A switch in regulatory T cells through farm exposure during immune maturation in childhood.

BACKGROUND: Farm exposure protects against development of allergies early in life. At 4.5 years, protection against asthma by farm-milk exposure was partially mediated by regulatory T cells (Tregs). The aim of this study was to investigate the critical time window of the 'asthma-protective' farm effect via Tregs during childhood immune maturation. METHODS: Tregs were assessed longitudinally at 4.5 and 6 years in 111 children (56 farm and 55 reference children) from the PASTURE/EFRAIM birth cohort (flow cytometry). Peripheral blood mononuclear cells were cultured unstimulated (U), with phorbol 12-myristate 13-acetate/ionomycin (PI) or lipopolysaccharide (LPS), and stained for Tregs (CD4+ CD25high FOXP3upper20% ). mRNA expression of Treg/Th1/Th2/Th17-associated cell markers was measured ex vivo. Suppressive capacity of Tregs on effector cells and cytokines was assessed. Detailed questionnaires assessing farm exposures and clinical phenotypes from birth until age 6 years were answered by the parents. RESULTS: Treg percentage before and after stimulation and FOXP3mRNA expression ex vivo decreased from age 4.5 to 6 years (P(U,LPS) < 0.001; P(PI) = 0.051; P(FOXP3) < 0.001). High vs low farm-milk and animal-stable exposure was associated with decreased LPS-stimulated Treg percentage at age 6 years (P(LPS) = 0.045). Elevated LPS-stimulated-Treg percentage at age 6 was associated with increased risk of asthma (aOR = 11.29, CI: 0.96-132.28, P = 0.053). Tregs from asthmatics vs nonasthmatics suppressed IFN-γ (P = 0.015) and IL-9 (P = 0.023) less efficiently. mRNA expression of Th1/Th2/Th17-associated cell markers decreased between 4.5 and 6 years (P < 0.001). CONCLUSIONS: Tregs at the age of 6 years were decreased with farm exposure and increased within asthmatics, opposite to age 4.5 years. This immunological switch defines a critical 'time window' for Treg-mediated asthma protection via environmental exposure before age 6 years.

STAT6 polymorphisms are associated with neonatal regulatory T cells and cytokines and atopic diseases at 3 years.

BACKGROUND: The transcription factor STAT6 is crucial for activation of the interleukin (IL)-4/IL-13 pathway and has been linked to regulatory T cells (Tregs). Associations of STAT6 polymorphisms with IgE levels were described; however, their impact on neonatal immune responses and early disease development is unknown. METHODS: STAT6 polymorphisms were genotyped in cord blood mononuclear cells by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Gene expression was assessed by real-time polymerase chain reaction (PCR) and cytokines by Multiplex. At age 3 years, atopic diseases were assessed by questionnaires. RESULTS: STAT6 rs324011 but not rs1059513 polymorphism was associated with significant or borderline significant decreased mRNA expression of Treg-associated genes (FOXP3, GITR, LAG3). Heterozygotes and minor allele homozygotes of rs324011 had low levels of tumor necrosis factor alpha (TNF-α) and increased interferon gamma (IFN-γ) (P ≤ 0.04), while heterozygotes and minor allele homozygotes of rs1059513 had increased TNF-α and Granulocyte-macrophage colony-stimulating factor (GM-CSF) (P ≤ 0.05). In minor allele homozygotes of rs324011, expression of Treg-associated genes was strongly inverse correlated with IFN-γ (unstimulated, r = -0.7, P = 0.111; LpA stimulation, r = -0.8, P = 0.011), but not in heterozygotes or major allele homozygotes. Heterozygotes and minor allele homozygotes of rs324011 presented a lower risk of atopic dermatitis and obstructive bronchitis until age 3 years. CONCLUSIONS: Two STAT6 polymorphisms were associated with altered immune responses already at birth. STAT6 rs324011 was associated with lower neonatal Treg and increased Th1 response. Those neonates had a lower risk of atopic dermatitis and obstructive bronchitis until 3 years. Our data suggest a role for STAT6 polymorphisms in early immune regulation and implications on early atopic disease development.