An inhaled dose of budesonide induces genes involved in transcription and signaling in the human airways: enhancement of anti- and proinflammatory effector genes

Abstract

Although inhaled glucocorticoids, or corticosteroids (ICS), are generally effective in asthma, understanding their anti-inflammatory actions in vivo remains incomplete. To characterize glucocorticoid-induced modulation of gene expression in the human airways, we performed a randomized placebo-controlled crossover study in healthy male volunteers. Six hours after placebo or budesonide inhalation, whole blood, bronchial brushings, and endobronchial biopsies were collected. Microarray analysis of biopsy RNA, using stringent (≥2-fold, 5% false discovery rate) or less stringent (≥1.25-fold, P ≤0.05) criteria, identified 46 and 588 budesonide-induced genes, respectively. Approximately two third of these genes are transcriptional regulators (KLF9, PER1, TSC22D3, ZBTB16), receptors (CD163, CNR1, CXCR4, LIFR, TLR2), or signaling genes (DUSP1, NFKBIA, RGS1, RGS2, ZFP36). Listed genes were qPCR verified. Expression of anti-inflammatory and other potentially beneficial genes is therefore confirmed and consistent with gene ontology (GO) terms for negative regulation of transcription and gene expression. However, GO terms for transcription, signaling, metabolism, proliferation, inflammatory responses, and cell movement were also associated with the budesonide-induced genes. The most enriched functional cluster indicates positive regulation of proliferation, locomotion, movement, and migration. Moreover, comparison with the budesonide-induced expression profile in primary human airway epithelial cells shows considerable cell type specificity. In conclusion, increased expression of multiple genes, including the transcriptional repressor, ZBTB16, that reduce inflammatory signaling and gene expression, occurs in the airways and blood and may contribute to the therapeutic efficacy of ICS. This provides a previously lacking insight into the in vivo effects of ICS and should promote strategies to improve glucocorticoid efficacy in inflammatory diseases.

Keywords: Anti‐inflammatory; asthma; corticosteroid; gene expression; transactivation.

Figures

Figure 1

Gene expression in bronchial biopsies following budesonide inhalation. RNA was prepared from biopsy samples taken 5–6 h after placebo (Plac) or budesonide (1600 μg) (Bud) inhalation. (A) RNA was subjected to expression profiling using Affymetrix PrimeView microarrays. Analysis (n = 11 individuals) was performed with Partek Genomics Suite (v6.6). Resultant fold expression and P values were used to generate a volcano plot. Probe sets with coloring (red = 109 increased probe sets, blue = 35 repressed probe sets) indicate fold ≥2 or ≤0.5 with P ≤ 0.05. (B) i, Budesonide‐induced probe sets meeting the expression criteria; ≥2‐fold, FDR 0.05, or ≥1.25‐fold, P ≤ 0.05, were analyzed by the DAVID gene ID conversion tool to give: 46 (Table 2) and 588 upregulated genes (Table S5), respectively. ii, Budesonide‐repressed probe sets (≤0.5‐fold, P ≤ 0.05) were analyzed by the DAVID gene ID conversion tool to give 28 repressed genes (Table 2). Using gene ontology (GO) and NCBI Gene information, genes were assigned to one of six groups: 1, Transcriptional control; 2, Membrane receptors (Receptors); 3, Signaling and regulation of signaling (Signaling); 4, Metabolic and metabolism (Metabolic); 5, Other function; or 6, Not assigned, where insufficient data were available to assign a particular function or activity and were plotted as pie charts. (C) Real‐time PCR was performed for the indicated genes. Data from the 12 individuals are expressed as the gene of interest/GAPDH and are plotted (arbitrary units) as means ±SE. Significance was assessed by Wilcoxon signed rank test. ** P ≤ 0.01, *** P ≤ 0.001.

Figure 2

Gene Ontology (GO) analysis of biopsy microarray data. (A) GO analysis of biological process: the 588 (≥1.25‐fold, P ≤ 0.05) budesonide‐induced gene list was analyzed by DAVID functional annotation to produce gene clusters (≥2 genes/cluster) corresponding to 647 GO annotation terms. GO terms corresponding to biological process (GOTERM_BP_FAT and KEGG_PATHWAY) were extracted. Those significantly associated (P ≤ 0.05) with the gene list are plotted with the numbers of genes (as a percentage of the list total) for each term along with the fold enrichment for each term. GO terms with less than 2% of the total genes are not plotted unless significantly enriched (Benjamini ≤0.05). Many terms were excluded as being redundant or having wide meaning (Table S7). (B) Functional cluster analysis, using DAVID, of the 588 (≥1.25‐fold, P ≤ 0.05) gene list produced 183 gene clusters. The top 20 clusters are listed (Table S8). The most highly enriched cluster, containing GO terms for cell proliferation and movement, is depicted. GO terms are represented by filled circles where size is proportional to the number of genes. Individual genes are color coded to indicate expression induced by budesonide. GO terms are linked to the respective genes by lines. qPCR was performed for four genes in the biopsy samples as shown in Figure 1C. (C) The 28 budesonide‐repressed genes (≤0.5‐fold, P ≤ 0.05) (Table 2) were functionally annotated using DAVID as in A. Abbreviations are as follows: +ve, positive; ‐ve, negative, reg, regulation; proc, process; metab, metabolism; transn, transcription. A number of GO terms are further abbreviated: +ve reg nucleic acid metabolic process = GO:0045935~positive regulation of nucleobase, nucleoside, nucleotide, and nucleic acid metabolic process; +ve reg nucleic acid metabolic process = GO:0045934~negative regulation of nucleobase, nucleoside, nucleotide, and nucleic acid metabolic process; transmembrane receptor PTK pathway GO:0007169 ~transmembrane receptor protein tyrosine kinase signaling pathway; enzyme‐linked receptor pathway, GO:0007167~enzyme‐linked receptor protein signaling pathway.

