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Inflammatory bowel disease
Genetic polymorphisms of IL-23R and IL-17A and novel insights into their associations with inflammatory bowel disease
  1. Seung Won Kim1,2,
  2. Eun Soo Kim1,
  3. Chang Mo Moon1,
  4. Jae Jun Park1,
  5. Tae Il Kim1,
  6. Won Ho Kim1,2,
  7. Jae Hee Cheon1,2
  1. 1Department of Internal Medicine and Institute of Gastroenterology, Yonsei University College of Medicine, Seoul, Korea
  2. 2Brain Korea 21 Project for Medical Science, Yonsei University, Seoul, Korea
  1. Correspondence to Professor Jae Hee Cheon, Division of Gastroenterology, Department of Internal Medicine, Yonsei University College of Medicine, 250 Seongsanno, Seodaemun-gu, Seoul 120-752, Korea; geniushee{at}yuhs.ac Professor Won Ho Kim, Division of Gastroenterology, Department of Internal Medicine, Yonsei University College of Medicine, 250 Seongsanno, Seodaemun-gu, Seoul 120-752, Korea; kimwonho{at}yuhs.ac

Abstract

Background and Aims To identify the associations of genetic and epigenetic variations in IL-23R and IL-17A with inflammatory bowel diseases (IBD).

Methods The promoter and exon regions of IL-23R and IL-17A were analysed in 727 subjects (201 Crohn's disease, 268 ulcerative colitis and 258 healthy controls) using DNA sequencing and denaturing high performance liquid chromatography. Transcription factor binding affinity, IL-17A mRNA expression and methylation of the IL-17A promoter were evaluated in peripheral blood mononuclear cells (PBMC) and Jurkat cells.

Results A case–control analysis showed that development of Crohn's disease is associated with the IL-23R variant G149R (OR 0.32, 95% CI 0.15 to 0.68) and IL-17A variant IVS1+18G>C (OR 10.65, 95% CI 1.32 to 85.89). Ulcerative colitis patients showed an association with IL-23R variants G149R (OR 0.41, 95% CI 0.21 to 0.76), IVS4+17C>T (OR 2.89, 95% CI 1.20 to 6.96) and Q3H (OR 0.61, 95% CI 0.38 to 0.99), and IL-17A variants −737C>T (OR 1.50, 95% CI 1.06 to 2.13), −197G>A (OR 0.63, 95% CI 0.40 to 0.97) and IVS1+18 G>C (OR 8.93, 95% CI 1.12 to 70.99). The −877G, −737T and −444A risk alleles of IL-17A displayed higher binding affinities with the transcription factor complex and higher expression levels of IL-17A transcripts. DNA hypomethylation of the IL-17A promoter was observed in PBMC from IBD patients with a significant inverse correlation between methylation extent of IVS1+17 and IL-17A mRNA level. Finally, Jurkat cells recovered IL-17A mRNA expression after exposure to demethylating agent.

Conclusions The results provide insights into the genetic and epigenetic interactions in the IL-23R/IL-17 axis that are associated with elevated expression of IL-17 and IBD pathogenesis.

  • Genetic polymorphism
  • IL-17A
  • IL-23R
  • inflammatory bowel disease

https://doi.org/10.1136/gut.2011.238477

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    Significance of this study

    What is already known about this subject?

    • The IL-23/IL-17 axis plays an important pathological role in the IBD development.

    • A strong association of functional variants in the IL-23R gene and IBD was found in Caucasians.

    • There are a few reports on the associations of IL-17A gene polymorphisms with several autoimmune diseases.

    What are the new findings?

    • New functional variations of the IL-23R and IL-17A genes were associated with susceptibility to IBD development in Koreans.

    • The hypermethylation status of the IL-17A promoter in PBMC of healthy controls was different from that of IBD patients.

    • Functional consequences of the IL-17A variants have allele-specific effects on the gene expression in PBMC by modifying their affinities for transcription factor complexes or CpG methylation profiles.

    • IL-17A expression in Jurkat cells was markedly restored by 5-Aza-dC, a DNA methyltransferase inhibitor.

    How might it impact on clinical practice in the foreseeable future?

    • Polymorphisms of both the IL-23R and IL-17A gene are fundamentally associated with the aetiology of IBD.

    • New mechanistic insights into the IL-23/IL-17 axis demonstrated that genetic and epigenetic interactions in the IL-17A gene regulation are the basis for IBD pathophysiology, which could highlight a potential target for the treatment of IBD.

