Epigenomic variability is associated with age-specific naïve CD4 T cell response to activation in infants and adolescents.

Childhood is a critical period of immune development. During this time, naïve CD4 (nCD4) T cells undergo programmed cell differentiation, mediated by epigenetic changes, in response to external stimuli leading to a baseline homeostatic state that may determine lifelong disease risk. However, the ontogeny of epigenetic signatures associated with CD4 T cell activation during key developmental periods are yet to be described. We investigated genome-wide DNA methylation (DNAm) changes associated with nCD4 T activation following 72 h culture in media+anti-CD3/CD28 beads in healthy infants (aged 12 months, n = 18) and adolescents (aged 10-15 years, n = 15). We integrated these data with transcriptomic and cytokine profiling from the same samples. nCD4 T cells from both age groups show similar extensive epigenetic reprogramming following activation, with the majority of genes involved in the T cell receptor signaling pathway associated with differential methylation. Additionally, we identified differentially methylated probes showing age-specific responses, that is, responses in only infants or adolescents, including within a cluster of T cell receptor (TCR) genes. These encoded several TCR alpha joining (TRAJ), and TCR alpha variable (TRAV) genes. Cytokine data analysis following stimulation revealed enhanced release of IFN-γ, IL-2 and IL-10, in nCD4 T cells from adolescents compared with infants. Overlapping differential methylation and cytokine responses identified four probes potentially underpinning these age-specific responses. We show that DNAm in nCD4T cells in response to activation is dynamic in infancy and adolescence, with additional evidence for age-specific effects potentially driving variation in cytokine responses between these ages.

Children of Asian ethnicity in Australia have higher risk of food allergy and early-onset eczema than those in Singapore.

BACKGROUND: In Western countries, Asian children have higher food allergy risk than Caucasian children. The early-life environmental exposures for this discrepancy are unclear. We aimed to compare prevalence of food allergy and associated risk factors between Asian children in Singapore and Australia. METHODS: We studied children in the Growing Up in Singapore Towards healthy Outcomes (GUSTO) birth cohort (n = 878) and children of Asian ancestry in the HealthNuts cohort (n = 314). Food allergy was defined as a positive SPT ≥3 mm to egg or peanut AND either a convincing history of IgE-mediated reaction at 18 months (GUSTO) or a positive oral food challenge at 14-18 months (HealthNuts). Eczema was defined as parent-reported doctor diagnosis. RESULTS: Food allergy prevalence was 1.1% in Singapore and 15.0% in Australia (P<0.001). Egg introduction was more often delayed (>10 months) in Singapore (63.5%) than Australia (16.3%; P<0.001). Prevalence of early-onset eczema (<6 months) was lower in Singapore (8.4%) than Australia (30.5%) (P<0.001). Children with early-onset eczema were more likely to have food allergy than those without eczema in Australia [aOR 5.11 (2.34-11.14); P<0.001] and Singapore [aOR4.00 (0.62-25.8); P = 0.145]. CONCLUSIONS: Among Asian children, prevalence of early-onset eczema and food allergy was higher in Australia than Singapore. Further research with larger sample sizes and harmonized definitions of food allergy between cohorts is required to confirm and extend these findings. Research on environmental factors influencing eczema onset in Australia and Singapore may aid understanding of food allergy pathogenesis in different parts of the world.

Genetic determinants of paediatric food allergy: A systematic review.

