Abstract
Rationale: Respiratory infections in early life are associated with risk for wheezing bronchiolitis, especially in children at high risk of atopy. The underlying mechanisms are unknown, but are suspected to involve imbalance(s) in host defense responses against pathogens stemming from functional immaturity of the immune system in this age group.
Objectives: To assess the contribution of eosinophil-trophic IL-5, and the potent antiinflammatory cytokine IL-10, to risk for infection in early life.
Measurements and Main Results: We prospectively monitored a cohort of 198 high-risk children to age 5 years, recording every acute respiratory infection episode and classifying them by severity. We measured cord blood T-cell capacity to produce IL-10 and IL-5, and related these functions to subsequent infection history. IL-10 and IL-5 were associated, respectively, with resistance versus susceptibility to infections. The greatest contrasting effects of these two cytokines were seen when they were considered in combination by generating IL-10/IL-5 response ratios for each subject. The low IL-10/high IL-5 T-cell response phenotype was strongly associated with susceptibility to all grades of acute respiratory infection, relative to the more resistant high IL-10/low IL-5 phenotype.
Conclusions: Excessive production of IL-5 by T cells at birth is associated with heightened risk for subsequent severe respiratory infections, and this risk is attenuated by concomitant IL-10 production. The underlying mechanisms may involve IL-10–mediated feedback inhibition of IL-5–dependent eosinophil-induced inflammation, which is a common feature of host antiviral responses in early life.
Respiratory infections in early life are associated with risk for wheezing bronchiolitis, especially in children at high risk of atopy. The underlying mechanisms are unknown, but are suspected to involve imbalance(s) in host defense responses against pathogens stemming from functional immaturity of the immune system in this age group.
The balance between the capacity to produce the eosinophil-trophic cytokine IL-5 and the antiinflammatory cytokine IL-10 is strongly associated with infection risk in early life. High IL-5 is not a risk if balanced by sufficient IL-10 capacity.
It is clear from the results of many investigators that overall immune function during the early postnatal period is constitutively skewed toward Th2 cytokine production (8, 9), representing the persistence of a phenotype that serves in utero to protect the fetoplacental unit from the highly toxic effects of Th1 cytokines (10). However, one potential consequence of the postnatal persistence of this fetal immune phenotype is that host defense mechanisms that rely principally on Th1 effector mechanisms, particularly antiviral immunity, may become distorted during this life phase by the presence of an excessive component of Th2 cytokine production, which has the potential to antagonize the development of effective Th1-dependent sterilizing immunity (11). The relatively high frequency of bronchiolitis associated with eosinophilia in RSV-infected infants relative to other age groups (12–15) represents one likely example, whereby the prominent IL-5 component in the Th2-biased antiviral response of this age group appears to drive eosinophil responses in the airways (16, 17). Similarly, the results from infant mouse models of RSV infection (18, 19) have linked the degree of functional immaturity (and hence Th2 bias) of the immune system at the time of first infection with ensuing morbidity, and also with morbidity at subsequent infection; in both cases linking infection outcome with degrees of ensuing eosinophilia. These findings are consistent with the general model that inflammation resulting from Th2-driven airway eosinophilia represents a major cause of severe bronchiolitis in early life, and that susceptibility is directly related to the developmentally determined Th1/Th2 balance at the time of infection (8).
We aimed to extend this model to consider the converse immunologic process, the contribution from mechanisms associated with protection against inflammation. The archetypal antiinflammatory mechanism in the immune system is represented by the activities of the cytokine IL-10, which, in humans, is produced by multiple cell types within the innate and adaptive arms of the immune system (20). Adaptive immune responses in newborns are dominated by this cytokine, which is produced at disproportionately high levels by T cells during early life (21). We hypothesized that high IL-10 responsiveness during infancy may provide protection against the downstream inflammatory consequences of respiratory infections in Th2-biased neonates and infants, and that infection severity may be related to the balance between capacity to produce IL-10 and proinflammatory Th2 cytokines, exemplified by IL-5. We tested this hypothesis in a prospective cohort study of children at high genetic risk of atopic disease, in whom the degree of Th2 bias during early life would be expected to be high relative to the population at large.
Subjects comprised 238 children from a cohort of 263 recruited in a prospective study on acute respiratory infections (ARI), 198 of whom were monitored to age 5 years (detailed in Reference22). This cohort is at high risk for developing atopic disease, as at least one parent of each subject had doctor-diagnosed asthma, hay fever, or eczema.
As described previously (22), infectious episodes were prospectively recorded throughout the first 5 years of each child's life. ARIs were subclassified as febrile respiratory infection (FRI), lower respiratory infection (LRI), or wheezy lower respiratory infection (wLRI) (see the online supplement).
Production of IL-10 and IL-5 by cord blood mononuclear cells (CBMCs; from 175 of the subjects) stimulated with the T-cell mitogen phytohemagglutinin (PHA) or staphylococcal enterotoxin B (SEB) was determined as described previously (see Reference 23 and the online supplement). The limits of detection for both were 10 pg/ml.
