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Vol. 51. Issue 11.
Pages 571-578 (November 2015)
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Vol. 51. Issue 11.
Pages 571-578 (November 2015)
Original Article
DOI: 10.1016/j.arbr.2015.02.013
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Associations of IL-2 and IL-4 Expression and Polymorphisms With the Risks of Mycoplasma pneumoniae Infection and Asthma in Children
Relación entre la expresión de IL-2 e IL-4 y sus polimorfismos y los riesgos de padecer infección por Mycoplasma pneumoniae y asma en niños
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Rong-Shan Wanga, Hong-Xing Jina, Shi-Qiang Shangb,
Corresponding author
shiqiangshang945@gmail.com

Corresponding author.
, Xi-Yong Liua, Shu-Jun Chena, Zhi-Biao Jina
a Department of Pediatrics, Yiwu Maternity and Child Care Hospital, Zhejiang, China
b Department of Pediatrics, Laboratory of the Children's Hospital of Zhejiang Province, Zhejiang, China
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Tables (6)
Table 1. Detection of M. pneumoniae Infection Using Different Samples.
Table 2. Genotype Distribution and Risk Association in Asthmatic Children and Healthy Controls.
Table 3. Genotype Distribution and Risk Association in Asthmatic Children and Healthy Controls Based on GINA Classification of Asthma Control.
Table 4. Genotype Distribution and Risk Association in Asthmatic Children and Healthy Controls Based on GINA Classification of Asthma Severity.
Table 5. IL-2 and IL-4 Serum Expression Level in M. pneumoniae-negative and Positive Subjects.
Table 6. Genotype Distribution and Risk Association in all Subjects With Low and High M. pneumoniae Loads.
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Abstract
Introduction

Asthma is an inflammatory disorder of the airways and the symptoms of asthma could be exacerbated by Mycoplasma pneumoniae infection. Interleukin-2 and interleukin-4 have been implicated in immune and inflammatory reactions. We examined the associations of IL2 and IL4 polymorphisms and expression with the risks of asthma and M. pneumoniae infection in children.

Methods

A total of 392 asthmatic children and 849 controls were recruited into the study. Eight polymorphisms in IL2 and IL4 were genotyped with Sequenom MassARRAY platform. M. pneumoniae infection and copy number was determined with fluorescence PCR. IL-2 and IL-4 serum expression levels were determined by using ELISA.

Results

We found a significant association of IL2 rs6534349 polymorphism with increased asthma risk (heterozygotes, P=.029; homozygous variants; P=.013) and of IL4 rs2227284 polymorphism with reduced asthma risk (heterozygotes, P=.026; homozygous variants; P=.001). Besides, the association of other polymorphisms, except rs2070874 polymorphism, became apparent when the asthmatic children were grouped according to GINA classification of asthma control and severity. In addition, IL-2 and IL-4 serum expression levels were significantly higher in M. pneumoniae negative (P=.038) and positive (P=.011) subjects respectively. This observation holds true among asthmatic patients (P=.016 for IL-2 and P=.042 for IL-4), but only the IL-4 observation remained correct among non-asthmatic controls (P=.032). We also observed that the rs6534349 GG genotype was significantly associated with increased odds of getting high load M. pneumoniae infection (P=.0376).

Conclusions

IL2 and IL4 could be important biomarkers for estimating the risks of asthma and M. pneumoniae infection in children.

Keywords:
Asthma
Children
Interleukin-2
Interleukin-4
Mycoplasma pneumoniae
Resumen
Introducción

El asma es una afección inflamatoria de las vías respiratorias. Las infecciones por Mycoplasma pneumoniae pueden exacerbar los síntomas del asma. Se ha demostrado que la interleucina 2 y la interleucina 4 participan en las reacciones inmunitarias e inflamatorias. Hemos estudiado la relación entre los polimorfismos de la IL2 y la IL4 y su expresión y el riesgo de padecer asma e infección por M. pneumoniae en niños.

Métodos

Se reclutó a 392 niños asmáticos y 849 controles para el estudio. Se genotiparon 8 polimorfismos en IL2 e IL4 con la plataforma MassARRAY de Sequenom. La infección por M. pneumoniae y el número de copias se establecieron mediante PCR fluorescente. Los niveles séricos de expresión de IL-2 e IL-4 se midieron con ELISA.

