Interleukin 10 -1082 G/A Gene Polymorphism and Susceptibility to Bronchial Asthma in Children: A Single-Center Study

Abstract

Interleukin-10 (IL-10) is the key regulator of immune responses preventing the undesirable exaggerated ones. Genetic variation in the promoter region of IL-10 may influence its serum level and contribute to susceptibility to bronchial asthma in children. This is a case–control study including 100 patients and 100 healthy control children who had undergone skin prick test, estimation of total IgE and serum level of IL-10 by enzyme-linked immunosorbent assay, and polymerase chain reaction-restriction fragment length polymorphism for IL-10 gene polymorphism. A significant association between IL-10 polymorphism and susceptibility to pediatric asthma was found. AA genotype represented (66%) of the patient group compared to (6%) only of the control group, while AG genotype was detected in 20% of patients and 4% of control. In contrast, wild genotype GG was found in 14% of patients and 90% of control with a highly statistically significant difference among both groups (P < 0.001). The serum level of IL-10 was significantly elevated in the GG genotype in comparison to other genotypes (P < 0.001), and it was negatively correlated with the severity of asthma among the studied pediatric asthmatic group (P < 0.001). In conclusion, IL-10 polymorphism may play an important role in the development of bronchial asthma in children.

Introduction

Asthma is considered one of the most challenging chronic respiratory diseases in children, which are associated with airway hyper-responsiveness with subsequently recurrent episodes of wheezing, breathlessness, chest tightness, and/or cough varying over time (Quirt and others 2018). Moreover, its prevalence has been increasing worldwide especially in developed countries (Mirzaei and others 2017).

Based on pathogenesis, the association between cytokine levels and clinical features of asthmatic patients in different populations had been investigated (Bazzi and others 2013; Dimitrova and others 2019). It was found that interleukin-10 (IL-10) had a significant role in asthma pathogenesis. It is an anti-inflammatory factor that inhibits the production of pro-inflammatory cytokines resulting in the prevention of allergic airway hyperreactivity and inflammation (Saba and others 2017). Besides, cytokine single nucleotide polymorphisms can be used as genetic predictors of asthma susceptibility, clinical outcome, and as a tool for anthropological studies (Amirzargar and others 2008).

IL-10 is synthesized by activated CD4 and CD8 T lymphocytes. The gene encoding IL-10 is placed on chromosome 1 in the human genome. It consists of 5 exons and 4 introns (Jahromi and others 2015). IL-10 gene polymorphism was found to be associated with inflammatory diseases like deep venous thrombosis (Tang and others 2014), rheumatoid arthritis (Ying and others 2011), Helicobacter Pylori-induced gastroduodenal diseases (Kang and others 2009), and acute pancreatitis (Cai and others 2015). The level of IL-10 production is influenced by the polymorphism in the IL-10 gene. Among the polymorphisms of IL-10, the (rs1800870) seems to have an important role in the modulation of IL-10 production (Koponen and others 2014; Jahromi and others 2015).

Polymorphisms in IL-10 (-819, -592) were associated with the risk of pediatric asthma (Osman and Elsaid 2019; Alyasin and others 2021). However, there is a controversy regarding the role of IL-10 gene polymorphism (-1082G/A) in susceptibility to asthma. Previous studies showed that it had no impact on asthma susceptibility in adults or children (Karjalainen and others 2003; Kim and others 2011). Other studies found that it was associated with the risk of pediatric asthma (Hussein and others 2011; Osman and Elsaid 2019; Alyasin and others 2021). Hence, this study was carried out to evaluate the associations between polymorphism in IL-10 (-1082 G/A) and pediatric asthma in Egyptian children.

Patients and Methods

Study design

This case–control study was conducted at the outpatient clinic of Chest Department, Microbiology and Immunology Department, Allergy Unit of Zagazig University Hospitals in collaboration with Medical Biochemistry and Molecular Biology Department, Faculty of Medicine, Zagazig University, Zagazig, Egypt. It was carried out between November 2020 and April 2021. The study protocol was reviewed and approved by the Institutional Review Board (IRB) (no. 6455, 2020), Faculty of Medicine, Zagazig University, Egypt. The parents of all study participants agreed and signed informed consent.