Microarray profiling and quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) studies were conducted on groups of selected individuals with high IL-10/IL-5 ratios (from within the upper quintile of the ratios, designated IL-10hi/IL-5low, n = 15; at low risk for ARI) and low IL-10/IL-5 ratios (from the lower quintile of the ratios, n = 15; at high risk for ARI), using standardized methodology detailed previously (see Reference 24 and the online supplement). Genes identified as putatively hyperexpressed in CBMCs were validated in individual samples by qRT-PCR employing purified RNA from CD4+ and CD8+ T-cell subsets derived from each of the CBMC samples tested (see the online supplement).
The number, type, and timing of infection episodes were enumerated for each of the children during each year of the observation period and incidence rates (which are defined as new ARI episodes divided by person-years at risk) were calculated.
At first, the relationship between levels of cytokines in cord blood (IL-10 and IL-5) and infection incidence was assessed by Spearman correlation. Univariate Poisson regression models were used to investigate associations between infection incidence and the loge values of individual cord blood cytokines and cytokine response ratios, as well as cytokine response data derived from peripheral blood mononuclear cells collected at 6 and 12 months. To estimate the relative risk of infection as a function of cytokine response capacity, incidence rate ratios (analogous to odds ratios generated in logistic regression models) were computed via these Poisson regression models. Incidence rate ratios in this context provide a relative measure of the effect of a given level of cytokine response capacity on risk for infection, designated in the text below as relative risk (RR). To allow for changes of rates with age and repeated measures over time on the same children, Poisson regression with random effects was used to estimate RRs of the loge(IL-10/IL-5) ratios on respiratory infections in the first 5 years of life, including a categorical variable for each year of age and with each number of infections in each year as the dependent variable and time spent in the study in that year as denominator (or “offset”). Possible differences in effects on infection rates of the ratios with age were also investigated, using interaction terms. All analyses were also adjusted for the possible confounding effects of daycare attendance, number of children at home, and passive smoking. The statistical methods used for the analyses of the microarray data (25–27) are detailed in the online supplement. Descriptive analyses were done with SPSS software (SPSS, Chicago, IL) and all other analyses (except for microarray data) were performed in Stata (StataCorp, College Station, TX). Although we looked at many combinations of cytokine levels and disease outcomes, the results of all are presented, so no formal adjustment to the P values was performed (28).
A total of 238 children were included in the study, with 134 of them boys (56.3%). Table E1 in the online supplement and Figure 1 show infection incidence rates during the first 5 years of life. The incidence rate for ARI was highest during Years 1 and 2 (about four episodes per person-year) and declined thereafter. The proportion of infections spreading to the lower respiratory tract annually averaged one third, and this proportion remained relatively constant throughout the observation period.
Figure 1. Infection incidence rates in the cohort during the first 5 years of life. Rates were estimated on the basis of the Poisson regression model: acute respiratory infection (ARI; solid squares), febrile respiratory infection (FRI; open triangles), lower respiratory infection (LRI; open reverse triangles), and wheezy lower respiratory infection (wLRI; open diamonds).
[More] [Minimize]Table E2 illustrates the spectrum of IL-5 and IL-10 response capacities in CBMC samples from the study group after stimulation with the polyclonal T-cell mitogen PHA. Comparable variation (data not shown) was observed with a second T-cell stimulant, SEB. PHA-induced IL-5 and IL-10 responses were highly variable, and these variations in production levels were associated with fluctuations in risk for subsequent infections. Cord blood cytokine response capacity correlated with the number of ensuing respiratory infection episodes. For example, during the first year, positive correlations were found between the number of FRIs and levels of IL-5 production in response to the polyclonal T-cell stimulant PHA or SEB, with P values of 0.018 and 0.002, respectively. There was also a correlation between IL-5 (SEB) production and the number of wLRIs (P = 0.034). In the second year, significant positive correlations were found between FRI and SEB-stimulated IL-5 (P = 0.046). In contrast, negative correlations were observed between infections and IL-10 responses sporadically across the time course. Notably, a negative correlation was observed between ARI in Year 5 and PHA- and SEB-stimulated IL-10 (P = 0.001 for both), and between FRI and PHA-induced IL-10 (P = 0.014). A similar weak negative association was observed between LPS-stimulated IL-10 and ARI in Year 2.
We next assessed cumulative risk for infection associated with individual cord blood cytokine responses by calculating RR for infection over the full 5-year observation period, using Poisson regression (Table 1). Significant negative correlations were observed between PHA- and SEB-stimulated IL-10 responses and estimated RR for total ARI over the 5 years (P < 0.001). In contrast, IL-10 production in response to the innate immune stimulus LPS was unrelated to infection incidence (data not shown). The association with PHA- and SEB-induced IL-10 did not extend to LRI and/or symptoms, suggesting that it was related to susceptibility to primary infection as opposed to ensuing infection severity. In contrast, PHA- and SEB-stimulated IL-5 production were associated with increased risk for all categories of infection.
