title: Increased regulatory T-cell numbers are associated with farm milk exposure and lower atopic sensitization and asthma in childhood.
abstract: 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 doctor's 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.
authors.affiliation: LMU Munich, University Children's Hospital, Munich, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Lluis
authors.affiliation: LMU Munich, University Children's Hospital, Munich, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Depner
authors.affiliation: University Hospital of Besançon and Plateforme de Biomonitoring, CIC-BT506, EFS Bourgogne Franche-Comté, INSERM UMR1098, University of Franche-Comté, Besançon, France.
authors.forename: Beatrice
authors.initials: B
authors.lastname: Gaugler
authors.affiliation: University Hospital of Besançon and Plateforme de Biomonitoring, CIC-BT506, EFS Bourgogne Franche-Comté, INSERM UMR1098, University of Franche-Comté, Besançon, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Saas
authors.affiliation: LMU Munich, University Children's Hospital, Munich, Germany.
authors.forename: Vera Isabel
authors.initials: VI
authors.lastname: Casaca
authors.affiliation: LMU Munich, University Children's Hospital, Munich, Germany.
authors.forename: Diana
authors.initials: D
authors.lastname: Raedler
authors.affiliation: University Children's Hospital Eastern Bavaria (KUNO), Department of Pediatric Pneumology and Allergy, University of Regensburg, Regensburg, Germany.
authors.forename: Sven
authors.initials: S
authors.lastname: Michel
authors.affiliation: Laboratory for Epigenetics and Environment (LEE), Centre National de Genotypage, CEA-Institut de Genomique, Evry, France.
authors.forename: Jorg
authors.initials: J
authors.lastname: Tost
authors.affiliation: LMU Munich, University Children's Hospital, Munich, Germany; Second Hospital of JI LIN University, Department of Respiratory Medicine, Chang Chun, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
authors.affiliation: Institute of Epidemiology and Medical Biometry, Ulm University, Ulm, Germany.
authors.forename: Jon
authors.initials: J
authors.lastname: Genuneit
authors.affiliation: Department of Clinical Chemistry and Molecular Diagnostics, Philipps University of Marburg, Marburg, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Pfefferle
authors.affiliation: Department of Environmental Science, University of Eastern Finland, Kuopio, Finland.
authors.forename: Marjut
authors.initials: M
authors.lastname: Roponen
authors.affiliation: LMU Munich, University Children's Hospital, Munich, Germany.
authors.forename: Juliane
authors.initials: J
authors.lastname: Weber
authors.affiliation: Swiss Tropical and Public Health Institute, Basel, Switzerland; University of Basel, Basel, Switzerland.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Braun-Fahrländer
authors.affiliation: Children's Hospital Schwarzach, Schwarzach, Austria.
authors.forename: Josef
authors.initials: J
authors.lastname: Riedler
authors.affiliation: Christine Kühne-Center for Allergy Research and Education, Hochgebirgklinik Davos, Davos-Wolfgang, Switzerland.
authors.forename: Roger
authors.initials: R
authors.lastname: Lauener
authors.affiliation: University Hospital of Besançon, Research Unit Health and Rural Environment, University of Franche-Comté, Besançon, France.
authors.forename: Dominique Angèle
authors.initials: DA
authors.lastname: Vuitton
authors.affiliation: University Hospital of Besançon, Research Unit Health and Rural Environment, University of Franche-Comté, Besançon, France.
authors.forename: Jean-Charles
authors.initials: JC
authors.lastname: Dalphin
authors.affiliation: Department of Environmental Health, National Institute for Health and Welfare, Kuopio, Finland; Public Health and Clinical Nutrition, University of Eastern Finland, Kuopio, Finland.
authors.forename: Juha
authors.initials: J
authors.lastname: Pekkanen
authors.affiliation: LMU Munich, University Children's Hospital, Munich, Germany.
authors.forename: Erika
authors.initials: E
authors.lastname: von Mutius
authors.affiliation: LMU Munich, University Children's Hospital, Munich, Germany. Electronic address: Bianca.Schaub@med.uni-muenchen.de.
authors.forename: Bianca
authors.initials: B
authors.lastname: Schaub
mesh_terms: D000383:Agriculture; D000818:Animals; D001249:Asthma / Q000175:diagnosis / Q000276:immunology*; D018791:CD4 Lymphocyte Count; D002675:Child, Preschool; D019175:DNA Methylation; D005060:Europe; D005260:Female; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000276:immunology; D006801:Humans; D006969:Hypersensitivity, Immediate / Q000175:diagnosis / Q000276:immunology*; D007073:Immunoglobulin E / Q000097:blood; D007223:Infant; D008297:Male; D008892:Milk; D011247:Pregnancy; D011446:Prospective Studies; D050378:T-Lymphocytes, Regulatory / Q000166:cytology / Q000276:immunology*
chemical_list: C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D007073:Immunoglobulin E
keywords: ALEX; Allergy and Endotoxin Study; Asthma; EFRAIM; FITC; FOXP3; FOXP3 demethylation; Fluorescein isothiocyanate; Forkhead box protein 3; Mechanisms of Early Protective Exposures on Allergy Development; OR; Odds ratio; PARSIFAL; PASTURE; PE; PI; Phorbol 12-myristate 13-acetate/ionomycin; Phycoerythrin; Prevention of Allergy—Risk Factors for Sensitization In Children Related to Farming and Anthroposophic Lifestyle; Protection Against Allergy: Study in Rural Environments; Regulatory T; SIC; Specific IgE class; TLR; Toll-like receptor; Treg; atopic sensitization; farming; innate; milk; peripheral blood mononuclear cells; regulatory T cells