Association of Cytokines in Individuals Sensitive and Insensitive to Dust Mites in a Brazilian Population

Introduction Allergic reaction to dust mites is a relatively common condition among children, triggering cutaneous and respiratory responses that have a great impact on the health of this population. Anaphylactic hypersensitivity is characterized by an exacerbated response involving the production of regulatory cytokines responsible for stimulating the production of IgE antibodies. Objective To investigate an association of variants in cytokine genes (IL1A −889, IL1B −511, +3962, IL1R 1970, IL1RA 11100, IL4RA +1902, IL12 −1188, IFNG +874, TGFB1 codon 10, codon 25, TNFA −308, −238, IL2 −330, +166, IL4 −1098, −590, −33, IL6 −174, nt565, and IL10 −1082, −819, −592) between patients sensitive to dust mites and a control group. Methods A total of 254 patients were grouped as atopic and non-atopic according to sensitivity as evaluated by the Prick Test and to cytokine genotyping by the polymerase chain reaction-sequence specific primers (PCR-SSP) method using the Cytokine Genotyping Kit. Results A comparison between individuals allergic to Dermatophagoides farinae, Dermatophagoides pteronyssinus, and Blomia tropicalis and a non-atopic control group showed significant differences between allele and genotype frequencies in the regulatory regions of cytokine genes, with important evidence for IL4 −590 in T/C (10.2% vs. 43.1%, odd ratio [OR] = 0.15, p = 5.2 10−8, pc = 0.0000011, and 95% confidence interval [95%CI] = 0.07–0.32) and T/T genotypes (42.9% vs. 13.8%, OR = 4.69, p = 2.5 10−6, pc = 0.000055, and 95%CI = 2.42–9.09). Other associations were observed in the pro-inflammatory cytokines IL1A −889 (T/T, C, and T) and IL2 −330 (G/T and T/T) and the anti-inflammatory cytokines IL4RA +1902 (A and G), IL4 −590 (T/C, T/T, C, and T), and IL10 −592 (A/A, C/A, A, and C). Conclusion Our results suggest a possible association between single nucleotide polymorphisms (SNPs) in cytokine genes and hypersensitivity to dust mites.


Introduction
The prevalence of allergic diseases has experienced an important increase in industrialized countries over recent decades. Although this increase has been widely investigated, the reasons for such trend have not yet been elucidated [1,2]. According to the World Allergy Organization [3], children carry the greatest burden of the rising trend which has occurred over the past two decades. Despite this increase, in many countries there are no specialized services for patients with allergic diseases.
In respiratory allergies, such as asthma and rhinitis, there is inflammation of the airways as a result of an immune reaction, with release of cytoplasmic granules from active substances found in mast cells, basophils, and eosinophils [4,5]. Two main factors contribute to the development and severity of allergic disease: host-related (genetic) factors and environmental factors, such as specific allergens, elements present in the environment, and air pollutants. When combined, these factors may trigger sensitization [6,7].
In addition, there is the ''hygiene hypothesis'', which became popular in the late 1980s to explain the high prevalence of atopic diseases in developed countries. It supports the idea that a reduced exposure to infections during childhood would predispose individuals to sensitization [8,9,10].
Since the mid-1960s, dust mites have been associated with allergic processes and identified as a causative agent of airway diseases, being considered one of the most important sources of allergens for humans. Among asthmatic patients, sensitivity to dust mites is present in 50% of adults and 80% of children [11,12]. The term ''house dust mite'' has been adopted to describe mite species that can be found in the indoor environment and have the ability to elicit a response resulting in IgE antibodies [13].
The study of cytokines and sensitivity to dust mites is intended to corroborate the understanding of this relationship, given the high incidence of positive skin reactions to Dermatophagoides pteronyssinus, D. farinae, and Blomia tropicalis that has been commonly reported in the literature. It is noteworthy that these species belong to the class of arachnids (class Arachnida, subclass Acarina) [14,15,16] and have glycoproteins capable of inducing an anaphylactic response. However, little is known about the mechanism of action of these allergens against the development of immune response in certain individuals [17,18].
Given the key role of cytokines in allergic reactions, gene variability in their regulatory regions might induce changes in the immune response [19]. Regulatory regions have shown an influence on cytokine production and transcription [20,21,22,23]. Cytokines participate not only in the regulation of the immune response, but also directly in the inflammatory response [24]. There are proinflammatory cytokines ( [25,26,27]. The involvement of interleukins in the pathogenesis of a range of diseases, such as lupus erythematosus, diabetes, chronic periodontitis, and cancer, has been widely studied. However, little is known about the association between single nucleotide polymorphisms (SNPs) in cytokine genes and sensitivity to dust mites. Therefore, we conducted a genetic association study to investigate markers of immune response in polymorphic variants of cytokine genes IL1A 2889 (rs1800587),

