The airway epithelium contains ionocytes, a rare cell type with high expression of Forkhead Box I1 (FOXI1) transcription factor and Cystic Fibrosis Transmembrane conductance Regulator (CFTR), a chloride channel that is defective in cystic fibrosis (CF). Our aim was to verify if ionocyte development is altered in CF and to investigate the relationship between ionocytes and CFTR-dependent chloride secretion. We collected nasal cells by brushing to determine ionocyte abundance. Nasal and bronchial cells were also expanded in vitro and reprogrammed to differentiated epithelia for morphological and functional studies. We found a relatively high (~3%) ionocyte abundance in ex vivo nasal samples, with no difference between CF and control individuals. In bronchi, ionocytes instead appeared very rarely as previously reported, thus suggesting a possible proximal–distal gradient in human airways. The difference between nasal and bronchial epithelial cells was maintained in culture, which suggests an epigenetic control of ionocyte development. In the differentiation phase of the culture procedure, we used two media that resulted in a different pattern of CFTR expression: confined to ionocytes or more broadly expressed. CFTR function was similar in both conditions, thus indicating that chloride secretion equally occurs irrespective of CFTR expression pattern.

The fluid covering the surface of airway epithelia represents a first barrier against pathogens. The chemical and physical properties of the airway surface fluid are controlled by the activity of ion channels and transporters. In cystic fibrosis (CF), loss of CFTR chloride channel function causes airway surface dehydration, bacterial infection, and inflammation. We investigated the effects of IL-17A plus TNF-α, 2 cytokines with relevant roles in CF and other chronic lung diseases. Transcriptome analysis revealed a profound change with upregulation of several genes involved in ion transport, antibacterial defense, and neutrophil recruitment. At the functional level, bronchial epithelia treated in vitro with the cytokine combination showed upregulation of ENaC channel, ATP12A proton pump, ADRB2 β-adrenergic receptor, and SLC26A4 anion exchanger. The overall result of IL-17A/TNF-α treatment was hyperviscosity of the airway surface, as demonstrated by fluorescence recovery after photobleaching (FRAP) experiments. Importantly, stimulation with a β-adrenergic agonist switched airway surface to a low-viscosity state in non-CF but not in CF epithelia. Our study suggests that CF lung disease is sustained by a vicious cycle in which epithelia cannot exit from the hyperviscous state, thus perpetuating the proinflammatory airway surface condition.

Cystic fibrosis (CF) is a genetic disease caused by mutations that impair the function of the CFTR chloride channel. The most frequent mutation, F508del, causes misfolding and premature degradation of CFTR protein. This defect can be overcome with pharmacological agents named “correctors”. So far, at least three different classes of correctors have been identified based on the additive/synergistic effects that are obtained when compounds of different classes are combined together. The development of class 2 correctors has lagged behind that of compounds belonging to the other classes. It was shown that the efficacy of the prototypical class 2 corrector, the bithiazole corr-4a, could be improved by generating conformationally-locked bithiazoles. In the present study, we investigated the effect of tricyclic pyrrolothiazoles as analogues of constrained bithiazoles. Thirty-five compounds were tested using the functional assay based on the halide-sensitive yellow fluorescent protein (HS-YFP) that measured CFTR activity. One compound, having a six atom carbocyle central ring in the tricyclic pyrrolothiazole system and bearing a pivalamide group at the thiazole moiety and a 5-chloro-2-methoxyphenyl carboxamide at the pyrrole ring, significantly increased F508del-CFTR activity. This compound could lead to the synthesis of a novel class of CFTR correctors.