(406) Pulmonary ionocytes play a role in pH regulation in human airway epithelia

Background: Recent single-cell RNA sequencing analysis of airway epithelia revealed a previously undescribed cell type, the pulmonary ionocyte. Pulmonary ionocytes express CFTR mRNA at a very high level, which may be several hundred times greater than other airway epithelial cell types. However, pulmonary ionocytes are rare, and account for only about ~1% of all epithelial cells [1, 2]. Besides CFTR-rich ionocytes, a population of secretory cells, including club cells, also express CFTR mRNA, albeit a lower levels. [3]. These findings highlight that the airway epithelium represents an elaborate “cellular ecosystem” that is more complex than previously thought. The functions of the various cell types and their potential roles in CF pathophysiology are not fully understood and there is still controversy about which cell types are responsible for the development of CF lung disease. Understanding the cellular basis of CF lung disease is crucial for identifying cellular targets for molecular therapies, such as gene therapies, targeted at the CFTR gene. We hypothesize that different cell types specialize in performing distinct tasks for maintaining the integrity of the airway surface liquid layer.

Methods: Using a high-resolution self-referencing ion-selective microelectrode technique [4], we measured single-cell ion transport (Na⁺, Cl^-, and H⁺) properties of ionocytes and club cells in healthy and CF primary Human Bronchial Epithelial (HBE) cell cultures.

Results: We showed that activating CFTR with forskolin + IBMX elicited different responses in ionocyte and club cells in wild-type HBE cell cultures. After forskolin + IBMX stimulation, H⁺ was transported across ionocytes from the apical (lumen) to the basolateral side, making the lumen pH basic. This H⁺ transport can be reduced by 50% with the carbonic anhydrase inhibitor, acetazolamide. On the other hand, forskolin + IBMX triggered Na⁺ and Cl^- transport into the lumen via club cells. However, ionocytes and club cells from CF HBE cell cultures did not respond to forskolin + IBMX stimulation, suggesting that ionocytes and club cells function abnormally in CF airways.

Conclusions: Our results indicate that ionocytes play a role in pH regulation in the airway. Defective ionocyte-related airway pH changes may contribute to CF lung disease pathogenesis. Club cells contribute to ion and liquid transport. Abnormal club cell function would lead to reduced airway hydration and MCC in CF.

Acknowledgements: Supported by Cystic Fibrosis Canada, Cystic Fibrosis Foundation, Canadian Institutes of Health Research, and the University of Saskatchewan Respiratory Research Centre.

References: [1] Montoro DT, Haber AL, Rajagopal J. A revised airway epithelial hierarchy includes CFTR-expressing ionocytes. Nature. 2018;560:319–24. Doi: 10.1038/s41586-018-0393-7.

[2] Plasschaert LW, Žilionis R, AJaffe AB. A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte. Nature. 2018;560:377–81. doi: 10.1038/s41586-018-0394-6.

[3] Okuda K, Dang H, RBoucher RC. Secretory cells dominate airway CFTR expression and function in human airway superficial epithelia. Am J Respir Crit Care Med. 2021;203:1275–89. doi: 10.1164/rccm. 202008-3198OC.

[4] Luan X, Le Y, Jagadeeshan S, Murray B, Carmalt JL, Duke T, et al. cAMP triggers Na⁺ absorption by distal airway surface epithelium in cystic fibrosis swine. Cell Rep. 2021;37(1):109795. doi: 10.1016/j.celrep.2021.109795.