BACKGROUND: The genetic determinants of food allergy have not been systematically reviewed. We therefore systematically reviewed the literature on the genetic basis of food allergy, identifying areas for further investigation. METHODS: We searched three electronic databases (MEDLINE, EMBASE and PubMed) on 9 January 2018. Two authors screened retrieved articles for review according to inclusion criteria and extracted relevant information on study characteristics and measures of association. Eligible studies included those that reported an unaffected nonatopic control group, had genetic information and were carried out in children. RESULTS: Of the 2088 studies retrieved, 32 met our inclusion criteria. Five were genome-wide association studies, and the remaining were candidate gene studies. Twenty-two of the studies were carried out in a predominantly Caucasian population with the remaining 10 from Asian-specific populations or unspecified ethnicity. We found FLG, HLA, IL10, IL13, as well as some evidence for other variants (SPINK5, SERPINB and C11orf30) that are associated with food allergy. CONCLUSIONS: Little genetic research has been carried out in food allergy, with FLG, HLA and IL13 being the most reproducible genes for an association with food allergy. Despite promising results, existing genetic studies on food allergy are inundated with issues such as inadequate sample size and absence of multiple testing correction. Few included replication analyses or population stratification measures. Studies addressing these limitations along with functional studies are therefore needed to unravel the mechanisms of action of the identified genes.

Egg allergen specific IgE diversity predicts resolution of egg allergy in the population cohort HealthNuts.

BACKGROUND: IgE-mediated egg allergy presents as one of the most common food allergies in children. Measurement of egg white specific IgE (sIgE) levels in serum or skin prick test has been shown to be a poor predictor of clinical allergy to raw egg white, and also to baked or cooked egg. Recent developments in component resolved diagnostic (CRD) technology have enabled us to improve the way in which we diagnose and predict peanut allergy by examining IgE specificity to individual peptides. OBJECTIVES: We aimed to investigate whether egg CRD could improve current methods to diagnose various egg allergy phenotypes as well as predict the development of tolerance to egg. METHODS: Using the HealthNuts cohort of food challenge-proven egg allergic and egg-sensitized and egg-tolerant, age-matched 12-month infants with longitudinal follow-up at 2 and 4 years (n = 451), we measured serum egg white, Gal d 1, 2, 3 and 5 sIgE using ImmunoCAP. RESULTS: Gal d 1 sensitization increased the risk of persistent egg allergy by 2.5-fold. The production of sIgE to all four egg allergens (Gal d 1, 2, 3 or 5) increased the risk of having persistent raw egg allergy fourfold (OR 4.19 (95% CI: 1.25-14.07). We did not find any improvements of using Gal d 1, 2, 3 or 5 to diagnose current egg allergy compared to egg white sIgE. CONCLUSION: Sensitization to multiple egg allergens Gal d 1, 2, 3 or 5 may be a prognostic marker that could be useful for patient management and identifying individuals at risk of developing persistent egg allergy.

Early life innate immune signatures of persistent food allergy.

BACKGROUND: Food allergy naturally resolves in a proportion of food-allergic children without intervention; however the underlying mechanisms governing the persistence or resolution of food allergy in childhood are not understood. OBJECTIVES: This study aimed to define the innate immune profiles associated with egg allergy at age 1 year, determine the phenotypic changes that occur with the development of natural tolerance in childhood, and explore the relationship between early life innate immune function and serum vitamin D. METHODS: This study used longitudinally collected PBMC samples from a population-based cohort of challenge-confirmed egg-allergic infants with either persistent or transient egg allergy outcomes in childhood to phenotype and quantify the functional innate immune response associated with clinical phenotypes of egg allergy. RESULTS: We show that infants with persistent egg allergy exhibit a unique innate immune signature, characterized by increased numbers of circulating monocytes and dendritic cells that produce more inflammatory cytokines both at baseline and following endotoxin exposure when compared with infants with transient egg allergy. Follow-up analysis revealed that this unique innate immune signature continues into childhood in those with persistent egg allergy and that increased serum vitamin D levels correlate with changes in innate immune profiles observed in children who developed natural tolerance to egg. CONCLUSIONS: Early life innate immune dysfunction may represent a key immunological driver and predictor of persistent food allergy in childhood. Serum vitamin D may play an immune-modulatory role in the development of natural tolerance.

The DNA methylation landscape of CD4+ T cells in oligoarticular juvenile idiopathic arthritis.

