Introduction

Inflammatory bowel disease (IBD) is characterized by chronic relapsing inflammation of the gastrointestinal tract, and includes two major phenotypes: Crohn’s disease (CD) and ulcerative colitis (UC). IBD is currently presumed to result from the complex interaction of diverse genes conferring risk of disease and environmental factors [1]. Some genes are associated with UC or CD itself, whereas others increase the risk of IBD or are associated with disease phenotypes [2, 3]. Recently, the important role of the innate immune response in the pathogenesis of IBD has been reported [4], and the cytokine profile provides evidence of a high level of pro-inflammatory cytokine interleukin-17 (IL-17) in IBD [5].

IL-17 is a newly described cytokine that bridges the adaptive and innate immune systems. IL-17 not only is involved in neutrophil proliferation, maturation, and chemotaxis but also stimulates T cells and enhances the maturation of dendritic cells [6]. T-helper-17 cells (Th17) cells preferentially produce IL-17A, IL-17F, IL-21, and IL-22 [7], and of these, IL-17A and IL-17F are the two main effective factors. Recent studies revealed that IL-17A plays important roles in the development of allergic and autoimmune diseases as well as protective mechanisms against bacterial and fungal infections [4, 8]. IL-17R-deficient mice were highly susceptible to Klebsiella pneumoniae infection because of a significant reduction in chemokine production and a consequent significant delay in neutrophil recruitment into the alveolar space [9, 10]. In contrast, an overproduction of IL-17 in lungs led to chemokine expression and tissue inflammation caused by the infiltration of large numbers of leukocytes [10]. Although IL-17 is generated in the acquired immune response, it can be induced by innate immune mediators such as IL-6 and granulocyte colony-stimulating factor (G-CSF) and can play a role in innate immunity [11]. These results indicate that IL-17 has pleiotropic activities, and plays an important role in immune responses.

With the advent of genome-wide association studies, complex disease genetics has progressed in recent years. Twin studies and large-scale population studies have confirmed an increased sibling risk for both CD and UC. Unlike single gene disorders, IBD is thought to result from a complex interplay of multiple genes and environmental factors [12]. Because of the important role of IL-17 in the immune response, there are a few reports on the associations of IL-17A gene polymorphisms with several autoimmune diseases such as rheumatoid arthritis (RA) and bronchial asthma.

Arisawa et al. [13] found that the IL17A (rs2275913, G-197A) and IL17F (rs763780, 7488T/C) alleles were both significantly associated with increased risk for the development of UC in Japanese patients. The study from Chen et al. [14] showed that the IL17F (rs763780, 7488T/C) polymorphism was weakly associated with UC protection. The clinical phenotypes of UC were also affected by this polymorphism. However, there are few reports of the possible association of IL17A/F gene polymorphisms with IBD in Asia, especially in the Chinese Han population. Here we investigated the association between IBD and polymorphisms of IL17A and IL17F genes in the Chinese Han population.

Materials and methods

Study population

A total of 352 patients with IBD (270 UC and 82 CD) and 268 healthy individuals were recruited from four different hospitals (the First Affiliated Hospital of Nanjing Medical University, the First People’s Hospital of Nantong, the Fourth People’s Hospital of Wuxi and the First Hospital of Huaian) between 2008 and 2011. The diagnoses of UC and CD were based on standard clinical, endoscopic, radiological, and histological criteria [15]. The UC and CD locations were classified according to the Montreal classification [16]. The endoscopic severities of UC and CD were assessed using the Rachmilewitz endoscopic index (REI) score [17] and the Crohn’s Disease Endoscopic Index of Severity (CDEIS) score [18], respectively. Healthy controls consisted of randomly selected and ethnically matched healthy individuals without tumors, autoimmune diseases, or IBD family history. The main clinical characteristics of the study population are summarized in Table 1.

Table 1 Statistical data and basic characteristics of IBD patients
Full size table

Our study was approved by the ethics committees of all four hospitals (Ethics Committee of the First Affiliated Hospital of Nanjing Medical University, Ethics Committee of the First People’s Hospital of Nantong, Ethics Committee of the Fourth People’s Hospital of Wuxi, and Ethics Committee of the First Hospital of Huaian). Patients signed informed consent and clinical data were collected from the patients’ files and from questionnaires.

Genomic DNA extraction

Blood samples (2 ml) from the patients with IBD and the controls were collected in tubes containing EDTA. Within 4 h of blood collection, blood cells and plasma were separated by centrifugation at 4 °C and 3,000 rpm. Genomic DNA was extracted using the RelaxGene Blood DNA System (TianGen Biotech, Beijing, China).