Patient enrollment

This study included 200 participants, including 100 healthy nonatopic controls (negative skin prick tests) and 100 asthmatic children, who continued to use their medications as usual according to the stepwise approach (doses and drug selection adjusted according to the severity of asthma). The used drugs were inhaled corticosteroids (fluticasone, budesonide, and ciclesonide), leukotriene antagonists (montelukast), long-acting beta-agonists (salmeterol and formoterol), and theophylline.

Patients were diagnosed to have allergic asthma (positive skin prick test and high total IgE) and were classified as mild, moderate, and severe persistent bronchial asthma according to the referral of their clinical manifestations to the current Global Initiative for Asthma (GINA) criteria (Reddel and others 2019) and confirmed by spirometry (Spirotube, PC Spirometer; THOR Laboratories, Budapest, Hungary). The forced expiratory volume in the first second (FEV1) and the forced vital capacity (FVC) were measured with the reading of spirometry before and after a bronchodilator (2 puffs of inhaled salbutamol, each of 200 μg). The reversibility is considered when the FEV1 increment by (≥12%) approaches the prebronchodilator value.

Inclusion and exclusion criteria

The study pediatric age group ranged from 6 to 11 years old. Exclusion criteria included children <6 years old, those with acute exacerbation of bronchial asthma, and unconsented patients.

Samples

Five milliliters of blood were obtained from each participant. About 2.5 mL blood was collected on ethylenediamine tetraacetic acid (EDTA) containing tube for DNA extraction and 2.5 mL in a serum tube for the enzyme-linked immunosorbent assay (ELISA) tests. Serum was obtained by allowing samples to clot for 30 min followed by centrifugation for 10 min at ∼3000 × g. Sera were stored at −20°C until processed.

Skin prick test

A skin prick test was performed at the Allergy and Immunology Unit, Medical Microbiology and Immunology Department, Faculty of Medicine, Zagazig University. Allergen panel was used (homemade extracts of pollen, house dust, smoke, wool, cotton, mixed fungi, hay rice dust, clover, and maize). Histamine was used as a positive control and saline as a negative control. The test was read by measuring the size of the wheal after 20 min. A wheal diameter of 3 mm or more, associated with erythema, was considered a positive reaction (Ebbesen and others 2018).

Measuring of total IgE and IL-10 by ELISA

Quantitative determination of total IgE in human serum was performed according to manufacturer's instructions (Thermo Fisher Scientific, Austria; catalog no. BMS2097). IL-10 in human serum was measured according to the manufacturer's instructions (Thermo Fisher Scientific; Catalog no. BMS215-2). The optical density of each well was immediately determined using a microplate reader (Palm city) set to 450 nm.

Detection of IL-10 polymorphism by polymerase chain reaction-restriction fragment length polymorphism

DNA was extracted from blood using the QIAamp^® DNA Mini Kit (QIAGEN, Germany) according to the manufacturer's instructions and stored at −20°C until amplification was performed. Polymerase chain reaction (PCR) was performed using a thermal cycler (Veriti^® 96-Well Thermal Cycler; Applied Biosystems, Singapore). PCR was performed in a final volume of 20 μL, including 1 μL of the template DNA and1 μL of each of the oligonucleotide primers (Biolegio, Netherlands), and 17 μL of sterile deionized water was added to each PCR bead of Maxime PCR PreMix Beads (iNtron, Certified Company, Germany). The sequence of the primers was F: 5′-TCTGAAGAAGTCCTGATGTCACTG-3′; R: 5′-ACTTTCATCTTACCTATCCCTACTTCC-3′ (Cai and others 2015). The conditions of the reaction were initial denaturation at 95°C for 5 min followed by 35 cycles of denaturation at 94°C for 40 s; annealing at 55°C for 60 s; and extension at 72°C for 40 s. A final extension at 72°C for 10 min was performed (Jia and others 2015).