Resultados

Hallamos una relación significativa entre el polimorfismo rs6534349 de IL2 y el aumento de riesgo de sufrir asma (heterocigóticos, p=0,029; variantes homocigóticas, p=0,013), así como entre el polimorfismo rs2227284 de IL4 y una reducción del riesgo de padecer asma (heterocigóticos, p=0,026; variantes homocigóticas, p=0,001). Además, la relación con otros polimorfismos, excepto el rs2070874, se hizo evidente al agrupar a los niños asmáticos según la clasificación GINA de control y gravedad del asma. Asimismo, los niveles séricos de expresión de IL-2 e IL-4 fueron significativamente mayores en los sujetos no infectados (p=0,038) e infectados (p=0,011) por M. pneumoniae, respectivamente. Esta observación también se cumple entre los pacientes asmáticos (p=0,016 para IL-2 y p=0,042 para IL-4), pero en los controles no asmáticos solo se cumple en el caso de la IL-4 (p=0,032). Del mismo modo, observamos que el genotipo GG rs6534349 estaba claramente relacionado con un aumento de las posibilidades de tener una infección con alta carga de M. pneumoniae (p=0,0376).

Conclusiones

La IL2 y la IL4 podrían ser biomarcadores importantes para calcular el riesgo de padecer asma, así como infección por M. pneumoniae, en niños.

Palabras clave:
Asma
Niños
Interleucina-2
Interleucina-4
Mycoplasma pneumoniae
Full Text
Introduction

Asthma is a complex chronic inflammatory disease of the airways characterized by airflow obstruction and hyperresponsiveness. In children, asthma arises principally from allergic inflammation, and can lead to varying degrees of airway obstruction, including but not limited to dyspnea, coughing, chest tightness, and recurrent episodes of wheezing. In China and the rest of the world, asthma in children represents a major public health concern in pediatric pulmonology, and the prevalence of childhood asthma increases every year.1,2

Mycoplasma pneumoniae is an important pathogen which has been primarily recognized as a causative agent of community-acquired pneumonia, especially among children.3 The link between M. pneumoniae infection and asthma was first suspected several decades ago.4 More recently, various lines of evidence have emerged and M. pneumoniae infection has been implicated in the onset and exacerbation of asthma.5,6M. pneumoniae infection damages respiratory epithelial cells, thus increasing airway reactivity and resulting in the activation of a large number of T cells.7 T cells trigger the activation of B cells, which produce antibodies and release inflammatory cytokines, which in turn contribute to the development of asthma-related symptoms.

Despite the complexity of the mechanisms underlying the onset of asthma, interleukins (ILs), especially IL-2 and IL-4, are known to play a central role in this process.8 IL-2 promotes the maturation of primitive T cells, while IL-4, through its complex interaction with IL-12, helps to determine whether T cells should differentiate into Th1 or Th2 cells, thus producing different types of cytokines.9 These events cause major pathophysiological changes during asthma, and therefore play a key role in the development of the disease. In addition, the important role of IL-2 and IL-4 in the adaptive immune system suggests that cytokines could affect the ability of immune cells to fight off M. pneumoniae infection. We therefore hypothesized that variations in IL-2 and IL-4 levels and function could be associated with the risk of asthma and M. pneumoniae infection.10,11 A potential factor which could influence the interindividual variation in IL-2 and IL-4 levels and function, and therefore the risk of asthma, is single nucleotide polymorphisms (SNPs) within the genes encoding the cytokines.12,13 In this study, we aimed to establish how SNPs in IL-2 and IL-4 genes may be associated with the risk of asthma in children. In particular, we addressed the rs6822844, rs6534349, rs2069762 and rs3136534 polymorphisms of IL-2, and rs2243250, rs2070874, rs2227284 and rs2243290 polymorphisms of IL-4. We also aimed to investigate the presence of differential serum expression levels of IL-2 and IL-4 in M. pneumoniae-positive and negative subjects, and to examine the relationship between SNPs and the risk of high-load M. pneumoniae infection.

MethodsSubjects

The study was approved by the ethics committee of the Yiwu Maternity and Child Care Hospital, Zhejiang (Ref. No: 2009/PED/0215.045). A total of 392 children with asthma and 849 non-asthmatic controls aged between 4 and 15 years old were recruited from the Yiwu Maternity and Child Care Hospital and the Children's Hospital of Zhejiang Province between March 2009 and October 2013. Asthma in children was ascertained by routine diagnosis based on the Global Initiative for Asthma (GINA) guidelines. Controls were children without asthma or allergic symptoms who visited the same hospitals for other medical problems unrelated to asthma. Among the controls, a total of 83 had stridor, 89 had foreign body aspiration, 80 had persistent cough, 66 underwent diagnosis for pulmonary infections, and 531 were healthy children who attended the hospital for follow-up after diagnosis of a transient respiratory infection. Controls and cases were matched by frequency in terms of gender and age. All subjects were of Chinese Han ethnicity. Informed consent was obtained from the parents of the participating subjects before inclusion in the study.

