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.

DUSP1 Maintains IRF1 and Leads to Increased Expression of IRF1-dependent Genes

“…Although the data described here are primarily from A549 cells, this is likely to be physiologically and therapeutically relevant, as in primary HBE cells there was also little repressive effect of dexamethasone on IL1B-induced IRF1 expression. Furthermore, DUSP1 is significantly induced in the human airways after inhaled corticosteroid administration (64). Likewise data from DUSP1 knock-out mouse models directly support these findings, and taken together this emphasizes the in vivo relevance of the current findings (12,46).…”

Glucocorticoid and cytokine crosstalk: Feedback, feedforward, and co-regulatory interactions determine repression or resistance

“…Thus, glucocorticoids reduce proinflammatory stimulus-induced expression of a protein that destabilizes those inflammatory mRNAs, TNF, CSF2 (GM-CSF), PTGS2, and others, that are in fact repressed by the glucocorticoid. Conversely, in both in vivo inhaled glucocorticoid and cell culture, glucocorticoids alone up-regulate ZFP36 expression (16,(72)(73)(74). This is consistent with GR recruitment to the ZFP36 gene (75).…”

“…GR recruitment may, in the same way as the feedback regulator genes, actively maintain expression. This requires careful testing, but the identification of multiple genes with apparent effects on inflammation, proliferation, and cell migration that are all modestly glucocorticoid-induced in vivo raises the prospect that the maintenance by direct GR binding is widespread (16).…”

“…Indeed, the ability of glucocorticoids to induce and/or enhance the expression of inflammatory response genes, including cytokines, chemokines, receptors, and signaling components, is widely reported (6, 7, 9 -11, 13-15). For example, in human volunteers, budesonide inhalation not only induced the expression of numerous anti-inflammatory genes, but also enhanced the expression of genes apparently involved in pro-inflammatory, pro-proliferative, and migratory responses (16). Thus, glucocorticoids are not simply anti-inflammatory, but exert a spectrum of effects that may include promoting pro-inflammatory pathways (12).…”

Cortisol limits selected actions of synthetic glucocorticoids in the airway epithelium

“…Synthetic GCs, including inhaled corticosteroids (ICSs), exert profound influences on the airway epithelium (15)(16)(17). Glucocorticoid anti-inflammatory effects are mediated by transactivation of selected genes, such as glucocorticoid inducible leucine zipper (GILZ) and MAP kinase phosphatase 1 (MKP-1) (18), and by inhibition of synthesis and the release of proinflammatory cytokines, including granulocyte macrophage colony-stimulating ABBREVIATIONS: 11b-HSD, 11-b-hydroxysteroid dehydrogenase; ALI, air-liquid interface; CBG, corticosteroid-binding globulin; CDKN1C, cyclin-dependent kinase inhibitor 1C; COPD, chronic obstructive pulmonary disease; Dex, dexamethasone; ENaCa, epithelial sodium-channel a subunit; FCS, fetal calf serum; FP, fluticasone propionate; GC, glucocorticoid; GILZ, glucocorticoid-induced leucine zipper; GM-CSF, granulocyte macrophage colony-stimulating factor; GR, glucocorticoid receptor; HBEC, human bronchial epithelial cell; HC, hydrocortisone; HPA, hypothalamic-pituitary-adrenal; ICS, inhaled corticosteroid; KCNB1, potassium voltage-gated channel subfamily B member 1; MKP-1, MAP kinase phosphatase 1; MR, mineralocorticoid receptor; PLZF, promyelocytic leukaemia zinc finger; qRT-PCR, quantitative RT-PCR; SCNN1A, sodium channel epithelial 1 a subunit factor (GM-CSF), IL-6, and IL-8 (19).…”