CLDN8 expression remained strongly dysregulated. Genetic predispositions and expression changes may together induce barrier dysfunction of the intestinal epithelium in patients with IBD and result in an enhanced uptake of harmful luminal antigens and initiation of inflammation. Symbols within the mucus layer are explained below the figure. Structures within the epithelial cells are defined by the category names. Arrows indicate persistent up- or downregulation of the genes during inactive disease. aSignificant enrichment of variants associated with CD; bsignificant enrichment of variants associated with UC. This figure was created with images adapted from Servier Medical Art.
GWAS have previously identified multiple individual SNPs that are associated with the risk of IBD. Although the functional relevance of many of these SNPs is not known yet, the observed higher genetic barrier risk scores in patients with IBD compared with controls suggest that patients also more commonly have a combination of disease-associated variants in intestinal barrier genes which could cause an intensification of the small effects from the individual risk variants. Part of the patients with IBD may thus have a distinct genetic predisposition to have intestinal barrier defects and respond differently—most likely in combination with other predisposing factors—to common environmental stimuli triggering disease onset or relapse.
Enrichment analysis with associated barrier SNPs for CD showed that the most significantly enriched gene in this study was PTGER4. The PTGER4 locus has already been identified in several other studies as associated with CD.17,31 The gene encodes the prostaglandin receptor EP4, of which activation has been suggested to result in redistribution of junctional proteins and the cytoskeleton, with an increase in epithelial barrier disruption.32 The most significant enriched gene for UC was HNF4A, a transcription factor known for its essential role in the development and regulation of intestinal epithelial cells, and previously associated with a number of GWAS with UC.17,33,34 Ahn et al35 showed that mice with a conditional knockout of Hnf4a in intestinal epithelial cells had a markedly increased intestinal permeability and susceptibility to acute DSS colitis. Among the other enriched genes, MUC19 (secreted gel-forming mucin) and GNA12 (tight junction regulator) are also extensively described based on their association with large GWAS and meta-analyses, whereas reports on genetic evidence for MUC21, MUC22, and TFF1 in IBD are rather limited.17,18,34MUC21 encodes a recently identified transmembrane mucin protein, in which 1 particular SNP has been associated with UC by Achkar et al who looked into the major histocompatibility complex on chromosome 6p.36 In the context of lung diseases, both MUC21 and MUC22, another membrane-bound mucin at epithelial surfaces, have been proposed as candidates for association with asthma, although it could not be excluded that other genes in close proximity including HLA regions are responsible for these signals.37 Changes in integrity of the bronchial epithelium are believed to play a central role in the sensitization to allergens and the development of asthma, a chronic inflammatory disease of the airways that have a similar defense barrier as in the gut.38 The family of trefoil factors, including TFF1, has received considerable attention in a number of animal and intestinal expression studies but has so far not been associated with the risk of IBD or other immune-related disorders. Although its precise physiological function and regulation in the gut is not clear, TFF1 is believed to act in mucosal repair and reinforcement of the mucus layer by interaction with mucin molecules.39
In addition to the enrichment analysis at single-gene level, which searched for multiple risk signals within the same genomic location, a component-level analysis was performed where we evaluated which barrier components had the highest number of genes with at least 1 associated SNP. We showed that the regulating proteins were most overrepresented, with multiple significant genes for both disease types, although only significant at P < 0.05 for UC. We could assume that IBD barrier defects partly originate from effects of variants within different regulating barrier genes, together with some strong signals from individual genes of other barrier components such as mucus layer factors that showed high enrichment at single-gene level. Of note, the group of regulating proteins involved a broad mixture of scaffolding proteins, transcription factors, and previously associated regulatory genes, possibly creating a selection bias toward association. We are also aware that the genetic analysis had limited power to detect genome-wide significant findings. Given our current sample size and a significance level of 3.8 × 10−5, we only had 57% and 40% power to identify variants with an effect size of 1.5 and allele frequency of 0.1 for CD and UC, respectively. Still, some of the most significant signals that we found were already described in larger studies, as were the genes enriched in independent significant variants (e.g., MUC19 and HNF4A).
