Corticosteroid-induced gene expression in allergen-challenged asthmatic subjects taking inhaled budesonide

Abstract

Background and purpose: Inhaled corticosteroids (ICS) are the cornerstone of asthma pharmacotherapy and, acting via the glucocorticoid receptor (GR), reduce inflammatory gene expression. While this is often attributed to a direct inhibitory effect of the GR on inflammatory gene transcription, corticosteroids also induce the expression of anti-inflammatory genes in vitro. As there are no data to support this effect in asthmatic subjects taking ICS, we have assessed whether ICS induce anti-inflammatory gene expression in subjects with atopic asthma.

Experimental approach: Bronchial biopsies from allergen-challenged atopic asthmatic subjects taking inhaled budesonide or placebo were subjected to gene expression analysis using real-time reverse transcriptase-PCR for the corticosteroid-inducible genes (official gene symbols with aliases in parentheses): TSC22D3 [glucocorticoid-induced leucine zipper (GILZ)], dual-specificity phosphatase-1 (MAPK phosphatase-1), both anti-inflammatory effectors, and FKBP5 [FK506-binding protein 51 (FKBP51)], a regulator of GR function. Cultured pulmonary epithelial and smooth muscle cells were also treated with corticosteroids before gene expression analysis.

Key results: Compared with placebo, GILZ and FKBP51 mRNA expression was significantly elevated in budesonide-treated subjects. Budesonide also increased GILZ expression in human epithelial and smooth muscle cells in culture. Immunostaining of bronchial biopsies revealed GILZ expression in the airways epithelium and smooth muscle of asthmatic subjects.

Conclusions and implications: Expression of the corticosteroid-induced genes, GILZ and FKBP51, is up-regulated in the airways of allergen-challenged asthmatic subjects taking inhaled budesonide. Consequently, the biological effects of corticosteroid-induced genes should be considered when assessing the actions of ICS. Treatment modalities that increase or decrease GR-dependent transcription may correspondingly affect corticosteroid efficacy.

Figure 1

mRNA expression of corticosteroid-inducible genes in bronchial biopsies from mild atopic asthmatics taking inhaled budesonide. Mild asthmatics were entered into a previously described cross-over study (Kelly et al., 2010). Patients were subjected to an initial 3 week washout period before an inhaled challenge with diluent alone (saline). Following this, patients were subjected to a further 3 week washout period before treatment with placebo (Plac) or drugs for 11 consecutive days in a randomized cross-over design. Budesonide (Bud) was inhaled using a Pulmicort Turbuhaler (budesonide, 200 µg) with two inhalations taken twice daily and placebo was administered in a similar manner. On day 9, patients were subjected to a mild allergen inhalation (All). Bronchoscopy was performed on day 10 (24 h after the allergen challenge). Biopsies were analysed for mRNA expression of GAPDH, GILZ and FKBP51 using real-time RT-PCR. Data obtained from matched samples (n = 7 individuals) are plotted as a ratio of the gene of interest/GAPDH in arbitrary units as means ± SEM. Significance was tested by non-parametric anova (Friedman) with a Dunn's post-test. *P < 0.05.

