Gene editing in allergic diseases: Identification of novel pathways and impact of deleting allergen genes.

Gene editing technology has emerged as a powerful tool in all aspects of health research and continues to advance our understanding of critical and essential elements in disease pathophysiology. The clustered regularly interspaced short palindromic repeats (CRISPR) gene editing technology has been used with precision to generate gene knockouts, alter genes, and identify genes that cause disease. The full spectrum of allergic/atopic diseases, in part because of shared pathophysiology, is ripe for studies with this technology. In this way, novel culprit genes are being identified and allow for manipulation of triggering allergens to reduce allergenicity and disease. Notwithstanding current limitations on precision and potential off-target effects, newer approaches are rapidly being introduced to more fully understand specific gene functions as well as the consequences of genetic manipulation. In this review, we examine the impact of editing technologies of novel genes relevant to peanut allergy and asthma as well as how gene modification of common allergens may lead to the deletion of allergenic proteins.

Molecular networks in atopic mothers impact the risk of infant atopy.

BACKGROUND: The prevalence of atopic diseases has increased with atopic dermatitis (AD) as the earliest manifestation. We assessed if molecular risk factors in atopic mothers influence their infants' susceptibility to an atopic disease. METHODS: Pregnant women and their infants with (n = 174, high-risk) or without (n = 126, low-risk) parental atopy were enrolled in a prospective birth cohort. Global differentially methylated regions (DMRs) were determined in atopic (n = 92) and non-atopic (n = 82) mothers. Principal component analysis was used to predict atopy risk in children dependent on maternal atopy. Genome-wide transcriptomic analyses were performed in paired atopic (n = 20) and non-atopic (n = 15) mothers and cord blood. Integrative genomic analyses were conducted to define methylation-gene expression relationships. RESULTS: Atopic dermatitis was more prevalent in high-risk compared to low-risk children by age 2. Differential methylation analyses identified 165 DMRs distinguishing atopic from non-atopic mothers. Inclusion of DMRs in addition to maternal atopy significantly increased the odds ratio to develop AD in children from 2.56 to 4.26. In atopic compared to non-atopic mothers, 139 differentially expressed genes (DEGs) were identified significantly enriched of genes within the interferon signaling pathway. Expression quantitative trait methylation analyses dependent on maternal atopy identified 29 DEGs controlled by 136 trans-acting methylation marks, some located near transcription factors. Differential expression for the same nine genes, including MX1 and IFI6 within the interferon pathway, was identified in atopic and non-atopic mothers and high-risk and low-risk children. CONCLUSION: These data suggest that in utero epigenetic and transcriptomic mechanisms predominantly involving the interferon pathway may impact and predict the development of infant atopy.

17q12-21 risk-variants influence cord blood immune regulation and multitrigger-wheeze.

BACKGROUND: Childhood wheeze represents a first symptom of asthma. Early identification of children at risk for wheeze related to 17q12-21 variants and their underlying immunological mechanisms remain unknown. We aimed to assess the influence of 17q12-21 variants and mRNA expression at birth on the development of wheeze. METHODS: Children were classified as multitrigger/viral/no wheeze until six years of age. The PAULINA/PAULCHEN birth cohorts were genotyped (n = 216; GSA-chip). mRNA expression of 17q21 and innate/adaptive genes was measured (qRT-PCR) in cord blood mononuclear cells. Expression quantitative trait loci (eQTL) and mediation analyses were performed. Genetic variation of 17q12-21 asthma-single nucleotide polymorphisms (SNPs) was summarized as the first principal component (PC1) and used to classify single SNP effects on gene expression as (locus)-dependent/independent eQTL SNPs. RESULTS: Core region risk variants (IKZF3, ZPBP2, GSDMB, ORMDL3) were associated with multitrigger wheeze (OR: 3.05-5.43) and were locus-dependent eQTL SNPs with higher GSDMA, TLR2, TLR5, and lower TGFB1 expression. Increased risk of multitrigger wheeze with rs9303277 was in part mediated by TLR2 expression. Risk variants distal to the core region were mainly locus-independent eQTL SNPs with decreased CD209, CD86, TRAF6, RORA, and IL-9 expression. Distinct immune signatures in cord blood were associated either with multitrigger wheeze (increased innate genes, e.g., TLR2, IPS1, LY75) or viral wheeze (decreased NF-κB genes, e.g., TNFAIP3 and TNIP2). CONCLUSION: Locus-dependent eQTL SNPs (core region) associated with increased inflammatory genes (primarily TLR2) at birth and subsequent multitrigger wheeze indicate that early priming and imbalance may be crucial for asthma pathophysiology. Locus-independent eQTL SNPs (mainly distal region, rs1007654) may be involved in the initiation of dendritic cell activation/maturation (TRAF6) and interaction with T cells (CD209, CD86). Identifying potential mechanistic pathways at birth may point to critical key points during early immune development predisposing to asthma.