Juvenile idiopathic arthritis (JIA) is presumed to be driven by an adverse combination of genes and environment. Epigenetic processes, including DNA methylation, act as a conduit through which the environment can regulate gene activity. Altered DNA methylation has been associated with adult autoimmune rheumatic diseases such as rheumatoid arthritis, but studies are lacking for paediatric autoimmune rheumatic diseases including JIA. Here, we performed a genome-scale case-control analysis of CD4+ T cell DNA methylation from 56 oligoarticular JIA (oJIA) cases and 57 age and sex matched controls using Illumina HumanMethylation450 arrays. DNA methylation at each array probe was tested for association with oJIA using RUV (Remove Unwanted Variation) together with a moderated t-test. Further to this 'all-inclusive' analysis, we stratified by age at diagnosis (≤6yrs, >6yrs) and by sex as potential sources of heterogeneity. Following False Discovery Rate (FDR) adjustment, no probes were associated with oJIA in the all-inclusive, >6yrs-diagnosed, or sex-stratified analyses, and only one probe was associated with oJIA in the ≤6yrs-diagnosed analysis. We attempted technical validation and replication of 14 probes (punadj<0.01) at genes of known/potential relevance to disease. At VPS53, we demonstrated a regional shift towards higher methylation in oJIA (all-inclusive) compared to controls. At REEP3, where polymorphism has been previously associated with JIA, we demonstrated higher DNA methylation in male oJIA compared to male controls. This is the most comprehensive JIA case-control analysis of DNA methylation to date. While we have generated some evidence of altered methylation in oJIA, substantial differences are not apparent in CD4+ T cells. This may indicate a lesser relevance of DNA methylation levels in childhood, compared to adult, rheumatic disease.

Airway epithelium respiratory illnesses and allergy (AERIAL) birth cohort: study protocol.

Introduction: Recurrent wheezing disorders including asthma are complex and heterogeneous diseases that affect up to 30% of all children, contributing to a major burden on children, their families, and global healthcare systems. It is now recognized that a dysfunctional airway epithelium plays a central role in the pathogenesis of recurrent wheeze, although the underlying mechanisms are still not fully understood. This prospective birth cohort aims to bridge this knowledge gap by investigating the influence of intrinsic epithelial dysfunction on the risk for developing respiratory disorders and the modulation of this risk by maternal morbidities, in utero exposures, and respiratory exposures in the first year of life. Methods: The Airway Epithelium Respiratory Illnesses and Allergy (AERIAL) study is nested within the ORIGINS Project and will monitor 400 infants from birth to 5 years. The primary outcome of the AERIAL study will be the identification of epithelial endotypes and exposure variables that influence the development of recurrent wheezing, asthma, and allergic sensitisation. Nasal respiratory epithelium at birth to 6 weeks, 1, 3, and 5 years will be analysed by bulk RNA-seq and DNA methylation sequencing. Maternal morbidities and in utero exposures will be identified on maternal history and their effects measured through transcriptomic and epigenetic analyses of the amnion and newborn epithelium. Exposures within the first year of life will be identified based on infant medical history as well as on background and symptomatic nasal sampling for viral PCR and microbiome analysis. Daily temperatures and symptoms recorded in a study-specific Smartphone App will be used to identify symptomatic respiratory illnesses. Discussion: The AERIAL study will provide a comprehensive longitudinal assessment of factors influencing the association between epithelial dysfunction and respiratory morbidity in early life, and hopefully identify novel targets for diagnosis and early intervention.

Variable promoter methylation contributes to differential expression of key genes in human placenta-derived venous and arterial endothelial cells.

