Introduction

Asthma is the most heterogeneous respiratory disease with a growing prevalence in resource-rich and resource-poor countries. It is associated with airway hyper-responsiveness, reversible airway obstruction, and allergen-specific IgE production [1, 2]. Growing body of evidence suggests the role of genetics in etiology of asthma through interactions with environmental factors [3]. More than 100 genes have been associated with asthma in candidate gene studies, positional cloning, or linkage studies [4].

IL-10 was first identified in 1989 as cytokine synthesis inhibitory factor with a down-regulatory effect on inflammatory responses. The IL-10 gene is located on chromosome 1 both in human (1q31-32) and murine genomes and is consisted of 5 exons and 4 introns [5]. It inhibits the production of several cytokines, chemokines, chemokine receptors, and inflammatory enzymes [6]. IL-10 has a protective role in asthma. While, low levels of IL-10 have been associated with asthma [7, 8], overexpression of IL-10 in regulatory T cells can suppress airway hyper-responsiveness [9].

IL-17F is located on chromosome 6p, a region which is associated with asthma in several genome studies. Furthermore, gene expression studies have identified IL-17F as a novel candidate gene in asthma-related studies [10, 11]. The gene-encoding human IL-17F is 7742 bps in length, consisting of three exons and two introns. It is expressed in basophils and activated mast cells, mononuclear cells, T cells clones, and CD4+ cells, a group of cells which are involved in the pathogenesis of asthma [12, 13]. IL-17F is overexpressed in the asthmatic patients airways where the level of overexpression is correlated with the severity of the disease [14]. In asthmatic patients, the overexpression of IL-17F induces airway neutrophilia, pulmonary mucus hyper-secretion, and goblet cell hyperplasia [7].

As cytokine expression is genetically controlled at transcription level, polymorphism in promoter region can affect cytokine expression level by changing the binding site of the transcription factors. As IL-10 −1082A/G polymorphism is located within a negative putative ETS (E26 transformation-specific or E-twenty-six) -like transcription factor binding site and the −819C/T polymorphism is located within a putative positive regulatory region, these polymorphisms are considered as significant loci to investigate for their association with asthma which is affected by the expression level of this cytokine [15]. Several studies have shown the association between these polymorphisms and asthma [16, 17]. An association between IL-10 −1082G allele and asthma has been indicated in East Asian population including that of north India [17, 18]. Although the role of IL-17F overexpression in asthma and its severity has been established well, very few studies have investigated the association of this cytokine polymorphism with asthma, with none from India. Among the identified IL-17F promoter polymorphisms, rs1889570 polymorphism has shown the highest minor allele frequency in European Americans (MAF: 0.348), African Americans (MAF: 0.375), and East Asians (MAF: 0.362) [19, 20].

Although several studies have investigated the association between IL-10 and IL-17F polymorphisms and asthma, very few ones have evaluated the association of these polymorphisms with asthma clinical phenotypes. Furthermore, population diversities are noticeable and specific genetic markers exist for each population. This study was conducted to evaluate the association of IL-10 (rs1800871, rs1800896) and IL-17F (rs1889570) promoter polymorphisms with asthma and its clinical phenotypes including severity (GINA), atopic status, spirometric parameters, and response to treatment with ICS and LABA in south Indian population for the first time. A sub-study was conducted to assess these cytokine levels in subjects with different IL-10 and IL-17F variants.

Methods

Subjects

This study included 419 asthmatic patients and 393 non-asthmatic controls. While the asthmatic subjects were recruited from “Allergy, Asthma and Chest Center,” India, age and gender-matched controls were selected from the general population. We included controls who had no history of asthma or other respiratory diseases, were non-smokers, and showed normal lung function in Pulmonary Function Test (PFT).

Spirometry to verify asthma in patients was performed according to ATS (American Thoracic Society) standards [21]. Diagnosis of asthma and its severity was performed according to the current Global Initiative for Asthma (GINA) guidelines. Asthmatic patients were classified into three groups: mild persistent, moderate persistent, and severe persistent [22]. This study was approved by the ethical committee of the University of Mysore, with Institutional Human Ethical Committee numbered IHEC-UOM No.80 PhD/2012-13, and accordingly informed written consent was obtained from all subjects.

