Barbara J. Holt

Development of allergen-specific T-cell memory in atopic and normal children

BACKGROUND In the past 20-30 years, there has been an increase in prevalence of allergic respiratory diseases, particularly amongst children. This study is a prospective analysis of the postnatal maturation of T-helper cell (Th) responses to aeroallergens in atopic and non-atopic infants. METHODS We measured mononuclear-cell proliferative and cytokine responses to specific allergens and tetanus toxoid in blood samples from atopic and non-atopic infants every 6 months from birth to 2 years of age. Cytokine analyses of responses to housedust-mite allergen used ELISA and reverse-transcriptase PCR. We also measured responses to Fel d1 (cat allergen) and tetanus toxoid. FINDINGS Samples from 18 atopic and 13 non-atopic infants showed low-level Th2-skewed allergen-specific responses at birth, with little accompanying specific interferon-gamma production. Neonatal Th2 responses were lower in the atopic group than in the non-atopic group; the differences were significant for interleukin-4 (mRNA: beta-actin ratio 0.48 [SE 0.15] vs 0.15 [0.06], p=0.049), interleukin-6 (4750 [48] vs 1352 [51] pg/mL culture fluid, p=0.003), interleukin-10 (1162 [228] vs 485 [89], p=0.015), and interleukin-13 (7.1 [0.9] vs 0.9 [0.3], p=0.008). There was rapid suppression of Th2 responses during the first year of life in non-atopic children, but there was consolidation of responses in atopic children, associated with defective neonatal interferon-gamma production. INTERPRETATION The continuation of fetal allergen-specific Th2 responses during infancy is a defining feature of the inductive phase of atopic disease, and is associated with decreased capacity for production of the Th1 cytokine interferon y by atopic neonates. These findings provide a plausible mechanism for persistence of the fetal Th2 responses during early childhood in atopic individuals and subsequent expression of disease.

Differential Patterns of Methylation of the IFN-γ Promoter at CpG and Non-CpG Sites Underlie Differences in IFN-γ Gene Expression Between Human Neonatal and Adult CD45RO− T Cells

IFN-γ is a potent pleiotropic Th1 cytokine, the production of which is tightly regulated during fetal development. Negative control of fetal/neonatal IFN-γ production is generally attributed to the Th1-antagonistic effect of mediators produced by the placenta, but evidence exists of additional and more direct transcriptional regulation. We report that neonatal (cord blood) CD3+/CD45RO− T cells, in particular the CD4+/CD45RO− subset, are hypermethylated at CpG and non-CpG (CpA and CpT) sites within and adjacent to the IFN-γ promoter. In contrast, CpG methylation patterns in cord blood IFN-γ-producing CD8+/CD45RO− T cells and CD56+/CD16+/CD3− NK cells did not differ significantly from those in their adult counterparts. Consistent with this finding, IFN-γ production by stimulated naive cord blood CD4+ T cells is reduced 5- to 10-fold relative to adult CD4+ T cells, whereas production levels in neonatal and adult CD8+ T cells are of a similar order. Evidence of significant CpA and CpT methylation was not discovered in promoter sequence from other cytokines (IL-4, TNF-α, or IFN-γR α-chain). We additionally demonstrate that overexpression of DNA methyltransferase 3a in embryonic kidney carcinoma cells is accompanied by CpA methylation of the IFN-γ promoter.

An immunoepidemiological approach to asthma: identification of in-vitro T-cell response patterns associated with different wheezing phenotypes in children

BACKGROUND Increasing evidence suggests that patterns of T-cell immunity to inhalant allergens in genetically diverse human populations are more heterogeneous than previously assumed, and that covert differences in expression patterns might underlie variations in airway disease phenotypes. We tested this proposition in a community sample of children. METHODS We analysed data from 172 individuals who had been recruited antenatally to a longitudinal birth cohort study. Of the 194 birth cohort participants, data from the 147 probands (age range 8.6-13.5 years) who consented to blood collection were included along with data from 25 consenting siblings (mean age 11 years [range 7.4-17.4]). We ascertained clinical phenotypes related to asthma and allergy. We measured T-cell responses to allergens and mitogens, together with blood eosinophils and IgE/IgG antibodies, and assessed associations between these indices and clinical phenotypes. FINDINGS Atopy was associated with allergen-specific T-helper (Th)2 responses dominated by interleukin 4, interleukin 5, interleukin 9, interleukin 13, whereas interleukin 10, tumour necrosis factor alpha, and interferon gamma responses were common to both atopics and non-atopics. The wheal size from skin prick with allergen was positively associated with in-vitro interleukin 5 and interferon gamma responses, and negatively associated with interleukin 10. Asthma, especially in atopics, was strongly associated with eosinophilia/interleukin 5, and bronchial hyper-responsiveness (BHR) was associated with eosinophilia plus polyclonal interferon gamma production. BHR in non-atopics was associated with elevated allergen-specific and polyclonal interleukin 10 production. INTERPRETATION Parallel immunological and clinical profiling of children identified distinctive immune response patterns related to asthma and wheeze compared with BHR, in atopics non-atopics. Immunological hyper-responsiveness, including within the Th1 cytokine compartment, is identified as a hallmark of BHR. RELEVANCE TO PRACTICE These findings highlight the heterogeneity of immune response patterns in asthmatic children, including those with seemingly homogeneous Th2-driven atopic asthma. Further elucidation of the covert relationships between wheezing phenotypes and underlying immunophenotypes in this age group will potentially lead to more effective treatments for what is an unexpectedly heterogeneous collection of disease subtypes.

