Maternal Cytokine Profiles during Pregnancy Predict Asthma in Children of Mothers without Asthma.

Little is known about whether maternal immune status during pregnancy influences asthma development in the child. We measured cytokine production in supernatants from mitogen-stimulated peripheral blood immune cells collected during and after pregnancy from the mothers of children enrolled in the Tucson Infant Immune Study, a nonselected birth cohort. Physician-diagnosed active asthma in children through age 9 and a history of asthma in their mothers were assessed through questionnaires. Maternal production of each of the cytokines IL-13, IL-4, IL-5, IFN-γ, IL-10, and IL-17 during pregnancy was unrelated to childhood asthma. However, IFN-γ/IL-13 and IFN-γ/IL-4 ratios during pregnancy were associated with a decreased risk of childhood asthma (n = 381; odds ratio [OR], 0.33; 95% confidence interval [CI], 0.17-0.66; P = 0.002; and n = 368; OR, 0.36; 95% CI, 0.18-0.71; P = 0.003, respectively). The inverse relations of these two ratios with childhood asthma were only evident in mothers without asthma (n = 309; OR, 0.18; 95% CI, 0.08-0.42; P = 0.00007; and n = 299; OR, 0.17; 95% CI, 0.07-0.39; P = 0.00003, respectively) and not in mothers with asthma (n = 72 and 69, respectively; P for interaction by maternal asthma = 0.036 and 0.002, respectively). Paternal cytokine ratios were unrelated to childhood asthma. Maternal cytokine ratios in mothers without asthma were unrelated to the children's skin-test reactivity, total IgE, physician-confirmed allergic rhinitis at age 5, or eczema in infancy. To our knowledge, this study provides the first evidence that cytokine profiles in pregnant mothers without asthma relate to the risk for childhood asthma, but not allergy, and suggests a process of asthma development that begins in utero and is independent of allergy.

Epigenome-wide analysis links SMAD3 methylation at birth to asthma in children of asthmatic mothers.

BACKGROUND: The timing and mechanisms of asthma inception remain imprecisely defined. Although epigenetic mechanisms likely contribute to asthma pathogenesis, little is known about their role in asthma inception. OBJECTIVE: We sought to assess whether the trajectory to asthma begins already at birth and whether epigenetic mechanisms, specifically DNA methylation, contribute to asthma inception. METHODS: We used the Methylated CpG Island Recovery Assay chip to survey DNA methylation in cord blood mononuclear cells from 36 children (18 nonasthmatic and 18 asthmatic subjects by age 9 years) from the Infant Immune Study (IIS), an unselected birth cohort closely monitored for asthma for a decade. SMAD3 methylation in IIS (n = 60) and in 2 replication cohorts (the Manchester Asthma and Allergy Study [n = 30] and the Childhood Origins of Asthma Study [n = 28]) was analyzed by using bisulfite sequencing or Illumina 450K arrays. Cord blood mononuclear cell-derived IL-1ß levels were measured by means of ELISA. RESULTS: Neonatal immune cells harbored 589 differentially methylated regions that distinguished IIS children who did and did not have asthma by age 9 years. In all 3 cohorts methylation in SMAD3, the most connected node within the network of asthma-associated, differentially methylated regions, was selectively increased in asthmatic children of asthmatic mothers and was associated with childhood asthma risk. Moreover, SMAD3 methylation in IIS neonates with maternal asthma was strongly and positively associated with neonatal production of IL-1ß, an innate inflammatory mediator. CONCLUSIONS: The trajectory to childhood asthma begins at birth and involves epigenetic modifications in immunoregulatory and proinflammatory pathways. Maternal asthma influences epigenetic mechanisms that contribute to the inception of this trajectory.

Perinatal tumor necrosis factor-α production, influenced by maternal pregnancy weight gain, predicts childhood asthma.

RATIONALE: Innate immune responses marked by increases in tumor necrosis factor (TNF)-α have been associated with asthma but whether such alterations are evident before symptoms is not yet clear. OBJECTIVES: To determine if prevalence of childhood asthma or asthma-related traits is predicted by perinatal innate immune status and if maternal factors related to pregnancy influence asthma prevalence and innate immune status. METHODS: In the Tucson Infant Immune Study (a nonselected birth cohort), presence of eczema and wheezing in the child's first year and physician-diagnosed asthma through age 9 and asthma in the parents was obtained from parent-completed questionnaires. TNF-α, IL-6, IL-10, and IL-12 were measured in supernatants of LPS-stimulated peripheral blood mononuclear cells at birth and 3 months as was TNF-α in plasma. TNF-α single nucleotide polymorphisms were genotyped by Sequenom. Percent predicted FEV1/FVC was measured at age 9. Maternal weight gain during pregnancy and prepregnancy weight were ascertained from medical records. MEASUREMENTS AND MAIN RESULTS: Infants with persistently elevated LPS-induced TNF-α at birth and 3 months were at increased risk for childhood asthma (odds ratio [OR], 4.1; confidence interval [CI], 1.9-8.8; n = 233; P = 0.0003) and had decreased FEV1/FVC ratios at age 9. Children with mothers in the top tertile for pregnancy weight gain had increased risk for asthma (OR, 3.4; CI, 1.7-6.9; n = 225; P = 0.001) and persistently elevated TNF-α in early life (OR, 2.9; CI, 1.4-8.2; n = 195; P = 0.013). These relations were independent of maternal asthma and rhinitis. CONCLUSIONS: Persistently elevated LPS-induced TNF-α production early in life acts as a predictive biomarker for childhood asthma, and excess pregnancy weight gain in the mother seems to contribute to both.