Ethical considerations
This study was approved by the Research Ethics Committee of Universidade Estadual de Maringá (protocol no. 693/2009) and conducted in accordance with the provisions of the Declaration of Helsinki. Educators and children from ''Lar Escola de Maringá'' and ''Sistema de Apoio à Saúde São Rafael'', two local public institutions providing child care and health care respectively, were invited to participate in the study. All participants completed a registration form, were adequately informed about the project, and provided written informed consent prior to their inclusion in the study. In the case of minors, under 18 years of age, written consent was obtained from their parents/legal guardian.

Sample size
Sample size calculation was based on city demographic data from the 2013 Census, with a population of 386,000 inhabitants. Assuming a 10% margin of error, maximum prevalence of 50% and 95% confidence interval, a minimum sample of 96 participants (cases) was required. Considering a possible loss of 10%, the final sample size was estimated at 138 participants (cases). Controls were defined according to the approximate number of cases, totaling 116 participants. Sample size was calculated using Statdisk version 8.4 [28].

Inclusion and exclusion criteria
The study included all children and educators who voluntarily agreed to participate in the study.

Characterization of the sample
Blood samples (10 ml) were drawn from 138 mite-sensitive and 116 mite-insensitive individuals, with a mean age of 12.967.6 years (range, 4-48 years), including 138 females and 116 males. Blood samples were collected into tubes containing EDTA anticoagulant.

Allergy testing
A skin prick test (Prick Test) was used to check for immediate sensitization to antigens specific for dust mites. The participants were tested for the following allergens: Dermatophagoides farinae, Dermatophagoides pteronyssinus, and Blomia tropicalis. Dust mite and control extracts were applied at a distance of 2 cm from each other in a predetermined order. A lancet was used to gently prick the extracts onto the skin surface. Reactions were read after 15-20 min by two independent readers, and the size of the wheal or erythema was the classification criterion for the degree of allergic reaction. A reaction was recorded as positive if the diameter of the wheal or erythema was $5 mm and negative in the absence of these signs.

Biological samples
Blood samples were centrifuged at 2500 rpm for 20 min to obtain the nucleated cell layer (buffy-coat), from which DNA was extracted using the commercial kit BIOPUR according to the manufacturer's instructions.