Dichotomous role of TGF-ß controls inducible regulatory T-cell fate in allergic airway disease through Smad3 and TGF-ß-activated kinase 1.

BACKGROUND: Inducible CD4+CD25+ regulatory T (iTreg) cells can become pathogenic effector cells, enhancing lung allergic responses. OBJECTIVE: We aimed to define the underlying cellular and molecular pathways activated by TGF-ß, which determine the suppressor or enhancing activities of iTreg cells. METHODS: Sensitized wild-type and CD8-deficient (CD8-/-) mice were challenged with allergen. Isolated CD4+CD25- T cells were activated by using anti-CD3/anti-CD28. To generate suppressor iTreg cells, cells were then differentiated in the presence of TGF-ß, whereas IL-17-producing effector T cells were additionally exposed to IL-6. After TGF-ß, Smad3 and TGF-ß-activated kinase 1 (TAK1) kinase levels were monitored. The consequences of inhibiting either kinase were determined in vitro and after transfer into CD8-/- recipients. Quantitative PCR and chromatin immunoprecipitation were used to monitor gene expression and histone modifications at the retinoic acid-related orphan receptor γt (Rorγt) locus. RESULTS: In wild-type mice, iTreg cells suppressed lung allergic responses linked to Smad3-dependent forkhead box P3 (Foxp3) expression and IL-10 production. In the presence of IL-6, iTreg cells converted to TH17 cells, mediating a neutrophil-dependent enhancement of lung allergic responses in CD8-/- mice. Conversion was regulated by TAK1. Inhibition or silencing of TAK1 prevented expression of Rorγt and TH17 differentiation through histone modifications of Rorγt; Foxp3 expression and iTreg cell-mediated suppression remained intact. In the same cell, TGF-ß induced coexpression of Smad3 and TAK1 proteins; in the presence of IL-6, expression of Smad3 and Foxp3 but not TAK1 decreased. CONCLUSION: TGF-ß regulates iTreg cell outcomes through 2 distinct signal transduction pathways: one Smad3 dependent and the other TAK1 dependent. The balance of these pathways has important implications in TH17-mediated autoimmune diseases and neutrophil-dependent asthma.

Plasticity of Naturally Occurring Regulatory T Cells in Allergic Airway Disease Is Modulated by the Transcriptional Activity of Il-6.

The impact of naturally occurring regulatory T cells (nTregs) on the suppression or induction of lung allergic responses in mice depends on the nuclear environment and the production of the pro-inflammatory cytokine interleukin 6 (IL-6). These activities were shown to be different in nTregs derived from wild-type (WT) and CD8-deficient mice (CD8-/-), with increased IL-6 levels in nTregs from CD8-/- mice in comparison to WT nTregs. Thus, identification of the molecular mechanisms regulating IL-6 production is critical to understanding the phenotypic plasticity of nTregs. Electrophoretic mobility shift assays (EMSA) were performed to determine transcription factor binding to four Il-6 promoter loci using nuclear extracts from nTregs of WT and CD8-/- mice. Increased transcription factor binding for each of the Il-6 loci was identified in CD8-/- compared to WT nTregs. The impact of transcription factor binding and a novel short tandem repeat (STR) on Il-6 promoter activity was analyzed by luciferase reporter assays. The Il-6 promoter regions closer to the transcription start site (TSS) were more relevant to the regulation of Il-6 depending on NF-κB, c-Fos, and SP and USF family members. Two Il-6 promoter loci were most critical for the inducibility by lipopolysaccharide (LPS) and tumor necrosis factor α (TNFα). A novel STR of variable length in the Il-6 promoter was identified with diverging prevalence in nTregs from WT or CD8-/- mice. The predominant GT repeat in CD8-/- nTregs revealed the highest luciferase activity. These novel regulatory mechanisms controlling the transcriptional regulation of the Il-6 promoter are proposed to contribute to nTregs plasticity and may be central to disease pathogenesis.