Adaptive cytokine production in early life differentially predicts total IgE levels and asthma through age 5 years.

BACKGROUND: Although it has been postulated that allergic disease is associated with a predominance of T(H)2 cells, whether IgE levels and asthma might differ in their relation to early-life cytokine production is not known. OBJECTIVE: We sought to assess the relationship between first-year adaptive immune cytokine production with asthma and total IgE levels through age 5 years in a nonselected birth cohort. METHODS: Mitogen (concanavalin A/phorbol 12-myristate 13-acetate)-stimulated IL-4, IL-5, IL-13, and IFN-γ levels were measured in supernatants from cord blood mononuclear cells and PBMCs at birth, 3 months, and 12 months. Total serum IgE levels and physician-diagnosed active asthma were assessed at 1, 2, 3, and 5 years. Longitudinal models that adjust for both T(H)1 and T(H)2 cytokine production were used to determine relations of outcomes. RESULTS: Relations of cytokines to total IgE levels and asthma were strikingly different. Total IgE levels through age 5 years were positively associated with 12-month IL-4 (P < .001), IL-5 (P < .001), and IL-13 (P = .02) levels when adjusted for IFN-γ levels and inversely associated with 12-month IFN-γ levels after IL-4 adjustment (P = .01). Active asthma through age 5 years was positively associated with 3-month IL-13 levels adjusted for IFN-γ (odds ratio, 2.6; P < .001) and inversely associated with 3-month IFN-γ levels adjusted for IL-13 (odds ratio, 0.5; P = .001). These relations were strongest for nonatopic asthma. CONCLUSION: Total IgE levels and active asthma through age 5 years are associated with adaptive cytokine production in early life, although relations vary temporally and with regard to the relative importance of individual cytokines.

Th1/Th2 patterns and balance in cytokine production in the parents and infants of a large birth cohort.

Regulation of human immune cell cytokine production in vivo is not well understood due in part to limitations on imposing experimental conditions. We proposed that life-imposed conditions (pregnancy, birth, age, gender), combined with large sample size, repeat sampling, and family-based recruitment would serve to reveal peripheral blood cell-derived cytokine patterns reflective of in vivo regulation regarding Th1/Th2 balance and familial correlation. Mononuclear cells were obtained from 483 trios in the Tucson Infant Immune Study: from mothers pre- and postpartum, infants at birth and at 3 mo, and fathers. Con A/PMA-stimulated supernatants were assayed by ELISA for IFN-gamma, IL-4, IL-13, IL-5, and IL-10 and allergen-stimulated supernatants for IFN-gamma, IL-4, and IL-13. Mitogen-stimulated prepartum samples were not globally Th2 biased, differing from postpartum only by a modestly reduced IFN-gamma:IL-5 ratio. Prepartum samples actually produced less IL-10 and IL-13 although more IL-5 than paternal samples. Newborns were also not globally Th2 biased, with mitogen stimulation producing approximately 10-fold less IL-4, IL-5, and IFN-gamma than adults but only 2- to 3-fold less IL-13 and IL-10. Despite these group differences, all cytokines showed marked positive intraindividual correlations (all p < 0.001). Allergen stimulation gave results consistent with a lack of global Th2 bias. Mitogen stimulation revealed parent-child and parent-parent correlations. Thus, rather than a global Th2 bias, cytokine production in pregnant mothers and newborns appears regulated so as to maintain a relative balance among the cytokines, with the nature of the balance differing in mothers and infants and with production influenced by familial factors that include shared environment.

Effects of parental smoking on interferon gamma production in children.