Genotyping of polymorphic variants in cytokine genes
A NanoDrop (2000c/2000 UV-Vis) spectrophotometer was used to evaluate DNA purity and adjust its concentration. The polymerase chain reaction-sequence specific primer (PCR-SSP) technique was used to genotype allelic variants in cytokine genes, using a commercially available genotyping kit. The method consists of a sequence of specific primers capable of amplifying the product (or not) based on the presence (or absence) of SNP in each well. According to information provided by the manufacturer, in addition to determining genotypes, it is also possible to define the haplotypes of TGFB1, TNFA, IL2, IL4, IL6, and IL10. The 22 selected SNPs are listed in Table 1, including chromosomal locations and associated diseases as reported in previous studies.
Each DNA sample, adjusted to a concentration of 20 to 200 gg/mL, was added to the amplification solution provided in the kit, along with the Taq DNA polymerase enzyme, according to the volumes recommended by the manufacturer.
After the solution was homogenized, aliquots were poured onto polyethylene plates containing 48 wells with specific primers. Plates were then transferred to a thermal cycler for gene amplification, according to the manufacturer's instructions.
Fragments of the amplified DNA were separated by electrophoresis in a micro SSP gel System in a 3% gel solution and homogenized with 2 mL of SYBR safe stain at 90 volts for 15 min. DNA bands exposed to ultraviolet light were visualized by SYBR staining and the results were interpreted based on the presence or absence of the specific fragment of DNA. All 48 wells had an internal control of known size as informed by the manufacturer. The samples were photo-documented for reading of results.

Statistical analysis
Allele, genotype and haplotype frequencies were determined by direct counting. Observed and expected frequencies of patients and controls were compared using a 262 contingency table. The significance of differences in allele, genotype and haplotype frequencies was evaluated using two-tailed Fisher's exact test with Yates' correction, followed by Bonferroni correction. The risk of developing sensitivity to dust mites was calculated by determining odds ratio (OR), according to Woolf [29], using Epi Info version 7. Hardy-Weinberg equilibrium (HWE) was tested using the method described by Guo and Thompson [30] with the Arlequin software version 3.0 (http://cmpg.unibe.ch/software/arlequin3) [31]. An age-and sex-adjusted logistic regression analysis was performed using SAS version 9.1 [32]. The level of significance for all analyses was set at p,0.05.
A significant difference was observed in genotype and allele frequency between cases and controls at position 2889 of the IL1A gene. The genotype T/T showed a negative association with sensitivity to dust mites (5.1% vs. 14.7%, OR = 0.31, p = 0.016, and 95% confidence interval [95% CI] = 0.12-0. 78). An analysis of T allele variant revealed a negative association (23.5% vs. 33.2%, OR = 0.62, p = 0.017, and 95% CI = 0.42-0.91) with sensitivity to at least one of the three types of dust mites. The positions IL2 2330, +166 also showed significant frequency in the GG/TT haplotype and the G/T. The GG/TT haplotype and the G/T genotype were indicated as risk factors, with a frequency of 18.8% vs. 7.8% and 42.7% vs. 27.6% in the allergic group vs. the control group, respectively (Table 3).
When comparing the frequency of cytokine SNPs between 123 patients sensitive to dust mite 1 (D. farinae) and 116 controls, the IL1A 2889 SNP showed a statistically significant frequency in the T/T genotype (5.7% vs. 14.7%, OR = 0.35, p = 0.029, and 95% CI = 0.14-0.88) and in the T allele (23.6% vs. 33.2%, OR = 0.62, p = 0.025, and 95% CI = 0.41-0.93), with a negative association. The IL4RA gene at position +1902 also showed a significant frequency in the A and G alleles. While the A allele was indicated as a risk factor, the G allele showed a protective effect, with a frequency of 74.4% vs. 63.8% and 25.6% vs. 36.2% in the allergic . These data suggest that individuals who express the A allele are at risk of developing hypersensitivity to dust mite 1, and those who express the C allele have a protective factor against this development ( Table 4). The analysis of the frequency of cytokine SNPs in 98 individuals sensitive to dust mite 2 (D. pteronyssinus) and 116 controls showed a significant difference in IL2 2330, +166 SNPs in the GG/TT haplotype (18.4% vs. 7.8%, OR = 2.67, p = 0.0234, and 95% CI = 1.14-6.26) and in the G/T genotype (42.9% vs. 27.6%, OR = 1.97, p = 0.0215, and 95% CI = 1.11-3.48). A positive association with sensitivity to dust mite 2 was found only at position 2330. There was also a significant statistical difference in the IL4 2590 SNP when analyzing the distribution of genotypic and allelic variants. The T/C genotype (10.2% vs. 43.1%, OR = 0.15, p = 0.000000052, pc = 0.0000011, and 95% CI = 0.07-0.32) showed a negative association with sensitivity to dust mite 2, while the T/T genotype (42.9% vs. 13.8%, OR = 4.69, p = 0.0000025, pc = 0.000055, and 95% CI = 2.42-9.09) showed a positive association. Additionally, C and T alleles were indicated as protective and risk factors, respectively, for the development of sensitivity to dust mite 2, with a frequency of 52.0% vs. 64.7% and 48.0% vs. 35.3%, respectively (Table 5).
As for the influence of regulatory regions on cytokine production, we found no significant difference in the association of TGFB1, TNFA and IL10 genotypes and haplotypes between cases and controls as selected using the Cytokine Genotyping Kit (Invitrogen).