Detection of Mycoplasma pneumoniae Infection

DNA was extracted from respiratory specimens (throat swab, No.=528; bronchoalveolar lavage, No.=432; sputum specimens, No.=204; bronchial aspirate, No.=77) obtained from the subjects. Bronchoalveolar lavage was performed on 318 controls (i.e. those who had stridor, foreign body aspiration, persistent cough and who underwent diagnosis for pulmonary infections) as part of their diagnosis or treatment, and on 114 asthmatic children for identification of infectious agents, cytokines and cellular profiles (either for diagnostic purposes or as part of another ongoing research study). A single bronchoalveolar lavage was performed on each subject. Detection of Mycoplasma pneumoniae infection among the subjects was performed with fluorescence PCR Mycoplasma pneumoniae Detection Kit (Acon Biotech, Hangzhou, China), according to the manufacturer's instructions.

ELISA Quantification of IL-2 and IL-4

Serum was isolated from the blood samples collected and diluted 1:4 in sample diluent before being used for ELISA quantification of IL-2 and IL-4. The serum IL-2 and IL-4 concentrations were determined with the Human Interleukin 2/IL-2 ELISA Kit and Human Interleukin 4/IL-4 ELISA Kit (Xinqidi Biological Technology Co. Ltd., Wuhan, China), which have a sensitivity of <0.40pg/ml and <0.20pg/ml respectively, according to the manufacturer's protocols.

SNP Genotyping

DNA was isolated from subjects’ blood samples using TIANamp blood DNA kit (Tiangen Biotech, Beijing, China). Polymorphisms were genotyped on the DNA isolated using the Sequenom MassARRAY platform (Sequenom, San Diego, USA), according to the manufacturer's instructions. Genotyping was repeated in 10% of the samples for confirmation purposes. The reproducibility rate of the genotypes was 100%.

Statistical Analysis

The distribution of genotypes in asthmatic children and non-asthmatic controls was compared using a χ2 test, and the association between the polymorphisms and asthma risk was evaluated by logistic regression analysis. To examine the relationship between polymorphisms and the risk of predisposition to high-load M. pneumoniae infection, the genotype distribution was compared using the χ2 test followed by logistic regression analysis. Expression of IL-2 and IL-4 in M. pneumoniae-positive and M. pneumoniae-negative patients was compared using a t-test to determine any significant difference in the expression between the two groups. For all analyses, P<.05 was considered significant.

ResultsSubjects Characteristics and M. pneumoniae Infection

Cases and controls were frequency-matched in terms of gender and age. No significant differences were found for either gender (P=.99) or mean age (P=.98). Based on GINA classification, 177 cases were controlled asthma, 118 were partially controlled asthma and 97 were uncontrolled asthma. In addition, 102 were classified as severe, 80 moderate and 210 mild.

Detection of M. pneumoniae Infection

A total of 344 subjects tested positive for M. pneumoniae infection, of which 130 were children with asthma and 214 were non-asthmatic controls. Of the 897 M. pneumoniae-negative subjects, 262 were children with asthma and 635 were non-asthmatic controls. Table 1 shows the prevalence of M. pneumoniae detected in different types of specimens. The type of specimen used did not significantly affect the detection of M. pneumoniae (P=.70).

Table 1.

Detection of M. pneumoniae Infection Using Different Samples.

Specimen  M. pneumoniae positive  M. pneumoniae negative  P-value 
Throat swab (No.=528)  154 (29.17%)  374 (70.83%)  .70
Bronchoalveolar lavage (No.=432)  113 (26.16%)  319 (73.84%) 
Sputum specimens (No.=204)  54 (26.47%)  150 (73.53%) 
Bronchial aspirate (No.=77)  23 (29.87%)  54 (70.13%) 
Total  344 (27.72%)  897 (72.28%) 

The number of bacterial copies in the M. pneumoniae-positive subjects ranged from 6.43×103 to 6.48×107organismsml−1, with a median and mean of 3.96×105 and 1.67×107organismsml−1, respectively. A total of 206 and 138 subjects had bacterial load lower and higher than mean, respectively.

Genotype Distribution in Asthmatic Children and Healthy Controls

Of the 8 polymorphisms studied, significant differences between cases and controls were observed in only 2, namely rs6534349 polymorphism of IL-2 (P=.006) and rs2227284 polymorphism of IL-4 (P=.001) (Table 2). No deviation from Hardy-Weinberg equilibrium was observed for any of the 8 polymorphisms (P>.05).

Table 2.

Genotype Distribution and Risk Association in Asthmatic Children and Healthy Controls.