The PCR product (139 bp) was digested with restriction enzyme (Mn II) according to the manufacturer's protocol (Thermo Scientific™). Briefly, a total reaction mixture of 30 μL was formed of 10 μL of PCR amplicon, 17 μL nuclease-free water, 2 μL 10 × FastDigest Green Buffer, and 1 μL (Mn II). The reaction mixture was incubated at 37°C for 5 min. Then, 10 μL of digested products were loaded on 2% agarose gel and analyzed by electrophoresis under ultraviolet light by ethidium-bromide staining. Genotype GG was digested into 2 fragments:106 bp and 33 bp. Genotype AA remained undigested (139 bp), and genotype AG was represented by 3 fragments; 33, 106, and 139 bp.

Statistical analysis

The sample size was calculated using Epi Info program 6 (Atlanta, GA). The case–control StatCalc method was used for analysis with a 95% confidence limit, 80% power, and equal allocation ratios for the study groups (Hammad and others 2018). The statistical package SPSS Version 20, Inc. (Chicago) was used to analyze collected data. Quantitative data were represented as mean, median, standard deviation, and range. The chi-square test (χ ²) was used to compare proportions as appropriate. t-test was used for detection of the difference between 2 groups and 1-way analysis of variance (ANOVA) for multigroup comparisons. A P value <0.05 was considered statistically significant at a 95% confidence interval.

Results

A total of 200 participants were enrolled in this study and were divided into 2 groups matched in age and sex. Group 1 included 100 children with asthma with a mean age of 9.5 ± 1.5 and a boys/girls ratio of 34/66. Group 2 included 100 controls with a mean age of 9.2 ± 2.2 and a boys/girls ratio of 38/62. Serum IgE was significantly increased in the pediatric asthmatic group compared to the controls (P < 0.001), while serum IL-10 was found to be significantly decreased in the pediatric asthmatic group compared to the controls (P < 0.001) (Table 1). IL-10 was markedly diminished in severe asthmatic pediatric patients (Table 2).

Table 1.

Demographic and Biochemical Characters Among the Study Groups

	Group 1 (n = 100) Patients, mean ± SD	Group 2 (n = 100) Control, mean ± SD	t-test	P
Age (years)	9.5 ± 1.5	9.2 ± 2.2	2.629	0.26
IgE (IU/mL)	180.5 ± 25.2	31 ± 9.5	55.51	<0.001^a
IL-10 (pg/mL)	4.33 + 2.55	11.65 + 4.22	−14.846	<0.001^a

	n (%)	n (%)	X ²	P
Sex
Boys	34 (34%)	38 (38%)	0.174	0.339
Girls	66 (66%)	62 (62%)

Highly significant.

IL-10, interleukin-10; SD, standard deviation; t-test, student t-test; X ², chi-square test.

Table 2.

Relationship Between Serum Level of Interleukin-10 and Severity of Asthma in Patients' Group

Serum level of IL-10 (mean ± SD)	The severity of asthma (n = 100)	ANOVA test	P
5.7 ± 1.1	Mild (n = 34)
3.8 ± 1.6	Moderate (n = 48)	60.246	<0.001^a
1.5 ± 0.8	Severe (n = 18)

Highly significant.

ANOVA, analysis of variance.

As regard pulmonary function tests, all tests were significantly diminished in patients compared to controls (P < 0.001) (Table 3). Regarding IL-10 polymorphism, AA was more prevalent in patient group (66%) than control 1 (6%), while heterozygous mutant AG was detected in 20% of patients and 4% of the control. In contrast, wild genotype GG was found in 14% of patients and 90% of control with a highly statistically significant difference among both groups (P < 0.001) (Table 4).

Table 3.

