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Glucocorticoid-induced TNFR family–related protein (GITR)–mediated activation of JNK was shown to regulate the suppressive activity of CD4+CD25+ naturally occurring T regulatory cells (nTregs) in wild-type (WT) hosts. In this study, CD4+CD25+ T cells were shown to be capable of becoming pathogenic effector cells in sensitized and challenged CD8−/− recipient mice. Only GITR-expressing CD4+CD25+ T cells, but neither GITR knocked-in CD4+CD25− T cells nor GITR-silenced CD4+CD25+ T cells, enhanced development of lung allergic responses. Inhibition of JNK in WT nTregs or nTregs from GITR−/−and JNK2−/− mice failed to enhance lung allergic responses in sensitized and challenged CD8−/− recipient mice. The failure to enhance responses was associated with increased bronchoalveolar lavage fluid levels of IL-10 and TGF-β and decreased levels of IL-5, IL-6, and IL-13. In contrast, nTregs from JNK1−/− mice, similar to WT nTregs, were fully effective in enhancing responses. Thus, GITR stimulation of nTregs and signaling through JNK2, but not JNK1, triggered the loss of regulatory function while concomitantly gaining pathogenic CD4+ T effector cell function responsible for exacerbating asthma-like immunopathology.

Background Previous studies suggested that maternal farm exposure during pregnancy modulates early immune development toward an allergy-protective status potentially mediated by T H 1 or regulatory T (Treg) cells. However, the underlying mechanisms might involve immune modulation of additional T-cell populations, such as T H 17 cells, influenced by genetic predisposition. Objective We examined the role of maternal farm exposure and genetic predisposition on T H 17 cell responses to innate and adaptive immune stimulation in cord blood. Methods Eighty-four pregnant mothers were recruited before delivery. Detailed questionnaires (60 nonfarming mother, 22 farming mothers, and 2 exclusions) assessed farming exposures. Cord blood was stimulated with lipid A, peptidoglycan (Ppg), or PHA. T H 17 lineage (retinoic acid receptor–related orphan receptor C [RORC] , retinoic acid receptor–related orphan receptor α [RORA] , IL-23 receptor [IL23R] , IL17 , IL17F , and IL22 ) and Treg cell markers (forkhead box protein 3 [FOXP3] , lymphocyte activation gene 3 [LAG3] , and glucocorticoid-induced TNF receptor [GITR] ) were assessed at the mRNA level. T H 17/Treg/T H 1/T H 2 cytokines and 7 single nucleotide polymorphisms within the T H 17 lineage ( RORC , IL23R , and IL17 ) were examined. Results T H 17 lineage mRNA markers were expressed at birth at low concentrations independent of maternal farm exposure. A positive correlation between T H 17 lineage markers and FOXP3 (mRNA) was observed on stimulation (nonfarming mothers: lipid A, Ppg, and PHA; farming mothers: Ppg and PHA), influenced by maternal farming. Specific single nucleotide polymorphisms within the T H 17 lineage genes influenced gene expression of T H 17 and Treg cell markers and cytokine secretion. Conclusions Gene expression of T H 17 lineage markers in cord blood was not influenced by maternal farming. Yet T H 17 and Treg cell markers were positively correlated and influenced by maternal farm exposure. Our data suggest that prenatal exposures and genetic predisposition play a role during early T H 17 immune maturation, potentially regulating the development of immune-mediated diseases, such as childhood asthma.

