Abstract

Previously, we reported genetic associations between severe respiratory syncytial virus (RSV) bronchiolitis in infants and polymorphisms in the interleukin (IL)–4 and IL-4 receptor α (IL-4Rα) genes, providing evidence for involvement of T helper type 2 cytokines in the pathogenesis of RSV bronchiolitis. We expanded our studies to polymorphisms in genes encoding IL-9, IL-10, and tumor necrosis factor (TNF)–α, using both a transmission/disequilibrium test and a case-control approach. Children homozygous for the IL-10 −592C or −592A allele had a higher risk of hospitalization for RSV bronchiolitis than did heterozygous carriers (odds ratio [OR], 1.73 vs. 2.55; 95% confidence interval [CI], 1.13–2.66 vs. 1.21–5.39). In children hospitalized at ⩽6 months of age, a significant association between RSV bronchiolitis and the IL-10 −592C allele was found (OR, 1.61; 95% CI, 1.10–2.35). No significant associations of TNF-α and IL-9 polymorphisms with RSV bronchiolitis were observed. We also explored the interactions between different polymorphisms and found an interaction between the IL-4Rα Q551R and IL-10 C−592A polymorphisms

Respiratory syncytial virus (RSV) is a common cause of severe lower respiratory tract infection in infants and children. The severity of disease is influenced by risk factors such as congenital heart disease, bronchopulmonary dysplasia, and prematurity. In addition, we and others have found evidence that host genetic factors influence the severity of RSV bronchiolitis [1–7]. Identification of such genetic factors may point to disease pathways involved in the pathogenesis of infection and may thus lead to novel targets for prevention and therapy

In a previous study, we found evidence of an association between severe RSV bronchiolitis and 2 gain-of-function polymorphisms in the genes encoding interleukin (IL)–4 and IL-4 receptor α (IL-4Rα), which suggested that Th2 cytokines were involved in the pathogenesis of this disease in the white population studied [1]. In an independent study, an association between IL-4 and severe RSV-induced disease was also found among Korean children [6]. Other gene variants that influence the course of RSV-induced disease are IL-8 and surfactant protein A and D variants [2–5]. Because severe RSV-induced disease is a complex disease in which many pathogenic pathways, either in interaction or not, may determine the final outcome of disease, it is likely that more loci account for disease outcome after infection. For comparison, it has been estimated that asthma, another complex disease, may be influenced by ⩾18 loci [8]

Our general hypothesis is that disease outcome of RSV bronchiolitis is determined by genetic heterogeneity in immune response genes, especially those that affect the Th1/Th2 balance. We therefore expanded our study of RSV genetics to other loci that might affect RSV disease, especially the IL-10, IL-9, and tumor necrosis factor (TNF)–α genes. IL-10 is a pleiotropic anti-inflammatory cytokine that could affect RSV bronchiolitis by inhibiting Th1 responses and by influencing antigen presentation and mast cell proliferation. IL-9 also is a pleiotropic cytokine. It promotes proliferation of Th2 cells and activates bronchial epithelial cells to produce mucin and different chemokines. In the present study, we examined the promoter polymorphisms IL-10 C−592A and IL-9 A−345G. Finally, because many inflammatory and infectious diseases are influenced by polymorphisms in the gene encoding TNF-α, we studied the TNF-α G−308A polymorphism. TNF-α is mainly produced by activated macrophages, B cells, and NK cells and is one of the major proinflammatory cytokines. We found evidence that IL-10, but not IL-9 and TNF-α variants, plays a role in the pathogenesis of severe RSV bronchiolitis, which further underlines the significance of immune regulation in this disease

Methods

Study designChildren, parents, and control subjects were selected from subjects included in our previous study [1]. In brief, DNA samples were obtained from 207 children hospitalized for RSV infection in 2 regions of The Netherlands and from their parents. Parents completed a questionnaire that gathered pregnancy and medical data for the hospitalized child and information about the ethnic origin of the family. The control population of 447 individuals was a random sample from an adult Dutch population study. Informed consent was obtained from the parents of each study subject, in accordance with the guidelines of the medical ethical committees of the Wilhelmina Children’s Hospital (Utrecht) and the Sophia Children’s Hospital (Rotterdam). For the control population, informed consent was obtained in accordance with the medical ethical committee of TNO Leiden

DNA isolation and genotypingDNA was isolated from blood samples or buccal swabs as described in our previous study [1]. The polymorphisms were genotyped by polymerase chain reaction (PCR) restriction fragment–length polymorphism (RFLP) analysis under the experimental conditions listed in table 1. PCR and RFLP analyses were done as described in our previous study [1]. Internal control samples (representing each genotype) and an empty sample were included on each plate. Samples from children and parents were genotyped blindly. Two different persons independently scored the plates

Table 1

Primers, polymerase chain reaction (PCR) conditions, and restriction enzymes used to determine genetic variation at the studied polymorphic sites in the interleukin (IL)–10, IL-9, and tumor necrosis factor (TNF)–α genes

Table 1

Primers, polymerase chain reaction (PCR) conditions, and restriction enzymes used to determine genetic variation at the studied polymorphic sites in the interleukin (IL)–10, IL-9, and tumor necrosis factor (TNF)–α genes