SNP  Genotype  Cases (No.)  Controls (No.)  P  Odds ratio  P 
rs6822844GG  277  70.66  562  66.20  .595  Reference  – 
GT  104  26.53  249  29.33    0.847 (0.647–1.110)  .230 
TT  11  2.81  38  4.48    0.587 (0.296–1.167)  .129 
rs6534349AA  279  71.17  665  78.33  .006  Reference  – 
AG  98  25.00  170  20.02    1.374 (1.033–1.828)  .029 
GG  15  3.83  14  1.65    2.554 (1.216–5.361)  .013 
rs2069762GG  41  10.46  78  9.19  .545  Reference  – 
GT  153  39.03  357  42.05    0.815 (0.534–1.245)  .344 
TT  198  50.51  414  48.76    0.910 (0.601–1.377)  .655 
rs3136534AA  176  44.90  346  40.75  .223  Reference  – 
AC  180  45.92  402  47.35    0.880 (0.684–1.133)  .322 
CC  36  9.18  101  11.90    0.701 (0.460–1.068)  .098 
rs2243250TT  165  42.09  333  39.22  .536  Reference  – 
TC  177  45.15  412  48.53    0.867 (0.671–1.121)  .276 
CC  50  12.76  104  12.25    0.970 (0.660–1.427)  .878 
rs2070874TT  167  42.60  337  39.69  .531  Reference  – 
TC  176  44.90  410  48.29    0.866 (0.671–1.119)  .272 
CC  49  12.50  102  12.02    0.969 (0.658–1.429)  .875 
rs2227284TT  225  57.40  408  48.06  .001  Reference  – 
TG  146  37.24  353  41.58    0.750 (0.583–0.965)  .026 
GG  21  5.36  88  10.37    0.433 (0.262–0.716)  .001 
rs2243290AA  174  44.39  331  38.99  .190  Reference  – 
AC  173  44.13  416  49.00    0.791 (0.613–1.021)  .072 
CC  45  11.48  102  12.01    0.839 (0.565–1.247)  .386 
Association of the Polymorphisms and Asthma Risk

Significant risk association was only observed for the IL-2 rs6534349 and IL-4 rs2227284 polymorphisms (Table 2). For the IL-2 rs6534349 polymorphism, the heterozygous AG genotype and homozygous variant GG genotype resulted in a 1.374-fold (95% CI=1.033–1.828, P=.029) and a 2.554-fold (95% CI=1.216–5.361, P=.013) increase in risk, respectively. Heterozygous TG genotype of the IL-4 rs2227284 polymorphism, meanwhile, showed an odds ratio of 0.750 (95% CI=0.583–0.965, P=.026), while the homozygous GG genotype showed an odds ratio of 0.433 (95% CI=0.262–0.716, P=.001).

Asthmatic Risk Association Based on GINA Classification of Asthma Control

The genotype distribution and risk association of the polymorphisms based on GINA classification of asthma control are shown in Table 3. For controlled asthma, only the IL-2 rs2069762 polymorphism was found to be associated with risk of the disorder (P=.047). None of the polymorphisms in the IL-4 gene appeared to confer a risk or protect its carriers from asthma. In the partially controlled asthma group, however, 3 IL-2 polymorphisms (rs6822822, rs2069762, rs6534349) and 1 IL-4 polymorphism (rs2227284) showed a risk association with asthma (P=.040, .001, .020 and .032, respectively). In the uncontrolled group, 5 polymorphisms, i.e. IL-2 rs6534349 (P=.001), rs2069762 (P=.038), and rs3136543 (P=.038), as well as IL-4 rs2243250 (P=.031) and rs2227284 (P=.004), showed significant asthmatic risk association.

Table 3.

Genotype Distribution and Risk Association in Asthmatic Children and Healthy Controls Based on GINA Classification of Asthma Control.

