Increased expression of nuclear factor of activated T cells 1 drives IL-9–mediated allergic asthma
Authors: Sonja Koch, Anna Graser, Hooman Mirzakhani, Theodor Zimmermann, Volker O. Melichar, Marco Wölfel, Damien C. Croteau-Chonka, Benjamin A. Raby, Scott T. Weiss, Susetta Finotto
Journal: Journal of Allergy and Clinical Immunology (2016)

Nuclear factor of activated T cells (NFAT) is a family of transcription factors activated by dephosphorylation mediated by Ca++-activated calcineurin. NFAT coordinates different aspects of T-cell development and activation of T, B, natural killer, and mast cells and is the target of the immunosuppressive drug cyclosporin A.1Muller M.R. Rao A. NFAT, immunity and cancer: a transcription factor comes of age.Nat Rev Immunol. 2010; 10: 645-656Crossref PubMed Scopus (206) Google Scholar We reported recently that targeted deletion of NFATc1 in T cells resulted in inhibition of TH2 and TH17 differentiation.2Koch S. Reppert S. Finotto S. NFATc1 deletion in T lymphocytes inhibits the allergic trait in a murine model of asthma.Clin Exp Allergy. 2015; 45: 1356-1366Crossref PubMed Google Scholar Here we first investigated NFATc1 mRNA expression in PBMCs isolated from healthy and asthmatic children from the PreDicta cohort. Children with allergic asthma expressed significantly more NFATc1 mRNA than healthy control subjects (Fig 1, A-C, and Tables I and II). Furthermore, we found that asthmatic children with a positive skin test result had significantly increased expression of NFATc1 mRNA compared with healthy control subjects (Fig 1, D, and Tables I and II). These results suggested that NFATc1 might have a role in allergic asthma. Consistent with a TH2-inducing function of NFATc1 and a TH2 cytokine inhibitory property of the TH1 cytokine IFN-γ (IFNG), levels of this TH1 cytokine were found to be downregulated in children with asthma and a positive skin test result (Fig 1, E).Table IClinical outcome of children with asthma participating in the WP1-UKER cohort in the European PreDicta studyGroup/patientAge (y)SexPBMCs (106 cells/mL of blood)Skin prick testFEV1 actual value (%)Asthma controlAsthma severityCigarette exposure/dNo. of symptomatic episodes before B0Asthma 16M1.75Positive126CMOPA00 26M2.27Positive80PCMOPA03 35F2.05Positive108PCMIPA25— 46M1.98Positive128CMIPA01 55M2.58Positive102PCI04 65F2.62Positive129CI01 75M2.32Positive143PCI04 86F1.57Positive94PCI03 94M1.96Positive115PCMOPA03 105F1.93Positive92UMOPA015 116F2.05Positive111CI02 125M1.14Positive99CI00 134F2.89Negative135CI04 144M1.91ND96CI54 155M1.29Positive80CI02 165M1.83Positive86CI01 175M2.50Positive107CI02 184M1.54Positive71PCI03 194M2.20Negative86CI03 205F2.27ND98CI01 215F1.77Negative—UMIPA020 225M1.39Positive81CMIPA03-5Mean5.0014 M1.99103.191.36SEM0.148 F0.094.201.15B0, Baseline visit; C, controlled; F, female; I, intermittent; M, male; MIPA, mild persistent asthma; MOPA, moderate persistent asthma; ND, not done; PC, partially controlled; U, uncontrolled; UKER, Uniklinikum Erlangen; WP1, Work Program 1. Open table in a new tab Table IIClinical outcome of control children participating in the WP1-UKER cohort in the European PreDicta studyGroup/patientAge (y)SexPBMCs (106 cells/mL of blood)Skin prick testFEV1 actual value (%)Cigarette exposure/dNo. of symptomatic episodes before B0Healthy subjects 16M1.14ND—0— 26F2.95ND1210— 35F1.79Negative810— 44M2.01ND—0— 56M1.41ND1050— 64F2.14ND1090— 76M2.86ND870— 84M1.73Negative1000— 95F1.67ND1120— 105F2.01Positive1190— 114M2.43Positive1160— 125M2.32ND1110— 134M2.40Negative1090— 144F1.60Negative1090— 155M2.22Negative920— 164M2.88Negative—0— 174M1.38Negative1090— 185M1.13ND1100— 194M1.77ND1180— 205F1.50ND—0—Mean4.7513 M1.97106.750SEM0.177 F0.122.480B0, Baseline visit; F, female; M, male; ND, not done; UKER, Uniklinikum Erlangen; WP1, Work Program 1. Open table in a new tab B0, Baseline visit; C, controlled; F, female; I, intermittent; M, male; MIPA, mild persistent asthma; MOPA, moderate persistent asthma; ND, not done; PC, partially controlled; U, uncontrolled; UKER, Uniklinikum Erlangen; WP1, Work Program 1. B0, Baseline visit; F, female; M, male; ND, not done; UKER, Uniklinikum Erlangen; WP1, Work Program 1. As in PBMCs from the PreDicta cohort, NFATc1 showed altered expression levels in