BACKGROUND: The endothelial compartment, comprising arterial, venous and lymphatic cell types, is established prenatally in association with rapid phenotypic and functional changes. The molecular mechanisms underpinning this process in utero have yet to be fully elucidated. The aim of this study was to investigate the potential for DNA methylation to act as a driver of the specific gene expression profiles of arterial and venous endothelial cells. RESULTS: Placenta-derived venous and arterial endothelial cells were collected at birth prior to culturing. DNA methylation was measured at >450,000 CpG sites in parallel with expression measurements taken from 25,000 annotated genes. A consistent set of genomic loci was found to show coordinate differential methylation between the arterial and venous cell types. This included many loci previously not investigated in relation to endothelial function. An inverse relationship was observed between gene expression and promoter methylation levels for a limited subset of genes implicated in endothelial function, including NOS3, encoding endothelial Nitric Oxide Synthase. CONCLUSION: Endothelial cells derived from the placental vasculature at birth contain widespread methylation of key regulatory genes. These are candidates involved in the specification of different endothelial cell types and represent potential target genes for environmentally mediated epigenetic disruption in utero in association with cardiovascular disease risk later in life.

Progress in understanding the epigenetic basis for immune development, immune function, and the rising incidence of allergic disease.

The profile of allergic disease worldwide continues to change as the number of severe IgE-mediated allergies increases. This phenomenon is thought to reflect the outcome of combined genetic/environmental/developmental/stochastic effects on immune development, but understanding this remains a challenge. Epigenetic disruption at key immune genes during development has been proposed as a potential explanation for how environmental exposures may alter immune cell development and function. This represents an emerging area of research with the potential to yield new understanding of how disease risk is modified. Here, we examine recent developments in this field that are defining new epigenetic paradigms of allergic disease.

Airway Epithelium Respiratory Illnesses and Allergy (AERIAL) birth cohort: study protocol.

Introduction: Recurrent wheezing disorders including asthma are complex and heterogeneous diseases that affect up to 30% of all children, contributing to a major burden on children, their families, and global healthcare systems. It is now recognized that a dysfunctional airway epithelium plays a central role in the pathogenesis of recurrent wheeze, although the underlying mechanisms are still not fully understood. This prospective birth cohort aims to bridge this knowledge gap by investigating the influence of intrinsic epithelial dysfunction on the risk for developing respiratory disorders and the modulation of this risk by maternal morbidities, in utero exposures, and respiratory exposures in the first year of life. Methods and Analysis: The Airway Epithelium Respiratory Illnesses and Allergy (AERIAL) study is nested within the ORIGINS Project and will monitor 400 infants from birth to five years. The primary outcome of the AERIAL study will be the identification of epithelial endotypes and exposure variables that influence the development of recurrent wheezing, asthma, and allergic sensitisation. Nasal respiratory epithelium at birth to six weeks, one, three, and five years will be analysed by bulk RNA-seq and DNA methylation sequencing. Maternal morbidities and in utero exposures will be identified on maternal history and their effects measured through transcriptomic and epigenetic analyses of the amnion and newborn epithelium. Exposures within the first year of life will be identified based on infant medical history as well as on background and symptomatic nasal sampling for viral PCR and microbiome analysis. Daily temperatures and symptoms recorded in a study-specific Smartphone App will be used to identify symptomatic respiratory illnesses. Ethics and Dissemination: Ethical approval has been obtained from Ramsey Health Care HREC WA-SA (#1908). Results will be disseminated through open-access peer-reviewed manuscripts, conference presentations, and through different media channels to consumers, ORIGINS families, and the wider community.

Children with East Asian-Born Parents Have an Increased Risk of Allergy but May Not Have More Asthma in Early Childhood.