95% CI | |||||
|---|---|---|---|---|---|
| Mitogen/Cytokine | RR | Lower | Upper | P Value | |
| PHA/IL-10 | |||||
| ARI | 0.92 | 0.89 | 0.96 | <0.001 | |
| FRI | 1.00 | 0.93 | 1.08 | 0.96 | |
| LRI | 0.96 | 0.89 | 1.02 | 0.18 | |
| wLRI | 0.97 | 0.88 | 1.07 | 0.52 | |
| SEB/IL-10 | |||||
| ARI | 0.89 | 0.85 | 0.93 | <0.001 | |
| FRI | 0.97 | 0.89 | 1.06 | 0.54 | |
| LRI | 1.01 | 0.94 | 1.10 | 0.75 | |
| wLRI | 1.03 | 0.92 | 1.16 | 0.57 | |
| PHA/IL-5 | |||||
| ARI | 1.04 | 1.00 | 1.08 | 0.055 | |
| FRI | 1.08 | 1.00 | 1.16 | 0.046 | |
| LRI | 1.13 | 1.05 | 1.21 | 0.002 | |
| wLRI | 1.06 | 0.96 | 1.17 | 0.265 | |
| SEB/IL-5 | |||||
| ARI | 1.05 | 1.00 | 1.09 | 0.036 | |
| FRI | 1.17 | 1.07 | 1.27 | 0.001 | |
| LRI | 1.18 | 1.08 | 1.28 | <0.001 | |
| wLRI | 1.16 | 1.03 | 1.30 | 0.014 | |
The contrasting positive and negative associations observed between IL-5 and IL-10 response capacity and ARI suggested that susceptibility to respiratory infection may be related to the balance between these two cytokines during host defense responses. In absolute terms IL-10 was produced at higher levels in all subjects, but the relativity between IL-10 and IL-5 levels varied widely within the population (Table E2). To address questions concerning the quantitative relationship between these two cytokine responses in individual children and their subsequent infection histories, we expressed individual IL-10 and IL-5 production levels as ratios that were then loge transformed to estimate RR in terms of log(IL-10/IL-5) response ratios.
Figure 2 shows the RR of respiratory infections for the loge ratios of PHA-stimulated (Figure 2A) versus SEB-stimulated (Figure 2B) IL-10/IL-5. Children with the higher IL-10/IL-5 ratios appeared to have lower risks for virtually all of the four categories of respiratory infections in each of the five 1-year periods spanning the study. Notably, these analyses generated 40 subgroups within the overall study population (Figure 2), and the IL-10/IL-5 ratios were protective against respiratory infections with 39 RRs in the 40 subgroups being below the reference value of 1.0 and among them 16 RRs being significant. The most consistent infection category yielding statistically significant P values across the observation period was ARI, but FRI and LRI additionally displayed repeated significant associations, suggesting that relative IL-10/IL-5 production capacity may also be related to infection severity as well as to overall infection susceptibility. When these analyses were repeated with IL-10 response data obtained from the innate immune stimulus LPS in conjunction with PHA-induced IL-5, no comparable associations were observed (data not shown).
Figure 2. Relative risks and 95% confidence intervals of acute respiratory infections for natural log values of (A) phytohemagglutinin (PHA)-stimulated versus (B) staphylococcal enterotoxin B (SEB)–stimulated IL-10/IL-5 ratios: ARI (solid squares), FRI (open triangles), LRI (open reverse triangles), and wLRI (open diamonds). *P < 0.05, **P < 0.01, ***P < 0.001.
[More] [Minimize]We also tested IFN-γ, IL-13, and tumor necrosis factor-α responses in combination with IL-10, and the combination of IL-5 and IFN-γ, but observed only weak and inconsistent relationships and these cytokines were not pursued further.
To further investigate the association between relative IL-10 and IL-5 response capacity and the incidence of ARI in the first 5 years of life, we reestimated RR for cumulative infections across the 5-year observation period in relation to cord blood loge(IL-10/IL-5) response ratios (Table 2). IL-10/IL-5 response ratios were strongly associated with reduced overall risk for respiratory infections over the first 5 years of life. Similar findings resulted from follow-up analyses on T-cell IL-10 versus IL-5 response profiles in samples collected from the cohort at 6 and 12 months of age (data not shown). In contrast to T-cell cytokine response profiles, ratios of IL-10 to IL-5 production elicited by the innate immune stimulus LPS were not predictive of subsequent infection risk (data not shown). To confirm the findings, Poisson regression models with random effects were used to estimate RRs for respiratory infections in the first 5 years of life (see Table 2), allowing for changes over time in infection rates. The RRs changed slightly and P values increased, as expected. However, the protective effects of the IL-10/IL-5 ratios on respiratory infections were still significant in the IL-10/IL-5 [PHA] ratios for ARI (P = 0.017) and LRI (P = 0.046) and in the IL-10/IL-5 [SEB] ratios for both ARI and FRI (P = 0.012 and 0.013, respectively). The protective effects were consistent across all categorical infections, with some insignificant RRs due to insufficient statistical power. We also tested the interactive effects of these ratios with age on respiratory infections and these interactions were not large or significant (all P values > 0.09).