For confirmation, 59 samples containing all genotypes of the IL-10 −592 polymorphism were also determined by pyrosequencing. Using this method, the reverse PCR primer was biotin labeled, and the biotinylated PCR products were immobilized on streptavidin-coated Sepharose beads (Amersham Biosciences). A sodium hydroxide solution was added to generate single-stranded DNA, and samples were washed and dissolved in annealing buffer. After addition of the sequence primer, annealing was performed at 80°C for 3 min, and the sequence reaction was performed automatically with a PSQ 96 system (Pyrosequencing AB) using an SNP reagent kit (Pyrosequencing AB). In all cases, the genotype obtained was the same as that detected by RFLP analysis (data not shown)

Statistical analysisWe analyzed our data using 2 different tests: the transmission/disequilibrium test (TDT), which explores the transmission of the risk allele from (heterozygous) parents to their affected children [12], and a case-control approach, which compared the genotype and allele distribution among the children with the distribution in a control group, using the χ2 or Mantel-Haenszel trend test. In the latter approach, only Dutch children and control subjects were included, to prevent population admixture effects. Both tests were done for the total group of children. For the IL-10 polymorphism only, the tests were also performed for children with differing severities of RSV bronchiolitis (children who needed mechanical ventilatory support and those who did not), for children with known risk factors (premature birth, RSV infection at ⩽6 months of age, and cardiac or lung disease), and for allergic children (eczema or food allergy)

Where appropriate (when the results of the TDT or case-control analysis were P<.05 and the same allele was overrepresented in both tests), we combined information from the TDT and the case-control approach. Because the TDT and the case-control approach used data from the same children, the 2 tests were not statistically independent. However, the comparison (made using a case-control approach) of parents of case patients with control subjects is statistically independent of the TDT [13, 14]. The overall estimate of the effect of an allele can be obtained by statistically combining both TDT and case-control data for parents with that for control subjects (authors’ unpublished data) [1]; this is referred to as the “combination test.”

Interactions (“genetic interactions”) between the different polymorphisms in the IL-10, IL-9, and TNF-α (this study) and IL-4 and IL-4Rα (previous study [1]) genes were explored using 2 approaches to genetic interaction. In the TDT, genetic interaction was construed as the effect of a particular genotype at one locus on the probability of transmission of an allele at another locus. For this, we used logistic regression analysis. Clearly, this interaction is asymmetrical at the 2 loci. In the case-control approach, by contrast, genetic interaction was construed symmetrically as a nonmultiplicative effect of the odds ratios (ORs) of 2 alleles. For this, the simultaneous effect of polymorphisms at different loci was analyzed by means of logistic regression analysis. Variables with predicted value were selected using backward selection (SAS software, version 8.2 [SAS Institute]). Throughout, we used a 2-sided significance level (α) of 0.05 (i.e., P<.05 was considered to be statistically significant)

Results

IL-10Table 2 summarizes the frequency with which alleles of the IL-10 C−592A polymorphism were transmitted from (heterozygous) parents to their RSV-hospitalized children (TDT) and the genotype and allele distributions among case patients and control subjects (case-control approach). In the TDT, the C allele was found to be transmitted more often than the expected 50% among children who were hospitalized at ⩽6 months of age (OR, 1.61; 95% confidence interval [CI], 1.10–2.35; P=.014). In the case-control approach, a borderline-significant result was found for the allele distribution among children who were hospitalized at ⩽6 months of age; the C allele was also overrepresented, compared with control subjects. In contrast, among children who were hospitalized at >6 months of age, overrepresentation of the A allele was found, although this was not statistically significant. Interestingly, the genotype distribution in the different groups of affected children (all children, those who did not require mechanical ventilatory support, those without recurrent wheezing, those who were not born prematurely, those without cardiac or lung disease, and those without eczema or food allergy) was significantly different from the genotype distribution in the control group. Both CC and AA homozygotes were found more often among RSV-hospitalized children than in the control group (for the CC homozygote, OR, 1.73; 95% CI, 1.13–2.66; P⩽.01; for the AA homozygote, OR, 2.55; 95% CI, 1.21–5.39; P⩽.01)

Table 2

Results of a transmission/disequilibrium test, a case-control study, and a combination test examining interleukin (IL)–10 C−592A polymorphism in children hospitalized for respiratory syncytial virus (RSV) bronchiolitis and several subgroups

Table 2

Results of a transmission/disequilibrium test, a case-control study, and a combination test examining interleukin (IL)–10 C−592A polymorphism in children hospitalized for respiratory syncytial virus (RSV) bronchiolitis and several subgroups

For the groups of children with a (borderline) significant result in the TDT or the case-control approach, all available information was included in the combination test. For children who were hospitalized at ⩽6 months of age, the combination test yielded a significant result (OR, 1.42; 95% CI, 1.04–1.92; P=.02), which confirms the potential role of the C allele in enhancing RSV disease in these children

