Marie-Laure Dalphin

Increased regulatory T-cell numbers are associated with farm milk exposure and lower atopic sensitization and asthma in childhood

BACKGROUND European cross-sectional studies have suggested that prenatal and postnatal farm exposure decreases the risk of allergic diseases in childhood. Underlying immunologic mechanisms are still not understood but might be modulated by immune-regulatory cells early in life, such as regulatory T (Treg) cells. OBJECTIVE We sought to assess whether Treg cells from 4.5-year-old children from the Protection against Allergy: Study in Rural Environments birth cohort study are critical in the atopy and asthma-protective effect of farm exposure and which specific exposures might be relevant. METHODS From 1133 children, 298 children were included in this study (149 farm and 149 reference children). Detailed questionnaires until 4 years of age assessed farming exposures over time. Treg cells were characterized as upper 20% CD4(+)CD25(+) forkhead box protein 3 (FOXP3)(+) (intracellular) in PBMCs before and after stimulation (with phorbol 12-myristate 13-acetate/ionomycin or LPS), and FOXP3 demethylation was assessed. Atopic sensitization was defined by specific IgE measurements; asthma was defined by a doctors diagnosis. RESULTS Treg cells were significantly increased in farm-exposed children after phorbol 12-myristate 13-acetate/ionomycin and LPS stimulation. Exposure to farm milk was defined as a relevant independent farm-related exposure supported by higher FOXP3 demethylation. Treg cell (upper 20% CD4(+)CD25(+), FOXP3(+) T cells) numbers were significantly negatively associated with doctor-diagnosed asthma (LPS stimulated: adjusted odds ratio, 0.26; 95% CI, 0.08-0.88) and perennial IgE (unstimulated: adjusted odds ratio, 0.21; 95% CI, 0.08-0.59). Protection against asthma by farm milk exposure was partially mediated by Treg cells. CONCLUSIONS Farm milk exposure was associated with increased Treg cell numbers on stimulation in 4.5-year-old children and might induce a regulatory phenotype early in life, potentially contributing to a protective effect for the development of childhood allergic diseases.

Prenatal and early-life exposures alter expression of innate immunity genes: The PASTURE cohort study

BACKGROUND There is evidence that gene expression of innate immunity receptors is upregulated by farming-related exposures. OBJECTIVE We sought to determine environmental and nutritional exposures associated with the gene expression of innate immunity receptors during pregnancy and the first year of a childs life. METHODS For the Protection Against Allergy: Study in Rural Environments (PASTURE) birth cohort study, 1133 pregnant women were recruited in rural areas of Austria, Finland, France, Germany, and Switzerland. mRNA expression of the Toll-like receptor (TLR) 1 through TLR9 and CD14 was assessed in blood samples at birth (n= 938) and year 1 (n= 752). Environmental exposures, as assessed by using questionnaires and a diary kept during year 1, and polymorphisms in innate receptor genes were related to gene expression of innate immunity receptors by using ANOVA and multivariate regression analysis. RESULTS Gene expression of innate immunity receptors in cord blood was overall higher in neonates of farmers (P for multifactorial multivariate ANOVA= .041), significantly so for TLR7 (adjusted geometric means ratio [aGMR], 1.15; 95% CI, 1.02-1.30) and TLR8 (aGMR, 1.15; 95% CI, 1.04-1.26). Unboiled farm milk consumption during the first year of life showed the strongest association with mRNA expression at year 1, taking the diversity of other foods introduced during that period into account: TLR4 (aGMR, 1.22; 95% CI, 1.03-1.45), TLR5 (aGMR, 1.19; 95% CI, 1.01-1.41), and TLR6 (aGMR, 1.20; 95% CI, 1.04-1.38). A previously described modification of the association between farm milk consumption and CD14 gene expression by the single nucleotide polymorphism CD14/C-1721T was not found. CONCLUSION Farming-related exposures, such as raw farm milk consumption, that were previously reported to decrease the risk for allergic outcomes were associated with a change in gene expression of innate immunity receptors in early life.