Background: IL-33 polymorphisms influence the susceptibility to asthma. IL-33 indirectly induces Th2-immune responses via dendritic cell activation, being important for development of atopic diseases. Furthermore, IL-33 upregulates regulatory T cells (Tregs), which are critical for healthy immune homeostasis. This study investigates associations between IL-33 polymorphisms during the development of childhood atopic diseases and underlying mechanisms including immune regulation of Tregs. Methods: Genotyping of IL-33-polymorphisms (rs928413, rs1342326) was performed by MALDI-TOF-MS in 880 of 1133 PASTURE/EFRAIM children. In 4.5-year-old German PASTURE/EFRAIM children (n = 99), CD4(+) CD25(high)FOXP3(+) Tregs were assessed by flow cytometry following 24-h incubation of PBMCs with PMA/ionomycin, LPS or without stimuli (U). SOCS3, IL1RL1, TLR4 mRNA expression and sST2 protein levels ex vivo were measured in PASTURE/EFRAIM children by real-time PCR or ELISA, respectively. Health outcomes (hay fever, asthma) were assessed by questionnaires at the age of 6 years. Results: rs928413 and rs1342326 were positively associated with hay fever (OR = 1.77, 95% CI = 1.02-3.08;OR = 1.79, 95% CI = 1.04-3.11) and CD4(+) CD25(high)FOXP3(+) Tregs (%) decreased in minor allele homozygotes/heterozygotes compared to major allele homozygotes (p(U) = 0.004;p(LPS) = 0.005;p(U) = 0.001;p(LPS) = 0.012). SOCS3 mRNA expression increased in minor allele homozygotes and heterozygotes compared with major allele homozygotes for both IL-33-polymorphisms (p (rs928413) = 0.032, p(rs1342326) = 0.019) and negatively correlated to Tregs. Conclusions: IL-33-polymorphisms rs928413 and rs1342326 may account for an increased risk of hay fever with the age of 6 years. Lower Tregs and increased SOCS3 in combined heterozygotes and minor allele homozygotes may be relevant for hay fever development, pointing towards dysbalanced immune regulation and insufficient control of allergic inflammation.

International audience; 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.

Background: For several immune-mediated diseases, immunological analysis will become more complex in the future with datasets in which cytokine and gene expression data play a major role. These data have certain characteristics that require sophisticated statistical analysis such as strategies for non-normal distribution and censoring. Additionally, complex and multiple immunological relationships need to be adjusted for potential confounding and interaction effects. Objective: We aimed to introduce and apply different methods for statistical analysis of non-normal censored cytokine and gene expression data. Furthermore, we assessed the performance and accuracy of a novel regression approach in order to allow adjusting for covariates and potential confounding. Methods: For non-normally distributed censored data traditional means such as the Kaplan-Meier method or the generalized Wilcoxon test are described. In order to adjust for covariates the novel approach named Tobit regression on ranks was introduced. Its performance and accuracy for analysis of non-normal censored cytokine/gene expression data was evaluated by a simulation study and a statistical experiment applying permutation and bootstrapping. Results: If adjustment for covariates is not necessary traditional statistical methods are adequate for non-normal censored data. Comparable with these and appropriate if additional adjustment is required, Tobit regression on ranks is a valid method. Its power, type-I error rate and accuracy were comparable to the classical Tobit regression. Conclusion: Non-normally distributed censored immunological data require appropriate statistical methods. Tobit regression on ranks meets these requirements and can be used for adjustment for covariates and potential confounding in large and complex immunological datasets.

Several population-based studies have replicated the finding that exposure to a farm environment is protective against the development of atopic diseases. From these studies, novel insights into potential allergy-protective mechanisms were retrieved. This review focuses on consistent and novel findings of immune mechanisms involved in the 'farm effect'.The most recent studies suggest that the 'farm effect' mediated by microbial exposure may be attributed to both microbial diversity and species specificity. There is convincing evidence that farm milk components and grass arabinogalactan, commonly found in cowshed, may be important. Furthermore, early exposure to a farming environment, in particular in utero, showed stronger effects than exposure later in life, potentially through modulation of the immature immune system by microbes, also involving epigenetic changes. This protective 'farm effect' remains in later adulthood. Regarding gene-environment interactions, polymorphisms in GRM1 interacted with farming in a genome-wide interaction scan for asthma.The novel studies strengthen the role of microbial exposure and farm milk and grass components, especially early in life, in the modulation of the immune system towards a Th1/Treg predominance. This may subsequently lead to a long-lasting lower risk of developing atopic diseases.