IL-9 and TNF-αThe results of the TDT and the genotype and allele distribution of the IL-9 A−345G and TNF-α G−308A polymorphisms are shown in table 3. When all children were included in the analysis, none of the tests had significant results. Because the frequencies of the allele variants were low (7% and 17%), subgroup analyses were considered to lack adequate power to yield reliable results

Table 3

Results of a transmission/disequilibrium test and a case-control study examining the interleukin (IL)–9 A−345G and tumor necrosis factor (TNF)–α G−308A polymorphisms in children hospitalized for respiratory syncytial virus (RSV) bronchiolitis

Table 3

Results of a transmission/disequilibrium test and a case-control study examining the interleukin (IL)–9 A−345G and tumor necrosis factor (TNF)–α G−308A polymorphisms in children hospitalized for respiratory syncytial virus (RSV) bronchiolitis

Hardy-Weinberg equilibrium and genetic interaction The Hardy-Weinberg equilibrium of the IL-10, TNF-α, and IL-9 polymorphisms in both the control group and among RSV-hospitalized children were consistent with a random allele distribution (data not shown). However, we found a significant (P<.01) Hardy-Weinberg disequilibrium for the IL-10 genotype distribution among children hospitalized because of RSV bronchiolitis

We calculated interactions between the IL-10, TNF-α, and IL-9 polymorphisms and the IL-4 and 2 IL-4Rα polymorphisms described elsewhere [1], both for the TDT and for the case-control approach. Statistically significant results were obtained in the TDT for the transmission of the IL-10 −592 and TNF-α −308 alleles with the IL-4 −590 genotypes and for the transmission of the IL-10 −592 alleles with the IL-4Rα 551 genotypes (table 4). For example, in the presence of an RR genotype at the IL-4Rα locus in a RSV-hospitalized child, the IL-10 A allele was preferentially inherited from a heterozygous parent, whereas, in the presence of the QQ genotype at the IL-4Rα locus, the IL-10 C allele was preferentially inherited (P=.02 for interaction)

Table 4

Genetic interaction (transmission/disequilibrium test) between the interleukin (IL)–4 C−590T genotypes in children and transmission of −592 alleles of the IL-10 gene and −308 alleles of the tumor necrosis factor (TNF)–α gene and between the IL-4 receptor α (IL-4Rα) Q551R genotypes and transmission of the −592 alleles of the IL-10 gene

Table 4

Genetic interaction (transmission/disequilibrium test) between the interleukin (IL)–4 C−590T genotypes in children and transmission of −592 alleles of the IL-10 gene and −308 alleles of the tumor necrosis factor (TNF)–α gene and between the IL-4 receptor α (IL-4Rα) Q551R genotypes and transmission of the −592 alleles of the IL-10 gene

In the case-control approach, there was genetic interaction between the IL-10 −592 and IL-4Rα 551 polymorphisms (P=.01 for interaction) and between the TNF-α −308 and IL-4Rα 551 polymorphisms (P=.01 for interaction) (table 5). Because an interaction between IL-4Rα Q551R and IL-10 C−592A was found in both the TDT and the case-control study, we assume that this finding is unlikely to be spurious. For this particular combination, we also tested the genotype distribution between case patients and control subjects, which was significantly different (P=.01; table 6). By contrast, interactions that were found in 1 of the 2 approaches but not in both may need confirmation

Table 5

Genetic interaction (case-control study) between the interleukin (IL)–10 C−592A and IL-4Rα Q551R genotypes and between the tumor necrosis factor (TNF)–α G−308A and IL-4 receptor α (IL-4Rα) Q551R genotypes, calculated using logistic regression

Table 5

Genetic interaction (case-control study) between the interleukin (IL)–10 C−592A and IL-4Rα Q551R genotypes and between the tumor necrosis factor (TNF)–α G−308A and IL-4 receptor α (IL-4Rα) Q551R genotypes, calculated using logistic regression

Table 6

Distribution of the interleukin-4 receptor α (IL-4Rα) Q551R genotype and the IL-10 C−592A genotype among case patients and control subjects

Table 6

Distribution of the interleukin-4 receptor α (IL-4Rα) Q551R genotype and the IL-10 C−592A genotype among case patients and control subjects

Discussion

The course of RSV infection in young infants who encounter their first infection with RSV varies from no symptoms at all to upper respiratory tract infection, more or less severe lower respiratory tract infection with or without wheezing, and even death. We hypothesized that genetic heterogeneity in the immune response explains some of the variation in the severity of RSV bronchiolitis and focused our studies on variants in genes involved in the Th2 response or affecting the Th1/Th2 balance. A contribution of Th2-mediated pathology in RSV disease was suggested by the finding that enhanced disease following natural infection of children immunized with formalin-inactivated vaccine was accompanied by a Th2 response [15–17]. Several, but not all, studies subsequently found evidence for the involvement of a Th2 response in severe RSV-associated disease [18–22]