Genotyping analysis

Eleven single nucleotide polymorphisms (SNPs) were genotyped by ligase detection reaction allelic (LDR) technology (Shanghai ZJ Bio-Tech Co., Ltd, China). The PCR was carried out in a total volume of 15 μl that included 1 μl of genomic DNA, 1.5 μl 10× buffer, 1.5 μl MgCl2, 0.3 μl dNTP (10 mM), 0.5 μl primer and water. The DNA was denatured at 94 °C for 2 min, followed by 35 cycles of 94 °C for 20 s, 56 °C for 20 s, and 72 °C for 40 s, with the final extension at 72 °C for 3 min. Thereafter, 3 μl of the PCR product was mixed with 1 μl of 10× Taq DNA ligase buffer, 0.125 μl Taq DNA ligase (40 U/μl), and 0.01 μl probe (10p), and finally water was added to give a total volume of 10 μl. Then PCR amplified for 30 cycles of 94 °C for 30 s and 56 °C for 3 min. Next, 1 μl of ligation product was added to 10 μl of sample loading solution (already mixed with Mark), denatured at 95 °C for 3 min, and immediately placed in an ice bath. The denatured single-stranded DNA bands were then detected using an ABI 3730 sequencer (Applied Biosystems, CA, USA).

Statistical analysis

The Hardy–Weinberg test was used to calculate the distribution genotypes of the IL17A and IL17F polymorphisms in the patients and controls. The mean ages of the patients and controls were compared with Student’s t test. Genotype and allele associations were calculated using the SPSS 13.0 software package. Comparisons between the genotype distributions and allele frequencies in patients and controls were evaluated by the Chi-squared (χ2) test. The strength of association between allele frequencies and UC or CD was assessed by calculating the odds ratio (OR) and 95 % confidence intervals (95 % CI) by logistic regression analysis using the number of mutational alleles as a regressor. Haplotype frequencies and linkage disequilibrium (LD) were estimated and visualized by Haploview 4.2 (http://www.broadinstitute.org//haploview/haploview-downloads). SNPs with minor allele frequencies (MAF) below 5 % were excluded. For all the analyses, the level of significance was set at P < 0.05.

Results

Frequency distribution of the IL17A and IL17F genotypes and variant alleles in IBD patients

The variant allele frequencies of the SNPs in IL17A and IL17F are shown in Table 2. The genotype frequencies in IBD patients and controls conformed to the Hardy–Weinberg equilibrium (P > 0.05, data not shown). In our study, compared with the controls, the frequency of mutant allele C for IL17F rs763780 was significant higher in CD patients (14.0 vs 8.4 %, P = 0.033, OR 1.18, 95 % CI 1.41–3.04). A disease risk mutation in the IL17F polymorphism was found to be associated with CD susceptibility. There were only wild alleles on IL17A rs10484879 of all the participants, and IL17F rs2397084 in patients and controls were mostly homozygous wild genotype TT, the mutation rate being less than 0.02.

Table 2 Allele frequency variants in IL17A and IL17F
Full size table

Association between the IL17A and IL17F polymorphisms and IBD phenotypes

Non-conditional logistic regression analysis indicated that the IL17F (rs763780, 7488T/C) polymorphism was associated with lesion location of CD (P = 0.01, OR 5.67, 95 % CI 1.45–12.07) and also had a weak association with age onset of UC (P = 0.05, OR 0.97, 95 % CI 0.94–1.00). After genotype–phenotype analysis, we found that the mutant allele C frequency for rs763780 in the CD patients with ileocolic phenotype was significantly higher than that in the control groups (22.2 vs 7.5 %, P = 0.02, OR 3.60, 95 % CI 1.51–8.60). The frequency of the combined heterozygous C/T and mutant homozygous C/C for rs763780 in CD patients with ileocolic phenotype was also significantly higher (45.0 vs 14.1 %, P = 0.003, OR 4.95, 95 % CI 1.81–13.54). These results indicated that SNP rs763780 may be responsible for the ileocolic phenotype in CD patients by altering the activity of the gene product (Table 3). This may be due to rs763780 (7488T/C) causing a His-to-Arg substitution at amino acid 161 (H161R), and the H161R variant of IL-17F affecting cytokine and chemokine production [19].