Poisson Regression Model | Random Effects Poisson Model* | |||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 95% CI | 95% CI | |||||||||||||
| RR | Lower | Upper | P Value | RR | Lower | Upper | P Value | |||||||
| IL-10/IL-5 (PHA) | ||||||||||||||
| ARI | 0.90 | 0.88 | 0.93 | <0.001 | 0.91 | 0.84 | 0.98 | 0.017 | ||||||
| FRI | 0.93 | 0.88 | 0.99 | 0.034 | 0.93 | 0.83 | 1.03 | 0.18 | ||||||
| LRI | 0.88 | 0.83 | 0.93 | <0.001 | 0.89 | 0.79 | 1.00 | 0.046 | ||||||
| wLRI | 0.93 | 0.85 | 1.01 | 0.075 | 0.93 | 0.78 | 1.10 | 0.38 | ||||||
| IL-10/IL-5 (SEB) | ||||||||||||||
| ARI | 0.86 | 0.82 | 0.90 | <0.001 | 0.88 | 0.80 | 0.97 | 0.012 | ||||||
| FRI | 0.83 | 0.76 | 0.91 | <0.001 | 0.84 | 0.73 | 0.96 | 0.013 | ||||||
| LRI | 0.86 | 0.79 | 0.94 | <0.001 | 0.87 | 0.74 | 1.01 | 0.074 | ||||||
| wLRI | 0.84 | 0.74 | 0.95 | 0.005 | 0.84 | 0.67 | 1.05 | 0.14 | ||||||
Microarray analysis was used to investigate whether additional genes were hyperexpressed in mitogen-stimulated CBMCs from the infection-resistant IL-10hi/IL-5low individuals. With a 2.0-fold–change cutoff, hyperexpressed genes were identified in PHA responses from this group after an empirical Bayes t test (see the online supplement), including a subset of genes that, in addition to IL-10, are known to be associated with immunologic function. These were selected for additional qRT-PCR analysis within the individual RNA samples employed for microarray analysis, employing material from unfractionated CBMCs and from CD4+ and CD8+ cells purified from the CBMCs. Among these, three major immune function–associated genes in addition to IL-10 differed significantly between the groups (Table 3), notably genes encoding the transcription factor c-Maf, which plays a number of roles in T-cell differentiation; and the pleiotrophic cytokines IL-2 and IL-21. Expression of c-Maf in purified CD4+ T cells correlated strongly with IL-10 (Pearson r2 = 0.86, P < 0.0001) and also with IL-2 (r2 = 0.47, P = 0.0095) and IL-21 (r2 = 0.88, P < 0.0001).
CBMCs | CD4+ T Cells | CD8+ T Cells | T-cell–depleted Cells | |||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Hyperexpressed Gene | High Risk (n = 14) | Low Risk (n = 15) | P Value | High Risk (n = 14) | Low Risk (n = 15) | P Value | High Risk (n = 14) | Low Risk (n = 15) | P Value | High risk (n = 14) | Low Risk (n = 15) | P Value | ||||||||
| IL-10 | −0.38 ± 0.90 | 5.41 ± 2.90 | 0.102 | 0.43 ± 0.31 | 4.06 ± 1.39 | 0.028 | −0.34 ± 0.15 | 1.60 ± 0.94 | 0.006 | −0.49 ± 0.17 | 1.06 ± 0.68 | 0.038 | ||||||||
| c-Maf | 1.86 ± 0.38 | 0.07 ± 0.96 | 0.077 | −1.14 ± 0.67 | 4.31 ± 2.09 | 0.040 | −3.30 ± 0.54 | −1.26 ± 0.95 | 0.014 | 4.48 ± 0.52 | −2.42 ± 0.86 | 0.052 | ||||||||
| IL-2 | 7.40 ± 0.73 | 16.84 ± 3.40 | 0.041 | 19.33 ± 2.42 | 43.35 ± 9.87 | 0.077 | 4.85 ± 0.78 | 10.16 ± 2.02 | 0.093 | 5.76 ± 1.96 | 15.96 ± 3.56 | 0.015 | ||||||||
| IL-21 | 2.47 ± 0.51 | 6.69 ± 2.49 | 0.217 | 9.02 ± 1.62 | 20.25 ± 7.09 | 0.285 | 1.19 ± 0.23 | 3.40 ± 0.98 | 0.038 | 1.63 ± 0.66 | 6.55 ± 1.33 | 0.006 | ||||||||
This study has shown that risk for repeated respiratory infections during infancy and early childhood is markedly influenced by the relativity between IL-10 and IL-5 response capacity. Moreover, the relevant cytokine responses in this context are within the adaptive immune system, as substitution of T-cell–derived IL-10 response data in these analyses with data obtained with the innate immune stimulus LPS did not yield similar findings. The summary data in Figure 3 illustrate this tripartite relationship between T-cell–derived IL-10 and IL-5 and cumulative infection risk over the first 5 years of life. Figure 3A presents the predicted infection incidence rates of ARI estimated with a Poisson regression model using loge PHA-induced cord blood IL-10 and IL-5 response data (as individual independent variables) and 5 years of ARI episodes from individual children (as the dependent variable). It can be seen that relative risk for ARI during the period varies over at least a twofold range between the two extremes of the cytokine response spectrum, namely, IL-10hi/IL-5lo versus IL-10lo/IL-5hi. For LRI (Figure 3B) it varies over at least a threefold range across the response spectrum.
Figure 3. Predicted incidence rates (episodes/5 yr) of (A) ARI and (B) LRI. Poisson regression model was used for the calculation of incidence rates and natural log values of IL-10 and IL-5 stimulated with phytohemagglutinin were fitted in the model. Figures were drawn with Graphis (Kylebank Software, Ayr, UK).