We found, in our previous study [1], an overrepresentation of the IL-4 −590T allele in the group of all children with RSV-induced bronchiolitis (OR, 1.4). Among children who were hospitalized for RSV bronchiolitis at ⩾6 months of age, the effect of the IL-4 −590T allele was even more pronounced (OR, 2.1), and higher frequencies of another polymorphism, the IL-4Rα 551R allele, were also detected in this group of children (OR, 1.7). Furthermore, an overrepresentation of the IL-4 T allele (here called “−589T allele”) was also observed in a study of Korean children hospitalized for RSV lower respiratory tract illness who were <24 months of age (OR, 1.71, for the TT genotype), which independently confirms the role of IL-4 in RSV-induced bronchiolitis. These observations suggest that gain-in-function variation in genes known to play a role in allergic disorders also may contribute to severity of RSV infections. Enhanced functions of IL-4, as well as the IL-4 receptor, thus may well be a relevant factor in the pathogenesis of RSV, probably via an impact on immunopathologic and inflammatory processes in the small airways. Because these genes are recognized to be important in allergy and asthma, this throws new light on the interaction between RSV and asthma

In the present study, we examined polymorphisms in the genes encoding IL-10, IL-9, and TNF-α. The investigated polymorphisms in the IL-10 gene (C−592A) and the TNF-α gene (G−308A) have been examined in a large number of studies, including in studies of the association of these polymorphisms with asthma and allergy [23–26]. The polymorphism in the IL-9 gene (A−345G) was also mentioned as a candidate gene variant for study in relation to asthma and allergy [27]. Although the 3 polymorphisms are located in the promoter regions and may influence cytokine expression levels, any apparent effect of one of these polymorphisms may be attributable to the polymorphism itself, or it may function as a marker for a nearby functional polymorphism

IL-10 is a multifunctional anti-inflammatory cytokine that plays an important role in balancing immune clearance of pathogens and immune-mediated cellular injury [28–30]. It is secreted by T cells (mainly Th2 cells), macrophages, B cells, mast cells, and keratinocytes and binds to the IL-10 receptor that is present on a large number of cells. IL-10 inhibits expression of most inducible cytokines involved in inflammation, such as interferon-γ, IL-1, IL-6, IL-8, IL-12, macrophage inflammatory protein–1α, and TNF-α, in monocytes and neutrophils and inhibits antigen presentation by down-regulating the major histocompatibility complex class II expression on the surface of antigen-presenting cells. In B cells, IL-10 promotes cell growth and differentiation, immunoglobulin synthesis, and class switching. The IL-10 gene is located on chromosome 1q31-31 [31, 32]. A large number of polymorphic sites in the 5′ flanking region of IL-10 have been described. Five polymorphic sites have been extensively studied: 2 short tandem repeats, located 4 and 1 kb upstream of the translation start, and 3 single-nucleotide polymorphisms (SNPs), G−1082A, C−819T, and C−592A. The C−592A polymorphism was the one examined in this study. It has been shown that in a white population the 3 SNPs form 3 common haplotypes, GCC, ACC, and ATA, at a frequency of 51%, 28%, and 21%, respectively [33]. The effect of these SNPs, alone or in the context of the 3 haplotypes, on IL-10 production has been the subject of a number of studies [27, 33–38], but no clear answer has been obtained. The IL-10 production that was measured depended not only on genetic variations at the IL-10 locus but also on the test system used and environmental factors, such as viral infection and age [37, 39]. Therefore, at present it is not possible to unambiguously conclude whether the −592C allele or the −592A allele is associated with higher levels of IL-10 production in children hospitalized for RSV-induced bronchiolitis. To obtain direct evidence of the role of the above-mentioned polymorphisms and haplotypes in IL-10 production in relation to RSV-induced bronchiolitis, IL-10 levels should be measured in relevant samples during the disease. In this study, biological samples other than DNA were not available; therefore, such samples should be the subject of future studies

We found an underrepresentation of the IL-10 C−592A heterozygous genotype in children who were hospitalized for RSV bronchiolitis, compared with the control group and the Dutch parents (data not shown). Although both homozygous genotypes were more common among RSV-hospitalized children, the allele distribution among these children was not significantly different from that in the control group. The RSV-hospitalized children were also not in Hardy-Weinberg equilibrium (P=.01), in contrast to the control subjects. The genotype distribution of our control population resembled that of white control populations investigated by Howell et al. [40] (58% for CC, 37% for CA, and 5% for AA) and Koss et al. [41] (58% for CC, 35% for CA, and 7% for AA, in a UK population, and 62% for CC, 32% for CA, and 6% for AA, in a Polish population). This finding rules out the possibility that our control group was enriched for heterozygotes. Another possible explanation for the underrepresentation of heterozygotes among the children included in our study might be the use of RFLP analysis for genotyping. However, we have taken all precautions, such as including controls for every genotype, including a negative control, and using independent scoring by 2 different persons. We nonetheless reanalyzed 59 samples of all genotypes with pyrosequencing. In all cases, the same genotype was detected, which indicates that genotyping with the RFLP method is reliable. Our results suggest that children who are heterozygous for the −592 IL-10 polymorphism have a smaller chance of being hospitalized for RSV bronchiolitis and that there is a heterozygote advantage at the IL-10 locus in RSV infection. As far as we know, heterozygote advantage at the IL-10 locus has not been described before. Because IL-10 is a pleiotropic cytokine involved in immune clearance of pathogens, immune regulation, and immune-mediated cellular injury, heterozygote advantage might be the result of better balancing of these processes. However, to rule out the possibility that these results are a chance finding, an independently obtained cohort should be tested for heterozygote advantage at the IL-10 locus

