Cell-intrinsic differences between human airway epithelial cells from children and adults

Summary The airway epithelium is a protective barrier that is maintained by the self-renewal and differentiation of basal stem cells. Increasing age is a principle risk factor for chronic lung diseases, but few studies have explored age-related molecular or functional changes in the airway epithelium. We retrieved epithelial biopsies from histologically normal tracheobronchial sites from pediatric and adult donors and compared their cellular composition and gene expression profile (in laser capture-microdissected whole epithelium, fluorescence-activated cell-sorted basal cells and basal cells in cell culture). Histologically, pediatric and adult tracheobronchial epithelium were similar in composition. We observed age-associated changes in RNA sequencing studies, including higher interferon-associated gene expression in pediatric epithelium. In cell culture, pediatric cells had higher colony-formation ability, sustained in vitro growth and out-competed adult cells in a direct competitive proliferation assay. Our results demonstrate cell-intrinsic differences between airway epithelial cells from children and adults in both homeostatic and proliferative states

Fluticasone Particles Bind to Motile Respiratory Cilia:A Mechanism for Enhanced Lung and Systemic Exposure?

Background: Inhaled corticosteroids (ICSs) are the main prophylactic treatment for asthma and are used in other diseases, including chronic pulmonary obstructive disease, yet the interaction of ICS particles with the ciliated epithelium remains unclear. The aim of this study was to investigate the earliest interaction of aerosolized fluticasone propionate (FP) particles with human ciliated respiratory epithelium. Methods: A bespoke system was developed to allow aerosolized FP particles to be delivered to ciliated epithelial cultures by nebulization and from a pressurized metered-dose inhaler (pMDI) through a spacer with interactions observed in real time using high-speed video microscopy. Interaction with nonrespiratory cilia was investigated using steroids on brain ependymal ciliary cultures. The dissolution rate of steroid particles was determined. Results: FP particles delivered by aerosol attached to the tips of rapidly beating cilia. Within 2 hours, 8.7% ± 1.8% (nebulization) and 12.1% ± 2.1% (pMDI through spacer) of ciliated cells had one or more particles attached to motile cilia. These levels decreased to 5.8% ± 1.6% (p = 0.59; nebulization) and 5.3% ± 2.2% (p = 0.14; pMDI through spacer) at 24 hours. Particle attachment did not affect ciliary beat frequency (p > 0.05) but significantly (p 2 particles bound p < 0.001). Dissolution of FP particles was slow with only 22.8% ± 1.3% of nebulized and 12.8% ± 0.5% of pMDI-delivered drug dissolving by 24 hours. Conclusions: FP particles adhere to the tips of rapidly moving cilia with significant numbers remaining bound at 24 hours, resisting the shear stress generated by ciliary beating. In vivo, this mechanism may predispose to high local drug concentrations and enhance respiratory and systemic corticosteroid exposure

Higher throughput drug screening for rare respiratory diseases: Readthrough therapy in primary ciliary dyskinesia.

Development of therapeutic approaches for rare respiratory diseases is hampered by the lack of systems that allow medium-to-high-throughput screening of fully differentiated respiratory epithelium from affected patients. This is a particular problem for primary ciliary dyskinesia (PCD), a rare genetic disease caused by mutations in genes that adversely affect ciliary movement and consequently mucociliary transport. Primary cell culture of basal epithelial cells from nasal brush biopsies, followed by ciliated differentiation at air-liquid interface (ALI) has proven to be a useful tool in PCD diagnostics but the technique's broader utility, including in pre-clinical PCD research, has been restricted by the limited number of basal cells that it is possible to expand from such biopsies. Here, we describe an immunofluorescence screening method, enabled by extensive expansion of PCD patient basal cells and their culture into differentiated respiratory epithelium in miniaturised 96-well transwell format ALI cultures. Analyses of ciliary ultrastructure, beat pattern and beat frequency indicate that a range of different PCD defects can be retained in these cultures. As proof-of-principle, we performed a personalised investigation in a patient with a rare and severe form of PCD (reduced generation of motile cilia, RGMC), in this case caused by a homozygous nonsense mutation in the MCIDAS gene. The screening system allowed drugs that induce translational readthrough to be evaluated alone or in combination with nonsense-mediated decay inhibitors. Restoration of basal body formation in the patient's nasal epithelial cells was seen in vitro, suggesting a novel avenue for drug evaluation and development in PCD