[More] [Minimize]The inference from these findings is that risk for infection during early life is associated with excessive IL-5 production and that this is attenuated by parallel production of IL-10, and further that IL-10 itself may have a positive role in resistance to early infections. It is possible that increased infection risk in the IL-5hi group may be due in part to underlying IL-4 production, which is a necessary prerequisite for IL-5 responses in these children, that is, via IL-4–mediated immune deviation (and hence attenuation) of antiviral Th1 responses. However, it is also feasible that IL-5 may contribute directly to the high-risk phenotype via its effects on eosinophil recruitment and activation. Eosinophil function during infancy is known to be increased throughout the whole population relative to that in older age groups (29, 30), reflecting the generalized Th2 bias characteristic of the immune system during this life phase (10), and the subset of IL-5hi children identified here as at maximal risk of infection are at the extreme of the Th2 bias spectrum. As such, the contribution of eosinophils to host defense responses (including to infections) within this subgroup is likely to be larger than within the population at large, and if this occurs in the context of respiratory viral infections it may translate into the intense symptomatology that is observed in susceptible infants (12–15).
However, findings suggest that the appearance of eosinophils at infection sites may not always represent an immunologic aberration per se. Notably, one animal model study has demonstrated accelerated respiratory viral clearance in hypereosinophilic (IL-5 transgenic) mice, which suggests that inflammatory effector mechanisms used by these cells can also potentially contribute positively to host defense in the airways (31). This contribution may be particularly significant during early infancy, when many other inflammatory effector mechanisms are developmentally attenuated, in contrast to the relative hyperactivity of the eosinophil pathway (29, 30). Nevertheless, findings linking airway eosinophilia with bronchiolitis symptoms during early life (12–15) indicate that if this alternative pathway for viral elimination indeed operates in human infants then it is prone to dysregulation; it has intrinsic potential to overshoot in intensity (i.e., airway eosinophilia), and as a consequence can itself contribute to local inflammation and resulting pathology. This suggests a failure of whatever endogenous homeostatic mechanisms normally operate to limit local inflammation. In this context, one of the key functions of IL-10 is downregulation of inflammation (20), including IL-5–dependent eosinophil-associated airways inflammation (32). The relationship between these two cytokines within the T-cell compartment and infection risk, which is illustrated in Figure 3, is consistent with such a damping role for IL-10 during infancy.
The data in Figure 3 also leave open the possibility that relative resistance to infection in the IL-10hi group may be due in part to other and more direct effects of IL-10 on viral elimination. IL-10 is a highly pleiotrophic cytokine with multiple immunoregulatory functions, which also include direct or indirect stimulation of a range of important immunoinflammatory effector mechanisms that are central to host defense. In particular, IL-10 drives the functional maturation of CD8+ cytotoxic T cells (33–36) and associated allograft rejection responses (37), and also activation of CD8+ NK cells (26, 32, 35, 36). Of particular note are reports demonstrating the role of IL-10 in enhancing the IFN-γ production capacity of these cell types (20, 33, 38, 39), which are the major cellular sources of this cytokine among neonatal CBMCs (40). We (41) and others (42) have demonstrated that reduced capacity for IFN-γ production during infancy contributes significantly toward risk for respiratory viral infection, and it is feasible that the putative protective role of IL-10 observed here may be mediated via enhancement of this function.
As noted previously, we also acknowledge the possibility that the covert expression of additional effector genes may be central to the relative resistance to infection observed in children expressing the IL-10hi/IL-5lo phenotype. As a preliminary step to investigate this possibility we have performed a case–control investigation to compare gene expression profiles in stimulated CBMCs from two groups of neonates at the extremes of the IL-10/IL-5 response spectrum, focusing on genes with known immune-associated functions that are differentially expressed in the resistant IL-10hi/IL-5lo subgroup. Prominent among the hyperexpressed genes were those encoding transcription factor c-Maf, and the pleiotrophic cytokines IL-2 and IL-21. IL-2 has multiple immunoregulatory functions of relevance to this discussion, in particular in the promotion of antiviral immunity via enhancement of NK-cell and CD8+ T-cell activity (43, 44) and in expansion of T-regulatory cells (45), which play a central role in limiting the intensity and duration of inflammatory responses within the airway mucosa. IL-21, which is itself IL-2 dependent (32), is also hyperexpressed in the IL-10hi/IL-5lo subgroup. This cytokine has potent effects in stimulation of NK-cell and CD8+ T-cell effector functions (46–50), and has also been implicated in the regulation of dendritic cells (51). One report has also demonstrated that IL-21 can mediate an alternative pathway for induction of proinflammatory Th17 cells (52).
Of additional interest was the finding that production of these three pleiotrophic cytokines in CBMCs from the IL-10hi/IL-5lo neonates was highly correlated with expression of the transcription factor c-Maf. Earlier interest in this transcription factor has focused mainly on its role in the regulation of IL-4/IL-5 balance in Th2 differentiation (53), but more recently it has become evident that it plays a broader role in T-cell function. In particular, c-Maf responsive elements have been demonstrated in the IL-10 promoter (54), overproduction of IL-10 is observed in c-Maf transgenic T cells (55, 56), and elevated expression of c-Maf is a feature of IL-10–producing regulatory T cells (57).