    Inflammatory bowel disease (IBD), which includes mainly ulcerative colitis (UC) and Crohn's disease (CD), is characterised by chronic relapsing intestinal inflammation of unknown aetiology.1 Currently, IBD is considered to be caused by complex interactions of genetic, environmental and other processes involving immunoregulatory factors.2 Of these, studies in twins and family members suggest that genetic factors play a principal role in the pathogenesis of IBD.3–5 Since the caspase-recruitment domain 15 gene (NOD2) was identified as the first CD susceptibility gene in 2001, genome-wide association studies or linkage analyses have uncovered several susceptibility genes related to IBD3–14 and undertaken several meta-analyses of genome-wide association scanning studies to date.8 15 16 However, the genetic polymorphisms located within these genes do not fully explain the pathogenesis of IBD or the variations in disease phenotypes. The precise genetic pathogeneses of IBD still remain poorly understood.

    Recently, a subset of T-helper 17 (Th17) cells characterised by interleukin (IL) 17A (or further referred to as IL-17) production was implicated as a critical mediator of autoimmune diseases such as experimental autoimmune encephalomyelitis, rheumatoid arthritis and IBD.17 18 As for IBD, an accumulating body of evidence has demonstrated that IL-17 plays an important pathological role in the development of the disease.12 13 IL-23 is essential for maintaining the Th17 response and is characteristically associated with Th17 cell lineage differentiation.19 20 Moreover, IL-23 receptors (IL-23R) are expressed on a variety of cells and may directly activate a subset of macrophages, natural killer cells, monocytes and dendritic cells that secrete IL-17.21 22 Interestingly, although functional variants in the IL-23R gene were identified as susceptibility loci in Caucasian IBD patients, these variants have not been detected in Asian individuals.9 23–26 These results reveal that there are distinct ethnic differences in the genetic background of IBD between Asian and Caucasian individuals. Therefore, it is necessary to identify new functional variations in the coding regions of IL-23R in Asian patients with IBD. Moreover, while there are a few reports on the associations of the IL-17A gene polymorphisms with several autoimmune diseases such as RA and bronchial asthma,27 28 it is still unknown whether IL-17A single-nucleotide polymorphisms (SNP) are associated with IBD susceptibility and, if so, how exactly these IL-17A SNP modulate IBD susceptibility.

    In this study, we assessed the roles of IL-23R and IL-17A SNP in the genetic susceptibility to IBD in the Korean population. We also investigated the functional consequences of genetic and epigenetic factors that influence the susceptibility to IBD.

    Materials and methods

    Study subjects

    We surveyed a total of 727 unrelated Korean participants: 201 patients with CD, 268 with UC and 258 healthy controls. All patients were diagnosed and managed at the gastroenterology clinics of Yonsei University College of Medicine, Severance Hospital in Seoul, Korea, between June 2006 and February 2008. The demographic and clinical characteristics of the patients with CD and UC are summarised in supplementary table 1, available online only. Healthy subjects had no gastrointestinal symptoms, took no regular medications and had a normal complete blood count and a normal biochemical profile, including erythrocyte sedimentation rate and C-reactive protein. All control subjects underwent colonoscopy for routine check-up, which revealed grossly normal findings.

    This study was conducted according to the ethical guidelines of the Declaration of Helsinki and was approved by the institutional review board of Yonsei University College of Medicine. Written informed consent was obtained from all participants or their guardians.

    DNA extraction and PCR

    Genomic DNA was isolated from whole blood samples from each subject using commercially available kits (Qiagen, Hilden, Germany). Specific primers were designed to amplify the promoter and exons spanning splice junctions (see supplementary table 2, available online only). Genotyping was performed using PCR (i-StarmaxTM II DNA Taq Polymerase; Intron Biotechnology, Seoul, Korea).

    Genotyping

    All subjects were genotyped using full DNA sequencing for promoter regions and denaturing high performance liquid chromatography (DHPLC) for all exons and splice junctions (see supplementary figure 1, available online only). The shapes of the DHPLC elution peaks compared with sequence-confirmed wild-type controls were used as references (see supplementary figure 2, available online only).