We found no indication that one of the IL-10 −592 alleles was overrepresented in the group of all RSV-hospitalized children, although the 95% CI (0.92–1.58; combination test) does not exclude the possibility that there was some effect. However, the allele and genotype distribution among children hospitalized for RSV bronchiolitis at ⩽6 months of age was significantly different from that among children hospitalized at >6 months of age (P=.01 vs. P=.03; data not shown). We found an overrepresentation of the C allele among children hospitalized for RSV bronchiolitis at ⩽6 months of age, whereas the A allele was overrepresented among children hospitalized at >6 months of age (the results of comparison with control subjects were not significant). In our previous study [1], we also found a different allele distribution for the IL-4 C−590T and IL-4Rα Q551R polymorphisms among children hospitalized for RSV-induced bronchiolitis at ⩽6 months of age, compared with those who were hospitalized at >6 months of age. We were therefore surprised to find an association in this study between the IL-10 −592C allele and RSV bronchiolitis in children ⩽6 months of age. Together, these findings support the idea that RSV pathogenesis is age dependent and that IL-4, IL-4Rα, and IL-10 may play different roles in RSV pathogenesis in children in these age groups. Thus, age, decreasing levels of maternal antibodies [42], differences in IL-4 and IL-10 cytokine production during the first 12 months of life [43, 44], and promoter polymorphisms in cytokine genes may all affect RSV disease severity

IL-9 is a pleiotropic cytokine produced by Th2 cells, mast cells, and eosinophils that promotes proliferation of Th2 cells and the production of IgE by B cells [45, 46]. In bronchial epithelial cells, it activates different chemokines and genes involved in mucin production [46, 47]. Transgenic mice overexpressing IL-9 show bronchial hyperresponsiveness, whereas IL-9–deficient mice have reduced mucus production and mast cell proliferation [48–50]. In the present study, we investigated the genetic association between the IL-9 A−345G polymorphic site and RSV-induced bronchiolitis. No significant results were obtained. Bear in mind, however, that the A−345G IL-9 polymorphism is rare and that our negative finding cannot exclude the possibility that a genetic effect exists, because the 95% CI is wide (0.82–2.07; combination test [data not shown]). Nonetheless, thus far we have concluded that the IL-9 A−345G polymorphism likely is not involved in the susceptibility of RSV-induced bronchiolitis in these young children

The TNF-α gene is located on chromosome 6p21.3 in a region containing the major histocompatibility complex genes [51, 52]. Many polymorphisms occur in the TNF-α gene region, of which the G−308A polymorphism located in the promoter region is best studied. There is some debate about the functionality of this particular polymorphic site, which might reflect the complex genetic situation in this genomic region. In our present study, we could not detect any association between the G−308A TNF-α polymorphism and RSV bronchiolitis in any group of children. Again, the power of our analysis may have been insufficient to detect small effects, but the 95% CI is rather narrow (0.70–1.32; combination test [data not shown]), and the existence of a major genetic effect is, therefore, highly unlikely. The same polymorphism was examined in another study that searched for an association of RSV illness severity and complications with cytokine gene polymorphisms [7]. In that study, too, no associations were found for this particular polymorphic site. In conclusion, the genetic variation on position −308 of the TNF-α gene plays no important role in the severity of RSV-induced bronchiolitis

Interactions were found between the IL-10 −592 and IL-4Rα 551 polymorphisms. In the TDT, the C allele of IL-10 was more often found in homozygous carriers of the IL-4Rα 551Q allele, and the A allele of IL-10 was more often found in homozygous carriers of the IL-4Rα 551R allele. In the case-control approach, the combination of the CC homozygote of IL-10 with the QQ homozygote of IL-4Rα 551 and of the AA homozygote with the RR homozygote were overrepresented among RSV-hospitalized children, compared with the control group (table 6). Thus, such a genotype combination was associated with risk of severe disease. It is interesting to speculate about the mechanisms by which the IL-10 and IL-4Rα alleles could interact. It might reflect a biological interaction of these molecules in RSV bronchiolitis, for example in mucus production. Excess mucus production contributes to the airway obstruction seen in severe RSV disease. In mice and in vitro, it was demonstrated that both IL-10 and IL-4 induce mucus production in airways in an IL-4Rα–dependent way [53–55]. IL-10–induced mucus production was independent of IL-4 but dependent on IL-13, which binds to a receptor formed by IL-4Rα and IL-13Rα [55]. It is likely that the IL-10/IL-4Rα pathway plays a role in RSV disease, because IL-10 deficiency has been found to reduce RSV-induced mucin expression [55]. In addition, we may postulate that IL-10 influences the expression of IL-4Rα. In a study by Lang et al. [56], bone marrow–derived macrophages of IL-10−/− mice were stimulated with IL-10, and RNA was analyzed on microarrays. In that experiment, IL-10 up-regulated the expression of the IL-4Rα gene