Latent class analysis reveals clinically relevant atopy phenotypes in 2 birth cohorts

Background Phenotypes of childhood‐onset asthma are characterized by distinct trajectories and functional features. For atopy, definition of phenotypes during childhood is less clear. Objective We sought to define phenotypes of atopic sensitization over the first 6 years of life using a latent class analysis (LCA) integrating 3 dimensions of atopy: allergen specificity, time course, and levels of specific IgE (sIgE). Methods Phenotypes were defined by means of LCA in 680 children of the Multizentrische Allergiestudie (MAS) and 766 children of the Protection against allergy: Study in Rural Environments (PASTURE) birth cohorts and compared with classical nondisjunctive definitions of seasonal, perennial, and food sensitization with respect to atopic diseases and lung function. Cytokine levels were measured in the PASTURE cohort. Results The LCA classified predominantly by type and multiplicity of sensitization (food vs inhalant), allergen combinations, and sIgE levels. Latent classes were related to atopic disease manifestations with higher sensitivity and specificity than the classical definitions. LCA detected consistently in both cohorts a distinct group of children with severe atopy characterized by high seasonal sIgE levels and a strong propensity for asthma; hay fever; eczema; and impaired lung function, also in children without an established asthma diagnosis. Severe atopy was associated with an increased IL‐5/IFN‐&ggr; ratio. A path analysis among sensitized children revealed that among all features of severe atopy, only excessive sIgE production early in life affected asthma risk. Conclusions LCA revealed a set of benign, symptomatic, and severe atopy phenotypes. The severe phenotype emerged as a latent condition with signs of a dysbalanced immune response. It determined high asthma risk through excessive sIgE production and directly affected impaired lung function. Graphical abstract Figure. No caption available.

Disagreement between Skin Prick Tests and Specific IgE in Early Childhood.

Background: Accurate diagnosis of allergic sensitization is essential in clinical practice and allergy research, and the choice of assessment method may have an important impact. The PASTURE study (Protection against Allergy: Study of Rural Environment) examines the influence of exposure to a dairy farm environment on the occurrence of allergy in a cohort of rural European children from birth to 10 years. The aim of our study was to analyze agreement between skin prick tests (SPTs), to aeroallergens and food allergens, and specific IgE and to evaluate the association of SPT with atopic dermatitis in the 204 French children of the PASTURE study. Methods: SPT, atopic dermatitis assessment, and specific IgE measurements were performed at 1, 4.5, and 6 years. Results: A total of 137 children attended all three visits. The agreement between SPTs and specific IgE was poor except for perennial aeroallergens at 6 years and for an IgE cutoff greater than 0.7 IU/ml (κ = 0.69, 0.5202 - 0.8621). The prevalence of positive SPTs increased with age. Positive SPTs were transient at 1 year, whereas they were persistent between 4.5 and 6 years. Positive SPTs at 1 year were predictive of the occurrence of atopic dermatitis during follow-up. Conclusion: SPTs did not have good agreement with serum-specific IgE in early childhood. Both tests (SPT and specific IgE) should be used. Skin allergenic reactivity increased with age and was transient at 1 year but associated with the occurrence of atopic dermatitis.

Asthmatic farm children show increased CD3+ CD8low T-cells compared to non-asthmatic farm children

Several studies report an important role of CD8+ cytotoxic T-cells in atopy. Farm children show protection against atopy development, partly explained by CD4+ T-cell subtypes. Additional effects of CD8+ T-cells are unknown being investigated in this study within the PASTURE/EFRAIM birth cohort in PBMCs from farming and non-farming 6-year-old (N = 76) German children. CD3+ CD8+ CD25+ T-cells were analyzed by flow cytometry. Genotyping of 17q21 locus-SNPs associated with childhood asthma was performed. No differences in CD8+ T-cell subsets were seen between farmers and non-farmers regardless of asthma. Among farm children, asthmatics displayed increased CD3+ CD8low(CD25+) T-cells compared to non-asthmatics. Asthmatic farm children exhibited a lower PI-induced stimulatory capacity of CD3+ CD8low(CD25+) cells and a lower IFN-γ secretion than non-asthmatic farm children. Among farm children with GSDMB and ORMDL3 risk alleles, asthmatics displayed higher CD3+ CD8low cells than non-asthmatics. Our data indicates a specific role of CD8low T-cells in asthmatic farm children.

Skin prick tests and specific IgE in 10-year-old children: Agreement and association with allergic diseases

Accurate assessment of atopic sensitization is pivotal to clinical practice and research. Skin prick test (SPT) and specific IgE (sIgE) are often used interchangeably. Some studies have suggested a disagreement between these two methods, and little is known about their association with allergic diseases. The aims of our study were to evaluate agreement between SPT and sIgE, and to compare their association with allergic diseases in 10‐year‐old children.