    PBMC isolation and cell culture

    Peripheral blood mononuclear cells (PBMC) were isolated using Ficoll-paque plus density gradient centrifugation (GE Health Care, Piscataway, New Jersey, USA) from healthy donors and IBD patients. Jurkat cells (American Type Culture Collection, Manassas, Virginia, USA) or PBMC were then activated with anti-CD3 monoclonal antibody (eBioscience, San Diego, California, USA) and anti-CD28 monoclonal antibody (eBioscience) for 48 h and were stimulated with transforming growth factor beta (R&D Systems, Minneapolis, Minnesota, USA), interleukin (IL) 6 (R&D Systems), IL-23 (R&D Systems), or with phorbol 12-myristate 13-acetate (Sigma-Aldrich, St Louis, Missouri, USA) and ionomycin (Sigma-Aldrich) for 12 h.

    Electrophoretic mobility shift assay

    Nuclear and cytoplasmic extracts were prepared as described previously.29 Electrophoretic mobility shift assay (EMSA) was performed using a Lightshift chemiluminiscent EMSA kit (Pierce, Rockford, Illinois, USA) following the manufacturer's protocol. Complementary oligonucleotides (see supplementary table 3, available online only) were biotin-labelled separately using the biotin end-labelling kit (Pierce) and annealed before use. RAR-related orphan receptor C (RORC) antibody (Abcam, Cambridge, Massachusetts, USA) was used for the supershift assay.

    Quantitative real-time reverse-transcriptase PCR

    Quantitative real-time reverse-transcriptase (RT)–PCR was performed using primers described in supplementary table 3 (available online only). Quantitative analysis was performed using the relative standard curve method, and the results were reported as the relative expression or fold change compared with that of the calibrator after normalisation of the transcript level against the control, namely GAPDH, or RORC.

    Determination of DNA methylation using bisulfate sequencing and pyrosequencing

    To determine the methylation status of CpG sequences in the IL-17A gene promoter, bisulfite modification was performed using an Epitect Bisulfite Kit (Qiagen) according to the manufacturer's instructions. The promoter region of the IL-17A gene from PBMC was PCR amplified with primers (see supplementary table 4, available online only), which were designed using a PSQ assay design program. Bisulfite sequencing analyses were performed as described previously,30 and the pyrosequencing reactions were performed on a PyroMark Q24 system (Qiagen) following the manufacturer's instructions. The subsequent data were analysed using methylation-analysis software.

    Statistical analysis

    All SNP investigated in this study were tested for Hardy–Weinberg equilibrium in controls, and associations of SNP with the disease susceptibility were determined by comparing allele and genotype frequencies between cases and controls using the χ2 test. Logistic regression analyses were performed to calculate the OR, 95% CI and corresponding p values of each SNP under four alternative models (additive, codominant, dominant and recessive). For multiple testing, the false discovery rate method was applied.31

    Gene–gene interactions were tested using an interaction test in logistic regression models.32 For all calculations, SPSS V.12.0 was used. A two-tailed test was used for all analyses, and two-sided p values of less than 0.05 were considered significant.

    Results

    Association of IL-23R polymorphisms with IBD susceptibility

    According to DHPLC and DNA sequencing of the exon regions, including exon/intron junctions of IL-23R to uncover functional variations, we found seven SNP in Hardy–Weinberg equilibrium (p>0.05), of which five (intron 1 IVS1-26G>C; exon 4 V134D, K150E, K160L; intron 4 IVS4+17C>T) had never been reported (Table 1 and supplementary table 5, available online only). CD showed a significant association with one marker in exon 4, G149R (p=0.002, 95% CI 0.15 to 0.68 in codominant model) and UC with three markers, rs1884444 (named ‘Q3H’ hereafter) in exon 2 (p=0.045, 95% CI 0.38 to 0.99 in recessive), G149R (p=0.004, 95% CI 0.21 to 0.76 in additive and dominant), and IVS4+17C>T (p=0.012, 95% CI 1.20 to 6.96 in codominant). G149R and Q3H showed a protective association, and IVS4+17C>T was responsible for the disease risk. The SNP rs11209026, which was reported to be responsible for CD in Caucasians, appeared to be absent in the Korean population.