GINA classification of control  SNP  Genotype  Cases (No.)  Controls (No.)  Odds ratio  P 
Controlledrs6822844GG  64  562  Reference  – 
GT  30  249  1.058 (0.668–1.673)  .809 
TT  38  0.693 (0.208–2.309)  .550 
rs6534349AA  73  665  Reference  – 
AG  20  170  1.071 (0.635–1.807)  .795 
GG  14  2.602 (0.834–8.115)  .099 
rs2069762GG  78  Reference  – 
GT  32  357  1.747 (0.600–5.085)  .305 
TT  61  414  2.873 (1.101–8.130)  .047 
rs3136534AA  42  346  Reference  – 
AC  46  402  0.945 (0.607–1.470)  .802 
CC  101  0.734 (0.345–1.559)  .421 
rs2243250TT  40  333  Reference  – 
TC  42  412  0.848 (0.537–1.339)  .481 
CC  15  104  1.200 (0.637–2.261)  .571 
rs2070874TT  47  337  Reference  – 
TC  43  410  0.752 (0.485–1.165)  .202 
CC  102  0.492 (0.215–1.122)  .091 
rs2227284TT  57  408  Reference  – 
TG  33  353  0.669 (0.425–1.051)  .081 
GG  88  0.569 (0.251–1.290)  .177 
rs2243290AA  44  331  Reference  – 
AC  41  416  0.741 (0.473–1.161)  .191 
CC  12  102  0.885 (0.450–1.739)  .723 
Partially controlledrs6822844GG  89  562  Reference  – 
GT  24  249  0.608 (0.378–0.978)  .040 
TT  38  0.830 (0.318–2.167)  .704 
rs6534349AA  89  665  Reference  – 
AG  23  170  1.010 (0.620–1.647)  .965 
GG  6  14  3.202 (1.199–8.546)  .020 
rs2069762GG  17  78  Reference  – 
GT  27  357  0.347 (0.180–0.667)  .001 
TT  74  414  0.820 (0.459–1.464)  .502 
rs3136534AA  48  346  Reference  – 
AC  56  402  1.004 (0.665–1.515)  .984 
CC  14  101  0.999 (0.529–1.886)  .998 
rs2243250TT  40  333  Reference  – 
TC  63  412  1.273 (0.835–1.940)  .262 
CC  15  104  1.200 (0.637–2.261)  .571 
rs2070874TT  53  337  Reference  – 
TC  47  410  0.728 (0.479–1.107)  .138 
CC  18  102  1.122 (0.629–2.001)  .696 
rs2227284TT  71  408  Reference  – 
TG  39  353  0.634 (0.418–0.962)  .032 
GG  88  0.522 (0.242–1.124)  .096 
rs2243290AA  47  331  Reference  – 
AC  54  416  0.914 (0.602–1.386)  .673 
CC  17  102  1.173 (0.645–2.133)  .599 
Uncontrolledrs6822844GG  124  562  Reference  – 
GT  50  249  0.910 (0.634–1.305)  .608 
TT  38  0.357 (0.108–1.177)  .090 
rs6534349AA  117  665  Reference  – 
AG  55  170  1.838 (1.280–2.641)  .001 
GG  14  2.029 (0.717–5.742)  .182 
rs2069762GG  20  78  Reference  – 
GT  50  357  0.546 (0.307–0.969)  .038 
TT  107  414  1.008 (0.590–1.721)  .976 
rs3136534AA  86  346  Reference  – 
AC  78  402  0.780 (0.556–1.095)  .151 
CC  13  101  0.517 (0.277–0.966)  .038 
rs2243250TT  85  333  Reference  – 
TC  72  412  0.684 (0.484–0.967)  .031 
CC  20  104  0.753 (0.441–1.285)  .299 
rs2070874TT  67  337  Reference  – 
TC  86  410  1.055 (0.743–1.497)  .764 
CC  24  102  1.183 (0.706–1.983)  .522 
rs2227284TT  97  408  Reference  – 
TG  74  353  0.881 (0.631–1.231)  .460 
GG  6  88  0.286 (0.121–0.675)  .004 
rs2243290AA  83  331  Reference  – 
AC  78  416  0.747 (0.531–1.051)  .094 
CC  16  102  0.625 (0.350–1.116)  .112 
Asthmatic Risk Association Based on GINA Classification of Asthma Severity

The distribution of polymorphism genotypes in patients with different levels of asthma severity is shown in Table 4, along with the corresponding risk association. For individuals with severe asthma, significant associations were observed for rs2243290 (P=.002). For moderate asthma, only rs6534349 was significant (P=.007). Finally, rs2069762 and rs2227284 polymorphisms were associated with a risk of mild asthma (P=.007 and P=.008, respectively).

Table 4.

Genotype Distribution and Risk Association in Asthmatic Children and Healthy Controls Based on GINA Classification of Asthma Severity.

