Abstract Background Intraluminal obstruction creates hypoxia, which promotes mucus dehydration by activating hypoxia-inducible factors (HIF1α, EPAS1), which upregulate transepithelial Na+/fluid absorption (e.g., γENaC). However, in muco-obstructive diseases, hypoxia co-exists with bacterially derived intense inflammation. It remains unknown how hypoxia and bacterially driven inflammatory pathways interact in airway epithelia to produce disease. We investigated the interconnections between chronic hypoxia and inflammatory stimuli in human airway epithelial (HAE) cultures and the relative roles of HIF1α and EPAS1 in muco-inflammatory disease pathogenesis. Materials and Methods HAE cultures were exposed to normoxia or hypoxia (1% O2) and/or supernatants from cystic fibrosis lung-derived mucopurulent material (SMM). Bulk RNA-sequencing, histologic, and ion transport studies were performed. HIF1α and EPAS1-mediated transcriptional responses were characterized using CRISPR knockdown HAE cells. Results Hypoxia and SMM regulated largely distinct transcriptional programs, indicating they act additively in disease pathogenesis. A small number were coordinately regulated by both stimuli, e.g., CXCL8 and PTGS2. Importantly, SMM administration did not attenuate the hypoxia-induced upregulation of SCNN1G. RNA in situ hybridization studies detected in lungs excised from bronchiectasis subjects the expression of genes selectively induced in vitro by SMM (SLC26A4) or hypoxia (IGFBP3). CRISPR-Cas9 studies revealed HIF1α and EPAS1 mediated distinct transcriptional responses to hypoxia: HIF1α dominated regulation of glycolytic pathways and IL1α-mediated inflammation pathways, whereas EPAS1 dominated regulation of SCNN1G and MMP7. Functionally, hypoxia-induced Na+ transport was effectively blocked by a selective EPAS1 inhibitor. Conclusion Chronic hypoxia and inflammation produce distinct RNA regulatory responses in human airway epithelia that add to the pathogenesis of muco-obstructive disease. Similarly, HIF1α and EPAS1 regulate largely distinct RNA responses to hypoxia. The hypoxia-EPAS1-SCNN1G axis is a robust driver of Na+ hyperabsorption, and its transcriptional activation is not suppressed by inflammatory stimuli. EPAS1 inhibition may be a therapeutic strategy to target mucus dehydration in muco-obstructive diseases. This abstract is funded by: CFF, JSPS
Mikami et al. (Fri,) studied this question.