DNA Extraction and SNP Genotyping

Two milliliter of venous blood was collected from the participants and stored in EDTA tubes at −20 °C for further analysis. DNA was extracted using Promega Wizard genomic DNA purification kit. The test procedure was conducted according to the manufacturer's guidelines. Three promoter polymorphisms [IL-10 −819C/T (rs1800871), IL-10 −1082A/G (rs1800896) and IL-17F −1507G/A (rs1889570)] were selected for genotyping using Mass ARRAY technique (eTable 1 in Electronic Supplementary Material).

Comparison of Serum Cytokine Levels in Subjects with Different IL-10 and IL-17F SNPs

Serum levels of IL-10 and IL-17F were assessed in 44 asthmatic and 44 non-asthmatic subjects [23]. Commercial ELISA kits (Ray Biotech Inc., USA) were used to measure serum levels of IL-10 and IL-17F. The test procedure was performed according to the manufacturer's guidelines. IL-10 and IL-17F serum levels were compared among subjects with different IL-10 and IL-17F SNPs.

Association of IL-10 and IL-17F SNPs to Different Clinical Variables

Total serum IgE was measured using ELFA (Enzyme-Linked Fluorescent Assay) method (Minividas, France). In skin prick test (SPT), sensitization to 36 common allergens (HollisterStier Allergy, USA) was tested where the wheal size of 3 mm greater than saline control was assumed as positive [24]. Severity of sensitization was specified by comparing the largest allergen wheal size with that of histamine as positive control. Severity of sensitization was tested according to Aas et al. (1973) where allergen wheal diameter of greater than histamine is graded as 4+ [25].

Comparison of FVC (Forced Vital Capacity) and FEV1 (Forced Expiratory Volume in 1 s) Improvements Following Two Months of ICS and LABA Treatment in Patients with Different IL-10 and IL-17F SNPs

232 asthmatic patients were followed up after 2 months of ICS and LABA treatment. The improvement in FVC and FEV1 was compared among subjects with different IL-10 and IL-17F SNPs. The rest of 187 patients were referred to their family practitioner and were not available for follow-up.

Statistical Analysis

Hardy–Weinberg equilibrium (HWE) was calculated using FINETTI program [26]. The statistical power of the study was confirmed by Quanto (http://hydra.usc.edu/gxe) software V.1.2.4 [27]. Genotype and allelic frequencies were assessed using Chi-square, Odds Ratio (OR), and 95 % Confidence Interval (CI). Further analysis was performed by assessment of genotype frequencies under dominant, recessive, and additive genetic models [28]. Haplotype–disease association was assessed using Haploview software V.4.0 [29]. The association between SNPs and clinical variables was tested using SPSS software V.19 (SPSS, Inc, USA). As multiple comparisons were carried out on the same sample, Bonferroni correction was applied and P <0.016 was considered as significant.

Results

Demographic characteristics and mean of the spirometric variables of the study population are presented in Table 1. IL-10 (rs1800871, rs1800896) and IL-17F (rs1889570) promoter polymorphisms were genotyped in 419 cases and 393 controls. All the SNPs were in Hardy–Weinberg equilibrium in both the cases and controls (Table 2). Our results on power analysis showed the desirable power of 80 % with the relative risk ratio of 1.33 for IL-10 rs1800871 and IL-17F rs1889570 SNPs, and 1.39 for IL-10 rs1800896 SNP. Assessment of genotype and allele distributions of IL-10 and IL-17F SNPs showed no significant difference between asthmatics and controls (Table 2). On subgroup analysis based on asthma severity, IL-10 rs1800871 SNP in both homozygous and heterozygous conditions as well as allelic distribution and IL-10 rs1800896 SNP in homozygous condition as well as allelic distribution were significantly different in mild asthmatics as compared to controls, indicating an association (Table 3). There was no association with any of three SNPs in moderate to severe asthma. Comparison of IL-10 and IL-17F genotypes frequencies between asthmatics and controls under dominant, recessive, and additive genetic models showed nonsignificant differences (Table 4). On haplotype analysis, our results showed no significant difference in IL-10 haplotypes frequencies in asthmatics as compared to controls (eTable 2 in Electronic Supplementary Material). The association of IL-10 and IL-17F SNPs to their serum levels showed no significant difference in cytokine serum levels in subjects with different IL-10 and IL-17F SNPs (eTable 3 in Electronic Supplementary Material). Comparison of genotype distribution of IL-17F rs1887570 AA variant among atopic and non-atopic patients showed a significant difference (p = 0.024, eTable 4 in Electronic Supplementary Material). Comparison of the number of allergens sensitized among atopic patients with different IL-17F rs1887570 genotypes showed a significant difference (p = 0.008, eFig. 1 in Electronic Supplementary Material). The least (47 patients) and the most (247 patients) amount of sensitization was shown to helminthosporium and house dust mite mixed allergens, respectively. Correlation analysis of SNPs to different clinical variables showed a significant correlation between IL-17F rs1889570 SNP and the number of allergens sensitized (Table 5, rs = 0.142, p = 0.004). Significant improvement in lung function was observed after 2 months of ICS and LABA treatment with no statistically significant difference between SNPs variants (Table 6).