GINA classification of severity  SNP  Genotype  Cases (No.)  Controls (No.)  Odds ratio  P 
Severers6822844GG  73  562  Reference  – 
GT  28  249  0.865 (0.546–1.372)  .539 
TT  38  0.202 (0.027–1.497)  .117 
rs6534349AA  73  665  Reference  – 
AG  26  170  1.393 (0.863–2.247)  .174 
GG  14  1.952 (0.548–6.952)  .302 
rs2069762GG  10  78  Reference  – 
GT  24  357  0.524 (0.241–1.140)  .103 
TT  68  414  1.281 (0.632–2.596)  .491 
rs3136534AA  46  346  Reference  – 
AC  46  402  0.860 (0.558–1.327)  .497 
CC  10  101  0.744 (0.362–1.528)  .421 
rs2243250TT  44  333  Reference  – 
TC  45  412  0.826 (0.532–1.283)  .396 
CC  13  104  0.946 (0.490–1.824)  .868 
rs2070874TT  44  337  Reference  – 
TC  47  410  0.878 (0.567–1.357)  .558 
CC  11  102  0.826 (0.411–1.658)  .590 
rs2227284TT  60  408  Reference  – 
TG  36  353  0.693 (0.447–1.073)  .100 
GG  88  0.463 (0.194–1.107)  .083 
rs2243290AA  54  331  Reference  – 
AC  34  416  0.501 (0.318–0.787)  .002 
CC  14  102  0.841 (0.448–1.577)  .589 
Moderaters6822844GG  53  562  Reference  – 
GT  24  249  1.022 (0.616–1.693)  .932 
TT  38  0.837 (0.250–2.803)  .773 
rs6534349AA  56  665  Reference  – 
AG  19  170  1.327 (0.768–2.293)  .310 
GG  5  14  4.241 (1.473–12.208)  .007 
rs2069762GG  78  Reference  – 
GT  24  357  1.310 (0.442–3.885)  .625 
TT  52  414  2.449 (0.861–6.966)  .093 
rs3136534AA  29  346  Reference  – 
AC  42  402  1.246 (0.760–2.044)  .382 
CC  101  1.063 (0.487–2.319)  .877 
rs2243250TT  33  333  Reference  – 
TC  36  412  0.881 (0.538–1.445)  .617 
CC  11  104  1.067 (0.521–2.186)  .858 
rs2070874TT  35  337  Reference  – 
TC  34  410  0.798 (0.487–1.307)  .371 
CC  11  102  1.038 (0.509–2.117)  .917 
rs2227284TT  42  408  Reference  – 
TG  34  353  0.935 (0.582–1.503)  .783 
GG  88  0.441 (0.154–1.263)  .127 
rs2243290AA  38  331  Reference  – 
AC  35  416  0.732 (0.452–1.185)  .205 
CC  102  0.597 (0.259–1.379)  .227 
Mildrs6822844GG  151  562  Reference  – 
GT  52  249  0.777 (0.548–1.101)  .156 
TT  38  0.685 (0.300–1.565)  .370 
rs6534349AA  150  665  Reference  – 
AG  53  170  1.382 (0.968–1.972)  .074 
GG  14  2.216 (0.879–5.587)  .091 
rs2069762GG  27  78  Reference  – 
GT  61  357  0.493 (0.294–0.826)  .007 
TT  122  414  0.851 (0.525–1.378)  .512 
rs3136534AA  101  346  Reference  – 
AC  92  402  0.784 (0.571–1.076)  .132 
CC  17  101  0.576 (0.329–1.009)  .053 
rs2243250TT  88  333  Reference  – 
TC  96  412  0.881 (0.538–1.445)  .617 
CC  26  104  0.946 (0.579–1.543)  .824 
rs2070874TT  88  337  Reference  – 
TC  95  410  0.887 (0.641–1.226)  .469 
CC  27  102  1.013 (0.624–1.646)  .956 
rs2227284TT  123  408  Reference  – 
TG  76  353  0.714 (0.518–0.983)  .038 
GG  11  88  0.414 (0.214–0.801)  .008 
rs2243290AA  82  331  Reference  – 
AC  104  416  1.009 (0.730–1.394)  .956 
CC  24  102  0.949 (0.572–1.575)  .841 
IL-2 and IL-4 Expression in M. pneumoniae-negative and Positive Subjects

Table 5 shows serum expression levels (in terms of concentration) of IL-2 and IL-4 in M. pneumoniae-negative and positive subjects. Mean IL-2 concentration in M. pneumoniae-negative subjects was significantly higher than in M. pneumoniae-positive subjects (P=.038). The opposite was observed for IL-4 (P=.011).

Table 5.

IL-2 and IL-4 Serum Expression Level in M. pneumoniae-negative and Positive Subjects.

Subjects  Gene  Expression  M. pneumoniae negative  M. Pneumoniae positive  P 
OverallIL-2Range (pg/ml)  0.85–156.36  0.46–144.36   
Mean±SD (pg/ml)  80.23±45.98  74.55±41.98  .038 
IL-4Range (pg/ml)  0.22–15.34  0.83–15.89   
Mean±SD (pg/ml)  7.72±4.34  8.41±4.28  .011 
AsthmaticsIL-2Range (pg/ml)  1.56–156.36  0.52–143.99   
Mean±SD (pg/ml)  81.74±45.69  74.58±41.48  .016 
IL-4Range (pg/ml)  0.46–15.34  1.13–15.89   
Mean±SD (pg/ml)  7.93±4.29  8.61±4.32  .042 
ControlIL-2Range (pg/ml)  0.85–151.18  0.46–144.36   
Mean±SD (pg/ml)  77.73±46.51  74.46±43.25  .492 
IL-4Range (pg/ml)  0.22–11.42  0.83–13.97   
Mean±SD (pg/ml)  7.50±4.38  8.21±4.23  .032 

When the subjects were classified according to presence of asthma, a similar trend was observed for asthmatic patients (P=.016 for IL-2; P=.042 for IL-4). Among non-asthmatic controls, however, IL-2 levels in M. pneumoniae-positive and negative subjects did not differ significantly (P=.492). Nonetheless, as in asthmatic subjects, a significantly lower mean concentration of IL-4 was observed among M. pneumoniae-negative subjects compared to M. pneumoniae-positive subjects (P=.032).