    View this table:
    Table 1

    Distributions of genotype and allele frequencies of IL-23R SNP

    Associations of IL-17A polymorphisms with IBD susceptibility

    Based on DNA sequencing and DHPLC of the promoter and exon regions of IL-17A, we found 14 SNP from the promoter region, and seven of them (−861G>A, −851C>G, −759C>G, −604delT, −265G>A, −168C>G, and −112G>A) had never been reported (Table 2 and supplementary table 6, available online only). A χ2 test of homogeneity with regard to allelic distributions between cases and controls showed significant associations with CD for one marker, rs3819025 (‘IVS1+18’; p=0.009, 95% CI 1.23 to 80.56 in codominant model; p=0.004, 95% CI 1.32 to 85.89 in recessive), and with UC for three markers, rs8193036 (‘737’; p=0.033, 95% CI 1.02 to 1.74 in additive; p=0.020, 95% CI 1.06 to 2.13 in dominant), rs2275913 (‘−197’; p=0.036, 95% CI 0.40 to 0.97 in recessive), and IVS1+18G>A (p=0.027, 95% CI 1.13 to 72.21 in codominant; p=0.007, 95% CI 1.12 to 70.99 in recessive).

    View this table:
    Table 2

    Distributions of genotype and allele frequencies of IL-17A SNP

    Haplotypes and gene–gene interaction and risk of IBD

    The linkage disequilibriums (LD) of the analysed SNP were calculated and plotted (figure 1A). A single LD block between the G149R and IVS4+17C>T was identified at IL23-R. In addition, we calculated the haplotype frequencies and found both protective (H1) and risk (H2) haplotypes. The H2 haplotype showed a significant association with CD (p=0.005) and UC (p=0.005) (see supplementary table 7, available online only). One LD block among −197, rs8193037 (‘−121’) and IVS1+18 was identified at IL-17A, but any correlation between haplotype patterns and disease susceptibility to CD or UC was not observed (data not shown).

    Figure 1
    Figure 1

    Genetic analysis of IL-23R and IL-17A in inflammatory bowel disease (IBD). (A) Linkage disequilibrium (LD) and haplotype block structures of IL-23R and IL-17A for the combined case–control. The single-nucleotide polymorphism (SNP) with D′ values were ordered according to their position in the gene. The direction of transcription is shown above, and internal references were used for polymorphisms not present in dbSNP. A single LD block between the G149R and IVS4+17C>T (D′=1.0, r2=0.310) was identified at IL23-R. One LD block was identified in IL-17A; −197 showed an association only with ulcerative colitis (UC), and it was located on the same strong LD block with rs8193037 (‘−121’) and IVS1+18 (D′=0.864, r2=0.090 between −197 and −121; D′=0.895, r2=0.142 between −197 and IVS1+18; D′=1.0, r2=0.033 between −121 and IVS1+18). (B) OR for UC with increasing protective (G149R or Q3H) or risk (IVS1+18A, −877G, or −737T) alleles in IL-23R or IL-17A, respectively. The x-axis shows the accumulations of corresponding alleles in order of frequency. (C) OR for UC with protective alleles from IL-23R with and without risk alleles from IL-17A. A dominant model was applied to (B) and (C), and the IL-23R gene was considered dominant.

    Significant increases of UC risk were found in a dose-dependent manner with decreasing protective or increasing risk alleles (figure 1B). Consequently, cumulative risk alleles of IL-17A diminished the protective OR of IL-23R in IBD patients, demonstrating an additive risk of UC along with an increase in the number of risk alleles in IL-17A (figure 1C).

    As further evidence of the synergistic effects of the genes in this pathway in the development of IBD, a significant statistical gene–gene interaction between Q3H in IL-23R and IVS1+18G>A in IL-17A was observed in UC patients (p=0.003 in codominant model; p=0.003 in dominant model; Table 3) but not CD patients (see supplementary table 8, available online only). These data suggest that there is a gene dosage effect on the IL23R risk associations of UC patients with other genetic variants within IL-17A.

    View this table:
    Table 3

    Synergistic interactions between IL-23R and IL-17A genotypes in UC

    Moreover, the genotype–phenotype association analysis was conducted (see supplementary tables 9, 10 and 11, available online only). A significant correlation between the Q3H genotype and ileal involvement (p=0.029, 95% CI 0.17 to 0.94 in codominant model; p=0.003, 95% CI 0.01 to 0.58 in recessive model) was observed in CD patients after adjustment for all other covariates.