OBJECTIVES: Environmental tobacco smoke is associated with several negative health outcomes in children, including an increased susceptibility to infections. One of the postulated mechanisms for these effects is the impairment of the immune system function and/or development. Yet, it remains unknown whether cumulative exposure to parental smoking is associated with altered immune responses in childhood and whether these effects are independent of in utero exposure to maternal smoking. In a population-based birth cohort, we sought to determine the relation of parental smoking, as assessed prospectively since pregnancy, to the child's interferon gamma and interleukin 4 production at 11 years of age. PATIENTS AND METHODS: We used data on 512 children and their parents from the Tucson Children's Respiratory Study cohort. Information on maternal and paternal smoking was collected prospectively by questionnaire, and pack-years for mother, father, and both parents combined were assessed prospectively between the prenatal period and year 11. At age 11 years, children's interferon gamma and interleukin 4 production from mitogen-stimulated peripheral blood mononuclear cells was measured. RESULTS: Children of parents who smoked between the prenatal period and year 11 were more likely to be in lower quartiles of interferon gamma production than children of nonsmoking parents. In addition, maternal, paternal, and parental pack-years showed significant inverse dose-response relationships with interferon gamma production in the child. These dose-response relationships with interferon gamma remained significant for both paternal and parental pack-years among children of mothers who did not smoke during pregnancy, suggesting the existence of specific postnatal effects of environmental tobacco smoke exposure. In contrast, no significant effects of parental smoking were found on interleukin 4 production. CONCLUSIONS: Interferon gamma responses of school-aged children are impacted by parental smoking.

Influence of early day-care exposure on total IgE levels through age 3 years.

BACKGROUND: Early day care is inversely associated with asthma and atopy in later childhood, but its association with early immunologic markers of asthma risk is not known. OBJECTIVE: We sought to assess the relation of day care by 3 months to total IgE levels through age 3 years. METHODS: Day care was assessed prospectively among 362 nonselected infants enrolled in the Infant Immune Study. Children were categorized based on day-care status by 3 months of age as follows: no day care, day care inside the home with other children, day care outside the home with no other children, or day care outside the home with other children. Total IgE levels were measured in blood obtained at 3, 12, 24, and 36 months. Relations between day care and IgE levels were assessed at each age and longitudinally, with stratification by maternal asthma and atopy. RESULTS: Day care by 3 months was associated with decreased IgE levels through age 3 years (coefficient: -0.19 log IU/mL, P = .001). The greatest effect was evident for children cared for outside the home. Stratified analyses indicated that the relation existed primarily among children who had atopic or asthmatic mothers. Day-care entry after 3 months showed no relation with IgE levels. CONCLUSION: Day-care attendance by 3 months is associated with decreased total IgE levels in the first 3 years of life in children of mothers who are atopic, asthmatic, or both. CLINICAL IMPLICATIONS: Early day-care exposure can reduce IgE levels, which in turn might reflect a reduced risk of allergic disease in predisposed children.

Association of IL-5 cytokine production and in vivo IgE levels in infants and parents.

BACKGROUND: Total IgE in human subjects tracks strongly from birth onward through unknown mechanisms. Regulation of IgE might occur in relation to adaptive immune cytokine production. In vitro studies have assessed the role of individual cytokines in regulating IgE production in human subjects. OBJECTIVE: We sought to investigate the association between IgE levels in vivo and the capacity of the individuals to produce adaptive immune cytokines. METHODS: Blood samples from participants in the Tucson Infant Immune Study (children at birth and at 3 and 12 months of age, fathers, and mothers before and after delivery) were assessed for percentage of eosinophils and plasma total IgE levels. IFN-gamma, IL-4, IL-5, IL-13, and IL-10 levels were measured in supernatants of mitogen-stimulated PBMCs and examined cross-sectionally for relation to cytokine production by using simple regression, multiple regression with cytokines only and with other known predictors of IgE levels, and longitudinally by means of random effects modeling. RESULTS: After adjusting for eosinophils and other predictors, IL-5 production (but not that of other cytokines) was associated directly with total IgE levels in children at 3 months (P = .009) and 12 months (P = .011) of age but not at birth. The IL-5/IgE association was present also in fathers (P = .040) and in mothers, both during pregnancy (P < .001) and after delivery (P = .030). CONCLUSIONS: This study indicates that mitogen-stimulated IL-5 production is associated with in vivo total IgE levels, independent of the production of other cytokines and circulating eosinophils. CLINICAL IMPLICATIONS: Understanding the regulation of IgE in vivo might help elucidate the development of allergic responses in individuals.

Th2 cell-selective enhancement of human IL13 transcription by IL13-1112C>T, a polymorphism associated with allergic inflammation.