Pulmonary Function Tests Among the Study Groups

Pulmonary function tests	Group 1 (n = 100) Patients, mean ± SD	Group 2 (n = 100) Control, mean ± SD	t-test	P
FEV1/FVC ratio	47.21 ± 9.31	72.01 ± 12.49	15.919	<0.001^a
FEV1% predicted	52.07 ± 8.24	79.21 ± 8.06	23.545	<0.001^a
FVC (L)	1.80 ± 0.64	2.86 ± 0.96	9.187	<0.001^a

Highly significant.

FEV1/FVC, forced expiratory volume in the first second/forced vital capacity; FEV1% predicted, percent of predicted forced expiratory volume.

Table 4.

Polymorphism of Interleukin-10 Among the Study Groups

PCR genotypes	Group 1 (n = 100) Patients, n (%)	Group 2 (n = 100) Control, n (%)	X ²	P
AA	66 (66%)	6 (6%)
AG	20 (20%)	4 (4%)	116.2	<0.001^a
GG	14 (14%)	90 (90%)

Highly significant.

PCR, polymerase chain reaction.

Among the different PCR genotypes, the serum level of IL-10 was significantly elevated in the GG genotype in comparison to other genotypes (P < 0.001) (Table 5). The serum level of IgE was significantly lower in the GG genotype in comparison to other genotypes (P < 0.001) (Table 6).

Table 5.

The Relationship Between Interleukin-10 (IL-10) Genotypes and Serum Level of IL-10 in Patients' Group

PCR genotypes	Group 1 (n = 100) Patients, mean ± SD IL-10 serum level	ANOVA test	P
AA	1.2 ± 1.2
AG	4.1 ± 2.3	88.119	<0.001^a
GG	6.5 ± 1.2

Highly significant.

Table 6.

Relationship Between Serum Level IgE and Interleukin-10 Genotypes in Patients' Group

PCR genotypes	IgE serum level, mean ± SD	ANOVA test	P
AA	183.5 ± 23.5
AG	161.7 ± 18.3	18.427	<0.001^a
GG	151.3 ± 4.5

Highly significant.

Discussion

Pediatric asthma is one of the distressing conditions with worldwide spread. In the past 20 years, there was a significant increase in the prevalence of asthma especially in children (Bilal and others 2016). The pathogenesis of asthma is a mixture of risk factors, including environmental factors and genetic factors among which were the polymorphisms in the IL-10 (Favarato and others 2014). So, the present study aimed to evaluate the association between polymorphism in IL-10 (-1082 G/A) and pediatric asthma in Egyptian children.

Concerning IgE level, this study reported that there was a highly significant increase in IgE level in asthmatic children compared to the control group (P < 0.001). This result agreed with previous studies that denoted that IgE was a major contributing factor for the development of bronchial hyperresponsiveness in asthma (Lama and others 2013; Hameed and others 2019). Moreover, Abo-Shanab and others (2020) reported the same result in their study explaining the role of T helper 2 cytokines and IgE in Egyptian asthmatic children.

IL-10 is a potent regulator of inflammatory responses and plays a critical role in controlling allergic airway inflammation. Polymorphisms in the IL-10 gene resulting in low IL-10 production have been associated with severe asthma, suggesting that IL-10 is a major determinant in the development and severity of airway allergy (Coomes and others 2017). The serum level of IL-10 in our study was found to be significantly decreased in the pediatric asthmatic group compared to the controls (P < 0.001). This was following the results of Jahromi and others (2014), Tomiita and others (2015), Osman and Elsaid (2019), and Zedan and others (2021). In contrast, Hussein and others (2014) found no significant difference in IL-10 serum levels between patients and controls. They claimed that IL-10 function is not only related to its level but also its receptor binding affinity.

In contrast, a higher serum level of IL-10 was detected in asthmatics compared with controls by other studies performed by Al-Hilali and Atta (2016), AlShehri and others (2017), and Bi and others (2018). Stelmach and others (2002) explained that elevated serum level of IL-10 in asthmatic patients was attributed to treatment with steroids, which enhances T cell secretion of IL-10.