    Allele-specific binding activities of nuclear proteins and their effects on gene expression in the IL-17A promoter region

    Gene expression is tightly regulated by a complex of transcriptional activators or inhibitors with transcriptional machinery, and may be influenced by SNP. We thus performed EMSA to investigate whether the allelic differences between the risk and the non-risk alleles of IL-17A are attributable to their binding with nuclear proteins using activated Jurkat cells. As shown in figure 2A, a higher level of protein binding to the risk alleles than to the non-risk alleles was detected, supporting the roles of the risk alleles in IL-17A gene expression. However, we found no binding to the −197 site at any concentration of the nuclear extract. Moreover, we investigated binding of RORC (human orthologue of RORγt), a master regulator of IL-17,33 with a nuclear protein complex. Antibodies against RORC supershifted the bands of the DNA–protein complex, implicating the binding of RORC to the complex (figure 2B).

    Figure 2
    Figure 2

    The preferential binding of transcription factor (TF) complex to the risk alleles and its correlations with the expression of IL-17A. Binding profiles of TF complex to the IL-17A promoter with a major or minor allele. (A) Electrophoretic mobility shift assay showing binding profiles of nuclear proteins to major and minor allele probes. Risk alleles increase TF binding affinity in the promoter of IL-17A binding in activated Jurkat cells that were incubated for 6 h in media with IL-23 (10 ng/μl), transforming growth factor beta (TGF-β; 10 ng/μl), and IL-6 (10 ng/μl) after activation with CD3 (1 mg/ml) and CD28 (1 μg/ml) for 48 h. *Risk alleles. (B) Competition experiments and a supershift assay of the probes were performed using a cold probe consensus sequence and antibodies against RAR-related orphan receptor C (RORC), respectively. The bands were competed against a 200-fold molar excess of unlabelled probe, which clearly demonstrates the specific binding of probes to the nuclear protein. The results of one representative experiment (out of five that produced identical results) are shown. (C) IL-17A mRNA expression in peripheral blood mononuclear cells (PBMC) from inflammatory bowel disease (IBD) patients was measured using real-time reverse transcriptase PCR and is expressed as normalised units according to RORC mRNA level. Controls had undetectable levels of IL-17A. Dots represent triplicate assays, and the bars indicate the mean expression level of each specimen group. p Values were obtained by t-test analysis.

    Although some genetic polymorphisms have been partly characterised, no biological explanation has been provided for the observed associations of IL-17A genotypes with diseases. Expression levels of IL-17A messenger RNA were elevated in the colonic mucosa of IBD patients (see supplementary figure 3, available online only).34 Moreover, high IL-17A mRNA expression in PBMC was observed in IBD patients.34 Therefore, we further evaluated the in-vivo mRNA expression levels according to IL-17A variants by comparing the expression patterns of IL-17A in PBMC of normal and IBD patients using quantitative real-time RT–PCR. To understand how risk alleles might influence IBD predisposition, we evaluated the effects of variants at −877, −737, −444 and −197 on IL-17A mRNA expression levels. Although a large variability was found among individuals, the expressions of IL-17A transcripts were generally higher in patients with the risk alleles, variant −737T (p=0.004) or −444A (p=0.016), compared with those with no risk alleles (figure 2C).

    Associations of aberrant hypomethylation status of IVS1+18 in IL-17A and its implication in IBD

    Sequencing results revealed that a cluster of CpG sites were located near the translation start codon that includes a considerable number of regulatory sites (data not shown). However, the DNA methylation status of the exact CpG sites of IL-17A remains unknown. Therefore, to identify the difference in methylation status of the CpG sites between control and IBD patients and to confirm the likelihood of the IVS1+18G/A-mediated regulation of IL-17A, we performed bisulfite sequencing of 12 CpG sites in the IL-17A locus (−969 to −684 bp and −234 to IVS1+21), including the variations. Interestingly, we found that two CpG sites (IVS1+17 and IVS1+21) at −234 of the IVS1+21 region were significantly hypermethylated in healthy controls and hypomethylated in IBD patients, based on the results from both bisulfite DNA sequencing (figure 3A and supplementary figure 4A, available online only) and pyrosequencing (figure 3B and supplementary figure 4B, available online only). In particular, IVS1+17 was significantly less methylated in CD patients (2.9%; n=20) and UC patients (4.3%; n=21) than it was in controls (44%; n=21) (figure 3B). IVS1+17 was strongly correlated with IVS1+18G>A conversion in IBD patients but not significantly correlated in controls (figure 3C). We also investigated whether IVS1+18G>A conversion is correlated with IL-17A expression in PBMC of IBD. A significant inverse correlation was observed between the IL-17A mRNA levels and IVS1+18G>A conversion in IBD patients (figure 3D). Based on these results, we propose that IL-17A is downregulated via hypermethylation of CpG sites in the promoter, which might be critical for transcriptional silencing of IL-17A. We also examined the relationships between the methylation status and phenotypes of CD and UC, including age at diagnosis, total follow-up duration, disease location and behaviour, use of immunosuppressive drugs and the need for surgery. However, the methylation levels at IVS1+21 and IVS1+17 showed no significant association with the disease phenotypes (see supplementary table 12, available online only).