In conclusion, although the balance between the pro- and antiinflammatory consequences of IL-5 and IL-10 production may play a direct role in resistance to (and subsequent intensity of) respiratory infections in the IL-10hi/IL-5lo subgroup in this cohort, it appears likely that genes encoding other cytokines and associated regulators, exemplified by this group of IL-2, IL-21, and c-Maf with functions overlapping and complementing IL-10, may also be involved in the expression of this phenotype. Such findings are not unexpected, given the accumulating evidence from genetic studies indicating the involvement of multiple genes in the pathogenesis of airway diseases.
It is also pertinent to note the report relating to the Childhood Origins of Asthma (COAST) Project on high-risk children in the United States, which has also investigated relationships between cord blood cytokine responses and susceptibility to viral infections during the first years of life. No comparable relationships were detected between IL-10 and IL-5 response capacity and susceptibility to subsequent infection in that cohort (42). However, in that study a clinical score was used to determine whether episodes would be logged as infections and specimens collected. Symptoms needed to achieve a score of 5 (note: wheeze scored 5, and therefore all wheeze-associated infections would have been logged) before specimens were collected. Thus, infections in the COAST project excluded milder and upper respiratory infections and were more similar to those classified as severe (i.e., LRI/wLRI) in our study. In addition, IL-5 responses above the limits of detection were observed in only approximately 50% of the COAST cohort (58), in contrast to the present study in which responses were detected in at least 80% of subjects. These differences, which may be due to variations in cell-handling/culture techniques and/or to differing patterns of gene × environment interactions within the relevant Australian/U.S. populations, limit the capacity to make direct comparisons between the studies and underscore the necessity for follow-ups in other independent cohorts.
| 1. | Siegrist CA. Vaccination in the neonatal period and early infancy. Int Rev Immunol 2000;19:195–219. |
| 2. | Hoffjan S, Nicolae D, Ostrovnaya I, Roberg K, Evans M, Mirel DB, Steiner L, Walker K, Shult P, Gangnon RE, et al. Gene–environment interaction effects on the development of immune responses in the 1st year of life. Am J Hum Genet 2005;76:696–704. |
| 3. | Kusel MM, de Klerk NH, Holt PG, Kebadze T, Johnston SL, Sly PD. Role of respiratory viruses in acute upper and lower respiratory tract illness in the first year of life: a birth cohort study. Pediatr Infect Dis J 2006;25:680–686. |
| 4. | Hall CB. Respiratory syncytial virus and parainfluenza virus. N Engl J Med 2001;344:1917–1928. |
| 5. | Oddy WH, de Klerk NH, Sly PD, Holt PG. The effects of respiratory infections, atopy, and breastfeeding on childhood asthma. Eur Respir J 2002;19:899–905. |
| 6. | Stein RT, Sherrill D, Morgan WJ, Holberg CJ, Halonen M, Taussig LM, Wright AL, Martinez FD. Respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years. Lancet 1999;354:541–545. |
| 7. | Holt PG, Clough JB, Holt BJ, Baron-Hay MJ, Rose AH, Robinson BWS, Thomas WR. Genetic “risk” for atopy is associated with delayed postnatal maturation of T-cell competence. Clin Exp Allergy 1992;22:1093–1099. |
| 8. | Martinez FD, Holt PG. The role of microbial burden in the aetiology of allergy and asthma. Lancet 1999;354:12–15. |
| 9. | Holt PG, Macaubas C. Development of long term tolerance versus sensitisation to environmental allergens during the perinatal period. Curr Opin Immunol 1997;9:782–787. |
| 10. | Wegmann TG, Lin H, Guilbert L, Mosmann TR. Bidirectional cytokine interactions in the maternal–fetal relationship: is successful pregnancy a Th2 phenomenon? Immunol Today 1993;14:353–356. |
| 11. | Holt PG, Sly PD. Interactions between RSV infection, asthma, and atopy: unraveling the complexities. J Exp Med 2002;196:1271–1275. |
| 12. | Dimova-Yaneva D, Russell D, Main M, Brooker RJ, Helms PJ. Eosinophil activation and cysteinyl leukotriene production in infants with respiratory syncytial virus bronchiolitis. Clin Exp Allergy 2004;34:555–558. |
| 13. | Harrison AM, Bonville CA, Rosenberg HF, Domachowske JB. Respiratory syncytical virus–induced chemokine expression in the lower airways: eosinophil recruitment and degranulation. Am J Respir Crit Care Med 1999;159:1918–1924. |