Table 1 General characteristics of the study population
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Table 2 Hardy–Weinberg equilibrium test, genotype, and allelic distributions of IL-10 and IL-17F SNPs in asthmatic patients and healthy controls
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Table 3 Genotype and allele frequency distributions of IL-10 and IL-17F SNPs among controls (CT) and patients with mild asthma (MI), moderate asthma (MO), and severe asthma (SE)
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Table 4 Comparison of genotype frequencies between controls and asthmatic cases by dominant, recessive, and additive models
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Table 5 Haplotype analysis of IL-10 rs1800871 and rs1800896 SNPs among asthmatic patients and healthy controls
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Table 6 Mean concentration of IL-10 and IL-17F in different genotypes of the corresponding genes variants
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Discussion

One of the major goals of SNP studies is to perceive the genetic basis of human phenotype variation, especially of complex diseases [30]. Identification of asthma-associated genetic variants may be clinically useful for identification of the patients at risk [31]. Our results showed an association between IL-10 rs1800871 and rs1800896 SNPs and mild asthma in south Indian population (Table 3). There was no association with any of the three SNPs in moderate to severe asthma. Comparison of IL-17F rs1887570 AA genotype distribution among atopic and non-atopic patients showed significant difference (p = 0.024, eTable 4 in Electronic Supplementary Material). Comparison of the number of allergens sensitized among atopic patients with different IL-17F rs1887570 genotypes showed a significant difference (p = 0.008, eFig. 1 in Electronic Supplementary Material). Correlation analysis of IL-10 and IL-17F SNPs to clinical variables showed a correlation (rs = 0.142, p = 0.004) between IL-17F SNP and the number of allergen sensitized (Table 5). No difference was observed when serum cytokines levels were compared between subjects with different IL-10 and IL-17F SNPs (eTable 3 in Electronic Supplementary Material). Significant improvement in lung function was observed after 2 months of ICS and LABA treatment with no statistically significant difference between SNPs variants (Table 6).

Most of the interest in IL-10 has focused on the promoter region to illustrate the variation in IL-10 levels by altering the binding site of the transcription factors [32]. Our results showed an association between IL-10 rs1800871 and rs1800896 polymorphisms and mild asthma, where a significant difference was noticed in T and G allele frequencies in controls than mild asthmatics (Table 3). No association was found with IL-10 SNPs in moderate to severe asthma. Several studies have investigated the association between IL-10 promoter polymorphism and asthma (eTable 5 in Electronic Supplementary Material), but the results are inconsistent [17, 18]. While some have reported the association between IL-10 polymorphism and asthma [33, 34], others have reported negative results [35–37]. Such discrepancies are because of differences in patient demographics, sample size, environmental factors, genetic background, study design, asthma definition, gene–gene and gene–environment interactions.

We observed an association of both IL-10 SNPs with mild asthma, but not with moderate and severe asthma. This observation should be confirmed in other studies. Unlike this study, most of the studies on IL-10 SNPs and asthma have not assessed the association of IL-10 SNPs with different subgroups of asthma severity (GINA), possibly containing a heterogeneous severity groups. Reports suggest different pathogenesis mechanisms, pathways, and cytokine profiles in mild as compared to severe asthma [38]. There is a need for further analysis of our subjects with different asthma severities to understand the pathogenetic mechanism of IL-10 in mild asthma.

