Inducible expression of the proallergic cytokine thymic stromal lymphopoietin in airway epithelial cells is controlled by NFkappaB

Abstract

The epithelial-derived cytokine thymic stromal lymphopoietin (TSLP) is important for the initiation of allergic airway inflammation through a dendritic cell-mediated T helper 2 response. To identify the factors that control TSLP expression, we examined the ability of inflammatory mediators to regulate TSLP production in human airway epithelial cells. We found that both IL-1beta and TNF-alpha were capable of inducing rapid TSLP production in primary human bronchial airway epithelial cells. We further characterized the human TSLP gene promoter, using two human epithelial cell lines, 16HBEo(-) and A549, and showed that IL-1beta- and TNF-alpha-mediated human TSLP promoter activation in these cells was mediated by an upstream NFkappaB site. Mutation of this NFkappaB site abolished activation, as did overexpression of a dominant-negative version of IkappaB kinase (IKK)beta (a kinase acting on IkappaB, the inhibitor of NFkappaB). Interestingly, human TSLP mRNA levels were also increased after exposure to Toll-like receptor (TLR) 2, TLR8, and TLR9 ligands, further supporting an important role for NFkappaB in TSLP gene regulation. Similarly, analysis of the mouse TSLP gene promoter revealed the presence of a similarly situated NFkappaB site that was also critical for IL-1beta-inducible expression of mouse TSLP. Taken together, these results demonstrate that the inflammatory mediators IL-1beta and TNF-alpha regulate human TSLP gene expression in an NFkappaB-dependent manner.

Fig. 1.

IL-1β and TNF-α induce human TSLP expression on NHBE cells. NHBE cells were cultured with IL-1β (1 ng/ml), TNF-α (10 ng/ml), or LPS (10 μg/ml) for the indicated time course. Total RNA was extracted, and human TSLP mRNA was measured by real-time quantitative PCR. TSLP mRNA level was normalized with GAPDH mRNA. Data are the mean ± SE of triplicate data points from a representative experiment. Results are representative of at least three independent experiments.

Fig. 2.

Transcriptional activation on 5′ deletion of the human promoter by IL-1β and TNF-α. Transfection of 16HBEo⁻ (a) and A549 (b) cells was performed with the mixture of the luciferase constructs containing various 5′ deletions of the human TSLP promoter and pRSV-β-Gal luciferase control vector. Schematic representations of promoter elements used are shown on the left. Nineteen hours after transfection, cells were incubated for 5 h in IL-1β (1 ng/ml) or TNF-α (10 ng/ml). At that time, cells were harvested, and lysates were prepared for determination of luciferase activity. Luciferase activity in the whole-cell lysate was normalized with β-galactosidase activity. Data are the mean ± SE of triplicate data points from a representative experiment of five independent experiments performed.

Fig. 3.

Identification of an upstream cis-positive element involved in the activation of human TSLP transcription by IL-1β stimulation. The 16HBEo⁻ cells were transfected with the mixture of the luciferase constructs containing additional 5′ deletion constructs (a) in −4.0 to −3.3 kb of the human TSLP promoter or various mutation constructs (c) and pRSV-β-Gal luciferase control vector. The experiments were carried out as described in the legend to Fig. 2. Schematic representations of human TSLP promoter reporter constructs are shown on the left. (b) Nucleotide sequence from −3.86 to −3.74 kb relative to the start site of human TSLP. The position of the NFκB, IRF-1, Opaque-2, and AP-1 binding site are indicated. All results are representative of three independent experiments.

Fig. 4.

NFκB binds to the NFκB motif of the human TSLP promoter. Nuclear extracts prepared from A549 cells stimulated with IL-1β were incubated with labeled oligonucleotide probes and subjected to EMSA. Arrows indicate specific binding activity. For supershift assay (a), nuclear extract was preincubated with 1 μg of normal rabbit IgG, rabbit anti-NFκB p65, or rabbit anti-NFκB p50 for 1 h before the addition of labeled oligonucleotide probe. For competition assay (b), the reaction was preincubated with 12.5- to 50-fold molar excess of unlabeled oligonucleotide for 5 min before the addition of labeled oligonucleotide probe.

Fig. 5.

IL-1β regulates NFκB activation and transcription from the human TSLP promoter via the IKK pathway. The A549 cells were transfected with the mixture of the luciferase constructs containing human TSLP promoter, NFκB expression plasmid (a) or dominant-negative mutant of IKKβ (b) and pRSV-β-Gal luciferase control vector. The experiments carried out as described in the legend to Fig. 2. The A549 cells were stimulated with IL-1β (1 ng/ml) for 5 h. All results are representative of three independent experiments.

Fig. 6.

TLR2, TLR8, and TLR9 induce human TSLP expression on NHBE cells. NHBE cells were incubated with the TLR2 agonist lipoteichoic acid (10 μg/ml), the TLR3 agonist poly(I·C) (5 μg/ml), the TLR4 agonist LPS (10 μg/ml), the TLR8 agonist poly(CpG) (5 μg/ml), and the TLR9 agonist CpG-B (5 μg/ml) for 2 or 20 h. Total RNA was extracted, and human TSLP mRNA was measured by real-time quantitative PCR. TSLP mRNA level was normalized with GAPDH. Data are the mean ± SE of triplicate data points from a representative experiment and represent cells treated with agonist for indicated TLR. Results are representative of three independent experiments.

Fig. 7.

Identification of an upstream cis-positive element involved in the activation of mouse TSLP transcription by IL-1β stimulation. The MLE12 cells were transfected with the mixture of the luciferase constructs containing −5.0, −4.0, or −3.0 kb of the mouse TSLP promoter (a), 5′ deletion constructs in −4.0 to −3.0 kb of the mouse TSLP promoter (b), NFκB mutation constructs (c), or retinoid X receptor (RXR) deletion constructs (d); in every case, pRSV-β-Gal luciferase control vector was added. The experiments were carried out as described in Fig. 2 legend. Schematic representations of mouse TSLP promoter reporter constructs are shown on the left. All results are representative of three independent experiments.