Figure 3

Expression of glucocorticoid‐induced genes in human cells. (A) Human bronchial epithelial (HBE) cells, airway smooth muscle (ASM) cells, lung fibroblasts, and endothelial cells were either not treated or treated with dexamethasone (1 μmol/L) (Dex) or budesonide (Bud) at 0.3 μmol/L (or 0.1 μmol/L for HBE cells). RNA was subjected to qPCR for the indicated genes and GAPDH. Where threshold cycle (CT) values were ≥34, then the gene was deemed not to be expressed. Data, n = 7–8 individuals (HBE), n = 8 individuals (ASM), n = 7 individuals (fibroblasts), n = 6 individuals (endothelial), expressed as the gene of interest/GAPDH, are plotted as log2‐fold relative to not treated as means ±SE. Significance was assessed by paired analysis of variance (ANOVA) (Dunn's Multiple Comparison Test). * P < 0.05, ** P < 0.01, *** P < 0.001. (B) Primary HBE cells from six individuals were not treated or treated with budesonide (0.1 μmol/L). RNA was prepared and microarray analysis performed using Affymetrix Primeview arrays. Analysis was performed with Partek Genomics Suite (v6.6) and resultant fold expression and P values were used to generate a volcano plot (Fig. S6). Budesonide‐induced and ‐repressed probe sets meeting the criteria; i, ≥2‐fold, P ≤ 0.05, or ii, ≤0.5‐fold, P ≤ 0.05, were analyzed by the DAVID gene ID conversion tool to give: i, 84 upregulated and ii, 114 downregulated genes, respectively (Table S10). Using gene ontology (GO) and NCBI gene information, genes were assigned to one of six groups: 1, Transcriptional control; 2, Membrane receptors (Receptors); 3, Signaling and regulation of signaling (Signaling); 4, Metabolic and metabolism (Metabolic); 5, Other function; or 6, Not assigned, where insufficient data were available to assign a particular function or activity, and were plotted as pie charts.

Figure 4

Comparison of budesonide‐modulated gene expression in bronchial biopsies with human bronchial epithelial cells in culture. (A) Fold change (budesonide/not treated) for all probe sets in human bronchial epithelial cells (HBE) treated with budesonide (Bud) (0.1 μmol/L) (data from Fig. 3B) was plotted against the fold change (budesonide/placebo) obtained with the bronchial biopsies (data from Fig. 1A). (B) Heat maps representing the data in panel A are shown for: i) genes (≥2‐fold or ≤0.5‐fold, P ≤ 0.05) ranked by fold (budesonide/placebo) in the biopsies; and for ii) genes (≥2‐fold or ≤0.5‐fold, P ≤ 0.05) by fold (budesonide/not treated) in the human bronchial epithelial cells. Genes with multiple probe sets are represented by the probe set with the greatest absolute value (modulus). Genes with probe sets showing ≥2‐fold and ≤0.5‐fold for each ranked set are shown. Repressed genes in panel ii) are listed in Table S10. (C) qPCR was performed for TLR2 in the biopsy samples as described in Figure 1C.

Figure 5

Elevated serum budesonide and budesonide‐induced gene expression in blood. (A) Blood was collected 5–6 h post placebo (Plac) or budesonide (Bud) (1600 μg) inhalation, immediately prior to bronchoscopy. Serum was prepared and samples analyzed by LC‐MS/MS to determine budesonide concentration. Data (n = 12 individuals) are plotted as mean ± SE. (B) RNA was prepared from whole blood taken 5–6 h post placebo or budesonide inhalation. Data, from all 12 individuals, are expressed as the gene of interest/GAPDH and are plotted as means ± SE. All genes investigated by qPCR in Figures 1, 2, 3 were analyzed, but only those showing significant changes are shown. Significance was assessed by Wilcoxon signed rank test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.