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.

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.

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.

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

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.

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