Spectrum of T-lymphocyte activities regulating allergic lung inflammation.

Despite advances in the treatment of asthma, optimization of symptom control remains an unmet need in many patients. These patients, labeled severe asthma, are responsible for a substantial fraction of the disease burden. In these patients, research is needed to define the cellular and molecular pathways contributing to disease which in large part are refractory to corticosteroid treatment. The causes of steroid-resistant asthma are multifactorial and result from complex interactions of genetics, environmental factors, and innate and adaptive immunity. Adaptive immunity, addressed here, integrates the activities of distinct T-cell subsets and by definition is dynamic and responsive to an ever-changing environment and the influences of epigenetic modifications. These T-cell subsets exhibit different susceptibilities to the actions of corticosteroids and, in some, corticosteroids enhance their functional activation. Moreover, these subsets are not fixed in lineage differentiation but can undergo transcriptional reprogramming in a bidirectional manner between protective and pathogenic effector states. Together, these factors contribute to asthma heterogeneity between patients but also in the same patient at different stages of their disease. Only by carefully defining mechanistic pathways, delineating their sensitivity to corticosteroids, and determining the balance between regulatory and effector pathways will precision medicine become a reality with selective and effective application of targeted therapies.

Hypoxia enhances CD8+ TC2 cell-dependent airway hyperresponsiveness and inflammation through hypoxia-inducible factor 1α.

BACKGROUND: CD8+ type 2 cytotoxic T (TC2) cells undergo transcriptional reprogramming to IL-13 production in the presence of IL-4 to become potent, steroid-insensitive, pathogenic effector cells in asthmatic patients and in mice in a model of experimental asthma. However, no studies have described the effects of hypoxia exposure on TC2 cell differentiation. OBJECTIVE: We determined the effects of hypoxia exposure on IL-13-producing CD8+ TC2 cells. METHODS: CD8+ transgenic OT-1 cells differentiated with IL-2 and IL-4 (TC2 cells) were exposed to normoxia (21% oxygen) or hypoxia (3% oxygen), and IL-13 production in vitro was monitored. After differentiation under these conditions, cells were adoptively transferred into CD8-deficient mice, and lung allergic responses, including airway hyperresponsiveness to inhaled methacholine, were assessed. The effects of pharmacologic inhibitors of hypoxia-inducible factor (HIF) 1α and HIF-2α were determined, as were responses in HIF-1α-deficient OT-1 cells. RESULTS: Under hypoxic conditioning, CD8+ TC2 cell differentiation was significantly enhanced, with increased numbers of IL-13+ T cells and increased production of IL-13 in vitro. Adoptive transfer of TC2 cells differentiated under hypoxic conditioning restored lung allergic responses in sensitized and challenged CD8-deficient recipients to a greater degree than seen in recipients of TC2 cells differentiated under normoxic conditioning. Pharmacologic inhibition of HIF-1α or genetic manipulation to reduce HIF-1α expression reduced the hypoxia-enhanced differentiation of TC2 cells, IL-13 production, and the capacity of transferred cells to restore lung allergic responses in vivo. IL-4-dependent, hypoxia-mediated increases in HIF-1α and TC2 cell differentiation were shown to be mediated through activation of Janus kinase 1/3 and GATA-3. CONCLUSIONS: Hypoxia enhances CD8+ TC2 cell-dependent airway hyperresponsiveness and inflammation through HIF-1α activation. These findings coupled with the known insensitivity of CD8+ T cells to corticosteroids suggests that activation of the IL-4-HIF-1α-IL-13 axis might play a role in the development of steroid-refractory asthma.

IRF-1 SNPs influence the risk for childhood allergic asthma: A critical role for pro-inflammatory immune regulation.