In conclusion, we found no genetic association of the IL-9 A−345G and TNF-α G−308A polymorphisms with hospitalization for RSV-induced bronchiolitis in children. Among children hospitalized for RSV bronchiolitis, heterozygotes for the IL-10 C−592A polymorphism were underrepresented, compared with the control group. This suggests that such heterozygous children might be protected against severe RSV bronchiolitis. For the IL-10 C−592A polymorphism, no association was found in the group of all hospitalized children, but the C allele was overrepresented among children ⩽6 months of age. Interestingly, this is the third polymorphism in which we found an age effect for genetic association. Apparently, RSV pathogenesis is different during the first 6 months of life, compared with older ages, or the role of cytokines IL-4, IL-4Rα, and IL-10 may be different at different ages. Finally, we found genetic interactions between the IL-10 −592 and IL-4Rα 551 polymorphisms

Acknowledgment

We thank Petra van Impelen for technical assistance

References

1
Hoebee
B
Rietveld
E
Bont
L
, et al.  . 
Association of severe respiratory syncytial virus bronchiolitis with interleukin-4 and interleukin-4 receptor α polymorphisms
J Infect Dis
 , vol. 
187
 (pg. 
2
-
11
)
2
Hull
J
Thomson
A
Kwiatkowski
D
Association of respiratory syncytial virus bronchiolitis with the interleukin 8 gene region in UK families
Thorax
 , vol. 
55
 (pg. 
1023
-
7
)
3
Hull
J
Ackerman
H
Isles
K
, et al.  . 
Unusual haplotypic structure of IL8, a susceptibility locus for a common respiratory virus
Am J Hum Genet
 , vol. 
69
 (pg. 
413
-
9
)
4
Lahti
M
Lofgren
J
Marttila
R
, et al.  . 
Surfactant protein D gene polymorphism associated with severe respiratory syncytial virus infection
Pediatr Res
 , vol. 
51
 (pg. 
696
-
9
)
5
Lofgren
J
Ramet
M
Renko
M
Marttila
R
Hallman
M
Association between surfactant protein A gene locus and severe respiratory syncytial virus infection in infants
J Infect Dis
 , vol. 
185
 (pg. 
283
-
9
)
6
Choi
EH
Lee
HJ
Yoo
T
Chanock
SJ
A common haplotype of interleukin-4 gene IL4 is associated with severe respiratory syncytial virus disease in Korean children
J Infect Dis
 , vol. 
186
 (pg. 
1207
-
11
)
7
Gentile
DA
Doyle
WJ
Zeevi
A
, et al.  . 
Cytokine gene polymorphisms moderate illness severity in infants with respiratory syncytial virus infection
Hum Immunol
 , vol. 
64
 (pg. 
338
-
44
)
8
Hoffjan
S
Ober
C
Present status on the genetic studies of asthma
Curr Opin Immunol
 , vol. 
14
 (pg. 
709
-
17
)
9
Kelleher
K
Bean
K
Clark
SC
, et al.  . 
Human interleukin-9: genomic sequence, chromosomal location, and sequences essential for its expression in human T-cell leukemia virus (HTLV)–I–transformed human T cells
Blood
 , vol. 
77
 (pg. 
1436
-
41
)
10
Wilson
AG
de Vries
N
Pociot
F
di Giovine
FS
van der Putte
LBA
An allelic polymorphism within the human tumor necrosis factor α promoter region is strongly associated with HLA A1, B8 and DR3 alleles
J Exp Med
 , vol. 
177
 (pg. 
557
-
60
)
11
Takashiba
S
Shapira
L
Amar
S
Van Dyke
TE
Cloning and characterization of human TNF alpha promoter region
Gene
 , vol. 
131
 (pg. 
307
-
8
)
12
Sham
PC
Statistics in human genetics
 