peripheral blood CD4+ T cells from 300 children and adults from the Asthma BRIDGE cohort (P < .05; Fig 1, F, and Table III). This cohort was described previously by Raby et al,3Raby B. Barnes K. Beaty T.H. Bosco A. Carey V.J. Castro M. et al.Asthma Bridge: the Asthma Biorepository for Integrative Genomic Exploration.Am J Respir Crit Care Med. 2011; 183: A6189Google Scholar and some of the microarray data regarding genes other than NFATc1 and IRF4 were described before.4Sharma S. Zhou X.B. Thibault D.M. Himes B.E. Liu A. Szefler S.J. et al.A genome-wide survey of CD4(+) lymphocyte regulatory genetic variants identifies novel asthma genes.J Allergy Clin Immunol. 2014; 134: 1153-1162Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar Both the PreDicta study and the Asthma BRIDGE study used the same phenotype definitions. The shorter isoform A (variant 1) lacks the C-terminal extension of approximately 245 amino acids that is present in all other NFAT proteins. This isoform of NFATc1 was found to be significantly overexpressed in peripheral blood CD4+ T cells from asthmatic patients with a positive skin test result compared with both asthmatic patients with a negative skin test result and nonasthmatic subjects with a negative skin test result (Table III). Moreover, the induced isoform D (variant 4) of NFATc1 was also found to be differentially expressed between asthmatic patients with a positive skin test result and nonasthmatic subjects with a negative skin test result (Table III). The log fold change (FC) for NFATc1 expression was 0.21 (1.2-fold increase), with an average expression of 7.44 (P = .013). The log FC for NFATc1 expression was 0.1 (1.1-fold increase), with an average expression of 7.83 (P = .013). Adding the 84 white asthmatic patients with positive skin test results to the model and adjusting for age, race, and sex (146 black plus 84 white vs 39 black subjects; female/male ratio, 127:106) did not change the NFATc1 result (log FC = 0.1, P = .018). Induction of the short isoform A of NFATc1 takes place after activation of T cells and is controlled by promoter 1 (Fig 1, A), a strong inducible promoter. This effect is autoregulated by the NFAT transcription factors.5Chuvpilo S. Jankevics E. Tyrsin D. Akimzhanov A. Moroz D. Jha M.K. et al.Autoregulation of NFATc1/A expression facilitates effector T cells to escape from rapid apoptosis.Immunity. 2002; 16: 881-895Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar NFATc1/A is thought to be needed for exerting effector functions in activated T cells. In contrast to NFATc1/C, NFATc2, and NFATc3 proteins, the short isoform A of NFATc1 is not able to promote cell apoptosis.5Chuvpilo S. Jankevics E. Tyrsin D. Akimzhanov A. Moroz D. Jha M.K. et al.Autoregulation of NFATc1/A expression facilitates effector T cells to escape from rapid apoptosis.Immunity. 2002; 16: 881-895Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar We presume that this function of NFATc1 leads to a prolonged survival of effector T cells in asthmatic patients with a positive skin test result. Moreover, it has been observed that NFATc1/αA (Fig 1, A) is the most prominent NFATc1 protein on receptor stimulation of peripheral B and T cells. In these cells the first activation, through the respective receptor, of the full induction of NFATc1/αA requires 24 hours. By contrast, this isoform is completely induced within a few hours after secondary stimulation.5Chuvpilo S. Jankevics E. Tyrsin D. Akimzhanov A. Moroz D. Jha M.K. et al.Autoregulation of NFATc1/A expression facilitates effector T cells to escape from rapid apoptosis.Immunity. 2002; 16: 881-895Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar This is in line with our findings that NFATc1/A is upregulated in CD4+ T cells of asthmatic patients with a positive skin test result because these patients were already sensitized to an allergen. Moreover, we observed that NFATc1 isoform D (variant 4) was upregulated in asthmatic patients with a positive skin test result. To our knowledge, the function of NFATc1/D has not been described yet.Table IIIComparison between different groups of children and adults with or without asthma and atopy (Asthma BRIDGE)Group comparisonsAsthma +, skin test + (n = 251)Asthma −, skin test + (n = 44)P = .1Asthma +, skin test − (n = 47)P = .006 (NFATc1 isoform A)Asthma −, skin test − (n = 75)P = .002 (NFATc1 isoform A);P = .05 (NFATc1 isoform D)For analysis using a microarray, there were 47,009 tag probes to target individual exons, including 4 exon sequences linked to specific NFATc1 isoforms. The particular isoforms of NFATc1 mRNA were as follows: NFATc1 isoform A = variant 1; NM 172390.2; NP 765978.1; NFATc1 isoform D = variant 4; NM 172388.2; NP 765976.1; Gene ID: 4772. P values for association of probes with phenotypes were adjusted by using the Benjamini-Hochberg method for multiple comparisons between groups. Open table in a new tab For analysis using a microarray, there were 47,009 tag probes to target individual exons, including 4 exon sequences linked to specific NFATc1 isoforms. The particular isoforms of NFATc1 mRNA were as follows: NFATc1 isoform A = variant 1; NM 172390.2; NP 765978.1; NFATc1 isoform D = variant 4; NM 172388.2; NP 765976.1; Gene ID: 4772. P values for association of probes with phenotypes were adjusted by using the Benjamini-Hochberg method for multiple comparisons between groups. In addition to NFATc1, the transcription factor interferon regulatory factor 4 (IRF4), which is encoded by the IRF4 gene, has been shown to play a role in the differentiation of various T-cell subsets known to have an effect on asthma pathology.6Staudt V. Bothur E. Klein M. Lingnau K. Reuter S. Grebe N. et al.Interferon-regulatory factor 4 is essential for the developmental program of T helper 9 cells.Immunity. 2010; 33: 192-202Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar Therefore we investigated IRF4 mRNA expression in whole blood from healthy and asthmatic children from PreDicta (Fig 2, A). We found that the asthmatic children had a significantly increased expression of IRF4 compared with that seen in healthy control subjects (Fig 2, A), especially when they also had a positive skin test result (Fig 2, B). This observation was further confirmed by analyzing total blood cells in Asthma BRIDGE asthmatic patients with positive skin test results (P = .02; Fig 2, C). IRF4 is known to be an important factor for innate and adaptive immune responses, cooperating with various other transcription factors, including NFAT family members, to act as both a transcriptional repressor and activator.7De Silva N.S. Simonetti G. Heise N. Klein U. The diverse roles of IRF4 in late germinal center B-cell differentiation.Immunol Rev. 2012; 247: 73-92Crossref PubMed Scopus (60) Google Scholar Therefore it is likely that IRF4 interacts with NFATc1 to prolong the survival of effector T cells in patients with allergic asthma. Namely, IRF4 would directly promote the production of IL-4, one of the TH2-associated cytokines that contributes to asthma pathogenesis, by binding to the IL-4 promoter in cooperation with NFATc1. In support of this notion, we found reduced IFNG mRNA expression in the group of children with positive skin test results who had higher levels of NFATc1 and IRF4. Consistently, we previously reported that asthmatic mice deficient in NFATc1 in T cells (NFATc1fl/flxCD4Cre) have increased numbers of IFN-γ+CD4+ T cells in their lungs and that these cells expressed less Batf, a transcription factor essential for immunoglobulin class-switching that cooperates with IRF4 at the promoter of different genes relevant for asthma8Glasmacher E. Agrawal S. Chang A.B. Murphy T.L. Zeng W.W. Vander Lugt B. et al.A genomic regulatory element that directs assembly and function of immune-specific AP-1-IRF complexes.Science. 2012; 338: 975-980Crossref PubMed Scopus (240) Google Scholar and that NFATc1fl/flxCD4Cre mice have reduced serum levels of ovalbumin (OVA)–specific IgE.2Koch S. Reppert S. Finotto S. NFATc1 deletion in T lymphocytes inhibits the allergic trait in a murine model of asthma.Clin Exp Allergy. 2015; 45: 1356-1366Crossref PubMed Google Scholar Moreover, these data are also supported by our findings in the basic leucine zipper transcription factor, ATF-like (BATF)–deficient mice, which do not induce IgE, and we demonstrated T cells that produce increased IFN-γ levels9Ubel C. Sopel N. Graser A. Hildner K. Reinhardt C. Zimmermann T. et al.The activating protein 1 transcription factor basic leucine zipper transcription factor, ATF-like (BATF), regulates lymphocyte- and mast cell-driven immune responses in the setting of allergic asthma.J Allergy Clin Immunol. 