BACKGROUND: Allergic and non-allergic childhood asthma has been characterized by distinct immune mechanisms. While interferon regulating factor 1 (IRF-1) polymorphisms (SNPs) influence atopy risk, the effect of SNPs on asthma phenotype-specific immune mechanisms is unclear. We assessed whether IRF-1 SNPs modify distinct immune-regulatory pathways in allergic and non-allergic childhood asthma (AA/NA). METHODS: In the CLARA study, asthma was characterized by doctor's diagnosis and AA vs NA by positive or negative specific IgE. Children were genotyped for four tagging SNPs within IRF-1 (n = 172). mRNA expression was measured with qRT-PCR. Gene expression was analyzed depending on genetic variants within IRF-1 and phenotype including haplotype estimation and an allelic risk score. RESULTS: Carrying the risk alleles of IRF-1 in rs10035166, rs2706384, or rs2070721 was associated with increased risk for AA. Carrying the non-risk allele in rs17622656 was associated with lower risk for AA but not NA. In AA carrying the risk alleles, an increased pro-inflammatory expression of ICAM3, IRF-8, XBP-1, IFN-γ, RGS13, RORC, and TSC2 was observed. NOD2 expression was decreased in AA with risk alleles in rs2706384 and rs10035166 and with risk haplotype. Further, AA with risk haplotype showed increased IL-13 secretion. NA with risk allele in rs2070721 compared to non-risk allele in rs17622656 showed significantly upregulated calcium, innate, mTOR, neutrophil, and inflammatory-associated genes. CONCLUSION: IRF-1 polymorphisms influence the risk for childhood allergic asthma being associated with increased pro-inflammatory gene regulation. Thus, it is critical to implement IRF-1 genetics in immune assessment for childhood asthma phenotypes.

Inducible and naturally occurring regulatory T cells enhance lung allergic responses through divergent transcriptional pathways.

BACKGROUND: Regulatory T cells attenuate development of asthma in wild-type (WT) mice, with both naturally occurring regulatory T (nTreg) cells and inducible regulatory T (iTreg) cells exhibiting suppressive activity. When transferred into CD8-deficient (CD8-/-) recipients, both cell types enhanced development of allergen-induced airway hyperresponsiveness. OBJECTIVE: We sought to determine whether the pathways leading to enhancement of lung allergic responses by transferred nTreg and iTreg cells differed. METHODS: nTreg cells (CD4+CD25+) were isolated from WT mice and iTreg cells were generated from WT CD4+CD25- T cells after activation in the presence of TGF-ß and transferred into sensitized CD8-/- recipients before challenge. Development of airway hyperresponsiveness, cytokine levels, and airway inflammation were monitored. RESULTS: Transfer of nTreg cells enhanced lung allergic responses, as did transfer of iTreg cells. Although anti-IL-13 reduced nTreg cell-mediated enhancement, it was ineffective in iTreg cell-mediated enhancement; conversely, anti-IL-17, but not anti-IL-13, attenuated the enhancement by iTreg cells. Recovered iTreg cells from the lungs of CD8-/- recipients were capable of IL-17 production and expressed high levels of signature genes of the TH17 pathway, RORγt and Il17, whereas reduced expression of the Treg cell key transcription factor forkhead box p3 (Foxp3) was observed. In vitro exogenous IL-6-induced IL-17 production in iTreg cells, and in vivo conversion of transferred iTreg cells was dependent on recipient IL-6. CONCLUSIONS: iTreg cells, similar to nTreg cells, exhibit functional plasticity and can be converted from suppressor cells to pathogenic effector cells, enhancing lung allergic responses, but these effects were mediated through different pathways.

JNK2 regulates the functional plasticity of naturally occurring T regulatory cells and the enhancement of lung allergic responses.

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-ß 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.

Polymorphisms related to ORMDL3 are associated with asthma susceptibility, alterations in transcriptional regulation of ORMDL3, and changes in TH2 cytokine levels.