13
Whittemore
AS
Tu
IP
Detection of disease genes by use of family data. I. Likelihood-based theory
Am J Hum Genet
 , vol. 
66
 (pg. 
1328
-
40
)
14
Tu
IP
Balise
RR
Whittemore
AS
Detection of disease genes by use of family data. II. Application to nuclear families
Am J Hum Genet
 , vol. 
66
 (pg. 
1341
-
50
)
15
Openshaw
PJ
Pemberton
RM
Ball
LA
Wertz
GW
Askonas
BA
Helper T cell recognition of respiratory syncytial virus in mice
J Gen Virol
 , vol. 
69
 (pg. 
305
-
12
)
16
Connors
M
Giese
NA
Kulkarni
AB
Firestone
C
Morse
HC
Enhanced pulmonary histopathology induced by respiratory syncytial virus (RSV) challenge of formalin-inactivated RSV-immunized BALB/c mice is abrogated by depletion of interleukin- 4 (IL-4) and IL-19
J Virol
 , vol. 
68
 (pg. 
5321
-
5
)
17
Boelen
A
Andeweg
A
Kwakkel
J
, et al.  . 
Both immunisation with a formalin-inactivated respiratory syncytial virus (RSV) vaccine and a mock antigen vaccine induce severe lung pathology and a Th2 cytokine profile in RSV-challenged mice
Vaccine
 , vol. 
19
 (pg. 
982
-
91
)
18
Bendelja
K
Gagro
K
Bace
A
, et al.  . 
Predominant type-2 response in infants with respiratory syncytial virus (RSV) infection demonstrated by cytokine flow cytometry
Clin Exp Immunol
 , vol. 
121
 (pg. 
332
-
8
)
19
Rabatic
S
Gagro
A
Lokar-Kolbas
R
Krsulovic-Hresic
V
Vrtar
A
Increase in CD23+ B cells in infants with bronchiolitis is accompanied by appearance of IgE and IgG4 antibodies specific for respiratory syncytial virus
J Infect Dis
 , vol. 
175
 (pg. 
32
-
7
)
20
Roman
M
Calhoen
WJ
Himton
KL
Respiratory syncytial virus infection in infants is associated with predominant Th-2 like response
Am J Respir Crit Care Med
 , vol. 
156
 (pg. 
190
-
5
)
21
Tripp
RA
Moore
D
Barskey A 4th
, et al.  . 
Peripheral blood mononuclear cells from infants hospitalized because of respiratory syncytial virus infection express T helper-1 and T helper-2 cytokines and CC chemokine messenger RNA
J Infect Dis
 , vol. 
185
 (pg. 
1388
-
94
)
22
Bont
L
Heijnen
CJ
Kavelaars
A
, et al.  . 
Peripheral blood cytokine responses and disease severity in respiratory syncytial virus bronchiolitis
Eur Respir J
 , vol. 
14
 (pg. 
144
-
9
)
23
Hobbs
K
Negri
J
Klinnert
M
Rosenwasser
LJ
Borish
L
Interleukin-10 and transforming growth factor-β promoter polymorphisms in allergies and asthma
Am J Respir Crit Care Med
 , vol. 
158
 (pg. 
1958
-
62
)
24
Bidwell
J
Keen
L
Gallagher
G
, et al.  . 
Cytokine gene polymorphism in human disease: on-line databases
Genes Immun
 , vol. 
1
 (pg. 
3
-
19
)
25
Bidwell
J
Keen
L
Gallagher
G
, et al.  . 
Cytokine gene polymorphism in human disease: on-line databases, supplement 1
Genes Immun
 , vol. 
2
 (pg. 
61
-
70
)
26
Haukim
N
Bidwell
JL
Smith
AJ
, et al.  . 
Cytokine gene polymorphism in human disease: on-line databases, supplement 2
Genes Immun
 , vol. 
3
 (pg. 
313
-
30
)
27
Rosenwasser
L
Promoter polymorphism in the candidate genes, IL-4, IL-9, TGF-β1, for atopy and asthma
Int Arch Allergy Immunol
 , vol. 
118
 (pg. 
268
-
70
)
28
Opal
SM
Huber
CE
The role of interleukin-10 in critical illness
Curr Opin Infect Dis
 , vol. 
13
 (pg. 
221
-
6
)
29
Moore
KW
de Waal Malefyt
R
Coffman
RL
O’Garra
A
Interleukin-10 and the interleukin-10 receptor
Annu Rev Immunol
 , vol. 
19
 (pg. 
683
-
765
)
30
Redpath
S
Ghazal
P
Gascoigne
NR
Hijacking and exploitation of IL-10 by intracellular pathogens
Trends Microbiol
 , vol. 
9
 (pg. 
86
-
92
)
31
Kim
JM
Brannan
CI
Copeland
NG
Jenkins
NA
Khan
TA
Structure of the mouse IL-10 gene and chromosomal localization of the mouse and human genes
J Immunol
 , vol. 
148
 (pg. 
3618
-
23
)
32
Eskdale
J
Kube
D
Tesch
H
Gallagher
G
Mapping of the human IL10 gene and further characterization of the 5′ flanking sequence
Immunogenetics
 , vol. 
46
 (pg. 
120
-
8
)
33
Turner
DM
Williams
DM
Sankaran
D
Lazarus
M
Sinnott
PJ
An investigation of polymorphism in the interleukin-10 gene promoter
Eur J Immunogenet
 , vol. 
24
 (pg. 
1
-
8
)
34
Crawley
E
Kay
R
Sillibourne
J
Patel
P
Hutchinson
I
Polymorphic haplotypes of the interleukin-10 5′ flanking region determine variable interleukin-10 transcription and are associated with particular phenotypes of juvenile rheumatoid arthritis
Arthritis Rheum
 , vol. 
42
 (pg. 
1101