Table 3 Associations between rs763780 genotypes and phenotypes of CD
Full size table

We analyzed the age of disease onset between rs763780 homozygous wild-type T/T and the mutant heterozygous C/T using the t test. This revealed that the average age of disease onset in patients with the T/T genotype was greater than that in patients with the C/T genotype (TT, \( \bar{X} \) = 48.48; CT, \( \bar{X} \) = 42.61; P = 0.046). These results indicated that the IL17F (rs763780 C/T) variant was associated with an earlier age of disease onset in UC patients. However, we did not find any significant relationship between these eleven SNPs of IL17A/F and the patients’ gender, smoking habits, or endoscopic severity score by non-conditional logistic regression analysis (P > 0.05).

The genotype–phenotype analysis showed that the occurrence of IL17A (rs2275913, G-197A) was correlated with disease severity in the UC patients. The genotype distribution in moderate to severe UC patients was significantly different from that in the control subjects (P = 0.036, P = 0.023, respectively). The frequency of the combined homozygous A/A and mutant heterozygous A/G for rs2275913 was significantly lower in moderate to severe UC patients compared with the control group (moderate vs controls: 63.4 vs 79.9 %, P = 0.01; severe vs controls: 44.4 vs 79.9 %, P = 0.04). Thus, there may be a negative relationship between this mutation and the severity of UC (Table 4).

Table 4 Associations between rs2275913 genotypes and phenotypes of UC
Full size table

Haplotypes of IL17A and the susceptibility of IBD

We measured the linkage disequilibrium (LD) patterns of the SNPs and identified one LD block of IL17A in UC and one block in CD patients (Fig. 1). Then we investigated whether any specific haplotype would confer a higher risk or protection for UC or CD. Analysis of the haplotype structure revealed four distinct haplotypes in UC and two haplotypes in CD patients (Table 5). The rare haplotype (GGTT) of IL17A formed with four SNPs (rs2275913, rs8193037, rs8193038, and rs3804513) was a risk haplotype, with a frequency of 3.3 % in UC patients and 0.7 % in controls (P = 0.034). However, the same results were not detected in CD patients.

Fig. 1
figure 1

Linkage disequilibrium patterns of the region around the IL17A gene in the Chinese UC (a) and CD (b) patients. D′ values are multiplied by 100, e.g. 77 in the square at the bottom means a D′ of 0.77. Square without a number has a value of 100 that equals to a D′ of 1. Dark color indicates a strong connection

Full size image
Table 5 Frequency of IL17A haplotypes in IBD patients and healthy controls
Full size table

Discussion

The objective of our study was to assess how IL-17A and IL-17F genetic polymorphisms contribute to susceptibility to IBD and the association of IL-17A/17F genetic polymorphism with the clinical characteristics. IL-17 is an important pro-inflammatory cytokine in the pathological injury of IBD [5, 20]. The IL-17 cytokine family is functionally specialized and consists of six structurally related members (IL-17A, B, C, D, E, and F) [21]. IL-17A was cloned first [22] and five additional members were found between 2000 and 2002 [23–25]. All the family members share between 20 and 50 % amino acid identity with IL-17A. Of these, IL-17A and IL-17F share the highest amino acid identity (50 %). In addition, IL-17A is located on 6p12.1, a genomic region containing a putative susceptibility locus (IBD3) for IBD. The IL17F gene is located close to the IL17A gene in the genomes of both human (chromosome 6) and mouse (chromosome 1) [11]. IL-17A and IL-17F can trigger pro-inflammatory responses and promote granulopoiesis and neutrophil mobilization to sites of infection [26, 27].

Th17 cells are named for their ability to preferentially produce IL-17. They mediate host defense against infectious pathogens and contribute to the development of autoimmune diseases including RA, multiple sclerosis, IBD, and psoriasis [28, 29]. The continued increase in IL-17-producing cells in the intestinal tissue of IBD patients has been reported. Over-expression of IL-17A or IL-17F in vivo increased neutrophil infiltration through modulation of cytokines and chemokines, resulting in inflammation [30].