BACKGROUND: Chromosome 17q21, harboring the orosomucoid 1-like 3 (ORMDL3) gene, has been consistently associated with childhood asthma in genome-wide association studies. OBJECTIVE: We investigated genetic variants in and around ORMDL3 that can change the function of ORMDL3 and thus contribute to asthma susceptibility. METHODS: We performed haplotype analyses and fine mapping of the ORMDL3 locus in a cross-sectional (International Study of Asthma and Allergies in Childhood Phase II, n = 3557 total subjects, n = 281 asthmatic patients) and case-control (Multicenter Asthma Genetics in Childhood Study/International Study of Asthma and Allergies in Childhood Phase II, n = 1446 total subjects, n = 763 asthmatic patients) data set to identify putative causal single nucleotide polymorphisms (SNPs) in the locus. Top asthma-associated polymorphisms were analyzed for allele-specific effects on transcription factor binding and promoter activity in vitro and gene expression in PBMCs after stimulation ex vivo. RESULTS: Two haplotypes (H1 and H2) were significantly associated with asthma in the cross-sectional (P = 9.9 × 10(-5) and P = .0035, respectively) and case-control (P = 3.15 × 10(-8) and P = .0021, respectively) populations. Polymorphisms rs8076131 and rs4065275 were identified to drive these effects. For rs4065275, a quantitative difference in transcription factor binding was found, whereas for rs8076131, changes in upstream stimulatory factor 1 and 2 transcription factor binding were observed in vitro by using different cell lines and PBMCs. This might contribute to detected alterations in luciferase activity paralleled with changes in ORMDL3 gene expression and IL-4 and IL-13 cytokine levels ex vivo in response to innate and adaptive stimuli in an allele-specific manner. Both SNPs were in strong linkage disequilibrium with asthma-associated 17q21 SNPs previously related to altered ORMDL3 gene expression. CONCLUSION: Polymorphisms in a putative promoter region of ORMDL3, which are associated with childhood asthma, alter transcriptional regulation of ORMDL3, correlate with changes in TH2 cytokines levels, and therefore might contribute to the childhood asthma susceptibility signal from 17q21.

Loss of T regulatory cell suppression following signaling through glucocorticoid-induced tumor necrosis receptor (GITR) is dependent on c-Jun N-terminal kinase activation.

Naturally occurring Foxp3(+)CD4(+)CD25(+) T regulatory cell (nTreg)-mediated suppression of lung allergic responses is abrogated following ligation of glucocorticoid-induced tumor necrosis receptor (GITR) family-related protein. In vitro stimulation of nTregs with GITR ligand increased phosphorylation of c-Jun N-terminal kinase (JNK) but not extracellular signal-regulated protein kinase (ERK) or p38 MAPK. SP600125, a known JNK inhibitor, prevented GITR-mediated phosphorylation of JNK. Activation of JNK was associated with increases in the upstream mitogen-activated protein kinase kinase 7 (MKK7) and the downstream transcription factor NF-κß. Phosphorylated c-Jun (p-c-Jun), indicative of the activation of JNK, was detected in the immunoprecipitates of nTregs from wild-type but not JNK- or GITR-deficient mice. Treatment with an inhibitor of JNK phosphorylation resulted in complete reversal of all GITR-induced changes in nTreg phenotype and function, with full restoration of suppression of in vivo lung allergic responses and in vitro proliferation of activated CD4(+)CD25(-) T cells. Thus, regulation of JNK phosphorylation plays a central role in T regulatory cell function with therapeutic implications for the treatment of asthma and autoimmune diseases.

CD8 regulates T regulatory cell production of IL-6 and maintains their suppressive phenotype in allergic lung disease.

Naturally occurring CD4(+)CD25(+)Foxp3(+) T regulatory cells (nTregs) regulate lung allergic responses through production of IL-10 and TGF-ß. nTregs from CD8(-/-) mice failed to suppress lung allergic responses and were characterized by reduced levels of Foxp3, IL-10, and TGF-ß, and high levels of IL-6. Administration of anti-IL-6 or anti-IL-6R to wild-type recipients prior to transfer of CD8(-/-) nTregs restored suppression. nTregs from IL-6(-/-) mice were suppressive, but lost this capability if incubated with IL-6 prior to transfer. The importance of CD8 in regulating the production of IL-6 in nTregs was demonstrated by the loss of suppression and increases in IL-6 following transfer of nTregs from wild-type donors depleted of CD8(+) cells. Transfer of nTregs from CD8(-/-) donors reconstituted with CD8(+) T cells was suppressive, and accordingly, IL-6 levels were reduced. These data identify the critical role of CD8-T regulatory cell interactions in regulating the suppressive phenotype of nTregs through control of IL-6 production.

Autophagy-associated immune dysregulation and hyperplasia in a patient with compound heterozygous mutations in ATG9A.