Discussion
Allergy is a multifactorial condition, with the onset and severity dependent on genetic and environmental factors. Hypersensitivity to house dust mites may trigger different cutaneous and respiratory responses, which have a great impact on the health of affected individuals [33].
The discoveries made in the 1950s about the mechanisms of gene regulation in prokaryotes, such as lac or lambda repressor, have allowed researchers to investigate DNA binding sites in the regulatory regions of eukaryotes. Since then, several regulatory regions have been detected upstream and downstream of the transcribed gene, mainly in SNP regions.
The development of molecular biology techniques has allowed the identification of genetic polymorphisms within regulatory regions of cytokine genes, and also the finding that the level of cytokine production differs among individuals. These considerations have prompted many authors to investigate the regulation of genes expressing these cytokines in relation to susceptibility to and severity of different diseases [34,35]. Although cytokines are usually related to allergic diseases, such as allergic rhinitis, allergic conjunctivitis, food allergy, atopic dermatitis, and asthma, they have been suggested as important biomarkers of various diseases, such as lupus erythematosus, pediatric ulcerative colitis [36,37,38], Alzheimer's disease [39,40,41], and ankylosing spondylitis [42].
There is an increasing number of studies devoted to this topic in the international literature. However, to date, there are no studies conducted in Brazil that address this issue. A cohort study of asthma in the Korean population suggested a possible involvement of IL18 polymorphisms in asthma [33]. A study involving 417 Costa Rican children and 503 children participating in a childhood asthma management program showed significant changes in the interaction between IgE and dust mite allergens at three IL10 SNP positions (rs1800896, rs3024492, and rs3024496), suggesting that exposure to these allergens significantly modified the effect of IL10 SNPs [43]. Recently, higher serum levels of TNFA have been found in genotypes G/A and G/ G of asthmatic children compared to controls (60.0% vs. 30%) [44].
There is considerable evidence associating interleukins and somatic complaints. Thompson and Barkhuizen [45] reported on alterations of IL2, IL2 receptor (IL2R), IL8, and IL1 receptor antagonist (IL1RA) in fibromyalgia and described the mechanism  Table 3. Significant allele, genotype and haplotype frequencies of cytokine SNPs in individuals allergic to at least one of the following dust mites: Dermatophagoides farinae, Dermatophagoides pteronyssinus, and Blomia tropicalis, and controls.      Table 6. Significant allele, genotype and haplotype frequencies of cytokine SNPs in individuals allergic to dust mite 3 (Blomia tropicalis) and controls. by which alterations in cytokines (in fibromyalgia and hepatitis C) may trigger hyperalgesia and other symptoms mediated by the presence of cytokine receptors on certain cells. They also pointed out the role of other cytokines, such as IL6, IL10, and TNFA in the relationship between chronic inflammation and mortality. A study carried out in Boston with 183 patients on hemodialysis showed a strong association between SNPs (IL6 2174 , IL10 21082 , and TNFA 2308 ) and patients with end-stage renal disease [46]. Moreover, cytokines play an important physiological role in immune regulation and inflammatory processes, and changes in the aberrant expression or failure in production may contribute to the development of certain diseases [20,21,22,23]. Because of the influence on cytokine production, these polymorphisms may provide flexibility to the immune response. In addition, the balance between pro-and anti-inflammatory cytokines is known to determine the intensity of inflammatory response [47].