There are a few reports that show the influence of polymorphisms of IL17A and IL17F on the risk of IBD. Of all the SNPs, the IL17F (rs763780, 7488T/C) polymorphism has been the most frequently reported. Arisawa et al. [13] found that the IL17A (rs2275913, G-197A) and IL17F (rs763780, 7488T/C) alleles were both significantly associated with an increased risk of the development of UC. The risk for the wild genotype T/T of the IL17F (rs763780, 7488T/C) polymorphism was significantly higher than for other genotypes, and the frequency of the wild genotype T/T was significantly higher. We found that the frequency of the wild genotype T/T of the IL17F (rs763780, 7488T/C) allele in UC patients was lower than in the control group (78.5 vs 85.8 %), and no association between the IL17A (rs2275913, G-197A) and IL17F (rs763780, 7488T/C) gene polymorphisms and the susceptibility of patients was detected. Seiderer et al. [31] suggested that the IL17F (rs763780, 7488T/C) allele was not associated with IBD susceptibility or with the severity of CD, but UC patients with the heterozygous genotype had a lower body mass index (BMI) and an earlier disease onset. Although our results were not entirely consistent with them, we had the same conclusion on the association with age of onset. Chen et al. [14] observed that the homozygous polymorphic C/C genotype in UC patients was lower than in healthy subjects, suggesting that this genotype may have a protective effect. The patients with the rare allele C were more likely to develop mild CD and mild UC. We determined that the distribution of the homozygous C/C genotype was not significantly different between UC or CD patients and the controls. Our results are different from those of Arisawa et al. [13] and Chen et al. [14]. The reasons for this discrepancy may be due to different races and regions, and sample size; large sample sizes are needed for further research.

Kawaguchi et al. [19] reported that the rare allele C of the IL17F (rs763780, 7488T/C) polymorphism was inversely associated with the development of asthma in a Japanese population. They recombined the wild-type and mutant IL17F proteins, and examined the functional consequences of the gene polymorphism, suggesting that expression and activity of rs763780 may be suppressed in carriers of the rare allele C. Paradowska et al. [32] found that the polymorphisms of the IL17F gene were not correlated with susceptibility to RA in a Polish population. However, the IL17F His161Arg variant was associated with some activity parameters of the disease.

The correlation of the IL17A (rs2275913) polymorphism with the development and course of human disorders, especially with IBD, has rarely been reported. In one study of UC from Japan [13], a significant relationship between IL17A polymorphisms and UC was found and the frequency of the IL17A (rs2275913, G-197A) allele was reported to be significantly correlated with the chronic relapsing and pancolitis phenotypes and to steroid-dependent cases. Recently a report from Kim et al. [33] showed that the IL17A (rs2275913, G-197A) variant was associated with UC susceptibility. Nordang et al. [34] investigated the IL17A gene by tagging the main genetic variation and found association with the IL17A (rs2275913) promoter SNP in Norwegian RA patients. This association was not confirmed in a smaller New Zealand cohort. From our results, we concluded that the IL17A (rs2275913, G-197A) variant had a weak association with disease severity; patients with the mutant allele A tended to suffer milder lesions. This indicates that it may be a protective factor for UC.

The other IL17A and IL17F polymorphisms in our study have mostly been reported to be associated with some other inflammatory and autoimmune diseases. For example, Paradowska et al. [32] studied the relationship between the IL17A (rs2397084, 7383A/G) Glu126Gly variant and the susceptibility to RA in a Polish population. Another study from Jang et al. [35] found that the IL17F (rs2397084, 7383T/C) heterozygote genotype was associated with susceptibility to Behçet’s disease (BD), whereas the homozygous polymorphic genotype (CC) was more dominant in control subjects and had a negative correlation with the development of BD in a Korean population. Wang et al. [36] found that there was a weak correlation between the IL17A (rs8193036) polymorphism and susceptibility to childhood asthma in Taiwan. Furuya et al. [37] found that the IL17A (rs3804513) polymorphism may be associated with radiographic progression in Japanese patients with early RA. Although IBD has some features in common with RA, BD, and multiple sclerosis in pathogenesis, there were no relationships between the SNPs of IL17A/IL17F which occurred in RA and BD patients and IBD in our current study.

From the LD analysis, we found that one haplotype of IL17A was significantly associated with UC in the Chinese Han population. The haplotype GGTT from IL17A was a risk haplotype that increased UC susceptibility. This data is novel and has not been reported before.

Our study has some limitations. First, our sample size was not large enough, which may reduce our ability to detect the association of genes with the factors that affect IBD. Second, our patients and controls were mainly from the Jiangsu Province. Thus, there exists a regional limit.

In conclusion, our current findings indicate that the IL17F rs763780 polymorphisms may influence the susceptibility to CD in the Chinese Han population. The pathophysiological features of UC and CD may be affected by these polymorphisms. There was a risk haplotype in IL17A associated with increased risk of UC, and the IL17A rs2275913 variant may be a protective factor for UC. Further studies involving in the mechanism underlying the IL-17A and IL-17F genes in the pathogenesis of IBD are needed, including functional experiments.