BACKGROUND: We previously reported that infants with Asian-born parents are 3 times more likely to have IgE-mediated food allergy than those with Australian-born parents. It is unknown whether this translates to the increased risk of other allergic diseases later in childhood and whether ancestry interacts with other risk factors for allergic disease development. OBJECTIVE: To compare prevalence and risk factors for allergic rhinitis, asthma, and aeroallergen sensitization at age 6 between children with East Asian-born and Caucasian-born parents. METHODS: A total of 5276 1-year-old infants were recruited into a population-based longitudinal study of allergy. A total of 4455 children participated in age 6 follow-up (84.4%), including 3015 with Caucasian-born parents and 415 with East Asian-born parents. Children underwent skin prick tests to aeroallergens and questionnaires captured data on asthma, eczema, and allergic rhinitis. RESULTS: Compared with children with Caucasian-born parents, children of East Asian-born parents had more allergic rhinitis (19.9% [95% confidence interval (CI) 14.9-26] vs 9.3% [95% CI 8-10.8], P < .001) and aeroallergen sensitization (64.3% [95% CI 57.5-70.5] vs 34.4% [95% CI 32.2-36.7], P < .001) at age 6. Asthma was similar in both groups (9.1% [95% CI 6.2-13.2] vs 11.7% [95% CI 10.4-13.1]), P = .21. Children with IgE-mediated food allergy and eczema in infancy were 3 times more likely to have asthma and 2 times more likely to have allergic rhinitis at age 6, irrespective of ancestry. CONCLUSIONS: Children of East Asian ancestry born in Australia have a higher burden of most allergic diseases in the first 6 years of life, whereas asthma may follow a different pattern. IgE-mediated food allergy and eczema at age 1 increase the risk of asthma and allergic rhinitis irrespective of ancestry.

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry.

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective tag single nucleotide polymorphism (SNP) approach using a multiplexed genotyping assay with mass spectrometry, to investigate gene pathway associations in clinical cohorts. We investigate the food allergy candidate locus Interleukin13 (IL13) as an example. This method efficiently maximizes the coverage by taking advantage of shared linkage disequilibrium (LD) within a region. Selected LD SNPs are then designed into a multiplexed assay, enabling up to 40 different SNPs to be analyzed simultaneously, boosting cost-effectiveness. Polymerase chain reaction (PCR) is used to amplify the target loci, followed by single nucleotide extension, and the amplicons are then measured using matrix-assisted laser desorption/ionization-time of flight(MALDI-TOF) mass spectrometry. The raw output is analyzed with the genotype calling software, using stringent quality control definitions and cut-offs, and high probability genotypes are determined and output for data analysis.

Epigenetic modifications: mechanisms of disease and biomarkers of food allergy.

The rise in IgE-mediated food allergy in recent times is the likely result of gene-environment interactions mediated via epigenetic pathways. As epigenetic modifications, including DNA methylation, are at the interface between the environment and the genome, they may be ideal biomarkers of modifiable disease pathways. High-throughput methylation profiling of immune cell subtypes or whole blood from patients allows the identification of disease specific epigenetic variants. If faithfully tracking with disease parameters, these 'signatures' may have clinical applications as biomarkers of disease or therapeutic response. Development of such tools will depend on a number of factors, including determining the most appropriate experimental approach, analysis methodology, patient groups, and informative target cells/tissues. Here we discuss these potential applications and their implications for food allergy practise.

The role of gene-environment interactions in the development of food allergy.

The rates of IgE-mediated food allergy have increased globally, particularly in developed countries. The rising incidence is occurring more rapidly than changes to the genome sequence would allow, suggesting that environmental exposures that alter the immune response play an important role. Genetic factors may also be used to predict an increased predisposition to these environmental risk factors, giving rise to the concept of gene-environment interactions, whereby differential risk of environmental exposures is mediated through the genome. Increasing evidence also suggests a role for epigenetic mechanisms, which are sensitive to environmental exposures, in the development of food allergy. This paper discusses the current state of knowledge regarding the environmental and genetic risk factors for food allergy and how environmental exposures may interact with immune genes to modify disease risk or outcome.

Epigenome-wide analysis of neonatal CD4(+) T-cell DNA methylation sites potentially affected by maternal fish oil supplementation.