The present results showed an important association between SNPs in cytokine genes and susceptibility to or protection from allergy to dust mites in IL1A at position 2889, IL4RA at position +1902, IL2 at positions 2330 and +166, IL4 at position 2590, and IL10 at position 2592. All loci showed a gene frequency distribution in accordance with expected frequencies after calculating HWE, whose p-values were greater than the significance level of 0.05, IL12 at position 21188 and IL10 at position 21082. Considering that no evolutionary factor (mutation, migration, drift, or selection) was observed in this study, there may have been an error in the interpretation of the results. In order to avoid a type I error, the analyses of these two loci were excluded.
IL1A is synthesized by different cell types, such as activated macrophages and B lymphocytes, and by non-immune cells, such as fibroblasts. In addition, this cytokine is considered a potent inflammatory mediator and is located on chromosome 2q13 in humans [48]. Karjalainen et al. [49] conducted a study of 245 asthmatic adults and 405 controls associating the IL1A genotype with atopy in non asthmatic adults. No significant association was found between IL1A genotypes and skin test findings in asthmatic patients. Among controls, however, the IL1A genotype distribution was significantly different (p = 0.006) in individuals with positive (n = 150) and negative (n = 247) skin test responses. In our study, the T/T genotype and T allele of IL1A gene (rs1800587) were associated with protection against dust mite allergy (OR, 0.31 and 0.62 respectively) when cases were compared to controls. Conversely, the C allele was positively associated with disease (OR, 1.61).
In a German study [50], the IL1A 2889 T allele was also associated with a protective effect, with a lower prevalence of dermatitis in a group exposed to a high risk of skin irritability. In that same study, the authors investigated the association between the IL1A 2889 SNP and IL1 cytokine levels in the stratum corneum. The IL1 concentration was 23% and 47% lower in subjects with IL1A C/T and T/T genotypes, respectively, when compared to the C/C genotype. The reduced subcutaneous level of this pro-inflammatory cytokine for IL1A 2889 T allele would explain the protective effect of this allele against dermatitis.
IL2, located on chromosome 4q27 in humans, is considered a key lymphokine, responsible for triggering the proliferation of Ly-T cells after interaction with peptide presentation [51,52], which consequently stimulates Th2 responses and antibody production. Due to this central role in regulating the immune response, IL2 is considered a strong candidate gene for allergic disease. Christensen et al. [53] investigated the involvement of IL2 and IL15 in the etiology of atopy and allergic diseases in a family-based association study of two Danish samples comprising a total of 235 families with at least two siblings with allergic disease. IL2 at position 2330 showed a significant association with three clinical forms of allergic disease (asthma, allergic rhinitis, and atopic dermatitis) in the group with 135 families, with p-values between 0.01 and 0.05, and IL2 at position +166 also showed a significant association (with a p-value of 0.0005) in the same group. IL15 showed no significant association. SNP at position +166 does not alter IL2 expression, because it does not interfere with the amino acid sequence, i.e., it is a silent mutation [54]. Therefore, the association occurs only at position 2330.
Differences in cytokine production between allelic variants and their corresponding phenotypes were compared using Jonckheere's trend test for high, intermediate, and low comparisons. Individuals that were homozygous for the G allele produced over three times the amount of IL2 than the T/T and T/G genotypes (p,0.001) [55]. In the present study, we observed significant differences in the frequency of the G/T and T/T genotypes, which are associated with low IL2 production. The mechanism by which this is accomplished is currently under study. Given its influence on cell cycle advancement and apoptosis, much attention has been focused on the events that regulate IL2 signal transduction. It is possible that the polymorphism at position 2330 interferes with the binding of a key silencer element, thereby resulting in enhanced (or not) IL2 production [55].