The −1082 A allele is associated with reduced IL-10 levels, whereas the −1082G allele is associated with elevated IL-10 levels [39]. Lack of association between IL-10 promoter polymorphism and its serum levels (eTable 3 in Electronic Supplementary Material) may show that these promoter sites does not affect IL-10 expression, both alleles are codominantly expressed and there are other genes which regulate IL-10 expression [40]. The presence of serum inhibitors, disparate assay sensitivities, ethnic discrepancies in impact of IL-10 polymorphism on its expression level [41] and possibility of false negatives due to small sample size are other factors to explain our nonsignificant results.

Comparison of the IL-10 SNPs among atopic and non-atopic patients showed nonsignificant differences (eTable 4 in Electronic Supplementary Material). Our results are in line with other studies showing no difference between atopic and non-atopic asthma with respect to IL-10 promoter polymorphisms [35, 37]. Correlation analysis of IL-10 SNPs to clinical variables showed nonsignificant results (Table 5). Karjalainen et al. [42] found no association between IL-10 polymorphism and lung function in asthmatic patients. As lung function is a complex trait, a couple of genes may not cause enough variation to change the spirometric parameters significantly [32].

IL-17F induces the expression of several proinflammatory cytokines, chemokines, and growth factors which are involved in leukocyte activation and airway remodeling in patients with asthma [43]. No association was found between IL-17F promoter polymorphism and asthma in our study population (Tables 2, 3). Our results are in line with the results of Kawaguchi et al. [20] who found no association between IL-17F rs1889570 polymorphism and asthma. Overall variation in IL-17F expression cannot be solely because of the variation in the promoter region. Polymorphisms in the gene located upstream in the inflammatory pathway may change IL-17F expression as opposed to SNPs within IL-17F itself.

Comparison of IL-17F serum level among subjects with different IL-17F variants showed nonsignificant differences (eTable 3 in Electronic Supplementary Material) showing that this promoter polymorphism may not affect the IL-17F expression or other genes may regulate the expression of this cytokine [44]. Comparison of IL-17F rs1887570 AA variant among atopic and non-atopic patients showed a significant difference (p = 0.024, eTable 4 in Electronic Supplementary Material), where the most number of allergens sensitized were observed in atopic patients with IL-17F rs1887570 AA variant. Correlation analysis of IL-17F rs1889570 variant to clinical variables showed a correlation (rs = 0.142, p = 0.004, Table 5) between IL-17F SNP and the number of allergen sensitized. Interestingly, comparison of the number of allergens sensitized among atopic patients with different IL-17F rs1887570 genotypes showed a significant difference (p = 0.008, eFig. 1 in Electronic Supplementary Material). The positive correlation between the number of allergens sensitized and IL-17F rs1889570 variants suggests the role of IL-17F in atopic sensitization. IL-17F was first established in BALF of atopic patients on stimulation with ragweed allergen [13].

Inhaled corticosteroids are the most common medications to control asthma. It is likely that the response to ICS treatment has a genetic background [45]. Our results showed significant improvement in lung function after 2 months of ICS treatment with no statistically significant difference between SNPs variants (Table 6). Lack of association between IL-10 and IL-17F SNPs and spirometric parameters can be because of the complex genetic basis of response to ICS treatment or not including other functional SNPs which may be associated in our population (Tables 7, 8).

Table 7 Correlation analysis of IL-10 and IL-17F SNPs to various clinical variables
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Table 8 Comparison of FVC and FEV1 improvements following 2 months of ICS and LABA treatment in patients with different IL-10 and IL-17F SNPs variants
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To the best of our knowledge, this is the first report on the association of asthma and IL-10 promoter polymorphism in south Indian population and on the association of asthma and IL-17F promoter polymorphism in India. Taking a desirable sample size, collection of control samples from the general population and assessment of various clinical variables are other advantages of this study. Our study has several potential limitations. Unlike the controls, the cases of our study were recruited from the hospital and not from the general population. Some of the subgroup analysis performed did not have sufficient sample size in the subgroups.

In conclusion, our results showed an association between IL-10 SNPs and mild asthma and IL-17F rs1887570 AA with number of allergens sensitized. Further large scale investigations along with in vitro functional experiments are required to assess the potential roles of these polymorphisms in asthma.