Asthma is a chronic airway inflammatory disease associated with structural and functional barrier alterations of the bronchial epithelium (BE). Synthetic glucocorticoids (GCs) are the mainstay treatment for asthma. The BE is a key orchestrator of lung inflammatory and immune responses. How the BE contributes to therapeutic outcomes of GCs and steroid resistance remains unclear.
GC genomic actions are mediated by the ligand-bound glucocorticoid receptor (GR). Each cell type expresses a distinctive pattern of GR isoforms that control partially overlapping sets of GC-sensitive genes. The enhancer landscape encountered by GRs is also an important player in shaping GC responses.
The genome-wide effects of GCs in human BE have predominantly been investigated using cellular monolayers and never under the clinically relevant scenario of a pre-established inflammatory milieu. For the first time this thesis examines the transcription-dependent actions of GCs (Dexamethasone) in both resting and inflamed cultures of patient-derived normal bronchial epithelial cells (BECs) differentiated at air-liquid interface (ALI). RNA sequencing of GC-only treated ALI cultures revealed a surprisingly small number of early steroid-regulated genes exhibiting overall modest expression changes. Twenty-eight of the genes (67%) were known GC targets in submerged lung epithelial cells. Bioinformatics analysis of Dexamethasone-responsive genes corroborated functional roles of GC drugs documented in monolayer models and bronchial biopsies. IL-1β pre-exposed ALI cultures showed more GC-evoked transcriptome alterations, with many instances of synergistic gene regulation by the pro- and anti-inflammatory stimuli. Attempts, for the first time in ALI-grown BECs, at profiling of GR occupancy via chromatin immunoprecipitation sequencing proved to be technically challenging.
Considering the findings from RNA sequencing of a model that more closely replicates the in vivo inflamed airway epithelium, further attempts to overcome the challenges and profile GR occupancy are warranted to obtain full elucidation of the molecular mechanisms behind GC genomic effects.