Transgenic ferret models define pulmonary ionocyte diversity and function.

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.

Development of an airway mucus defect in the cystic fibrosis rat.

The mechanisms underlying the development and natural progression of the airway mucus defect in cystic fibrosis (CF) remain largely unclear. New animal models of CF, coupled with imaging using micro-optical coherence tomography, can lead to insights regarding these questions. The Cftr-/- (KO) rat allows for longitudinal examination of the development and progression of airway mucus abnormalities. The KO rat exhibits decreased periciliary depth, hyperacidic pH, and increased mucus solid content percentage; however, the transport rates and viscoelastic properties of the mucus are unaffected until the KO rat ages. Airway submucosal gland hypertrophy develops in the KO rat by 6 months of age. Only then does it induce increased mucus viscosity, collapse of the periciliary layer, and delayed mucociliary transport; stimulation of gland secretion potentiates this evolution. These findings could be reversed by bicarbonate repletion but not pH correction without counterion donation. These studies demonstrate that abnormal surface epithelium in CF does not cause delayed mucus transport in the absence of functional gland secretions. Furthermore, abnormal bicarbonate transport represents a specific target for restoring mucus clearance, independent of effects on periciliary collapse. Thus, mature airway secretions are required to manifest the CF defect primed by airway dehydration and bicarbonate deficiency.

Particle-Tracking Microrheology Using Micro-Optical Coherence Tomography.

Clinical manifestations of cystic fibrosis (CF) result from an increase in the viscosity of the mucus secreted by epithelial cells that line the airways. Particle-tracking microrheology (PTM) is a widely accepted means of determining the viscoelastic properties of CF mucus, providing an improved understanding of this disease as well as an avenue to assess the efficacies of pharmacologic therapies aimed at decreasing mucus viscosity. Among its advantages, PTM allows the measurement of small volumes, which was recently utilized for an in situ study of CF mucus formed by airway cell cultures. Typically, particle tracks are obtained from fluorescence microscopy video images, although this limits one's ability to distinguish particles by depth in a heterogeneous environment. Here, by performing PTM with high-resolution micro-optical coherence tomography (µOCT), we were able to characterize the viscoelastic properties of mucus, which enables simultaneous measurement of rheology with mucociliary transport parameters that we previously determined using µOCT. We obtained an accurate characterization of dextran solutions and observed a statistically significant difference in the viscosities of mucus secreted by normal and CF human airway cell cultures. We further characterized the effects of noise and imaging parameters on the sensitivity of µOCT-PTM by performing theoretical and numerical analyses, which show that our system can accurately quantify viscosities over the range that is characteristic of CF mucus. As a sensitive rheometry technique that requires very small fluid quantities, µOCT-PTM could also be generally applied to interrogate the viscosity of biological media such as blood or the vitreous humor of the eye in situ.

Combination therapy with cystic fibrosis transmembrane conductance regulator modulators augment the airway functional microanatomy.

Recently approved therapies that modulate CFTR function have shown significant clinical benefit, but recent investigations regarding their molecular mechanism when used in combination have not been consistent with clinical results. We employed micro-optical coherence tomography as a novel means to assess the mechanism of action of CFTR modulators, focusing on the effects on mucociliary clearance. Primary human airway monolayers from patients with a G551D mutation responded to ivacaftor treatment with increased ion transport, airway surface liquid depth, ciliary beat frequency, and mucociliary transport rate, in addition to decreased effective viscosity of the mucus layer, a unique mechanism established by our findings. These endpoints are consistent with the benefit observed in G551D patients treated with ivacaftor, and identify a novel mechanism involving mucus viscosity. In monolayers derived from F508del patients, the situation is more complicated, compounded by disparate effects on CFTR expression and function. However, by combining ion transport measurements with functional imaging, we establish a crucial link between in vitro data and clinical benefit, a finding not explained by ion transport studies alone. We establish that F508del cells exhibit increased mucociliary transport and decreased mucus effective viscosity, but only when ivacaftor is added to the regimen. We further show that improvement in the functional microanatomy in vitro corresponds with lung function benefit observed in the clinical trials, whereas ion transport in vitro corresponds to changes in sweat chloride. Functional imaging reveals insights into clinical efficacy and CFTR biology that significantly impact our understanding of novel therapies.

An autoregulatory mechanism governing mucociliary transport is sensitive to mucus load.

Mucociliary clearance, characterized by mucus secretion and its conveyance by ciliary action, is a fundamental physiological process that plays an important role in host defense. Although it is known that ciliary activity changes with chemical and mechanical stimuli, the autoregulatory mechanisms that govern ciliary activity and mucus transport in response to normal and pathophysiological variations in mucus are not clear. We have developed a high-speed, 1-µm-resolution, cross-sectional imaging modality, termed micro-optical coherence tomography (µOCT), which provides the first integrated view of the functional microanatomy of the epithelial surface. We monitored invasion of the periciliary liquid (PCL) layer by mucus in fully differentiated human bronchial epithelial cultures and full thickness swine trachea using µOCT. We further monitored mucociliary transport (MCT) and intracellular calcium concentration simultaneously during invasion of the PCL layer by mucus using colocalized µOCT and confocal fluorescence microscopy in cell cultures. Ciliary beating and mucus transport are up-regulated via a calcium-dependent pathway when mucus causes a reduction in the PCL layer and cilia height. When the load exceeds a physiological limit of approximately 2 µm, this gravity-independent autoregulatory mechanism can no longer compensate, resulting in diminished ciliary motion and abrogation of stimulated MCT. A fundamental integrated mechanism with specific operating limits governs MCT in the lung and fails when periciliary layer compression and mucus viscosity exceeds normal physiologic limits.