The infant nasopharyngeal microbiome impacts severity of lower respiratory infection and risk of asthma development.

Summary The nasopharynx (NP) is a reservoir for microbes associated with acute respiratory infections (ARIs). Lung inflammation resulting from ARIs during infancy is linked to asthma development. We examined the NP microbiome during the critical first year of life in a prospective cohort of 234 children, capturing both the viral and bacterial communities and documenting all incidents of ARIs. Most infants were initially colonized with Staphylococcus or Corynebacterium before stable colonization with Alloiococcus or Moraxella. Transient incursions of Streptococcus, Moraxella, or Haemophilus marked virus-associated ARIs. Our data identify the NP microbiome as a determinant for infection spread to the lower airways, severity of accompanying inflammatory symptoms, and risk for future asthma development. Early asymptomatic colonization with Streptococcus was a strong asthma predictor, and antibiotic usage disrupted asymptomatic colonization patterns. In the absence of effective anti-viral therapies, targeting pathogenic bacteria within the NP microbiome could represent a prophylactic approach to asthma.

Development of Interleukin-12-Producing Capacity throughout Childhood

ABSTRACT Increasing evidence indicates that the capacity to induce protective Th1 immune responses is impaired in early childhood, an observation that can be partially attributed to deficiencies in antigen-presenting-cell function. Synthesis of interleukin 12 (IL-12), a key Th1-trophic cytokine, is markedly reduced in the neonatal period, though there is a paucity of knowledge concerning the ontogeny of IL-12-synthetic capacity throughout the childhood years. Hence, we examined the production of bioactive IL-12 p70 by circulating mononuclear cells in a population of healthy individuals. As expected, the capacity to synthesize IL-12 p70 in response to either lipopolysaccharide or heat-killed Staphylococcus aureus was markedly impaired at birth, even after priming of cells with gamma interferon. Surprisingly however, IL-12 p70 synthesis by peripheral blood mononuclear cells from both 5- and 12-year-old children was still substantially below that seen in adults, and this did not appear to be related to excessive production of IL-10. In contrast, dendritic cells from adults and neonates, derived from monocytes with granulocyte-macrophage colony-stimulating factor and IL-4, synthesized equivalent amounts of IL-12 p70 in response to microbial stimulation. This indicates that the impaired capacity for IL-12 synthesis in childhood is not an intrinsic property of circulating mononuclear cells but rather can be readily overcome in response to appropriate maturational stimuli. Because IL-12 arose predominantly from circulating HLA-DR+ cells that lacked B-cell- and monocyte-specific markers, we propose that the slow maturation of IL-12-synthetic capacity in the childhood years can be attributed to deficiencies in the number and/or function of dendritic cells.

TH2-polarized immunological memory to inhalant allergens in atopics is established during infancy and early childhood

Background There is increasing evidence that the T‐cell reactivity to environmental allergens underlying expression of allergic disease in adulthood, develops initially during childhood. However, there is little information available on the kinetics of these early responses, or on the patterns of cytokine production during this period.

Association of IL12B promoter polymorphism with severity of atopic and non-atopic asthma in children

BACKGROUND Severe asthma is a frequent cause of hospital admission, especially among children. The main environmental triggers of airway inflammation in asthma are viruses and aeroallergens. These agents elicit reciprocal immune responses, characterised by production of T helper 1 and T helper 2 cytokines, respectively. There is no genetic explanation for how hyper-responsiveness to these disparate environmental stimuli develops among individuals with asthma. Our aim was to assess relation between an IL12B promoter polymorphism and asthma. METHODS We did a cohort study in which we initially genotyped 411 6-year olds for the IL12B promoter polymorphism. We then assessed the relation between this polymorphism and asthma severity. A further 85 asthmatic children in an additional sample of 433 children from the same cohort were then assessed to confirm these findings. We also examined in-vitro interleukin-12 responses in a subgroup of individuals. FINDINGS Heterozygosity for the IL12B promoter polymorphism was observed in 76% (16) of atopic and non-atopic individuals with severe asthma in the initial sample. By comparison, heterozygotes comprised only 31% (17) of the moderate asthma group, and 48% (20) of individuals with mild asthma were heterozygous, as were unaffected controls. These findings were confirmed in the second sample (overall p<0.0001). Our data suggest that IL12B promoter heterozygosity contributes to asthma severity rather than susceptibility per se. The severity-predisposing genotype was associated with reduced interleukin 12 p40 gene transcription and decreased interleukin 12 p70 secretion. INTERPRETATION Interleukin 12 plays a key part in antagonism of T helper 2 differentiation, and in induction of antiviral host defense. Genetically determined attenuation of interleukin-12 response capacity would, therefore, provide a plausible common immunological pathway to disease severity for the two major forms of asthma.