2014; 133 (e1-9): 198-206Abstract Full Text Full Text PDF PubMed Google Scholar in a model of allergic asthma. Because IRF4 is also known to be crucial for IL-9 production,6Staudt V. Bothur E. Klein M. Lingnau K. Reuter S. Grebe N. et al.Interferon-regulatory factor 4 is essential for the developmental program of T helper 9 cells.Immunity. 2010; 33: 192-202Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar we also investigated IL-9 in cultured PBMCs of preschool children stimulated with PHA. We could not find any differences in IL-9 production when we compared asthmatic and healthy control children (Fig 2, D), but we observed a significant increase in IL-9 levels in the supernatants of PBMCs isolated from asthmatic children with an additional positive skin test result compared with those isolated from healthy children with a negative skin test result (Fig 2, E). Both NFATc1 and IRF4 also positively influence IL-9 production of TH9 cells.6Staudt V. Bothur E. Klein M. Lingnau K. Reuter S. Grebe N. et al.Interferon-regulatory factor 4 is essential for the developmental program of T helper 9 cells.Immunity. 2010; 33: 192-202Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar Consistent with the increased expression of both IRF4 and NFATc1 seen in asthmatic patients with positive skin test results, we also found increased IL-9 levels in their PBMCs. Overall, we found that in PBMCs and CD4+ T cells of asthmatic patients with a positive skin test result, expression of NFATc1 isoforms and IRF4 was increased, suggesting a fundamental role for both transcription factors in the immunologic switch in allergic asthma. In this survey we analyzed subjects from 2 different studies. Although the PreDicta study includes only 42 children, we obtained important indications regarding NFATc1, IRF4, and IL-9 expression in patients with allergic asthma. These results could be verified and confirmed in the bigger cohorts of the Asthma BRIDGE study, in which 300 asthmatic patients and 122 control subjects were included. The functional relevance of NFATc1 in asthmatic patients was supported by our observations in targeted conditional deletion of NFATc1 in T lymphocytes, where Nfatc1/A mRNA expression perfectly correlated with OVA-specific IgE levels (R2 = 0.94) in patients with experimental asthma (see Fig E1, D, and the Methods, Results, and Discussion sections in this article's Online Repository at www.jacionline.org) and resulted in downregulation of IL-9 (see Fig E1, B) and mast cell function (see Figs E2 and E3 and the Methods, Results, and Discussion sections in this article's Online Repository at www.jacionline.org). Therefore targeting NFATc1 in T lymphocytes might ameliorate the allergic phenotype seen in asthmatic patients. We thank Rebekka Springel and Sonja Trump at the Molecular Pneumology Department in Erlangen and Lena Schramm, Ines Yava, and Evelin Muschiol at the Children's Hospital in Erlangen for their technical assistance. Two cohorts of preschool-aged children with and without asthma at the age of 4 to 6 years were recruited at the Children's Hospital of Erlangen, Department of Pediatrics and Adolescent Medicine. This research was conducted through the European study “Post-infectious immune reprogramming and its association with persistence and chronicity of respiratory allergic diseases” (PreDicta), a multicenter prospective cohort study carried out in 5 different centers in Europe. The study was approved by the ethics committee of the Friedrich-Alexander University Erlangen-Nürnberg, Germany (reference no. 4435), and is also registered in the German Clinical Trial Register (registration no. DRKS00004914). Informed consent was obtained from the parents of all children of the PreDicta study. Twenty-two asthmatic patients and 20 healthy subjects were analyzed. Inclusion criteria for cases were age of 4 to 6 years, gestational age of 36 weeks or greater, and diagnosis of asthma within the last 2 years confirmed by a doctor of the Children's Hospital in Erlangen of mild-to-moderate persistent severity according to Global Initiative for Asthma (GINA) guidelines (2005). In this