The results from the gene expression study showed that IBD patients with active disease had major gene expression changes at different levels of the intestinal epithelial barrier validating many previous reports. Interestingly, there was a considerable overlap between genes dysregulated in the colon and ileum of both patients with CD and patients with UC during active disease (e.g., MUC1, MUC5B, TFF1, CLDN1, CLDN8, and F2RL2). This suggests that these barrier molecules are affected in a similar way and represent largely the same barrier defects at both tissue sites under the influence of inflammatory mediators. The most aberrant changes during inflammation were found for MUC1 in the ileum of patients with CD and CLDN8 in the colon of patients with UC and CD. MUC1 is synthesized by goblet and absorptive cells from the intestinal epithelium and is expressed as a membrane-bound glycoprotein in the mucus layer.40 Consistent with our results, different studies have previously implicated increased MUC1 gene and protein expression during inflammation.41–44 It was suggested by Kadayakkara et al that an increase in MUC1 gene expression may initially serve to protect the gut epithelium by strengthening the function of the mucus layer, whereas repetitive cycles of inflammation can induce an increased expression of an abnormal hypoglycosylated protein form of MUC1 which attracts innate inflammatory cells and promotes the development of chronic inflammation and oncogenesis.45,CLDN8 was the most downregulated gene in active patients with IBD, as also frequently described in previous studies.46–49 CLDN8 belongs to the “sealing” proteins of the claudin family which restrict paracellular flux and decrease intestinal permeability, in contrast to pore-forming claudins such as CLDN2 which increase permeability of the intestinal barrier.50 In a study of Zeissig and colleagues, downregulation of CLDN8 was accompanied by CLDN2 upregulation at the tight junctions, intensely enhancing tight junction permeability in active patients with CD.47 In our study, CLDN2 gene expression was increased in the colon of active patients with IBD compared with controls but not more than 2-fold different. When comparing the number of differentially expressed genes for IBD patients with active disease versus controls, the mucus layer genes were most enriched. Taken these results together with the findings from the genetic study, we suggest a key role for the mucus layer component in the pathogenesis of IBD. Future studies should look at protein levels of mucus layer genes to dissect their biological working mechanism and functional relevance for IBD.
Remarkably, the common barrier genes that remained dysregulated during inactive disease were MUC1 and MUC4 in CD and UC colon (qRT-PCR) and CD ileum (microarray and qRT-PCR). In inactive ileum of patients with CD, also CLDN8 expression remained strongly dysregulated according to both microarray and qRT-PCR analyses. Like MUC1, MUC4 is a membrane-bound mucin protein at the apical side of the intestinal epithelial cells and forms the glycocalyx which is situated just below the gel-like mucus layer.51 As opposed to studies of barrier gene expression changes during inflammation, little information is available on these barrier gene levels in quiescent disease.52,53 A recent study of Peloquin et al investigated a selection of 678 genes within previously identified IBD risk loci and found that uninflamed samples of patients with CD exhibited perturbed expression levels of particular genes with increased variances compared with healthy controls. They suggested that these genes are normally held under tight regulatory control, which is lost in the setting of CD.54 It could thus be that MUC1 and MUC4 are in a continuously, dysregulated state (primary or because of subclinical molecular inflammation) which can trigger disease onset and relapse in predisposed patients—and worsen with active inflammation. We should then suppose that high MUC1 and MUC4 levels have a detrimental effect on the intestinal barrier by expression of an aberrant form as suggested earlier for MUC1 or by causing a general imbalance in mucins which affects the mucus composition and function. An alternative hypothesis on persistent increases in MUC1 and MUC4 expression during inactive disease could be that they represent a secondary defense or repair mechanism to protect the gut and account for the damage of previous inflammation. Because CLDN8 encodes a pore-sealing protein, it is acceptable that its expression has not returned to normal levels in controls when secondary to inflammation, and thus is less dynamic than other barrier genes, again promoting chronic reactivation of the disease. Although not significant in our genetic study, Franke et al showed that the MUC1 locus was genetically associated with CD, which would be in favor of the hypothesis of a primary role for this gene.55 For MUC4 and CLDN8, no association reports are available in current literature.
In previous studies, our group has evaluated the effect of infliximab therapy on the mucosal expression of several genes involved in IBD.56–58 Albeit the primary goals of these studies were different, most dysregulated genes during active disease in the current study were also significantly dysregulated in the inflamed mucosa of patients with IBD before their first infusion of infliximab. Strikingly, MUC1 and MUC4 also remained significantly upregulated in the colon of CD responders after infliximab treatment compared with controls, and the same was found for MUC1 in the ileum of CD responders versus controls (see Methods [Supplemental Digital Content 1, http://links.lww.com/IBD/B619] for a description of this cohort, and see Table 10 [Supplemental Digital Content 1, http://links.lww.com/IBD/B619] for all comparisons before and after infliximab treatment). Both genes did not show significantly altered levels in UC responders compared with controls, which is similar to our microarray findings in uninflamed colon from patients with UC. Analysis with qRT-PCR, by contrast, did show persistent higher levels in the latter samples in our study, which might be explained by the higher sensitivity of qRT-PCR as opposed to microarray or a more pronounced effect in the phenotype of CD versus UC.