| 14. | Kristjansson S, Bjarnarson SP, Wennergren G, Palsdottir AH, Arnadottir T, Haraldsson A, Jonsdottir I. Respiratory syncytial virus and other respiratory viruses during the first 3 months of life promote a local TH2-like response. J Allergy Clin Immunol 2005;116:805–811. |
| 15. | Rakes GP, Arruda E, Ingram JM, Hoover GE, Zambrano JC, Hayden FG, Platts-Mills TA, Heymann PW. Rhinovirus and respiratory syncytial virus in wheezing children requiring emergency care: IgE and eosinophil analyses. Am J Respir Crit Care Med 1999;159:785–790. |
| 16. | Welliver RC. Respiratory syncytial virus and other respiratory viruses. Pediatr Infect Dis J 2003;22:S6–S10; discussion S10–S12. |
| 17. | Schwarze J, Cieslewicz G, Hamelmann E, Joetham A, Shultz LD, Lamers MC, Gelfand EW. IL-5 and eosinophils are essential for the development of airway hyperresponsiveness following acute respiratory syncytial virus infection. J Immunol 1999;162:2997–3004. |
| 18. | Dakhama A, Park JW, Taube C, Joetham A, Balhorn A, Miyahara N, Takeda K, Gelfand EW. The enhancement or prevention of airway hyperresponsiveness during reinfection with respiratory syncytial virus is critically dependent on the age at first infection and IL-13 production. J Immunol 2005;175:1876–1883. |
| 19. | Culley FJ, Pollott J, Openshaw PJ. Age at first viral infection determines the pattern of T cell–mediated disease during reinfection in adulthood. J Exp Med 2002;196:1381–1386. |
| 20. | Moore KW, O'Garra A, de Waal Malefyt R, Vieira P, Mosmann TR. Interleukin-10. Annu Rev Immunol 1993;11:165–190. |
| 21. | Prescott SL, Macaubas C, Holt BJ, Smallacombe T, Loh R, Sly PD, Holt PG. Transplacental priming of the human immune system to environmental allergens: universal skewing of initial T-cell responses towards the Th-2 cytokine profile. J Immunol 1998;160:4730–4737. |
| 22. | Kusel MM, de Klerk NH, Kebadze T, Vohma V, Holt PG, Johnston SL, Sly PD. Early-life respiratory viral infections, atopic sensitization and risk of subsequent development of persistent asthma. J Allergy Clin Immunol 2007;119:1105–1110. |
| 23. | Rowe J, Heaton T, Kusel MM, Suriyaarachchi D, Serralha M, Holt BJ, de Klerk N, Sly PD, Holt PG. High IFN-γ production by CD8+ T cells and early sensitization among infants at high risk of atopy. J Allergy Clin Immunol 2004;113:710–716. |
| 24. | Bosco A, McKenna KL, Devitt CJ, Firth MJ, Sly PD, Holt PG. Identification of novel Th2-associated genes in T memory responses to allergens. J Immunol 2006;176:4766–4777. |
| 25. | Smyth GK. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol 2004;3:Article 3. |
| 26. | Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U, Speed TP. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 2003;4:249–264. |
| 27. | Cui X, Churchill GA. Statistical tests for differential expression in cDNA microarray experiments. Genome Biol 2003;4:210. |
| 28. | Perneger TV. Adjusting for multiple testing in studies is less important than other concerns. BMJ 1999;318:1288. |
| 29. | Holt PG. Postnatal maturation of immune competence during infancy and early childhood. Pediatr Allergy Immunol 1995;6:59–70. |
| 30. | Fernandes R, Kusel MM, Cyr M, Sehmi R, Holt K, Holt B, Kebadze T, Johnston SL, Sly P, Denburg JA, et al. Cord blood hemopoietic progenitor profiles predict fever and wheeze during acute respiratory illness in infancy. Pediatr Allergy Immunol 2008;112:6968–6976. |
| 31. | Phipps S, Lam CE, Mahalingam S, Newhouse M, Ramirez R, Rosenberg HF, Foster PS, Matthaei KI. Eosinophils contribute to innate antiviral immunity and promote clearance of respiratory syncytial virus. Blood 2007;110:1578–1586. |
| 32. | Kearley J, Barker JE, Robinson DS, Lloyd CM. Resolution of airway inflammation and hyperreactivity after in vivo transfer of CD4+CD25+ regulatory T cells is interleukin 10 dependent. J Exp Med 2005;202:1539–1547. |
| 33. | Santin AD, Hermonat PL, Ravaggi A, Bellone S, Pecorelli S, Roman JJ, Parham GP, Cannon MJ. Interleukin-10 increases Th1 cytokine production and cytotoxic potential in human papillomavirus–specific CD8+ cytotoxic T lymphocytes. J Virol 2000;74:4729–4737. |
| 34. | Groux H, Bigler M, de Vries JE, Roncarolo MG. Inhibitory and stimulatory effects of IL-10 on human CD8+ T cells. J Immunol 1998;160:3188–3193. |
| 35. | Giovarelli M, Musiani P, Modesti A, Dellabona P, Casorati G, Allione A, Consalvo M, Cavallo F, di Pierro F, De Giovanni C, et al. Local release of IL-10 by transfected mouse mammary adenocarcinoma cells does not suppress but enhances antitumor reaction and elicits a strong cytotoxic lymphocyte and antibody-dependent immune memory. J Immunol 1995;155:3112–3123. |
