Caroline Roduit

Prenatal animal contact and gene expression of innate immunity receptors at birth are associated with atopic dermatitis

BACKGROUND Cross-sectional studies have suggested that prenatal farm exposures might protect against allergic disease and increase the expression of receptors of the innate immune system. However, epidemiologic evidence supporting the association with atopic dermatitis remains inconsistent. OBJECTIVE To study the association between prenatal farm-related exposures and atopic dermatitis in a prospective study. We further analyzed the association between the expression of innate immune genes at birth and atopic dermatitis. METHODS A total of 1063 children who participated in a birth cohort study, Protection against Allergy-Study in Rural Environments, were included in this study. Doctor diagnosis of atopic dermatitis was reported by the parents from 1 to 2 years of age by questionnaire. Gene expression of Toll-like receptors (TLRs) and CD14 was assessed in cord blood leukocytes by quantitative PCR. RESULTS Maternal contact with farm animals and cats during pregnancy had a significantly protective effect on atopic dermatitis in the first 2 years of life. The risk of atopic dermatitis was reduced by more than half among children with mothers having contact with 3 or more farm animal species during pregnancy compared with children with mothers without contact (adjusted odds ratio, 0.43; 95% CI, 0.19-0.97). Elevated expression of TLR5 and TLR9 in cord blood was associated with decreased doctor diagnosis of atopic dermatitis. A significant interaction between polymorphism in TLR2 and prenatal cat exposure was observed in atopic dermatitis. CONCLUSION Maternal contact with farm animals and cats during pregnancy has a protective effect on the development of atopic dermatitis in early life, which is associated with a lower expression of innate immune receptors at birth.

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.

Increased food diversity in the first year of life is inversely associated with allergic diseases

BACKGROUND The role of dietary factors in the development of allergies is a topic of debate, especially the potential associations between infant feeding practices and allergic diseases. Previously, we reported that increased food diversity introduced during the first year of life reduced the risk of atopic dermatitis. OBJECTIVE In this study we investigated the association between the introduction of food during the first year of life and the development of asthma, allergic rhinitis, food allergy, or atopic sensitization, taking precautions to address reverse causality. We further analyzed the association between food diversity and gene expression of T-cell markers and of Cε germline transcript, reflecting antibody isotype switching to IgE, measured at 6 years of age. METHODS Eight hundred fifty-six children who participated in a birth cohort study, Protection Against Allergy Study in Rural Environments/EFRAIM, were included. Feeding practices were reported by parents in monthly diaries during the first year of life. Data on environmental factors and allergic diseases were collected from questionnaires administered from birth up to 6 years of age. RESULTS An increased diversity of complementary food introduced in the first year of life was inversely associated with asthma with a dose-response effect (adjusted odds ratio with each additional food item introduced, 0.74 [95% CI, 0.61-0.89]). A similar effect was observed for food allergy and food sensitization. Furthermore, increased food diversity was significantly associated with an increased expression of forkhead box protein 3 and a decreased expression of Cε germline transcript. CONCLUSION An increased diversity of food within the first year of life might have a protective effect on asthma, food allergy, and food sensitization and is associated with increased expression of a marker for regulatory T cells.

Clinical and epidemiologic phenotypes of childhood asthma.

RATIONALE Clinical and epidemiologic approaches have identified two distinct sets of classifications for asthma and wheeze phenotypes. OBJECTIVES To compare epidemiologic phenotype definitions identified by latent class analysis (LCA) with clinical phenotypes based on patient histories, diagnostic work-up, and treatment responses. To relate phenotypes to genetic and environmental determinants as well as diagnostic and treatment-related parameters. METHODS LCA was performed in an international multicenter birth cohort based on yearly questions about current wheeze until age 6 years. Associations of wheeze classes and clinical phenotypes with asthma-related characteristics such as atopy, lung function, fraction of exhaled nitric oxide, and medication use were calculated using regression models. MEASUREMENTS AND MAIN RESULTS LCA identified five classes, which verified the clinically defined wheeze phenotypes with high sensitivity and specificity; the respective receiver operating characteristics curves displayed an area under the curve ranging from 84% (frequent wheeze) to 85% (asthma diagnosis) and 87% (unremitting wheeze) to 97% (recurrent unremitting wheeze). Recurrent unremitting wheeze was the most specific and unremitting wheeze at least once the most sensitive definition. The latter identified a subgroup of children with decreased lung function, increased genetic risk, and in utero smoke exposure (ODDS RATIO, 2.03; 95% CONFIDENCE INTERVAL, 1.12-3.68; P = 0.0191), but without established asthma diagnosis and treatment. CONCLUSIONS Clinical phenotypes were well supported by LCA analysis. The hypothesis-free LCA phenotypes were a useful reference for comparing clinical phenotypes. Thereby, we identified children with clinically conspicuous but undiagnosed disease. Because of their high area under the curve values, clinical phenotypes such as (recurrent) unremitting wheeze emerged as promising alternative asthma definitions for epidemiologic studies.

