Ionocytes are a new type of rare airway epithelial cells that express approximately 50% of Cftr-transcripts in the mouse airways. They are characterized by the expression of Ascl3 and Foxi1 transcription factors, which have been used to identify this cell type in the airway epithelium and submucosal glands of both humans and mice. However, their function and precise localization remain largely unknown. In this study, we aimed to investigate the distribution of ionocytes in the mouse airway epithelium.
Mice were bred in the C57Bl6/J background and maintained in the Specific Pathogen Free mouse facility of Centro de Estudios Científicos (CECs) with access to food and water ad libitum. We used 6 and 8-week-old wild-type and CftrΔF508/ΔF508 animals. The trachea was splitted in two sections: the upper trachea containing the submucosal glands and the rest of the lower trachea, down the cricoid cartilage, sliced in 5µm paraffin sections in the transverse and frontal plane respectively. Ionocytes were identified by immunofluorescence against the FOXI1 transcription factor. All values were expressed as mean±S.E.M. All animal procedures were approved by the institutional IACUC (CECS-2022-03).
We found that FOXI1+cells had a triangular shape with a basolateral process. In the lower trachea the number of cells decreased towards the distal part (proximal= 2.4 ± 0.3 vs distal= 0.8 ± 0.2 FOXI1+cells mm-1 basal lamina, n=5, p=0.005, t-test), and were not found in the intrapulmonary airways. FOXI1+cells were more often observed in the epithelia around the collecting duct exit, in the collecting duct epithelium and in the serous acini of the submucosal glands. The total number of FOXI1+cells per millimeter of basal lamina was higher in the airway epithelium of upper trachea than in the lower trachea (6.5± 0.6 vs 4.0 ± 0.6 FOXI1+ cells mm-1 basal lamina, respectively; n=3, p=0.004, Rank Sum test). In general, FOXI1+cells were often present in the epithelia on top of the annular ligaments (61.7% ± 3.0; n=5; p=0.008, Rank Sum Test).
Preliminary analysis of CftrΔ F508/ΔF508 tissues indicated that there were no differences in the amount of FOXI1+cells when compared to wild-type lower tracheas (2.0 ± 0.7 vs 1.9 ± 0.1 FOXI1+cells mm-1 basal lamina, respectively, n=2 each group). Unexpectedly, we observed that FOXI1+cells were lower in issues of 6-week-old than in those obtained from 8-week-oldwild type mice (2.0 ± 0.5 (n=2) vs 4.0 ± 0.6 (n=5) FOXI1+cells mm-1 basal lamina, respectively, p= 0.078; Rank Sum Test).
In conclusion, our study provides new insights into the localization and distribution of ionocytes in the mouse airway epithelium. Our results indicate that ionocytes may play a role in regulating mucus composition in upper airways. We suggest that age-dependent changes in cell quantity might reflect the need of increased CFTR function in adult stages. Further research is needed to fully understand the function of ionocytes in the airway and their potential role in respiratory diseases such as cystic fibrosis.