study we have assessed the degree of asthma as follows. Severity level I is defined as intermittent asthma. The children are free of asthma symptoms for at least 2 months with an FEV1 of greater than 80% and a mean expiratory flow (MEF) of greater than 60%. Severity level II is defined as mild persistent asthma. The children are symptom free for a period of less than 2 months. At the moment, they do not have asthmatic symptoms, with an FEV1 of greater than 80% and an MEF of greater than 60%. Severity level III is defined as moderate persistent asthma. The children have asthma symptoms several days a week with an FEV1 of less than 80% and an MEF of less than 65%. At the B0 visit, parents were asked to answer questions about asthma control according to 2009 GINA guidelines. Possible answers were as follows: (1) severity I, which means controlled asthma; (2) severity II, which means partly controlled asthma; and (3) severity III, which means uncontrolled asthma. The GINA guidelines take into account FEV1, limitation of physical activity, frequency of exacerbations, and use of asthma “reliever” medication. Exclusion criteria were severe or brittle asthma, children receiving immunotherapy or more than 6 courses of oral steroids during the preceding 12 months, and children with other chronic respiratory diseases (cystic fibrosis, bronchopulmonary dysplasia, and immunodeficiencies), except allergic rhinitis. Furthermore, children with other chronic diseases or chronic medication use, except atopic eczema, were excluded. The children performed a peak expiratory flow maneuver. Atopy was proved by at least 1 positive skin prick test response (wheal size, ≥3 mm; HAL Allergie GmbH, Düsseldorf, Germany). For skin prick tests, we analyzed allergic reactions to the following allergens: cat, house dust mite, Alternaria species, birch, grass, Ambrosia species, and mold. Asthma was confirmed by a physician's diagnosis of mucus production, airway hyperresponsiveness, and dyspnea. At visit B0, the doctor asked the mother several questions to evaluate the child's asthma symptoms during the last 12 months. The control subjects had no history of asthma, wheezing, or atopic illness. Peripheral blood from control or asthmatic children was collected into venous blood collection tubes with lithium heparin (BD Vacutainer Tubes; BD Biosciences, San Jose, Calif); the heparinized blood was transferred to a sterile 15-mL tube and diluted with an equal volume of warm saline and mixed well. The diluted blood was carefully stratified on top of the density gradient medium Biocoll separating solution (Biochrom AG, Berlin, Germany) and centrifuged. Afterward, the layer of PBMCs, which can be found between plasma and Biocoll, was transferred to a new sterile 15-mL tube. After washing the cells twice with RPMI 1640 medium, cells were used for RNA isolation, as described below. Some of the cells were cultured in complete culture medium for 48 hours with PHA (10 μg/mL). The complete culture medium consisted of RPMI-1640 supplemented with 25 mmol/L HEPES and l-Glutamine (Gibco, Invitrogen, Thermo Fisher Scientific, Waltham, Mass), 100 IU/mL penicillin (Sigma-Aldrich, St Louis, Mo), 100 μg/mL streptomycin (Sigma-Aldrich), 50 μmol/L β-mercaptoethanol (Sigma-Aldrich), 200 mmol/L 1% l-glutamine (Sigma-Aldrich), 1% MEM Vitamin (Sigma-Aldrich), 1% nonessential amino acids (Sigma-Aldrich), 1% sodium pyruvate (Sigma-Aldrich), and 10% H-FBS (Sigma-Aldrich). Additionally, whole blood from each child from the PreDicta cohort was collected into Tempus Blood RNA Tubes (Life Technologies, Carlsbad, Calif) and stored at −80°C. RNA was isolated and purified by using RNA binding beads with a MagMAX for Stabilized Blood Tube RNA Isolation Kit (Ambion, Life Technologies), according to the manufacturer's instructions. Asthma BRIDGE is an open-access biorepository for subjects participating in genetic studies of asthma in the EVE Consortium.E1Torgerson D.G. Ampleford E.J. Chiu G.Y. Gauderman W.J. Gignoux C.R. Graves P.E. et al.Meta-analysis of genome-wide association studies of asthma in ethnically diverse North American populations.Nat Genet. 