    Figure 3
    Figure 3

    Promoter methylation status of the IL-17A promoter and downregulation of IL-17A expression via DNA methylation. (A–D) Comparison of promoter CpG site methylation status in peripheral blood mononuclear cells (PBMC) from healthy controls and inflammatory bowel disease (IBD) patients. (A) Methylation status was analysed using bisulfite sequencing. Black, grey and white squares represent complete methylation, partial methylation and no methylation, respectively. Position +1 is determined by the start codon. (B) Methylation status was analysed using pyrosequencing. (C) Correlation between methylation status at IVS1+17C and allele types at IVS1+18 in IBD patients and healthy controls. (D) Allele types at IVS1+18 and IL-17A mRNA expression in IBD. (E) IL-17A is downregulated via DNA methylation in Jurkat cell lines. Jurkat cells were incubated for 12 h in media with IL-23/TGF-β/IL-6, or PMA (20 nM) and ionomycin (2 μM), or 5-Aza-dC (400 nM) for 72–96 h after activation with CD3 and CD28 for 48 h. IL-17A mRNA levels were measured using quantitative real-time reverse transcriptase PCR, in which RAR-related orphan receptor C (RORC) was used as the normalisation control. Error bars indicate means±SE.

    Association of IL-17A silencing with DNA methylation in Jurkat cells

    To determine the association between IL-17A induction and aberrant DNA methylation, we tested the effects of a demethylating agent 5-azadeoxycytidine (5-Aza-dC) on IL-17A expression in Jurkat cells, which initially displayed no IL-17A expression. IL-17A transcription was restored near a stimulated level after the cells were treated with 5-Aza-dC (figure 3E). Similar to the methylation patterns in clinical samples of PBMC, CpG sites at −969 to −684 bp were found to be extensively methylated in Jurkat cells. However, unexpectedly, CpG sites at −234 to IVS1+21 located at the transcriptional start site were unmethylated in 5-Aza-dC-treated and untreated samples, consistent with the increased IL-17A expression status in 5-Aza-dC-treated Jurkat cells and in contrast to that in PBMC of healthy controls (see supplementary figure 5, available online only). Although we cannot fully explain the direct effects of IVS1+18G>A conversion on IL-17A, these results imply that more complicated chromatin conformation changes or other CpG sites may be involved in the gene regulation via DNA methylation. The results also indicate that DNA methylation is a critical mechanism through which IL-17A expression is regulated.

    Discussion

    The main objective of our study was to assess how IL-23R and IL-17A genetic polymorphisms contribute to the susceptibility to IBD and to elucidate the underlying mechanisms. A growing body of evidence demonstrates that activation of the IL-23/IL-17 axis is fundamentally linked to the pathogenesis of autoimmune diseases including IBD.35–37 In this regard, recent studies in animal models and humans identified that Th17 cells promote intestinal inflammation38 and that IL-17 exerts multiple proinflammatory effects.39 40 IL-17 may be the key molecule to explain the mechanism through which common features of IBD are implicated in the autoimmunity that develops via different T-cell-dependent and independent aetiologies. Meanwhile, IL-23 is expressed on activated myeloid cells, and a subset of T cells that are the most potent inducers of IL-17 and terminal differentiator of Th17 cells and IL-23R serve as an initial sensor of IL-23 and as an important gate for the Th17 cell-mediated autoimmune responses.18 41