IL-13 is a central mediator of allergic inflammation. The single nucleotide polymorphism IL13-1112C>T (rs1800925) is associated with allergic phenotypes in ethnically distinct populations, but the underlying mechanism(s) remain unknown. Using in vivo, in vitro, and in silico analysis, we show that the IL13-1112T allele enhanced IL13 promoter activity in primary human and murine CD4(+) Th2 lymphocytes. Increased expression of IL13-1112T in Th2 cells was associated with the creation of a Yin-Yang 1 binding site that overlapped a STAT motif involved in negative regulation of IL13 expression and attenuated STAT6-mediated transcriptional repression. Because IL-13 secretion was increased in IL13-1112TT homozygotes, we propose that increased expression of IL13-1112T in vivo may underlie its association with susceptibility to allergic inflammation. Interestingly, IL13-1112T had opposite transcriptional effects in nonpolarized CD4(+) T cells, paralleled by distinct patterns of DNA-protein interactions at the IL13 promoter. Our findings suggest the nuclear milieu dictates the functional outcome of genetic variation.

Rethinking Th2 antibody responses and allergic sensitization.

Human Th2 cytokines (interleukins 4 and 13) induce co-expression of IgE and IgG4 through sequential switching. The regulation of IgG4 responses and the role of these responses in the pathogenesis of allergy have not been characterized. We are addressing these issues by comparing and contrasting the expression of allergen-specific IgE and IgG4 in a population of European children thoroughly defined for lifestyle, environmental exposures and allergic phenotypes. The current analysis focused exclusively on children from non-farming families (n=493) in order to avoid potential effects of exposure to microbial products abundant in farming environments. We found that allergens induce Th2-mediated IgG4 and/or IgE responses in the majority of the population. Approximately two-thirds of the children had allergen-specific IgG4 but not IgE, only a minority had both IgG4 and IgE, only a few were negative for both, and virtually none had only IgE. The prevalence of asthma and hay fever was dramatically higher in children with high IgG4 and IgE compared to children who only mounted IgG4 or low IgG4 and IgE responses. These results appear to recapitulate different stages of in vivo Th2-dependent sequential switching from IgG4 to IgE. These patterns of Th2-induced antibody responses may warrant a redefinition of the notion of allergen sensitization.

Reduced interferon gamma production and soluble CD14 levels in early life predict recurrent wheezing by 1 year of age.

It is unknown whether reduced production of IFNgamma in early life, before any lower respiratory tract illness, is a risk factor for recurrent wheezing in infancy. We followed 238 infants prospectively from birth to 1 year of age. At birth and at 3 months of age, IFNgamma production from polyclonally stimulated peripheral blood mononuclear cells and soluble CD14 (sCD14) levels in plasma were measured. The odds of developing recurrent wheezing (assessed by questionnaire) in the first year of life were up to 4.5 times higher for children in the lowest quartile of IFNgamma production at 3 months (p = 0.0005) and 3.2 times higher for children in the lowest quartile of sCD14 levels at birth (p = 0.004) as compared with children in the other 3 combined quartiles of IFNgamma and sCD14, respectively. Findings were confirmed in the multivariate analysis. IFNgamma production at 3 months and sCD14 levels at birth were correlated (r = 0.188, p = 0.031). Our findings from a longitudinal cohort suggest that impaired IFNgamma production at 3 months and reduced plasma-sCD14 levels at birth significantly increase the risk of developing recurrent wheezing in the first year of life.

TGF-beta in human milk is associated with wheeze in infancy.

BACKGROUND: Cytokines secreted in human milk might play important roles in newborn health and in the development of infant immune responses. We investigated the relationship of the concentration and dose of cytokines in human milk to infant wheeze at 1 year of age. OBJECTIVE: Our objective was to test whether the cytokines in milk could account for some of the apparent protective effect of breast-feeding against wheeze in the first year of life. METHODS: Data on breast-feeding and infant wheeze were collected prospectively from birth to 1 year from 243 mothers participating in the Infant Immune Study in Tucson, Arizona. Breast milk samples obtained at a mean age of 11 days postpartum were assayed by means of ELISA for concentrations of TGF-beta1, IL-10, TNF-alpha, and the soluble form of CD14. The dose of each cytokine was assessed for a relationship with wheeze in bivariate and logistic regression analyses. RESULTS: Increasing duration of breast-feeding was significantly associated with a decreased prevalence of wheeze (P =.039). There was wide variability in levels of each cytokine in milk, as well as variability between women in the amount of each cytokine produced. There was a significant inverse association between the dose of TGF-beta1 received through milk with the percentage of wheeze (P =.017), and the relationship was linear (P =.006). None of the other cytokines showed a linear relationship with wheeze. In multivariate analyses the risk of wheeze was significantly decreased (odds ratio, 0.22; 95% CI 0.05-0.89; P =.034) with increasing TGF-beta1 dose (long breast-feeding and medium-high TGF-beta1 level compared with short breast-feeding and low TGF-beta. CONCLUSION: This analysis shows that the dose of TGF-beta1 received from milk has a significant relationship with infant wheeze, which might account for at least some of the protective effect of breast-feeding against wheeze.