TLR3- and Th2 cytokine-dependent production of thymic stromal lymphopoietin in human airway epithelial cells

Abstract

Thymic stromal lymphopoietin (TSLP) is elevated in asthma and triggers dendritic cell-mediated activation of Th2 inflammatory responses. Although TSLP has been shown to be produced mainly by airway epithelial cells, the regulation of epithelial TSLP expression has not been extensively studied. We investigated the expression of TSLP in cytokine- or TLR ligand-treated normal human bronchial epithelial cells (NHBE). The mRNA for TSLP was significantly up-regulated by stimulation with IL-4 (5.5-fold) and IL-13 (5.3-fold), weakly up-regulated by TNF-alpha, TGF-beta, and IFN-beta, and not affected by IFN-gamma in NHBE. TSLP mRNA was only significantly up-regulated by the TLR3 ligand (dsRNA) among the TLR ligands tested (66.8-fold). TSLP was also induced by in vitro infection with rhinovirus. TSLP protein was detected after stimulation with dsRNA (120 +/- 23 pg/ml). The combination of TNF-alpha and IL-4 produced detectable levels of TSLP protein (40 +/- 13 pg/ml). In addition, TSLP was synergistically enhanced by a combination of IL-4 and dsRNA (mRNA; 207-fold, protein; 325 +/- 75 pg/ml). The induction of TSLP by dsRNA was dependent upon NF-kappaB and IFN regulatory factor 3 (IRF-3) signaling via TLR3 as indicated by a study with small interfering RNA. The potent topical glucocorticoid fluticasone propionate significantly suppressed dsRNA-dependent TSLP production in NHBE. These results suggest that the expression of TSLP is induced in airway epithelial cells by stimulation with the TLR3 ligand and Th2 cytokines and that this response is suppressed by glucocorticoid treatment. This implies that respiratory viral infection and the recruitment of Th2 cytokine producing cells may amplify Th2 inflammation via the induction of TSLP in the asthmatic airway.

FIGURE 1

Effect of cytokines and TLR ligands on the up-regulation of TSLP in airway epithelial cells. A, NHBE were incubated for 6 h with 100 ng/ml TNF-α, 100 ng/ml IL-4, 100 ng/ml IL-13, 1000 U/ml IFN-β, 100 ng/ml IFN-γ, 100 ng/ml IFN-λ1, 100 ng/ml TGF-β, 1 µg/ml Pam₃CSK₄, 10 µg/ml PGN, 10 µg/ml LTA, 25 µg/ml dsRNA, 1 µg/ml LPS, 10 ng/ml flagellin, 1 µg/ml FSL-1, 10 µg/ml R-837, and 4 µg/ml CpG-C as indicated and then mRNA was extracted and analyzed for TSLP using real-time PCR. B, NHBE were incubated for 6 h with 2.5–25,000 ng/ml dsRNA and then the expression of mRNA for TSLP was analyzed by real-time PCR. C, NHBE were incubated with 25 µg/ml dsRNA (●) or vehicle control (■) for 1–48 h and then the expression of mRNA for TSLP was analyzed by real-time PCR. The copy number is expressed as the number of transcripts per nanogram of total RNA. D, Detection of TSLP protein by ELISA in the culture supernatant of NHBE stimulated with cytokines and dsRNA for 24 h. Results shown are mean ± SEM of 4–7 independent experiments. N.D., not detectable. *, p < 0.05.

FIGURE 2

Effect of combination of cytokines and dsRNA on the up-regulation of TSLP. NHBE were incubated for 6 h (A) or 24 h (D) with 100 ng/ml IL-4, 100 ng/ml IL-13, 100 ng/ml IFN-γ, 1000 U/ml IFN-β, and 25 µg/ml dsRNA in the presence or absence of 100 ng/ml TNF-α as indicated. NHBE were incubated for 6 h (B) or 24 h (E) with 100 ng/ml IL-4, 25 µg/ml dsRNA, or a combination of IL-4 and dsRNA. C and F, NHBE were incubated for 1–48 h with medium control (◻), 100 ng/ml IL-4 plus 100 ng/ml TNF-α(◼), 25 µg/ml dsRNA (○), and 100 ng/ml IL-4 plus 25 µg/ml dsRNA (●). The level of TSLP mRNA was determined by real-time PCR (A–C). Concentrations of TSLP protein in the culture supernatant were measured by ELISA (D–F). Results shown are mean ± SEM of 4–7 independent experiments. NS, Not significant; N.D., not detectable. *, p < 0.05.

FIGURE 3

Effect of rhinovirus infection on the up-regulation of TSLP in airway epithelial cells. NHBE were infected with RV16 at a MOI of 2 or 10 and then cultured for 6 h at 33°C in the presence or absence of 100 ng/ml IL-4. The level of TSLP mRNA was determined by real-time PCR. The results are shown as the mean ± SEM of four independent experiments. NS, not significant. * p < 0.05.

FIGURE 4

Effect of the dsRNA-related molecules on the expression of TSLP by dsRNA. NHBE were transfected with siRNA against control RNA, TLR3, MDA5, RIG-1, PKR, RELA, NFKB1, IRF-3, IFNAR2, or STAT6 at 5 nM for 48 h (A) and then stimulated with 25 µg/ml dsRNA (B) or 100 ng/ml IL-4 plus 25 µg/ml dsRNA (C) for 6 h. The levels of mRNAs were determined by real-time PCR. Efficiency of siRNA against target molecules was expressed as a percentage of non-siRNA transfected cells (A). The results are shown as the mean ± SEM of four independent experiments. *, p < 0.05; **, p < 0.005.

FIGURE 5

Effect of glucocorticoids on the up-regulation of TSLP by dsRNA and IL-4 in airway epithelial cells. NHBE were preincubated with 0.01% DMSO, 100 nM FP (A, C, and D), or 10⁻¹¹ to 10⁻⁶ M FP (B) for 2 h and then stimulated with 100 ng/ml IL-14, 100 ng/ml TNF-α, and 25 µg/ml dsRNA for 6 h (A and B), 24 h (C), or 24–72 h (D). The level of TSLP mRNA was determined by real-time PCR (A and B). Concentrations of TSLP protein in the culture supernatant were measured by ELISA (C and D). The results are shown as the mean ± SEM of 4–7 independent experiments. N.D., Not detectable. *, p < 0.05.