| 36. | Chen WF, Zlotnik A. IL-10: a novel cytotoxic T cell differentiation factor. J Immunol 1991;147:528–534. |
| 37. | Qian S, Li W, Li Y, Fu F, Lu L, Fung JJ, Thomson AW. Systemic administration of cellular interleukin-10 can exacerbate cardiac allograft rejection in mice. Transplantation 1996;62:1709–1714. |
| 38. | Zheng LM, Ojcius DM, Garaud F, Roth C, Maxwell E, Li Z, Rong H, Chen J, Wang XY, Catino JJ, et al. Interleukin-10 inhibits tumor metastasis through an NK cell–dependent mechanism. J Exp Med 1996;184:579–584. |
| 39. | Lauw FN, Pajkrt D, Hack CE, Kurimoto M, van Deventer SJ, van der Poll T. Proinflammatory effects of IL-10 during human endotoxemia. J Immunol 2000;165:2783–2789. |
| 40. | White GP, Watt PM, Holt BJ, Holt PG. Differential patterns of methylation of the IFNγ promoter at CpG and non-CpG sites underlie differences in IFNγ gene expression between human neonatal and adult CD45RO− T-cells. J Immunol 2002;168:2820–2827. |
| 41. | Rowe J, Macaubas C, Monger T, Holt BJ, Harvey J, Poolman JT, Loh R, Sly PD, Holt PG. Heterogeneity in diphtheria–tetanus–acellular pertussis vaccine–specific cellular immunity during infancy: relationship to variations in the kinetics of postnatal maturation of systemic Th1 function. J Infect Dis 2001;184:80–88. |
| 42. | Copenhaver CC, Gern JE, Li Z, Shult PA, Rosenthal LA, Mikus LD, Kirk CJ, Roberg KA, Anderson EL, Tisler CJ, et al. Cytokine response patterns, exposure to viruses, and respiratory infections in the first year of life. Am J Respir Crit Care Med 2004;170:175–180. |
| 43. | Kundig TM, Schorle H, Bachmann MF, Hengartner H, Zinkernagel RM, Horak I. Immune responses in interleukin-2-deficient mice. Science 1993;262:1059–1061. |
| 44. | Cousens LP, Orange JS, Biron CA. Endogenous IL-2 contributes to T cell expansion and IFN-γ production during lymphocytic choriomeningitis virus infection. J Immunol 1995;155:5690–5699. |
| 45. | Nelson BH. IL-2, regulatory T cells, and tolerance. J Immunol 2004;172:3983–3988. |
| 46. | Perez SA, Mahaira LG, Sotiropoulou PA, Gritzapis AD, Iliopoulou EG, Niarchos DK, Cacoullos NT, Kavalakis YG, Antsaklis AI, Sotiriadou NN, et al. Effect of IL-21 on NK cells derived from different umbilical cord blood populations. Int Immunol 2006;18:49–58. |
| 47. | Kumano M, Hara I, Furukawa J, Oniki S, Nagai H, Miyake H, Fujisawa M. Interleukin-21 activates cytotoxic T lymphocytes and natural killer cells to generate antitumor response in mouse renal cell carcinoma. J Urol 2007;178:1504–1509. |
| 48. | Casey KA, Mescher MF. IL-21 promotes differentiation of naive CD8 T cells to a unique effector phenotype. J Immunol 2007;178:7640–7648. |
| 49. | Brady J, Hayakawa Y, Smyth MJ, Nutt SL. IL-21 induces the functional maturation of murine NK cells. J Immunol 2004;172:2048–2058. |
| 50. | Allard EL, Hardy MP, Leignadier J, Marquis M, Rooney J, Lehoux D, Labrecque N. Overexpression of IL-21 promotes massive CD8+ memory T cell accumulation. Eur J Immunol 2007;37:3069–3077. |
| 51. | Maeda M, Yanagawa Y, Iwabuchi K, Minami K, Nakamaru Y, Takagi D, Fukuda S, Onoe K. IL-21 enhances dendritic cell ability to induce interferon-γ production by natural killer T cells. Immunobiology 2007;212:537–547. |
| 52. | Korn T, Bettelli E, Gao W, Awasthi A, Jager A, Strom TB, Oukka M, Kuchroo VK. IL-21 initiates an alternative pathway to induce proinflammatory TH17 cells. Nature 2007;448:484–487. |
| 53. | Kim JI, Ho IC, Grusby MJ, Glimcher LH. The transcription factor c-Maf controls the production of interleukin-4 but not other Th2 cytokines. Immunity 1999;10:745–751. |
| 54. | Cao S, Liu J, Song L, Ma X. The protooncogene c-Maf is an essential transcription factor for IL-10 gene expression in macrophages. J Immunol 2005;174:3484–3492. |
| 55. | Pauza ME, Nguyen A, Wolfe T, Ho IC, Glimcher LH, von Herrath M, Lo D. Variable effects of transgenic c-Maf on autoimmune diabetes. Diabetes 2001;50:39–46. |
| 56. | Ho IC, Lo D, Glimcher LH. c-Maf promotes T helper cell type 2 (Th2) and attenuates Th1 differentiation by both interleukin 4–dependent and –independent mechanisms. J Exp Med 1998;188:1859–1866. |
| 57. | Shoemaker J, Saraiva M, O'Garra A. GATA-3 directly remodels the IL-10 locus independently of IL-4 in CD4+ T cells. J Immunol 2006;176:3470–3479. |
| 58. | Neaville WA, Tisler C, Bhattacharya A, Anklam K, Gilbertson-White S, Hamilton R, Adler K, Dasilva DF, Roberg KA, Carlson-Dakes KT, et al. Developmental cytokine response profiles and the clinical and immunologic expression of atopy during the first year of life. J Allergy Clin Immunol 2003;112:740–746. |