    Although the exact mechanisms by which IL-23R modulates IBD susceptibility are still unclear, the key role of IL-23 in IBD has been demonstrated by recent genetic studies in which genetic variants in the IL-23R gene were associated with IBD.9 23–26 Although the functional variants of IL-23R such as rs11209026 were not identified in Asian individuals, including Japanese and Korean populations, intronic SNP at intron 5 and at an intergenic region of IL-23R were recently found to be associated with CD in these populations.23 42 These results suggest that there are ethnic differences in IBD, and that entire exons of IL-23R need to be investigated in the Asian population. Therefore, we identified new functional SNP of IL-23R in the Korean population through whole exon and junction scanning instead of using common tag SNP. Although rs11209026 was not polymorphic in either IBD patients or healthy controls in this study, which was different from Caucasians, we found that the two novel SNP G149R (protective in CD and UC) and IVS4+17C>T (risk in UC) located in exon 4 showed significant associations with IBD development. It is interesting that the protective arginine 149 allele was present in approximately 13% of controls, 4% of CD patients and 15% of UC patients in our study, whereas the protective glutamine 381 allele is known to be present in approximately 7% of controls, approximately 2–4% of CD cases and approximately 4% of UC in Caucasians.9 43 However, the OR for IBD in Asians was similar to that in Caucasians. Q3H in exon 2 of IL-23R also showed a protective effect against UC. Furthermore, pairwise analysis suggests that there is a genetic interaction between Q3H of IL-23R and IVS1+18G>A of IL-17A. Functional variants, G149R and Q3H, may affect the structure and the biological function or stability of IL-23R, and nucleotide alteration in intron 4 (IVS4+17C>T) may alter the stability of mRNA. These findings support the idea that IL-23R is associated with IBD in both Caucasian and Asian populations even though there are ethnic differences in specific SNP affecting the IBD risk. Further investigations are required to validate our results.

    IL-17A is located on 6p12.1, a genomic region containing a putative susceptibility loci (IBD3) for IBD.44 Moreover, a few studies have recently reported the associations between the −197 in the IL-17A promoter and the UC phenotype45 and between that at −737 and paediatric asthma in Taiwanese children.35 However, it has not yet been clarified whether IL-17A polymorphisms truly affect the risk of IBD development or how these polymorphisms influence the activity and expression of IL-17A. Our results are the first findings to explain the mechanism underlying the disease development that is associated with the variants of −737 and IVS1+18 in addition to variant −197. Allele −737T was significantly associated with a higher level of IL-17A mRNA expression in IBD patients and displayed a higher activity to transcription factor complexes as a kind of regulatory mechanism. Although the alleles at −444 and −877 showed no significant association with IBD, they may also contribute to IBD susceptibility in terms of binding affinity to the transcription factor complex. RORC was recently identified as a master regulator of Th17 cytokine production.36However, transcription factors such as STAT3, RORα and IRF4 promote Th17 cytokine production,37–39 46 while Foxp3, Ets1, Gfi1, T-bet and Smad3 negatively regulate Th17.40 41 Nevertheless, the mechanisms underlying the roles of these factors in Th17 differentiation are not entirely clear, and more studies are required to understand the complex regulation system of IL-17A expression. Of note, the variant at −197 was previously the only identified SNP explaining a possible association with autoimmune diseases in IBD; however, it did not show any affinity for transcription factor complexes or methylation status and was located in LD with IVS1+18, an important position that affected the methylation status in our study. These results suggest that −197 may simply be a ‘linked SNP’ rather than an SNP with its own functional consequences.