ABBREVIATIONS: BCL2: BCL2 apoptosis regulator; BCL10: BCL10 immune signaling adaptor; CARD11: caspase recruitment domain family member 11; CBM: CARD11-BCL10-MALT1; CR2: complement C3d receptor 2; EBNA: Epstein Barr nuclear antigen; EBV: Epstein-Barr virus; FCGR3A; Fc gamma receptor IIIa; GLILD: granulomatous-lymphocytic interstitial lung disease; HV: healthy volunteer; IKBKB/IKB kinase: inhibitor of nuclear factor kappa B kinase subunit beta; IL2RA: interleukin 2 receptor subunit alpha; MALT1: MALT1 paracaspase; MS4A1: membrane spanning 4-domain A1; MTOR: mechanistic target of rapamycin kinase; MYC: MYC proto-oncogene, bHLH: transcription factor; NCAM1: neural cell adhesion molecule 1; NFKB: nuclear factor kappa B; NIAID: National Institute of Allergy and Infectious Diseases; NK: natural killer; PTPRC: protein tyrosine phosphatase receptor type C; SELL: selectin L; PBMCs: peripheral blood mononuclear cells; TR: T cell receptor; Tregs: regulatory T cells; WT: wild-type.

Asthma-associated polymorphisms in 17q21 influence cord blood ORMDL3 and GSDMA gene expression and IL-17 secretion.

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 < .05, respectively). No correlation between 17q21 polymorphisms and regulatory T/T(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.

An IgE-associated polymorphism in STAT6 alters NF-kappaB binding, STAT6 promoter activity, and mRNA expression.

BACKGROUND: The IL-4/IL-13 pathway is central for IgE regulation. Signal transducer and activator of transcription 6 (STAT6) is the major transcription factor within this pathway. STAT6 polymorphisms were recently associated with elevated total IgE levels in a genome-wide association study. OBJECTIVE: This study aimed to assess biological mechanisms by which an IgE-associated genetic variation in STAT6 may potentially influence gene expression. METHODS: STAT6 intron 2 carrying either the wild-type C or the polymorphic T allele of the putatively causal single nucleotide polymorphism rs324011 was cloned into STAT6 promoter vectors to investigate their influence on STAT6 promoter activity by in vitro luciferase assays. Transcription factor binding depending on rs324011 was examined by electrophoretic mobility shift assays in Jurkat T cells and primary CD4(+) T cells. Allele-specific STAT6 gene expression of 3 splice variants was studied ex vivo by real-time PCR in 239 individuals. RESULTS: STAT6 intron 2 acts as a silencer regulatory element. The polymorphic T allele at rs324011 (in linkage disequilibrium with the genome-wide association signal and consistently associated with elevated IgE levels in 3 previous studies) increases STAT6 promoter activity significantly in vitro (P < .00001) and gene expression of STAT6 splice variants ex vivo (P < .01) compared with the wild-type C allele. These effects correlate with the creation of a novel, T-allele-specific binding site for the transcription factor nuclear factor-kappaB in T cells. CONCLUSION: The consistently replicated effects of genetic variance in STAT6 on IgE regulation may be explained in part by allele-specific alterations in nuclear factor-kappaB binding at rs324011 and consecutive changes in STAT6 gene expression.

HLX1 gene variants influence the development of childhood asthma.

BACKGROUND: Major transcription factors controlling T(H)1 and T(H)2 development, such as T-box transcription factor and GATA3, might be centrally involved in asthma and atopic diseases. Only recently, the homeobox transcription factor H.20-like homeobox 1 (HLX1), interacting closely with T-box transcription factor, has been identified as an important regulator of T(H)1 differentiation and suppressor of T(H)2 commitment. OBJECTIVE: We investigated whether genetic variations in the HLX1 gene exist and whether these could affect the development of childhood asthma. METHODS: The HLX1 gene was resequenced in 80 chromosomes. Associations between identified polymorphisms, asthma, and atopic diseases were investigated in German children (total n = 3099) from the cross-sectional International Study of Asthma and Allergy in Childhood phase II. Functional properties of polymorphisms were studied by using luciferase reporter gene assays and electrophoretic mobility shift assays in T cells. All statistical analyses were performed with SAS/Genetics software (SAS Institute, Inc, Cary, NC). RESULTS: Nineteen polymorphisms were identified in the HLX1 gene, and 2 tagging single nucleotide polymorphisms representing 7 polymorphisms were associated with childhood asthma in our study population. Two promoter polymorphisms, C-1407T and C-742G, contained in 1 tagging block were associated with asthma (odds ratio, 1.44; 95% CI, 1.11-1.86; P = .0061), significantly decrease promoter transactivation, and disrupt specificity protein-transcription factor binding in in vitro experiments. CONCLUSIONS: Our data suggest that polymorphisms in the HLX1 gene increase the risk for childhood asthma. On the cellular level, altered binding of specificity protein-transcription factors to the HLX1 promoter and subsequent changes in HLX1 gene expression might contribute to these effects.