MHC Class II Molecules Enhance Toll-Like Receptor Mediated Innate Immune Responses

Background Major histocompatibility complex (MHC) class II molecules play crucial roles in immune activation by presenting foreign peptides to antigen-specific T helper cells and thereby inducing adaptive immune responses. Although adaptive immunity is a highly effective defense system, it takes several days to become fully operational and needs to be triggered by danger-signals generated during the preceding innate immune response. Here we show that MHC class II molecules synergize with Toll-like receptor (TLR) 2 and TLR4 in inducing an innate immune response. Methodology/Principal Findings We found that co-expression of MHC class II molecules and TLR2 or TLR4 in human embryonic kidney (HEK) cells 293 leads to enhanced production of the anti-microbial peptide human-β-defensin (hBD) 2 after treatment with TLR2 stimulus bacterial lipoprotein (BLP) or TLR4 ligand lipopolysaccharide (LPS), respectively. Furthermore, we found that peritoneal macrophages of MHC class II knock-out mice show a decreased responsiveness to TLR2 and TLR4 stimuli compared to macrophages of wild-type mice. Finally, we show that MHC class II molecules are physically and functionally associated with TLR2 in lipid raft domains of the cell membrane. Conclusions/Significance These results demonstrate that MHC class II molecules are, in addition to their central role in adaptive immunity, also implicated in generating optimal innate immune responses.

Davos declaration: allergy as a global problem.

Allergy and allergic diseases represent a major health problem not only in industrialized ‘modern’ societies, but worldwide. There has been an epidemic increase in prevalence of allergic diseases in the last few decades with 10–30% of the population affected. Apart from individual suffering, because of their life-threatening or chronic course, these diseases present a high socioeconomic burden. In many countries, patient care of affected individuals is insufficient and/or inadequate. In spite of great progress in research into the causes and treatment of allergy in the last decades, there are still many problems to be solved in moving to reach our goals of more effective therapies and eventual prevention (1–3). Therefore, a group of 40 scientists and clinicians from four continents and all fields of allergy and related disciplines gathered under the sponsorship of the Christine-Kühne Center of Allergy Research and Education (CK-CARE) in Davos, Switzerland from 17 to 20 July 2011 for the first ‘Global Allergy Forum’. Under the topic ‘Allergy and Allergic Diseases: Barriers to Cure’, the participants formed five working groups to discuss and define the most urgent problems in the field and seek solutions and recommend an action plan. There are numerous unmet clinical needs and millions of patients are undertreated or not treated with the most appropriate methods. Accessibility to and affordability of effective therapeutic regimens are not provided in many countries. The development of innovative therapies is slow compared to most other fields of medicine. Allergic diseases encompass broad fields of medicine and show a wide heterogeneity involving different organs such as eyes, respiratory tract, gut, and skin. Diseases include rhinoconjunctivitis, asthma, anaphylaxis, eczema, urticaria, and angioedema as well as drug and food allergies. Allergic diseases show variability in severity and clinical course which at the moment are only poorly defined. Much better definition of the subtypes of allergic patients (phenotyping) appears crucial and very much needed to address the right therapy to the right patient. A new integrative approach is needed to understand how a complex network of immunological, genetic, and environmental factors leads to a complex allergic phenotype (1). There is a tremendous lack of knowledge regarding many unsolved issues such as: • The causes of the epidemic increase in allergic diseases are unknown. Environmental exposures that appear to be critical factors include factors as diverse as air quality, diet and nutrition, climate, UV radiation, and direct skin contact as well as psycho-social interactions. Moreover, when genetic predisposition is taken into account, environment can provide either risk or protection. • The effects of changes in climate, urbanization, etc. have to be anticipated. Better ways to assess spatial and temporal environmental exposure at population and individual levels are much needed and should be related to the assessment of individual genetic susceptibility. • The interactions between microbes, pollutants, and the immune system are marginally understood. • There is inadequate understanding of the natural mechanisms that limit acute vs. chronic disease or spontaneous resolution. • There is a need for better subclassification of allergic disorders based on pathobiology. • There is a need for new agents acting on specific pathways in pathogenesis with regard to new therapeutic approaches. • There is a need for better preclinical models for translational research. • There is a need to develop better tools for complex data analysis. • There is a need for efficient strategies for primary and secondary allergy prevention. • There is a need for better approaches in diagnosis and prediction of treatment responses and the monitoring of therapeutic effectiveness. Apart from true lack of information, there is a tremendous gap between actual existing knowledge and its effective application for the millions of people in need. • There is a shortage of well-trained allergy specialists in most countries. • Education and training efforts should also be directed toward medical students at the curricular level and extended to primary care physicians who have to be involved in a strategy for diagnosing and managing allergic diseases with such high prevalence rates of 20% of the population. • Awareness campaigns for targeted public groups should be performed. Allied health professionals, such as nurses, school teachers, etc., should be included. Better and more effective tools to spread the available information should be developed. • Close cooperation with patient organizations is highly recommended. • Decision makers involved in developing and approving health policies and administration must be made more aware of the problem of allergic diseases. Action should be taken at various levels and through existing doctors, scientists, and lay organizations to solve these problems. The global expertise from clinical allergists, immunologists, microbiologists, biologists, nutritionists,