2011; 43: 887-892Crossref PubMed Scopus (621) Google Scholar Sample collection and processing were carried out at each institution according to standardized and validated protocols. These samples were centralized at the Data Coordinating Center at the Channing Division of Network Medicine at Brigham and Women's Hospital at the Harvard Medical School (Boston, Mass). Contributing centers isolating peripheral blood CD4+ T lymphocytes used a modified version of the protocol previously optimized for collections in the Childhood Asthma Management Program study by using anti-CD4+ microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and column separation. The modification includes isolating PBMCs and then stimulating them with PHA before CD4+ lymphocyte isolation. NFATc1 alone or in cooperation with IRF4 is known to be important for the differentiation of different subsets of lymphocytes that play a crucial role in asthma development.E2Koch S. Reppert S. Finotto S. NFATc1 deletion in T lymphocytes inhibits the allergic trait in a murine model of asthma.Clin Exp Allergy. 2015; 45: 1356-1366Crossref PubMed Scopus (15) Google Scholar, E3Muller M.R. Rao A. NFAT, immunity and cancer: a transcription factor comes of age.Nat Rev Immunol. 2010; 10: 645-656Crossref PubMed Scopus (426) Google Scholar, E4Robinson D.S. Hamid Q. Ying S. Tsicopoulos A. Barkans J. Bentley A.M. et al.Predominant TH2-like bronchoalveolar T-lymphocyte population in atopic asthma.N Engl J Med. 1992; 326: 298-304Crossref PubMed Scopus (2568) Google Scholar, E5Hu C.M. Jang S.Y. Fanzo J.C. Pernis A.B. Modulation of T cell cytokine production by interferon regulatory factor-4.J Biol Chem. 2002; 277: 49238-49246Crossref PubMed Scopus (90) Google Scholar, E6Rengarajan J. Tang B. Glimcher L.H. NFATc2 and NFATc3 regulate T(H)2 differentiation and modulate TCR-responsiveness of naive T(H)cells.Nat Immunol. 2002; 3: 48-54Crossref PubMed Scopus (130) Google Scholar To analyze these factors, microarray analysis was used within the Asthma BRIDGE study. Therefore samples for RNA extraction were collected with BD Vacutainer CPT tubes (BD Diagnostics, Franklin Lakes, NJ), placed on ice, and centrifuged within 1 hour (20 minutes at 1700 relative centrifugal force). Total RNA was extracted by using the RNeasy Mini Protocol (Qiagen, Valencia, Calif) and stored at −80°C. Analysis with the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, Calif) confirmed average total RNA yields of 2 μg per collection, with minimal evidence of RNA degradation and 28S/18S ratios approaching 2.0. Some of the microarray data regarding genes other than NFATc1 and IRF4 were described before.E7Sharma S. Zhou X.B. Thibault D.M. Himes B.E. Liu A. Szefler S.J. et al.A genome-wide survey of CD4(+) lymphocyte regulatory genetic variants identifies novel asthma genes.J Allergy Clin Immunol. 2014; 134: 1153-1162Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, E8Yan X.T. Chu J.H. Gomez J. Koenigs M. Holm C. He X.X. et al.Noninvasive analysis of the sputum transcriptome discriminates clinical phenotypes of asthma.Am J Respir Crit Care Med. 2015; 191: 1116-1125Crossref PubMed Scopus (65) Google Scholar For our differential expression analyses, we studied 300 asthmatic Asthma BRIDGE subjects with log2-transformed and quantile-normalized gene expression (n = 47,009 probes) from unstimulated blood-derived CD4+ T cells measured with Illumina Human HT-12 v4 BeadChips (Illumina, San Diego, Calif). Asthma was defined based on having a doctor's diagnosis of the disease with evidence of reversible airflow obstruction with a bronchodilator. The R Bioconductor limma package (version 3.20.1)E9Smyth G.K. Linear models and empirical Bayes methods for assessing differential expression in microarray experiments.Stat Appl Genet Mol Biol. 2004; 3: Article3Crossref PubMed Scopus (9178) Google Scholar was used to perform differential expression analysis. A linear model was fitted along with implementation of empiric Bayes statistics to assess whether expression levels of the candidate gene NFATc1 were significantly altered among asthmatic patients after stratifying by race and adjusting for age and sex. Two differential expression analyses were performed: (1) comparing asthmatic patients with positive skin test results (defined as a wheal ≥3 mm in size) with atopic dermatitis (eczema) with those without eczema and (2) comparing asthmatic patients with positive skin test results with those with negative skin test results. Eighty-seven white asthmatic patients with positive skin test results were identified within the Asthma BRIDGE study. After removing those asthmatic patients with missing information on unknown conditions of atopic eczema or skin tests, 82 subjects (female/male subjects, 34/48), consisting of 49 asthmatic patients with eczema and positive skin test results and 33 asthmatic patients without eczema and positive skin test results, remained for analysis. One hundred forty-seven black asthmatic patients with positive and negative skin test results were identified within the Asthma BRIDGE study. After removing 1 subject with missing information, 146 subjects (female/male subjects, 91/55), consisting of 107 asthmatic patients with positive skin test results and 39 asthmatic patients with negative skin test results, remained for analysis. NFATc1fl/fl control mice and NFATc1fl/flxCD4Cre mice were on a C57BL/6 background and kept in house under specific pathogen-free conditions. Mice were 6 to 8 weeks old in all experiments. The experiments were performed with approved licenses (23-177-07/G09-1-008 from the ethical review board Rheinland-Pfalz and 54-2532.1-2/10 and 54-2532.1-55/12 from the government of Mittelfranken, Bavaria). For the experimental asthma model, mice were sensitized with OVA (100 μg; Calbiochem, San Diego, Calif) complexed with alum (10%; Sigma-Aldrich, Steinheim, Germany) on days 0 and 7 administered intraperitoneally. Allergen challenge was performed with an intranasal treatment of OVA (2 mg/mL) on days 18, 19, and 20, as described previously.E10Finotto S. Buerke M. Lingnau K. Schmitt E. Galle P.R. Neurath M.F. Local administration of antisense phosphorothioate oligonucleotides to the c-kit ligand, stem cell factor, suppresses airway inflammation and IL-4 production in a murine model of asthma.J Allergy Clin Immunol. 2001; 107: 279-286Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar Mice were killed on day 21, and lung cells were isolated. CD4+ T cells were purified from isolated total lung cell suspensions, as previously described.E11Sauer K.A. Scholtes P. Karwot R. Finotto S. Isolation of CD4+ T cells from murine lungs: a method to analyze ongoing immune responses in the lung.Nat Protoc. 2006; 1: 2870-2875Crossref PubMed Scopus (52) Google Scholar Shortly, total lung cells were incubated with anti-mouse CD4 L3T4 microbeads and positively sorted in a magnetic cell sorter system (MACS; Miltenyi Biotech, Germany), according to the manufacturer's protocol. Afterward, CD4+ T cells were cultured in RPMI with plate-bound α-CD3 (2 μg/mL) and soluble α-CD28 (2 μg/mL) antibodies for 24 hours in a density of 106 cells/mL. Supernatants were analyzed by means of ELISA for protein production. Hind legs of the mice were prepared, and bone marrow cells were flushed from the tibias and femurs with sterile PBS. Afterward, cells were centrifuged for 5 minutes at 4°C and 1500 rpm. Cells were then resuspended in RPMI 1640 culture medium supplemented with 10% FBS, 1% penicillin/streptomycin, 1% L-glutamine, 1% HEPES, 50 μmol of β-mercaptoethanol, and 10 ng/mL IL-3 and stem cell factor (PeproTech, Hamburg, Germany) and cultured for 4 weeks in 10 mL of the medium in small culture flasks, as previously described.E12Ubel C. Sopel N. Graser A. Hildner K. Reinhardt C. Zimmermann T. et al.The activating protein 1 transcription factor basic leucine zipper transcription factor, ATF-like (BATF), regulates lymphocyte- and mast cell-driven immune responses in the setting of allergic asthma.J Allergy Clin Immunol. 2014; 133 (e1-9): 198-206Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar Some of the cells were additionally cultured with 10 ng/mL recombinant IL-9 (PeproTech). Every week, medium with cytokines was replaced, and fluorescence-activated cell sorting (FACS) analysis was performed to control mast cell differentiation. After 4 weeks of cell culture, mast cells were resuspended in warm Tyrode buffer (1 × 107 cells/mL of buffer) and incubated with 10 μg/mL purified mouse IgE, κ isotype control antibody (BioLegend, Fell, Germany) for 30 minutes at 37°C. Afterward, cells were washed twice with warm Tyrode buffer and centrifuged for 5 minutes at 4°