(391) Lef-1 Induces FoxI1-Dependent Ionocyte Specification From Submucosal Gland Progenitors

Background: The cystic fibrosis transmembrane conductance regulator (CFTR) is highly enriched in pulmonary ionocytes of the proximal airways. Pulmonary ionocytes are present at low frequencies (~0.5-1%) in the tracheal epithelium of mice and throughout the cartilaginous airways of humans. Previously published studies suggested that ablation of ionocytes in the mouse tracheal epithelium causes loss of expression of CFTR and disruption of airway fluid and mucus balance in polarized airway cultures. Thus, expanding pulmonary ionocyte populations in respiratory airways might improve surface airway fluid and mucus physiology and thus mucociliary clearance for patients with COPD and CF patients on CFTR modulators. To this end, we sought to characterize the regulatory pathways that promote specification of pulmonary ionocytes in the proximal airways. Ionocytes are significantly enriched in airway submucosal gland (SMG) ducts and the surrounding surface airway epithelium. On the airway surface, basal stem cells can give rise to ionocytes. However, little is known about the origins of glandular ionocytes and the progenitor cell population that maintains ionocytes within glands. Myoepithelial cells in mouse airway SMGs (GMECs) have been identified as reserve stem cells that can give rise to at least seven different cell types populating SMGs and surface airway epithelium (SAE): GMECs, serous cells, mucous cells, ductal cells, basal cells, ciliated cells, secretory cells. The Wnt signaling transcription factor, lymphoid enhancer-binding factor 1 (Lef-1), plays an important role in regulating GMEC lineage commitment and self-renewal following airway injury. Notably, scRNA-seq of human airway epithelia demonstrates that LEF-1 is enriched in pulmonary ionocytes. We hypothesized that Lef-1 might play an important role in ionocyte specification from GMECs.

Methods: Lef-1 overexpression (OE) was induced in GMECs of α-SMA^CreERT2::Rosa26 ^LoxPStop^LoxPhLef-1 mice by tamoxifen injection. Guide RNAs targeting LoxP and the first exon of FoxI1 were transfected into GMECs of H11^Cas9::Rosa26^LoxPStop^LoxPhLEF-1 mice to induce Lef-1 OE and knockout of FoxI1.

Results: Lef-1 OE in mouse GMECs induced ionocyte differentiation in vivo and in air-liquid interface (ALI) cultures as assessed by Bsnd and FoxI1 expression. Additionally, ionocyte abundance correlated with Cftr expression and cAMP-inducible chloride transport, both being enhanced in GMEC-derived ALI cultures OE Lef-1. Lef-1 OE in GMECs led to the formation of large, vesicular, extensively branching, and anastomosing organoids that exhibit enhanced swelling upon cAMP-stimulation of CFTR, while GMECs lacking Lef-1 OE were compact and did not swell. Results of mRNA analysis of these cultures demonstrated that Lef-1 OE in GMECs cultures drives specification toward ionocyte markers. Moreover CRISPR/Cas9-mediated knockout of FoxI1 in Lef-1 overexpressing GMECs abolished the enhanced chloride conductance and prevented ionocyte specification.

Conclusions: These studies demonstrate that GMECs are resident stem cells within SMGs capable of specifying pulmonary ionocytes. The finding that ionocytes are specified from GMECs in a Lef-1-dependent manner provides a therapeutic target for enhancing ionocyte specification which may be useful in stem cell therapies for CF.

Acknowledgements: This work was supported by Cystic Fibrosis Foundation [grant number 001478XX220]

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