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. 2018 Oct 5;50(10):1-12.
doi: 10.1038/s12276-018-0158-2.

IL-17A-associated IKK-α signaling induced TSLP production in epithelial cells of COPD patients

Affiliations

IL-17A-associated IKK-α signaling induced TSLP production in epithelial cells of COPD patients

Giulia Anzalone et al. Exp Mol Med. .

Abstract

Thymic stromal lymphopoietin (TSLP) is a cytokine expressed in the epithelium, involved in the pathogenesis of chronic disease. IL-17A regulates airway inflammation, oxidative stress, and reduction of steroid sensitivity in chronic obstructive pulmonary disease (COPD). TSLP and IL-17A were measured in induced sputum supernatants (ISs) from healthy controls (HC), healthy smokers (HS), and COPD patients by enzyme-linked immunosorbent assay. Human bronchial epithelial cell line (16HBE) and normal bronchial epithelial cells were stimulated with rhIL-17A or ISs from COPD patients to evaluate TSLP protein and mRNA expression. The effects of the depletion of IL-17A in ISs, an anticholinergic drug, and the silencing of inhibitor kappa kinase alpha (IKKα) on TSLP production were evaluated in 16HBE cells. Coimmunoprecipitation of acetyl-histone H3(Lys14)/IKKα was evaluated in 16HBE cells treated with rhIL-17A and in the presence of the drug. TSLP and IL-17A levels were higher in ISs from COPD patients and HS compared with HC. TSLP protein and mRNA increased in 16HBE cells and in normal bronchial epithelial cells stimulated with ISs from COPD patients compared with ISs from HC and untreated cells. IKKα silencing reduced TSLP production in 16HBE cells stimulated with rhIL-17A and ISs from COPD patients. RhIL-17A increased the IKKα/acetyl-histone H3 immunoprecipitation in 16HBE cells. The anticholinergic drug affects TSLP protein and mRNA levels in bronchial epithelial cells treated with rhIL-17A or with ISs from COPD patients, and IKKα mediated acetyl-histone H3(Lys14). IL-17A/IKKα signaling induced the mechanism of chromatin remodeling associated with acetyl-histone H3(Lys14) and TSLP production in bronchial epithelial cells. Anticholinergic drugs might target TSLP derived from epithelial cells during the treatment of COPD.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. TSLP and IL-17A concentrations in ISs from HC (n = 10), HS (n = 10), and COPD patients (n = 12).
a TSLP levels (pg/mL) and b IL-17A levels (pg/mL) were measured using specific commercially available kits as described in the “Materials and methods” section. Two technical replicates were performed. The bars represent the mean ± SD of the values (pg/mL). Statistical analysis was performed using the Kruskal–Wallis test followed by Bonferroni–Dunn correction for multiple comparisons. A p value < 0.05 was considered statistically significant
Fig. 2
Fig. 2. TSLP production in 16HBE cells stimulated with ISs from HC (n = 6), HS (n = 6), and COPD patients (n = 6).
TSLP was detected in protein extracts by western blot analysis as described in the “Materials and methods” section. Bars represent the mean ± SD of arbitrary densitometric units (A.D.U.), normalized to β-actin, which was used as the loading control. Representative gel images of the experiments are shown. ANOVA with Fisher’s test correction was used to analyze data. A p value < 0.05 was considered statistically significant
Fig. 3
Fig. 3. Effects of anticholinergic drugs on the production of TSLP in 16HBE cells stimulated with rhIL-17A or with ISs from COPD patients.
16HBE cells, in the presence or absence of Tiotropium (100 nM), were stimulated with rhIL-17A (20 ng/ml) or with ISs from COPD patients, untreated or treated with anti-IL-17A antibody, as well as with DTT (vehicle of ISs) (n = 6 for each experimental condition). a and b show TSLP protein detected by western blot analysis as described in the “Materials and methods” section. Representative gel images of the experiments are shown. Bars represent the mean ± SD of arbitrary densitometric units (A.D.U.), normalized to β-actin, which was used as the loading control. c and d show TSLP mRNA levels measured by real-time PCR as described in the Materials and Methods section (n = 3) and expressed as fold-change compared with untreated cells, which were chosen as the reference samples. ANOVA with Fisher’s test correction was used to analyze data. A p value < 0.05 was considered statistically significant
Fig. 4
Fig. 4. Effect of anticholinergic drugs on TSLP production in NHBE cells stimulated with rhIL-17A or with ISs from COPD patients.
NHBE cells, in the presence or absence of Tiotropium (100 nM), were stimulated with rhIL-17A (20 ng/ml) (n = 3) or with ISs from COPD patients untreated or treated with anti-IL-17A antibody (n = 3) for each experimental condition). a and b show TSLP protein detected by western blot analysis as described in the “Materials and methods” section. Representative gel images of the experiments are shown. Bars represent the mean ± SD of arbitrary densitometric units (A.D.U.), normalized to β-actin used as the loading control. c and d show TSLP mRNA levels measured by real-time PCR as described in the “Materials and Methods” section and expressed as fold-change compared with untreated cells, chosen as the reference sample. ANOVA with Fisher’s test correction was used to analyze data. A p value < 0.05 was considered statistically significant
Fig. 5
Fig. 5. Effect of IKKα silencing on TSLP protein in 16HBE cells stimulated with rhIL-17A or ISs from COPD patients.
a TSLP protein in unsilenced, silenced, and scrambled untreated cells (n = 3); b TSLP protein in unsilenced and silenced cells stimulated with rhIL-17A (n = 3); c TSLP protein in unsilenced and silenced cells stimulated with ISs from COPD (n = 3). TSLP protein was detected by western blot analysis as described in the “Materials and methods” section. Bars represent the mean ± SD of arbitrary densitometric units (A.D.U.), normalized to β-actin, which was used as the loading control. Representative gel images of the experiments are shown. ANOVA with Fisher’s test correction and the Student t test were used to analyze data. A p value < 0.05 was considered statistically significant
Fig. 6
Fig. 6. IKKα binding to acetyl-His H3 (Lys14) and acting on the promoter region of TSLP in epithelial cells.
a Coimmunoprecipitation of IKKα and Ac-His H3 (Lys14) was performed in cell extracts from 16HBE cells stimulated for 4 h with rhIL-17A (20 ng/ml) in the presence or absence of tiotropium (n = 3). ChiP assay was performed using primers spanning the His H3 binding site of the human TSLP gene promoter: primer 1: 5′–3′; primer 2: 3′–5′; b the cells were stimulated with rhIL-17A (20 ng/ml), alone or in combination, for 4 h. Lane 1: negative control of PCR; lane 2: negative control of immunoprecipitation; lane 3: positive control of PCR; lane 4: untreated cells; lane 5: rhIL-17A (20 ng/ml); lane 6, rhIL-17A + tiotropium (n = 3). c the cells were stimulated with ISs alone or in combination for 4 h. Lane 1: negative control of PCR; lane 2: negative control of immunoprecipitation; lane 3: positive control of PCR; lane 4: untreated cells; lane 5: ISs from COPD patients; lane 6: ISs from COPD patients + tiotropium; line 7: ISs from COPD patients treated with anti-IL-17A Ab. Purified DNA was analyzed by PCR, using control primers specific for the GAPDH promoter. Representative gel images of the experiments are shown
Fig. 7
Fig. 7. Graphical abstract.
Summary of the study

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