Maternal atopy is a strong predictor for the development of childhood allergic diseases. The underlying mechanisms are ill defined, yet regulatory T (Treg) and T(H)17 cells may play a key role potentially shaping the early immune system toward a proallergic or antiallergic immune regulation.We examined T(H)1/T(H)2, Treg, and T(H)17 cell responses to innate (lipid A/peptidoglycan) and mitogen/adaptive (phytohemagglutinin/Dermatophagoides pteronyssinus 1) immune stimulation in cord blood from offspring of atopic/nonatopic mothers.Cord blood mononuclear cells from 161 healthy neonates (59% nonatopic, 41% atopic mothers) were investigated regarding Treg and T(H)17 cells (mRNA/surface markers), suppressive function, and proliferation/cytokine secretion.Cord blood from offspring of atopic mothers showed fewer innate-induced Treg cells (CD4(+)CD25(+)high), lower mRNA expression of associated markers (glucocorticoid-induced tumor necrosis factor receptor-related protein/lymphocyte activation gene 3; P.05), and a trend toward lower Forkhead box transcription factor 3 (Foxp3) expression. Treg cell function was impaired in mitogen-induced suppression of T effector cells in cord blood of offspring from atopic mothers (P = .03). Furthermore, IL-10 and IFN-gamma secretion were decreased in innate-stimulated cord blood of offspring from atopic mothers (P = .04/.05). Innate-induced IL-17 was independent of maternal atopy and highly correlated with IL-13 secretion.In offspring of atopic mothers, Treg cell numbers, expression, and function were impaired at birth. T(H)17 cells were correlated with T(H)2 cells, independently of maternal atopy.

Background In a genome-wide association study, genetic variants on chromosome 17q21 were strongly associated with childhood asthma and orosomucoid 1–like 3 (ORMDL3) gene expression. Regulation of the 17q21 locus and its immunologic relevance early in life have not been well characterized. Objective We investigated the relation between polymorphisms and mRNA expression of 17q21 locus genes and their influence on T-cell subsets in cord blood. Methods In 200 children of our cord blood study, 17q21 polymorphisms were genotyped by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Gene expression was assessed for ORMDL3 ; gasdermin A ( GSDMA, alias GSDM1 ); gasdermin B ( GSDMB, alias GSDML ); Ikaros family zinc finger 3 ( ZNFN1A3 ), zona pellucida binding protein 2 ( ZPBP2 ); and proteasome (prosome, macropain) 26S subunit, non-ATPase, 3 ( PSMD3 ), in cord blood mononuclear cells (CBMCs) and for ORMDL3 in peripheral blood (real-time RT-PCR). Mononuclear cells were assessed before and after microbial (lipid A/peptidoglycan), phytohemagglutinin, or allergen (Der p 1) stimulation. Regulatory T–associated markers (forkhead box protein 3, glucocorticoid-induced TNF receptor, lymphocyte activation gene 3 mRNA expression) and T h 2/T h 1/T h 17 cytokines were examined. Results In CBMCs, single genetic risk variants within 17q21 were associated with increased ORMDL3 (Der p 1 stimulation; P ≤ .01) and GSDMA expression (phytohemagglutinin/Der p 1 stimulation; P ≤ .05). Children homozygous for all 4 risk alleles for 17q21 tagging single nucleotide polymorphisms showed increased expression for ORMDL3 (Der p 1; P = .002) and GSDMA (phytohemagglutinin; P = .0009/Der p 1; P = .004). CBMC ORMDL3 expression was lower compared with PBMCs ( P ≤ .0003) and increased in both CBMC and PBMC after stimulation (phytohemagglutinin/lipid A/peptidoglycan/Der p 1; P ≤ .006 and phytohemagglutinin/peptidoglycan; P h 2/T h 1 lineages was detectable. However, 17q21 risk allele carriers showed significantly increased IL-17 secretion (unstimulated, phytohemagglutinin-stimulated). Conclusion Our results suggest an association of 17q21 polymorphisms with ORMDL3, GSDMA expression, and IL-17 secretion early in life. These observations may imply a functional role of the 17q21 locus affecting T-cell development during immune maturation.