Functional maturation of CD4+CD25+CTLA4+CD45RA+ T regulatory cells in human neonatal T cell responses to environmental antigens/allergens.

A number of laboratories have reported cord blood T cell responses to ubiquitous environmental Ags, including allergens, by proliferation and cytokine secretion. Moreover, the magnitude of these responses has been linked with risk for subsequent expression of allergy. These findings have been widely interpreted as evidence for transplacental priming and the development of fetal T memory cells against Ags present in the maternal environment. However, we present findings below that suggest that neonatal T cell responses to allergens (and other Ags) differ markedly from those occurring in later life. Notably, in contrast to allergen-responsive adult CD4+ T cell cultures, responding neonatal T cell cultures display high levels of apoptosis. Comparable responses were observed against a range of microbial Ags and against a parasite Ag absent from the local environment, but not against autoantigen. A notable finding was the appearance in these cultures of CD4+CD25+CTLA4+ T cells that de novo develop MLR-suppressive activity. These cells moreover expressed CD45RA and CD38, hallmarks of recent thymic emigrants. CFSE-labeling studies indicate that the CD4+CD25+ cells observed at the end of the culture period were present in the day 0 starting populations, but they were not suppressive in MLR responses. Collectively, these findings suggest that a significant component of the reactivity of human neonatal CD4+ T cells toward nominal Ag (allergen) represents a default response by recent thymic emigrants, providing an initial burst of short-lived cellular immunity in the absence of conventional T cell memory, which is limited in intensity and duration via the parallel activation of regulatory T cells.

Vitamin D and atopy and asthma phenotypes in children: a longitudinal cohort study

Vitamin D has been linked in some studies with atopy- and asthma-associated phenotypes in children with established disease, but its role in disease inception at the community level is less clear. The aim of the present study was to investigate associations between vitamin D status and biological signatures indicative of allergy and asthma development in children aged 6 and 14 years in Perth, WA, Australia (latitude 32° S). Serum vitamin D was assayed in 989 6-yr-olds and 1,380 14-yr-olds from an unselected community birth cohort; 689 subjects were assessed at both ages. Vitamin D levels were assessed as a risk modifier for respiratory and allergic outcomes at both ages, using previously ascertained phenotypic data. The predictive value of vitamin D levels at age 6 yrs for development of clinical phenotypes at age 14 yrs was also examined. Serum vitamin D levels in children of both ages were negatively associated with concurrent allergic phenotypes; sex stratification revealed that this association was restricted mainly to males. Furthermore, vitamin D levels at age 6 yrs were significant predictors of subsequent atopy/asthma-associated phenotypes at age 14 yrs. In an unselected community setting, children (particularly males) with inadequate vitamin D are at increased risk of developing atopy, and subsequently bronchial hyperresponsiveness (BHR) and asthma. In a large unselected cohort, males with inadequate vitamin D at 6 and 14 yrs of age had increased atopy and BHR. Low vitamin D at age 6 yrs was a predictor of atopy and asthma at 14 yrs of age.

Maternal Serum Vitamin D Levels During Pregnancy and Offspring Neurocognitive Development

OBJECTIVES: To determine the association between maternal serum 25(OH)-vitamin D concentrations during a critical window of fetal neurodevelopment and behavioral, emotional, and language outcomes of offspring. METHODS: Serum 25(OH)-vitamin D concentrations of 743 Caucasian women in Perth, Western Australia (32°S) were measured at 18 weeks pregnancy and grouped into quartiles. Offspring behavior was measured with the Child Behavior Checklist at 2, 5, 8, 10, 14, and 17 years of age (n range = 412–652). Receptive language was assessed with the Peabody Picture Vocabulary Test—Revised at ages 5 (n = 534) and 10 (n = 474) years. Raw scores were converted to standardized scores, incorporating cutoffs for clinically significant levels of difficulty. RESULTS: χ2 analyses revealed no significant associations between maternal 25(OH)-vitamin D serum quartiles and offspring behavioral/emotional problems at any age. In contrast, there were significant linear trends between quartiles of maternal vitamin D levels and language impairment at 5 and 10 years of age. Multivariate regression analyses, incorporating a range of confounding variables, found that the risk of women with vitamin D insufficiency (≤46 nmol/L) during pregnancy having a child with clinically significant language difficulties was increased close to twofold compared with women with vitamin D levels >70 nmol/L. CONCLUSIONS: Maternal vitamin D insufficiency during pregnancy is significantly associated with offspring language impairment. Maternal vitamin D supplementation during pregnancy may reduce the risk of developmental language difficulties among their children.