    DNA hypermethylation is strongly associated with heterochromatin and transcriptional silencing, and hypomethylation at CpG sites in the promoter region is a well-defined epigenetic phenomenon generally associated with active gene expression.47 Differential methylation of DNA has been reported for T cells at different stages of cell differentiation. Moreover, Th17 cells show distinct chromatin remodelling of the IL-17A gene locus, consistent with the production of IL-17,48 which is not stable and is reversible through linkage with RORC.49 50 These epigenetic modifications undoubtedly serve as an important regulatory mechanism during maintenance of the lineage commitment in T cells. In our study, CpG sites of the proximal promoter regions of IL-17A in the controls were dramatically hypermethylated with a lower transcript level of IL-17A compared with that of IBD patients. In particular, we identified a significant polymorphism in the promoter region of IL-17A, IVS1+18G>A. As expected, changing a G nucleotide to an A at this site resulted in no DNA methylation and induced aberrant hypomethylation of cytosine residues. In addition, the −121G>A in the same block with −197 showed a change in methylation status at CpG, and recently it was found to be polymorphic in other diseases,27 45 which may also influence transcription through a similar mechanism. Interestingly, IVS1+17C, including −122C, is colocalised with 5′ untranslated regions of the IL-17A gene that are intensively marked by permissive modifications, histone H3 lysine residue 4 trimethylation (H3K4me3) in Th17 cells, but extensively marked by repressive H3K27me3 modification in other T-cell lineages.48 49 These data suggest that the irreversible hypomethylation of cytosine residues by IVS1+18G>A or −121G>A may create a far more highly ‘poised’ state than normal for sustained expression of IL-17A through the H3K4me3 mark, which loosens the chromatin and recruits transcription factors. IVS1+18G>A, including −121G>A, could stably and irreversibly block a binding site for methyl CpG binding protein that maintains the epigenetic silencing of transcriptional activity. These changes may also create potential transcription factor binding sites associated with the phenotypic or developmental plasticity of Th17 cells.49 50 Furthermore, chromatin remodelling of IL-17-IL-17F cytokine gene locus is specifically associated with inflammatory helper T-cell lineage differentiation.50 The methylation status altered by genetic changes may sustain the transcriptional competence of IL-17A in Th17 cells or induce lineage transitions to other IL-17 secreting cells. Moreover, the IL-17A expression was markedly restored by 5-Aza-dC in Jurkat cells that initially showed no expression of IL-17A. To the best of our knowledge, this is the first demonstration that a change in DNA methylation of the IL-17A promoter may play a critical role in IL-17A expression and pathogenesis of IBD. This novel mechanism could be a potential target for modifying inflammatory cell differentiation, including Th17 cells. However, contrary to our expectations, the nucleotide at position IVS1+18 in Jurkat cells was a cytosine, which suggests the involvement of an epigenetic modification of RORC or an unknown upstream repression site including a conserved non-coding sequence or changes in higher-order chromatin structure.49–51 Taken together, our results indicate that CpG dinucleotides and methylation in this region play an important role in the regulation of IL-17A transcription, although further research is needed to elucidate the exact mechanism fully. Such aberrant methylation may result in increased sensitivity in IBD patients through more sensitive IL-23R, which sends out more intensive signals. Methylation may also be involved in intense transcriptional activation by modification of transcription factor binding sites with unknown cis elements. Genetic changes in multiple sites of the promoter can lead to physiological changes including epigenetic changes and may result in stringent or relaxed regulation of transcription (figure 4), which influences predisposition to IBD. Our findings show for the first time that IL-17 expression is regulated by DNA methylation, which suggests novel mechanisms through which epigenetic change is coordinated with cis regulation in IBD.

    Figure 4
    Figure 4

    Proposed model depicting the interplay of the polymorphic sites in the IL-23R and IL-17A genes in response to IL-23-mediated signals. Hypomethylation of the IL-17A promoter in inflammatory bowel disease (IBD) patients compared with that in controls leads to sustained signalling (1). In healthy controls, the protective allele of IL-23R is less sensitive to IL-23, but the IL-23R risk allele in IBD patients is more sensitive (2). The strong affinity of IL-17A risk alleles to the transcription factor complexes results in higher transcriptional activity of the promoter, which strengthens and retains the signalling in IBD patients compared to that in healthy control subjects (3).

    In summary, we performed a case–control association study and identified new variants of IL-23R and IL-17A that are associated with the susceptibility to IBD development. Moreover, we characterised the functional consequences of the IL-17A variants with allele-specific effects on the gene expression in PBMC and effects on DNA methylation profiles. We also found that the hypomethylation status of IL-17A in PBMC of IBD patients is significantly different from that of healthy controls. Our findings suggest that the polymorphisms of both IL-23R and IL-17 genes affect IL-17A gene expression and are fundamentally associated with the aetiology of IBD. These findings provide new mechanistic insights into the IL-23/IL-17 axis by demonstrating that genetic and epigenetic interactions in the IL-17A gene regulation are the basis for the high IL-17 expression in IBD patients, which could highlight a potential target for the treatment of IBD, although additional, larger multicentre replicated studies are required to confirm our findings.

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    Supplementary materials

    Footnotes

    • See Commentary, p 1447

    • This paper was presented at the UEGW 2010 in Barcelona, Spain.

    • Funding This work was supported by a grant from the Korea Research Foundation under the basic research promotion fund of the Ministry of Education and Health Resources Development of Korea (KRF-2008-331-E00105).

    • Competing interests None.

    • Patient consent Obtained.

    • Ethics approval This study was conducted with the approval of the institutional review board of the Severance Hospital in Korea.

    • Provenance and peer review Not commissioned; externally peer reviewed.

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