Thymic stromal lymphopoietin gene promoter polymorphisms are associated with susceptibility to bronchial asthma

Abstract

Thymic stromal lymphopoietin (TSLP) triggers dendritic cell--mediated T helper (Th) 2 inflammatory responses. A single-nucleotide polymorphism (SNP), rs3806933, in the promoter region of the TSLP gene creates a binding site for the transcription factor activating protein (AP)-1. The variant enhances AP-1 binding to the regulatory element, and increases the promoter--reporter activity of TSLP in response to polyinosinic-polycytidylic acid (poly[I:C]) stimulation in normal human bronchial epithelium (NHBE). We investigated whether polymorphisms including the SNP rs3806933 could affect the susceptibility to and clinical phenotypes of bronchial asthma. We selected three representative (i.e., Tag) SNPs and conducted association studies of the TSLP gene, using two independent populations (639 patients with childhood atopic asthma and 838 control subjects, and 641 patients with adult asthma and 376 control subjects, respectively). We further examined the effects of corticosteroids and a long-acting β(2)-agonist (salmeterol) on the expression levels of the TSLP gene in response to poly(I:C) in NHBE. We found that the promoter polymorphisms rs3806933 and rs2289276 were significantly associated with disease susceptibility in both childhood atopic and adult asthma. The functional SNP rs3806933 was associated with asthma (meta-analysis, P = 0.000056; odds ratio, 1.29; 95% confidence interval, 1.14-1.47). A genotype of rs2289278 was correlated with pulmonary function. Moreover, the induction of TSLP mRNA and protein expression induced by poly(I:C) in NHBE was synergistically impaired by a corticosteroid and salmeterol. TSLP variants are significantly associated with bronchial asthma and pulmonary function. Thus, TSLP may serve as a therapeutic target molecule for combination therapy.

Figure 1.

Single-nucleotide polymorphisms (SNPs) and pairwise linkage disequilibrium (LD) map of the thymic stromal lymphoprotein (TSLP) gene. (A) Graphic overview of polymorphisms identified in relation to the exon/intron structure of the human TSLP gene. Three polymorphisms were genotyped in this study. The ATG (initiation codon), untranslated region (UTR), and the open reading frame (ORF) are indicated by solid triangles, open boxes, and solid boxes, respectively. (B) Pairwise D′/LOD (left) and r² (right) for all combinations of single-nucleotide polymorphism (SNP) pairs are shown.

Figure 2.

Relationship of TSLP rs3806933, rs2289276, and rs2289278 genotype with lung function in adult patients with asthma. The ratio of forced expiratory volume in 1 second (FEV₁) to forced vital capacity (FVC) is plotted, and horizontal bars represent the mean for each genotype group.

Figure 3.

(A) DNA sequences of transcription factor–binding motifs around rs2289276 SNP. The positions of potential AP-2α binding sites are shown in the open box. *SNPs. (B) Binding affinity of transcription factors to oligonucleotides (oligo) in vitro. Normal human bronchial epithelia (NHBEs) were stimulated with or without 10 μg/ml polyinosinic-polycytidylic acid (poly[I:C]) for 1 hour. Proteins interacting with the double-stranded oligonucleotides were precipitated and analyzed by immunoblotting with the indicated antibodies. Three independent experiments were performed with similar results. AP, activating protein. w.b., Western blotting.

Figure 4.

Suppression of TSLP production by dexamethasone (DEX) and salmeterol (SAL) in NHBE. (A) Quantitative RT-PCR assay of TSLP. P < 0.001, according to Student t test. (B) Effects of DEX and SAL on TSLP protein production in NHBE stimulated (Stim) by poly(I:C). Concentrations of TSLP in supernatants were measured by ELISA. *Not detectable.

Figure 5.

(A) Luciferase (Luc) constructs. Gray box indicates NF-κB regulatory region. (B) Effects of DEX and SAL on luciferase transcriptional activities of haplotypes of the long form of TSLP in NHBE. Data represent mean ± SD and are from three experiments in triplicate. *P < 0.0001 and ^†P = 0.0021, according to the Bonferroni-Dunn test with two-factor ANOVA.