When combining the results from the genetic and mucosal gene expression study, there was no direct link between variants in the epithelial barrier genes and differences in their expression. Neither single cis-acting variants in the barrier genes nor the combined barrier risk scores were associated with the expression of the barrier genes at an FDR level of 5%. These results could imply that other mechanisms are primarily involved for the genetic barrier risk and expression changes seen in patients with IBD in this cohort. For the MUC19 risk locus, for example, it has been suggested that associated SNPs in the gene region probably exert their effect by inducing changes in mRNA conformation, translational efficiency, or subcellular localization rather than gene expression.59 Gene expression alterations of the barrier genes could also be regulated by SNPs further away from the genes, but because of the limited sample size of the overlapping cohort, we only examined eQTLs acting in cis (including a strict window of 1 Mb). Trans-eQTLs (>1 Mb from the barrier gene start/end sites) were not described here, as it was shown that small effects of trans variants are harder to detect and much more sensitive to statistical power.60 Unmistakably, interesting signals could be missed in that way, indicating the need for larger sample sizes. Some recent studies have investigated genome-wide eQTLs in primary tissue cell types for IBD and their overlap with the known IBD susceptibility loci.61–65 None of the top signals from these studies correspond with 1 of our selected barrier genes, confirming that we should search for other regulating mechanisms in these regions.
Taken together, the data in this study allowed us to get a better view on which genes and components from the intestinal epithelial barrier pathway are most critical for IBD, based on their genetic and transcriptomic significance. Identification of the most critical molecules could be necessary to enhance the development of novel barrier-restoring therapeutics. Today, several agents that modify intestinal barrier integrity have been proposed, but their clinical application is still limited, mostly because of shortcomings in the mechanistic and functional understanding of the intestinal barrier. One of the most promising agents for UC currently includes phosphatidylcholine, a major class of phospholipids in the colonic mucus layer. The delayed release of phosphatidylcholine in the gut is believed to reinforce the mucus layer. Our data also support the intestinal mucus layer as a key therapeutic target within the intestinal barrier. The compound has been shown to be an effective and safe therapeutic option for patients with UC in phase II clinical trials, but more research is needed to understand its exact working mechanism and its lack of efficacy for CD.66–68
In conclusion, we provided an in-depth view on the genetic and transcriptomic bases of intestinal epithelial barrier defects in IBD. By using 3 different approaches, we identified a selection of barrier genes (e.g., MUC1, MUC4, and MUC22) and components (e.g., mucus layer and regulating proteins) that may be plausible candidates for the onset or perpetuation of chronic gastrointestinal inflammation in IBD (Fig. 4). Future studies focusing on the functional working mechanism of these genes, and categories are required to uncover their precise role in the disease pathogenesis and therapeutic potential.
Acknowledgments
This work was supported by the Research Foundation Flanders (FWO) [G.0440.06, G.0479.10] and the European Crohn's and Colitis Organisation [ECCO Grant 2013]. T. Vanuytsel, M. Ferrante, G. Van Assche, and S. Vermeire are senior clinical investigators of the FWO. T. Vanuytsel receives lecture fees from Will Pharma and consulting fees from Shire. M. Ferrante reports financial support for research from Takeda; lecture fees from Abbvie, Boehringer-Ingelheim, Chiesi, Falk, Ferring, Janssen, Mitsubishi Tanabe, MSD, Takeda, Tillotts, and Zeria; and consulting fees from Abbvie, Boehringer-Ingelheim, Ferring, Janssen, and MSD. G. Van Assche receives support for research from Abbvie and MSD; lecture fees from Abbvie, MSD, Ferring, Janssen, and Takeda; and consulting fees from Abbvie, MSD, and Takeda. P. Rutgeerts receives research support and lecture fees from Abbvie, Centocor, and Merck; and consulting fees from Abbvie, Centocor, Merck, UCB, Takeda, Genentech/Hoffman-LaRoche, Serono, Bristol Myers Squibb, Robarts, Tillotts, Pfizer, and Falk Pharma. S. Vermeire reports grant support from Abbvie, MSD, and Takeda; lecture fees from Abbvie, MSD, Takeda, Ferring, Falk Pharma, Hospira, and Tillotts; and consulting fees from Abbvie, MSD, Takeda, Ferring, Genentech/Roche, Shire, Pfizer, Galapagos, Mundipharma, Hospira, Celgene, Second Genome, and Janssen. The remaining authors have no conflict of interest to disclose. I. Arijs and I. Cleynen share last co-authorship.
The authors thank Karolien Claes, Nooshin Ardeshir Davani, Sophie Organe, and Willem-Jan Wollants for the technical support; and Vera Ballet for the patient inclusion and database management. They also thank Leentje Van Lommel from the Gene Expression Unit for technical assistance with the microarray and quantitative reverse transcription PCR experiments.
References
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Author notes
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