Muco-obstructive lung disease exhibits distinct hypoxic and inflammatory airway epithelial gene pathways

Background Intraluminal obstruction produces chronic airway epithelial hypoxia. We investigated how chronic airway epithelial hypoxia and inflammatory stimuli interact in human bronchial epithelial (HBE) cultures and resected human bronchiectatic lungs to produce an airway epithelial environment characteristic of muco-obstructive disease. Materials and Methods HBE cultures were exposed to normoxia or hypoxia (1% O 2 ) for 5 days, followed by administration of supernatants from cystic fibrosis (CF) lung-derived mucopurulent material (SMM) or PBS for 24 h. A reverse order protocol with SMM followed by hypoxia was also performed. Bulk RNA-sequencing and functional (Na + transport) studies were performed. HIF1A and EPAS1-mediated transcriptional responses were characterised in CRISPR knockdown HBE cells. RNA in situ hybridisation studies of excised bronchiectatic lungs and bioinformatic analyses of published datasets tested for relevance of in vitro findings to in vivo conditions. Results Hypoxia and SMM regulated largely separate airway epithelial transcriptional programmes regardless of treatment order. PTGS2 (COX-2), CCL20 , MUC5B , and CXCL8 (IL-8) represented a small number of genes upregulated by both hypoxia and SMM. HIF1A knockdown and EPAS1 knockdown produced distinct, often opposing, effects on hypoxia-responsive pathways. HIF1A dominated upregulation of glycolytic pathway signalling. EPAS1 regulated genes that increase mucus concentration under hypoxia ( e.g. , SCNN1G ). An EPAS1 inhibitor blocked hypoxia-induced upregulation of Na + transport. Conclusion Chronic hypoxia and inflammation regulated distinct genes/pathways that combine to produce a complex diseased environment in muco-obstructed airway epithelia. HIF1A and EPAS1 regulated distinct hypoxia-induced transcriptional responses. EPAS1 inhibition may exhibit a favourable therapeutic ratio for treatment of muco-obstructive lung diseases.