IL4 and IL4RA are candidate genes for atopic diseases, i.e., diseases that are clinically expressed as atopic asthma, allergic rhinitis, and atopic dermatitis or that can be defined by at least one positive specific IgE response to inhalant or food allergens [56,57]. A case-control study of 265 patients with persistent allergic rhinitis and 275 healthy controls revealed that the C/T and C/C genotypes in IL4 2590 were associated with a significantly decreased risk of mite-sensitized persistent allergic rhinitis (32.5% vs. 42.9%, OR = 0.64, 95%CI = 0.45-0.92) compared to the T/T genotype [56]. In the current study, a highly significant association was observed between the frequency of IL4 2590 SNP in 98 individuals allergic to D. pteronyssinus and 116 controls and the T/C genotype (p = 0.000000052) and the C allele (p = 0.010), suggesting a protective effect against sensitization to dust mites.
However, there are conflicting reports in the literature. Micheal et al. [57], in a case-control study of 214 atopic patients (108 with asthma and 106 with allergic rhinitis), found that the T/T genotype in the SNP rs2243250 was significantly associated with asthma (p = 0.004) and allergic rhinitis (p ,0.001), but playing a protective role rather than predisposing to respiratory allergy, as demonstrated by our data for the same genotype (p = 0.0000025). Our results are consistent with findings of studies conducted in China [56], India [58], and Iran [59].
In Germany, Liu et al. [60] conducted a case-control study to examine the increased risk of sensitization to food, dust mites, cat epithelium, and outdoor allergens (e.g., pollen) in six SNPs (C-590T in the IL4 gene, C-1055T and Arg130Gln in the IL13 gene, and Ile50Val, Ser478Pro, and Gln551Arg in the IL4RA gene) and reported that, as for sensitization to dust mites, tests performed with 108 sensitized children and 432 controls revealed no significant association between sensitization to dust mite allergens and the six SNPs. Their findings disagree with our results for the IL4RA SNP (rs1801275), which expressed the A allele as a risk factor (OR, 1.65) and the G allele as a protective factor (OR, 0.61). It is worth noting that significant differences were observed between allele frequencies only for dust mite 1 (D. farinae), even after Bonferroni correction. No differences were observed in the genotypes at this position, probably due to the low risk obtained for these alleles.
The IL10 2592 gene was significantly different both for the A/A (OR, 3.24) and C/A (OR, 0.37) genotypes and for the A (OR, 1.47) and C (OR, 0.68) alleles. In Costa Rica, a study of 417 children and 503 participants of a childhood asthma management program highlighted the role of IL10 in allergy and asthma exacerbations (when exposed to dust mites). Using a family-based approach to test for associations, the results showed that exposure to house dust mite allergens led to significant differences in the IL10 gene [43].
So far, the present discussion has been based on the data considered significant as compared with those obtained from similar studies. However, because of the paucity of specific studies, we applied the Bonferroni correction to our data to avoid type II errors. After this correction, only IL10 at position 2592 for D. farinae and IL4 at position 2590 for D. pteronyssinus remained significant. These findings may suggest that different causative agents may be regulated by different cytokine production pathways, and that both cytokines have a biological role in controlling the production of different classes of antibodies. We believe that these data will add useful evidence to this line of research.

Conclusions
Our results suggest an association of allelic and genotypic variants in the pro-inflammatory cytokines IL1A 2889 and IL2 2330 and anti-inflammatory cytokines IL4 2590 , IL4RA +1902 and IL10 2592 .
The detection of candidate genes in individuals sensitive to dust mites may reveal mechanisms of interaction between allergies and hosts, and this detection allows the development of new preventive, diagnostic and therapeutic strategies. Thus, further studies are needed to clarify the role of these genetic markers in the susceptibility to and/or protection against house dust mite sensitivity.