-
8
)
35
Cartwright
NH
Keen
LJ
Demaine
AG
, et al.  . 
A study of cytokine gene polymorphisms and protein secretion in renal transplantation
Transpl Immunol
 , vol. 
8
 (pg. 
237
-
44
)
36
Helminen
ME
Kilpinen
S
Virta
M
Hurme
M
Susceptibility to primary Epstein-Barr virus infection is associated with interleukin-10 gene promoter polymorphism
J Infect Dis
 , vol. 
184
 (pg. 
777
-
80
)
37
Warle
MC
Farhan
A
Metselaar
HJ
, et al.  . 
Are cytokine gene polymorphisms related to in vitro cytokine production profiles?
Liver Transpl
 , vol. 
9
 (pg. 
170
-
81
)
38
Suarez
A
Castro
P
Alonso
R
Mozo
L
Gutierrez
C
Interindividual variations in constitutive interleukin-10 messenger RNA and protein levels and their association with genetic polymorphisms
Transplantation
 , vol. 
75
 (pg. 
711
-
7
)
39
Reuss
E
Fimmers
R
Kruger
A
Becker
C
Rittner
C
Differential regulation of interleukin-10 production by genetic and environmental factors—a twin study
Genes Immun
 , vol. 
3
 (pg. 
407
-
13
)
40
Howell
WM
Turner
SJ
Bateman
AC
Theaker
JM
IL-10 promoter polymorphisms influence tumour development in cutaneous malignant melanoma
Genes Immun
 , vol. 
2
 (pg. 
25
-
31
)
41
Koss
K
Fanning
GC
Welsh
KI
Jewell
DP
Interleukin-10 gene promoter polymorphism in English and Polish healthy controls: polymerase chain reaction haplotyping using 3′ mismatches in forward and reverse primers
Genes Immun
 , vol. 
1
 (pg. 
321
-
4
)
42
Crowe
JE
Jr
Immune responses of infants to infection with respiratory viruses and live attenuated respiratory virus candidate vaccines
Vaccine
 , vol. 
16
 (pg. 
1423
-
32
)
43
Vigano
A
Esposito
S
Arienti
D
, et al.  . 
Differential development of type 1 and type 2 cytokines and β-chemokines in the ontogeny of healthy newborns
Biol Neonate
 , vol. 
75
 (pg. 
1
-
8
)
44
Prescott
SL
Macaubas
C
Smallacombe
T
Holt
BJ
Sly
PD
Development of allergen-specific T-cell memory in atopic and normal children
Lancet
 , vol. 
353
 (pg. 
196
-
200
)
45
Demoulin
JB
Renauld
JC
Interleukin 9 and its receptor: an overview of structure and function
Int Rev Immunol
 , vol. 
16
 (pg. 
345
-
64
)
46
Soussi-Gounni
A
Kontolemos
M
Hamid
Q
Role of IL-9 in the pathophysiology of allergic diseases
J Allergy Clin Immunol
 , vol. 
107
 (pg. 
575
-
82
)
47
Louahed
J
Toda
M
Jen
J
, et al.  . 
Interleukin-9 upregulates mucus expression in the airways
Am J Respir Cell Mol Biol
 , vol. 
22
 (pg. 
649
-
56
)
48
Temann
UA
Geba
GP
Rankin
JA
Flavell
RA
Expression of interleukin 9 in the lungs of transgenic mice causes airway inflammation, mast cell hyperplasia, and bronchial hyperresponsiveness
J Exp Med
 , vol. 
188
 (pg. 
1307
-
20
)
49
McLane
MP
Haczku
A
van de Rijn
M
, et al.  . 
Interleukin-9 promotes allergen-induced eosinophilic inflammation and airway hyperresponsiveness in transgenic mice
Am J Respir Cell Mol Biol
 , vol. 
19
 (pg. 
713
-
20
)
50
Townsend
JM
Fallon
GP
Matthews
JD
Smith
P
Jolin
EH
IL-9–deficient mice establish fundamental roles for IL-9 in pulmonary mastocytosis and goblet cell hyperplasia but not T cell development
Immunity
 , vol. 
13
 (pg. 
573
-
83
)
51
Spies
T
Morton
CC
Nedospasov
SA
Fiers
W
Pious
D
Genes for the tumor necrosis factors α and β are linked to the human major histocompatibility complex
Proc Natl Acad Sci USA
 , vol. 
83
 (pg. 
8699
-
702
)
52
Wilson
AG
di Giovine
FS
Duff
GW
Genetics of tumour necrosis factor-α in autoimmune, infectious, and neoplastic diseases
J Inflamm
 , vol. 
45
 (pg. 
1
-
12
)
53
Cohn
L
Homer
RJ
MacLeod
H
Mohrs
M
Brombacher
F
Th2-induced airway mucus production is dependent on IL-4Rα, but not on eosinophils
J Immunol
 , vol. 
162
 (pg. 
6178
-
83
)
54
Dabbagh
K
Takeyama
K
Lee H-M
Ueki
IF
Lausier
JA
IL-4 induces mucin gene expression and goblet cell metaplasia in vitro and in vivo
J Immunol
 , vol. 
162
 (pg. 
6233
-
7
)
55
Lee
CG
Homer
RJ
Cohn
L
, et al.  . 
Transgenic overexpression of interleukin (IL)–10 in the lung causes mucus metaplasia, tissue inflammation, and airway remodeling via IL-13–dependent and –independent pathways
J Biol Chem
 , vol. 
277
 (pg. 
35466
-
74
)
56
Lang
R
Patel
D
Morris
JJ
Rutschman
RL
Murray
PJ
Shaping gene expression in activated and resting primary macrophages by IL-10
J Immunol
 , vol. 
169
 (pg. 
2253
-
63
)