Figure 2

GILZ expression is induced by corticosteroids in A549 pulmonary cells and primary human bronchial epithelial cells. (A) A549 cells were treated with combinations of IL-1β (1 ng·mL⁻¹) and dexamethasone (1 µM) (Dex). Cells were harvested at the times indicated for real-time RT-PCR analysis of GILZ and GAPDH mRNA. Data (n = 7), as a ratio of GILZ/GAPDH, are expressed as fold relative to non-stimulated (NS) at 1 h and are plotted as means ± SEM. Significance, relative to NS and between groups, was tested at each time by paired non-parametric anova (Friedman) with a Dunn's post-test. (B) Cells treated as in (A) were harvested for total protein and Western blotting was performed for GILZ and GAPDH. Images are representative of seven such blots (See Supporting Information Figure S2A for densitometric analysis). (C) Primary human bronchial epithelial cells (HBE) cells were treated with dexamethasone (1 µM) (Dex) for the indicated times. RNA was extracted for real-time RT-PCR analysis of GILZ and GAPDH. Data (n = 4) are plotted as in (A). Significance, relative to NS at each time, was tested by non-parametric t-test (Mann–Whitney U). (D) A549 cells were treated with various concentrations of dexamethasone in the absence and presence of IL-1β (1 ng·mL⁻¹). After 6 h, RNA was extracted for real-time RT-PCR analysis of GILZ and GAPDH. Data (n = 6), as a ratio of GILZ/GAPDH, are expressed as fold relative to NS and are plotted as means ± SEM. EC₅₀ values of 1.2 × 10⁻⁸ M were obtained in both cases. (E) A549 cells were treated with various concentrations of budesonide or dexamethasone (1 µM) (Dex) and cells were harvested after 6 h. RNA was extracted for analysis of GILZ and GAPDH mRNA expression using real-time RT-PCR. Data (n = 4), as a ratio of GILZ/GAPDH, are expressed as fold relative to NS and are plotted as means ± SEM. In (D) and (E), significance was tested relative to the no corticosteroid control by paired non-parametric anova (Friedman) with a Dunn's post-test. (F) Cells treated as in (E), were subjected to Western blot analysis of GILZ and GAPDH. Blots shown are representative of seven such experiments (See Supporting Information Figure S2B for densitometric analysis). Significance is indicated where: *P < 0.05, **P < 0.01.

Figure 3

GILZ expression is induced by corticosteroid in primary human airway smooth muscle (ASM) cells. (A) ASM cells were treated with dexamethasone (1 µM) (Dex). Cells were harvested at the times indicated for real-time RT-PCR analysis of GILZ and GAPDH. Data (n = 6–7), as a ratio of GILZ/GAPDH, are expressed as fold of non-stimulated (NS) cells at 1 h and are plotted as means ± SEM. Significance, relative to untreated at each time point, was performed by non-parametric t-test (Mann–Whitney U). (B) ASM cells were treated with budesonide (0.1 µM) (Bud) and harvested at the times indicated for Western blot analysis of GILZ and GAPDH. Blots shown are representative of six such experiments (See Supporting Information Figure S3A for densitometric analysis). (C) ASM cells were either NS or treated with various concentrations of budesonide. Cells were harvested after 6 h for real-time RT-PCR analysis of GILZ and GAPDH. Data (n = 4), as a ratio of GILZ/GAPDH, are expressed as fold relative to NS cells and are plotted as means ± SEM. Significance was tested by paired non-parametric anova (Friedman's) with a Dunn's post-test. (D) ASM cells from experiments as in (C), were harvested for Western blot analysis of GILZ and GAPDH. Images are representative of 10 such blots (See Supporting Information Figure S3B for densitometric analysis). Significance is indicated where: *P < 0.05, **P < 0.01 or ***P < 0.001.

Figure 4

GILZ protein is expressed in the airways of asthmatic subjects. Biopsy samples were subjected to immunohistochemical detection of GILZ protein using a validated antibody (See Supporting Information Figures S3 and S4). Samples depicted are: (A) post-allergen challenge after treatment with placebo; (B) post-allergen challenge after treatment with budesonide; and (C) post-allergen challenge after treatment with budesonide. Magnification: (A) 400× (B, C) 600×. (D) Comparison of staining for GILZ protein in bronchial epithelium from eight subjects obtained, post-allergen challenge (All) after treatment with placebo (Plac) or budesonide (Bud). GILZ immunoreactivity staining was scored according to five categories where: 0 = no staining; 1, <25%; 2, 25–50%; 3, 50–75% and 4, >75% staining. Individual immunohistochemical (IHC) scores are plotted and medians are indicated as horizontal bars. Significance was tested by Wilcoxon signed rank test. *P < 0.05. Note: where symbols and lines would have overlapped, data have been adjusted solely to facilitate visualization. EP = epithelium, SM = smooth muscle.