Supplementation of fish oil rich in omega-3 polyunsaturated fatty acids (n-3 PUFA) during pregnancy has been shown to confer favorable health outcomes in the offspring. In a randomized controlled trial, we have previously shown that n-3 PUFA supplementation in pregnancy was associated with modified immune responses and some markers of immune maturation. However, the molecular mechanisms underlying these heritable effects are unclear. To determine whether the biological effects of maternal n-3 PUFA supplementation are mediated through DNA methylation, we analyzed CD4(+) T-cells purified from cryo-banked cord blood samples from a previously conducted clinical trial. Of the 80 mother-infant pairs that completed the initial trial, cord blood samples of 70 neonates were available for genome-wide DNA methylation profiling. Comparison of purified total CD4(+) T-cell DNA methylation profiles between the supplement and control groups did not reveal any statistically significant differences in CpG methylation, at the single-CpG or regional level. Effect sizes among top-ranked probes were lower than 5% and did not warrant further validation. Tests for association between methylation levels and key n-3 PUFA parameters, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), or total n-3 PUFAs were suggestive of dose-dependent effects, but these did not reach genome-wide significance. Our analysis of the microarray data did not suggest strong modifying effects of in utero n-3 PUFA exposure on CD4(+) T-cell methylation profiles, and no probes on the array met our criteria for further validation. Other epigenetic mechanisms may be more relevant mediators of functional effects induced by n-3 PUFA in early life.

In vitro exposure of human blood mononuclear cells to active vitamin D does not induce substantial change to DNA methylation on a genome-scale.

It is well-established that vitamin D impacts gene regulation via vitamin D response elements (VDREs) across the genome. Recent evidence, primarily at a locus-specific level, suggests that alterations to DNA methylation may also be a relevant mechanism through which vitamin D regulates gene expression. Given the intense interest in vitamin D, particularly as an immune modifier, we sought to examine the impact of vitamin D exposure on the immune cell methylome in vitro. We exposed primary human blood mononuclear cells with up to 100nM calcitriol for up to 120h, and measured genome-scale DNA methylation response using the Illumina Infinium HumanMethylation450 beadchip array. We observed that, while the expression of known vitamin D responsive genes was clearly altered by calcitriol exposure, substantial genome-scale changes to DNA methylation were not induced. Our data suggests that, over the exposure period measured, changes to DNA methylation may not be a predominant mechanism through which vitamin D impacts gene expression in human immune cells.

Evidence for age-related and individual-specific changes in DNA methylation profile of mononuclear cells during early immune development in humans.

Environment induced epigenetic effects on gene expression in early life are likely to play important roles in mediating the risk of several immune-related diseases. In order to investigate this fully, it is essential to first document temporal changes in epigenetic profile in disease-free individuals as a prelude to defining environmentally mediated changes. Mononuclear cells (MC) were collected longitudinally from a small number of females at birth, 1 year, 2.5 years and 5 years of age and examined for changes in genome-scale DNA methylation profiles using the Illumina Infinium HumanMethylation27 BeadChip array platform. MC from two males were included for comparative purposes. Flow cytometry was used to define MC cell populations in each sample in order to exclude this as the major driver of epigenetic change. The data underwent quality control and normalization within the R programming environment. Unsupervised hierarchical clustering of samples clearly delineated neonatal MC from all other ages. A further clear distinction was observed between 1 year and 5 year samples, with 2.5 year samples showing a mixed distribution between the 1 and 5 year groups. Gene ontology of probes significantly variable over the neonatal period revealed methylation changes in genes associated with cell surface receptor and signal transduction events. In the postnatal period, methylation changes were mostly associated with the development of effector immune responses and homeostasis. Unlike all other chromosomes tested, a predominantly genetic effect was identified as controlling maintenance of X-chromosome methylation profile in females, largely refractory to change over time. This data suggests that the primary driver of neonatal epigenome is determined in utero, whilst postnatally, multiple genetic and environmental factors are implicated in the development of MC epigenetic profile, particularly between the ages of 1-5 years, when the highest level of inter individual variation is apparent. This supports a model for differential sensitivity of specific individuals to disruption in the developing epigenome during the first years of life. Further studies are now needed to examine evolving epigenetic variations in specific cell populations in relation to environmental exposures, immune phenotype and subsequent disease susceptibility.