Regarding pulmonary function tests, there were statistically significant differences between our studied groups in FEV1/FVC ratio, FEV1%predicted, and FVC (L). This was following Nagarch and others (2015) and Zhou and others (2019) who reported that all the pulmonary function test parameter values were less in asthmatic patients than control.

Concerning polymorphism of IL-10, homozygous mutant genotype AA was more prevalent in the patient group than the control one, while heterozygous mutant AG was detected in patients more than control. In contrast, wild genotype GG was found in patients less than control with a highly statistically significant difference among both groups. In parallel to our results, Kadhem and Darweesh (2017) had concluded that the GG genotype might have a preventive role against asthma, while other genotypes were more significantly associated with asthma. Similarly, Hussein and others (2011) and Shahin and Gouda (2017) reported that IL-10 (-1082 G/A) polymorphism may be contributing factors to the susceptibility and severity of childhood asthma. Other studies found the same result in adult asthma (Saba and others 2017; Ali and Settin, 2013). On the contrary, Kim and others (2011), in a Korean study, revealed that the IL-10 (-1082 G/A) polymorphism was not associated with pediatric asthma.

Our current study demonstrated that the serum level of IL-10 was significantly elevated in GG genotype in comparison to other genotypes. Moreover, Serum IL-10 was negatively correlated with the severity of asthma among the studied pediatric asthmatic group. This was in agreement with Kadhem and Darweesh (2017) who showed that IL-10 serum levels were significantly lower in the asthmatic patients with genotypes AA and AG than its level in genotypes GG. In addition, this was hand in hand with Smolnikova and others (2013) who reported that the G allele in the -1082-position correlated with higher production of IL-10 than the alternative allele A-1082. However, Zonoobi and others (2018) found partial agreement with our results as they reported that IL-10 was up to 10-fold downregulated in the group of severe asthma, but its expression level was not correlated with the severity of asthma. Besides, Hussein and others (2014) reported that neither IL-10 gene polymorphism influenced its serum level nor serum level of IL-10 was correlated with the severity of atopic asthma in their case–control study.

The serum level of IgE was significantly lower in the GG genotype in comparison to other genotypes. This was like Grant and others (2011) who reported that IL-10 promoter SNPs rs1800896, rs1800871, and rs1800872 were associated with high total IgE levels in the same direction as in atopy populations. Another study concluded that there was a significant decrease in serum IgE in wild-type GG than AA and AG genotypes, indicating that excess production of IgE was associated with A alleles in asthmatic children (Shahin and Gouda, 2017). In addition, Hussein and others (2014) reported that the same results that increased total IgE secretion were not affected by IL-10 serum levels, but it was significantly associated with heterozygosity or homozygosity for the risk allele of the IL-10 (-1082 G/A). In contrast, Kim and others (2011) observed no correlation between IL-10 (-1082 G/A) genotypes and total IgE levels.

Limitation and recommendation

It is a single-center study. Further studies on large scale with a higher number of participants, multicenter cooperation, and different ethnic groups are required to emphasize the actual role of this polymorphism on bronchial asthma among different age groups. Besides, additional future studies are required to benefit from our findings by targeting IL-10 polymorphism as a therapeutic modality.

Conclusion

There is an association between IL-10 (-1082 G/A) polymorphism and the development of bronchial asthma in Egyptian children. The serum level of IL-10 was significantly elevated in the GG genotype, while the serum level of IgE was significantly lower in comparison to other genotypes which denotes a possible protective effect of GG genotype against pediatric asthma.

Footnotes

Authors' Contributions

H.M.E., A.L.A., and N.A.I. designed and directed the project. H.M.E., A.L.A., N.A.I., N.A.S., and S.H. performed the experiments, analyzed the data, and wrote the article which was revised by all coauthors.

Data Sharing Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for this article.

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