Genotype Distribution in M. pneumoniae-positive Subjects With Low- and High-load Infections

Table 6 summarizes the distribution of genotypes of the 8 polymorphisms in M. pneumoniae-positive subjects with low and high bacterial loads. No significant difference was observed between the 2 study groups in terms of the genotypic distributions of all 8 polymorphisms (P>.05). None of the genotypic distributions deviated significantly from the Hardy-Weinberg equilibrium (P>.05).

Table 6.

Genotype Distribution and Risk Association in all Subjects With Low and High M. pneumoniae Loads.

SNP  Genotype  Low M. pneumoniae load (No.)  High M. pneumoniae load (No.)  P  Odds ratio  P 
rs6822844GG  147  71.35  92  66.67  .6427  Reference  – 
GT  52  25.24  41  29.71    1.2598 (0.7755–2.0467)  .3509 
TT  3.40  3.62    1.1413 (0.3518–3.7026)  .8258 
rs6534349AA  145  70.39  109  78.99  .1488  Reference  – 
AG  56  27.18  28  20.29    0.6651 (0.3965–1.1157)  .1223 
GG  2.43  0.72    0.2661 (0.0306–2.3102)  .2299 
rs2069762GG  23  11.17  14  10.14  .686  Reference  – 
GT  77  37.38  58  42.03    1.2375 (0.5865–2.6111)  .5760 
TT  106  51.46  66  47.83    1.0229 (0.4919–2.1269)  .9516 
rs3136534AA  89  43.20  57  41.30  .9356  Reference  – 
AC  96  46.60  66  47.83    1.0735 (0.6797–1.6952)  .7611 
CC  21  10.19  15  10.87    1.1153 (0.5314–2.3407)  .7730 
rs2243250TT  87  42.23  54  39.13  .8211  Reference  – 
TC  98  47.57  68  49.28    1.1179 (0.7062–1.7697)  .6343 
CC  21  10.19  16  11.59    1.2275 (0.5894–2.5567)  .5840 
rs2070874TT  85  41.26  52  37.68  .794  Reference  – 
TC  98  47.57  69  50.00    1.1509 (0.7247–1.8278)  .5515 
CC  23  11.17  17  12.32    1.2082 (0.5907–2.4712)  .6044 
rs2227284TT  109  52.91  66  47.83  .1019  Reference  – 
TG  86  41.75  56  40.58    1.0754 (0.6825–1.6945)  .7540 
GG  11  5.34  16  11.59    2.4022 (1.0514–5.4887)  .0376 
rs2243290AA  87  42.23  54  39.13  .844  Reference  – 
AC  97  47.09  68  49.28    1.1294 (0.7132–1.7887)  .6038 
CC  22  10.68  16  11.59    1.1717 (0.5658–2.4266)  .6696 
Relationship of Polymorphisms With Risk of Predisposition to High-load M. pneumoniae Infection

The association between the 8 polymorphisms and the risk of predisposition to high-load M. pneumoniae infection is also shown in Table 6. Significant association was observed only in IL-4 rs2227284 polymorphism (P=.0376). No statistically significant association was observed for the other polymorphisms (P>.05).

Discussion

Asthma is a disorder of the airways which arises principally from chronic inflammation of the respiratory system, the symptoms of which are thought to be exacerbated by M. pneumoniae infection.5,6 Eradication of M. pneumoniae infection and the development of asthma may be influenced by cytokines, key mediators of immune and inflammatory reactions. Two important cytokines linked to the development of asthma are IL-2 and IL-4. We hypothesized that polymorphisms within IL-2 and IL-4 genes could be associated with a risk of asthma and M. pneumoniae infection in children.

We investigated the association of 4 IL-2 polymorphisms and 4 IL-4 polymorphisms with risk of asthma in children. Our results showed that the IL-2 rs6534349 polymorphism and IL-4 rs2227284 polymorphism could significantly increase and decrease asthma risk, respectively. The association appeared to be dose-dependent. In other words, the risk and protective effects of the polymorphisms were stronger when the variant alleles were present in 2 copies (homozygous variant) than in 1 copy (heterozygous). We also analyzed the association of the polymorphisms with different levels of asthma control and severity based on GINA classification. We found that among controlled patients, only one polymorphism (rs2069762) was associated with asthma risk, while four polymorphisms (rs6822844, rs6534349, rs2069762 and rs2227284) were associated with asthma risk among the partially controlled group, and five polymorphisms (rs6534349, rs2069762, rs3136534, rs2243250 and rs2227284) were associated with asthma risk among uncontrolled group. The association of increasing numbers of polymorphisms with increasing loss of control suggests the involvement of an extensive network of genetic interactions and highlights the complexity of the disease.

