(421) Sonic Hedgehog Signaling Is Required for Basal Cell Specification of Pulmonary Ionocytes and Ciliated Cells in Human and Ferret Airways

Background: Recently scRNA-seq analysis has identified ionocytes and goblet cells as two major luminal cell types that express CFTR in the proximal airway. It is widely accepted that the CFTR channel conducts Cl^– and HCO₃^– to regulate hydration and pH at the airway surface, however, the contribution of ionocytes and goblet cells to these processes remains unclear. Sonic Hedgehog (SHH) signaling is an important pathway that regulate progenitor cell specification during lung development and is reactivated during the regeneration of the airway following injury. We discovered that the SHH pathway is required for basal cell specification of ionocytes and ciliated cells. In this study, we sought to better understand how perturbation of this pathway during human and ferret airway basal cell differentiation alters the cell type composition in polarized airway epithelial cultures and CFTR function.

Methods: Primary human bronchial epithelial cells (HBECs) were seeded onto transwell filters and lifted to an air-liquid interface (ALI) in the presence of SHH signaling agonist (SAG) or SHH inhibitor (HPI-1) and differentiated for 3 weeks. Primary ferret tracheal basal cells (FBECs) with CRISPR/Cas9-mediated genetic lesions in PTCH1 or SMO were differentiated in the ALI for 3 weeks. These cultures were then evaluated for the following endpoints: 1) CFTR-mediated ion transport (Cl^– and HCO₃^–), 2) the abundance of ciliated cells, goblet cells, and ionocytes using by localizing epithelial markers, and 3) qPCR for mRNA markers of specific cellular lineages, CFTR, and Sonic Hedgehog (SHH) signaling effectors.

Results: Ussing chamber assays revealed a striking reduction in CFTR-mediated transepithelial Cl^– and HCO₃^– ion transport in HBEC ALI cultures differentiated in the presence of SHH inhibitor (HPI1) and ALI cultures derived from FBECs disrupted for SMO gene. By contrast, HBEC ALI cultures differentiated in the presence of SHH agonist (SAG) and FBEC ALI cultures with PTCH1 gene knockout (KO) demonstrated an increase in CFTR-mediated transepithelial Cl^– and HCO₃^– ion transport. Despite these functional changes, the level of CFTR mRNA was largely unchanged. Cellular phenotyping by immunolocalization and/or qPCR using cellular markers demonstrated that HPI1-treated HBEC ALI cultures and ferret SMO KO epithelia were significantly enriched in goblet cells, while the abundance of ciliated cells and ionocytes was significantly reduced. By contrast, SAG-treated human ALI epithelia and PTCH1 KO ferret cultures exhibited an opposite effect, with enrichment of ciliated cells and ionocytes and reductions in goblet cells. qRT-PCR for effectors of SHH signaling pathway demonstrated that the genetic disruption of PTCH1 led to increase expression SHH signaling targets, GLI1 and GLI2, while disruption of SMO resulted in reduced expression of GLI1 and GLI2. Notably, genetic disruption of PTCH1 or SMO impaired the FBEC cell proliferation and clonogenicity.


Conclusions: These results demonstrate that SHH signaling is required for airway basal cell specification of ciliated cells and ionocytes, but repressed basal cell commitment toward goblet cells. These findings help to clarify the differentiation of ionocytes and their contributions in transepithelial anion transport in airway epithelia.

Acknowledgements:

References: