Transgenic ferret models define pulmonary ionocyte diversity and function

Abstract

Speciation leads to adaptive changes in organ cellular physiology and creates challenges for studying rare cell-type functions that diverge between humans and mice. Rare cystic fibrosis transmembrane conductance regulator (CFTR)-rich pulmonary ionocytes exist throughout the cartilaginous airways of humans1,2, but limited presence and divergent biology in the proximal trachea of mice has prevented the use of traditional transgenic models to elucidate ionocyte functions in the airway. Here we describe the creation and use of conditional genetic ferret models to dissect pulmonary ionocyte biology and function by enabling ionocyte lineage tracing (FOXI1-CreERT2::ROSA-TG), ionocyte ablation (FOXI1-KO) and ionocyte-specific deletion of CFTR (FOXI1-CreERT2::CFTRL/L). By comparing these models with cystic fibrosis ferrets3,4, we demonstrate that ionocytes control airway surface liquid absorption, secretion, pH and mucus viscosity—leading to reduced airway surface liquid volume and impaired mucociliary clearance in cystic fibrosis, FOXI1-KO and FOXI1-CreERT2::CFTRL/L ferrets. These processes are regulated by CFTR-dependent ionocyte transport of Cl− and HCO3−. Single-cell transcriptomics and in vivo lineage tracing revealed three subtypes of pulmonary ionocytes and a FOXI1-lineage common rare cell progenitor for ionocytes, tuft cells and neuroendocrine cells during airway development. Thus, rare pulmonary ionocytes perform critical CFTR-dependent functions in the proximal airway that are hallmark features of cystic fibrosis airway disease. These studies provide a road map for using conditional genetics in the first non-rodent mammal to address gene function, cell biology and disease processes that have greater evolutionary conservation between humans and ferrets.

Cell type-specific regulation of CFTR trafficking—on the verge of progress

“…Using conditional genetics allowed the identification of three subtypes of pulmonary ionocytes and the confirmation of their CFTR-dependent function. In fact, this work highlights the role of ionocytes in supporting a movement of Cl − and HCO 3 − that was found to be cell-autonomous, thus providing unique layers in the regulation of ASL volume, PH and viscosity, thus contributing to effective mucociliary clearance ( Yuan et al, 2023 ).…”

CFTR dysfunction leads to defective bacterial eradication on cystic fibrosis airways

“…Lei et al ( Lei et al, 2023 ) demonstrated that apical membrane CFTR Cl − channels collaborate with basolateral membrane barttin/ClC-K Cl − channels in ionocytes for transepithelial Cl − absorption, leading to fluid absorption. Moreover, FOXI1-knockout and CF ferrets both display reduced ASL volume and impaired mucociliary clearance due to ASL abnormalities, including slow fluid absorption but also absent fluid secretion, lack of CFTR-mediated ASL alkalization and increased mucus viscosity ( Yuan et al, 2023 ). Therefore, albeit less than about 1% of total epithelial cells in airways ( Montoro et al, 2018 ; Plasschaert et al, 2018 ; Lei et al, 2023 ), ionocytes regulate ASL homeostasis.…”

Durable transgene expression and efficient re-administration after rAAV2.5T-mediated fCFTRΔR gene delivery to adult ferret lungs

“… 16 , 17 The CF ferret model manifests lung disease that resembles the pathologies found in people with CF. 18 , 19 , 20 , 21 , 22 Given the availability of CF ferrets for preclinical studies, a better understanding of rAAV2.5T immunology and durability of transgene expression in ferret lungs is needed prior to embarking on studies in diseased CF animals.…”