TBX21 gene variants increase childhood asthma risk in combination with HLX1 variants.

BACKGROUND: The T cell-specific T-box transcription factor (TBX21) plays a crucial role in the regulation of the immune system because this factor induces the differentiation of T(H)1 and blocks T(H)2 commitment together with the homeobox transcription factor HLX1. OBJECTIVE: The role of genetic variants in TBX21 alone and in combination with HLX1 polymorphisms was investigated in the development of T(H)2-associated atopy and asthma. METHODS: The TBX21 gene was resequenced in 37 adult volunteers. Polymorphisms identified were genotyped in a cross-sectional (N = 3099) and nested asthma case-control population (N = 1872) using mainly matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Effects of promoter polymorphisms on TBX21 gene expression were studied by reporter gene assays. Furthermore, the impact of combinations of TBX21 and HLX1 polymorphisms on the development of asthma was assessed by using a risk score model. Statistical analyses were performed by using SAS/Genetics. RESULTS: Forty-three polymorphisms were identified in the TBX21 gene. Considering a minor allele frequency of at least 10%, single nucleotide polymorphisms were assigned to 7 linkage disequilibrium blocks. Three tagging single nucleotide polymorphisms increased childhood asthma risk significantly (odds ratio [OR], 2.60, 95% CI, 1.34-5.03, P = .003; OR, 1.39, 95% CI, 1.02-1.90, P = .039; and OR, 1.97, 95% CI, 1.18-3.30, P = .009). TBX21 promoter polymorphisms contained in 2 blocks significantly influenced TBX21 promoter activity. In a risk score model, the combination of TBX21 and HLX1 polymorphisms increased the asthma risk by more than 3-fold. CONCLUSIONS: These data suggest that TBX21 polymorphisms contribute to the development of asthma, potentially by altering TBX21 promoter activity. A risk score model indicates that TBX21 and HLX1 polymorphisms may have synergistic effects on asthma risk.

IRF-1 gene variations influence IgE regulation and atopy.

RATIONALE: The development of atopic diseases is characterized by skewed immune responses to common allergens. Only recently, interferons have been identified to play a crucial role in these mechanisms. OBJECTIVES: Because interferon regulatory factor (IRF)-1 is critical for interferon expression, we tested the hypotheses that genetic changes in this essential transcription factor may have consequences for the development of atopy. METHODS: The IRF-1 gene locus was resequenced in 80 human chromosomes. Association and haplotype analyses were performed in a cross-sectional study population of German children from Dresden (n = 1,940), and results were replicated in a second population sample from Munich (n = 1,159), both part of the ISAAC (International Study of Asthma and Allergy in Childhood) phase II. Promoter polymorphism effects were studied using electrophoretic mobility shift assay and colorimetric binding assays. Allele-specific IRF-1 gene expression was studied in vitro using luciferase reporter assays, whereas we assessed ex vivo expression of IRF-1 by real-time polymerase chain reaction and IFN-gamma protein by Luminex technology (Bio-Rad, Hercules, CA). Statistical analyses were performed using SAS/Genetics (SAS 9.1.3; SAS Institute, Cary, NC). MEASUREMENTS AND MAIN RESULTS: By resequencing, 49 polymorphisms were identified within the IRF-1 gene. Four blocks containing 11 polymorphisms were significantly associated with atopy, total IgE levels, or specific IgE levels in both populations (P < 0.05). Two polymorphisms changed transcription factor binding of nuclear factor (NF)-kappaB and EGR1 (early growth response 1) to the IRF-1 promoter, altered gene expression in vitro (P = 0.0004), and altered IRF-1 mRNA and IFN-gamma protein expression ex vivo. CONCLUSIONS: Our results suggest that functionally relevant IRF-1 polymorphisms influence atopy risk, potentially by altering transcription factor binding, IRF-1 gene expression, and IFN-gamma regulation.