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.

Soluble immunoglobulin A in breast milk is inversely associated with atopic dermatitis at early age: the PASTURE cohort study

The role of breastfeeding for the development of atopic diseases in childhood is contradictory. This might be due to differences in the composition of breast milk and levels of antimicrobial and anti‐inflammatory components.

The Early Development of Wheeze. Environmental Determinants and Genetic Susceptibility at 17q21

RATIONALE Growing up on a farm protects from childhood asthma and early wheeze. Virus-triggered wheeze in infancy predicts asthma in individuals with a genetic asthma risk associated with chromosome 17q21. OBJECTIVES To test environmental determinants of infections and wheeze in the first year of life, potential modifications of these associations by 17q21, and the implications for different trajectories of wheeze. METHODS We followed 983 children in rural areas of Europe from birth until age 6 years. Symptoms of wheeze, rhinitis, fever, and environmental exposures were documented with weekly diaries during year 1. Asthma at age 6 was defined as ever having a reported doctors diagnosis. Single-nucleotide polymorphisms related to ORMDL3 (rs8076131) and GSDMB (rs7216389, rs2290400) at 17q21 were genotyped. MEASUREMENTS AND MAIN RESULTS Early wheeze was positively associated with presence of older siblings among carriers of known asthma risk alleles at 17q21 (e.g., rs8076131) (adjusted odds ratio [aOR], 1.53; 95% confidence interval [CI], 1.16-2.01). Exposure to farm animal sheds was inversely related to wheeze (aOR, 0.44; 95% CI, 0.33-0.60). Both effects were similarly observed in children with transient wheeze up to age 3 years without subsequent development of asthma (aOR, 1.71 [95% CI, 1.09-2.67]; and aOR, 0.48 [95% CI, 0.30-0.76], respectively). CONCLUSIONS These findings suggest that the chromosome 17q21 locus relates to episodes of acute airway obstruction common to both transient wheeze and asthma. The previously identified asthma risk alleles are the ones susceptible to environmental influences. Thus, this gene-environment interaction reveals two faces of 17q21: The same genotype constitutes genetic risk and allows for environmental protection, thereby providing options for prospective prevention strategies.

Atopic sensitization in the first year of life.

BACKGROUND There is conflicting evidence on whether allergen-specific memory is primed prenatally, whether this priming affects persistent immunologic effects, and whether it is modulated by the first environmental exposures in infancy. OBJECTIVE We sought to explore the course of atopic sensitization between birth and 12 months of age. METHODS Specific IgE levels for 6 food and 13 common inhalant allergens were assessed in cord blood and 1-year blood samples in the Protection against Allergy-Study in Rural Environments (PASTURE) birth cohort including 793 children from rural regions of 5 European countries. Detailed information on childrens health, nutrition, and farm-related exposures was gathered by using a pregnancy questionnaire, 2 questionnaires at 2 and 12 months of age, and a diary covering the time in between. RESULTS Sensitization was more common at 12 months of age than at birth for almost all specificities. On an individual level, persistent sensitization to the same allergens was rare (1%), whereas transient (only at birth, 11%) and incident (only at 12 months, 34%) sensitization was seen in substantial proportions of children. Associations of transient sensitization with maternal sensitization differed with the allergen specificities, with the strongest associations for food allergens (odds ratio [OR], 10.6; 95% CI, 6.0-18.6) and the weakest associations for seasonal allergens (OR, 1.64; 95% CI, 0.94-2.86). Associations of maternal sensitization with incident sensitization were also seen. Incident sensitization was related to distinct prenatal and postnatal environmental exposures of mother and child, such as consumption of cereals for incident sensitization to seasonal allergens (OR, 0.66; 95% CI, 0.50-0.88). CONCLUSION IgE sensitization patterns change between birth and 12 months and are related to maternal and environmental influences.