The number of polymorphisms significantly associated with asthma risk, however, was similar in patients with different levels of asthma severity. Mild asthma patients demonstrated risk associated with two polymorphisms (rs2069762 and rs2227284), whereas moderate and severe patients each demonstrated risk associated with one polymorphism (rs6534349 for moderate, rs2243290 for severe). It is interesting to note that entirely different polymorphisms were involved in asthmatic patients with different degrees of severity, suggesting that each polymorphism could exert a unique effect which contributes to the development of asthma. Nonetheless, it should be pointed out that when the analysis was performed according to levels of asthma severity, the sample size became too low to assure reliable data interpretation.14 This is one of the limitations of this study.

It may be that these polymorphisms could change the expression of protein products, thereby causing the risk modifications mentioned above.15 Specifically, it seems that the variant alleles of rs6822844, rs2069762 and rs3136534 polymorphisms could decrease the expression of protein products, while those of rs6534349, rs2243250, rs2227284 and rs2243290 polymorphisms could increase the expression of protein products. Our hypothesis is based on the fact that IL-2 is a pro-inflammatory cytokine, so increased expression of IL-2 may produce a higher level of inflammation, facilitating the development of asthma, and vice versa.16 In contrast, IL-4 is an anti-inflammatory protein, and increased IL-4 expression can lead to reduced inflammation, which in turn would protect the host from asthma, and vice versa. However, further research is needed to confirm this.

A lack of association was observed between rs2070874 polymorphism and the risk of asthma in all classifications of study subjects (i.e. overall, classification based on GINA control, and classification based on GINA severity), suggesting that this polymorphism is not involved in the development of asthma.

The difference in IL-2 and IL-4 serum expression levels was also compared between subjects with and without M. pneumoniae infection, who were seen to have significantly higher levels of IL-2 and IL-4 expression, respectively. This finding was not unexpected, as inflammation is one of the earliest responses of the immune system.17 It seems likely that a higher IL-2 level, which triggers inflammation, plays a role in fighting off pathogenic infections.18 Subjects with a higher level of IL-2 were therefore M. pneumoniae-negative. The relationship between IL-2 levels and M. pneumoniae infection appeared to be significant only among asthmatic patients, but not the controls. This observation concurs with the fact that IL-2 plays an important role in the pathogenesis of asthma.19 In contrast, IL-4 suppresses inflammation, and a high level of IL-4 does not allow the host immune system to function in an optimum state. Patients with higher IL-4 were therefore M. pneumoniae-positive, regardless of whether they were asthmatic or non-asthmatic.

A higher M. pneumoniae load has been associated with worse clinical severity in respiratory diseases.20 Therefore, we further compared the distribution of the 8 IL-2 and IL-4 polymorphisms in subjects with high-load and low-load M. pneumoniae to find out whether any of the polymorphisms is associated with low-load infection. No significant difference was observed in the distribution of the polymorphisms between the two groups, but significant risk association was observed for the homozygous variant genotype of rs2227284 polymorphism. This suggests that the homozygous variant genotype of rs2227284 polymorphism could predispose its carriers to high-load M. pneumoniae infection, and is in line with Wang et al.21 who showed that the rs2227284 polymorphism could affect humoral response.

In fact, several studies have investigated the association of various polymorphisms within IL-2 and IL-4 genes with asthma risk.22–24 However, the majority of these studies incorporated only one or two polymorphisms in their analysis. This may result in misleading interpretations, as the effect of a polymorphism could be compensated by the presence of the other polymorphisms within the same gene.25 The strength of our study lies in the incorporation of a large number of polymorphisms within two closely-related genes.

Conclusions

In this study, we identified two polymorphisms which could serve as predictive biomarkers for estimating asthma risk in children. We also showed that serum IL-2 and IL-4 levels differed significantly between subjects with and without M. pneumoniae infection. Among M. pneumoniae-positive subjects, the rs2227284 GG genotype was significantly linked to increased likelihood of presenting high-load infection. However, we recognize that this study has several limitations, such as the small sample size, especially in terms of subjects with M. pneumoniae infection. Further studies with larger sample sizes are needed in this area.

Funding

This study was supported by the personal funds of the authors.

Authors’ Contributions

R.S.W. and H.X.J. recruited the study subjects, extracted DNA from all the samples and performed detection of Mycoplasma pneumoniae infection. S.Q.S. genotyped the SNPs and drafted the manuscript. X.Y.L. and S.J.C. quantified the serum expression levels of IL-2 and IL-4 and performed statistical analysis. Z.B.J. was responsible for the study conception, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.

Conflict of Interests

The authors declare they have no conflict of interest directly or indirectly related to the manuscript contents.

Acknowledgements

We thank the nurses from our hospitals for their help in subject recruitment. This study was funded by the authors.

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Please cite this article as: Wang R-S, Jin H-X, Shang S-Q, Liu X-Y, Chen S-J, Jin Z-B. Relación entre la expresión de IL-2 e IL-4 y sus polimorfismos y los riesgos de padecer infección por Mycoplasma pneumoniae y asma en niños. Arch Bronconeumol. 2015;51:571–578.

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