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Am J Respir Crit Care Med
Am J Respir Crit Care Med
ajrccm
American Journal of Respiratory and Critical Care Medicine
1073-449X
1535-4970
American Thoracic Society

38701428
202404-0727ED
10.1164/rccm.202404-0727ED
Editorials
Pulmonary Ionocytes: What Are They Transporting and Which Way?
https://orcid.org/0000-0001-9341-2730
Okuda Kenichi 1 2
https://orcid.org/0000-0002-9435-0321
Gentzsch Martina 1 3
1 Marsico Lung Institute/Cystic Fibrosis Research Center
2 Department of Medicine
The University of North Carolina at Chapel Hill
Chapel Hill, North Carolina
3 Department of Pediatrics
The University of North Carolina at Chapel Hill
Chapel Hill, North Carolina
3 5 2024
15 9 2024
3 5 2024
210 6 705707
Copyright © 2024 by the American Thoracic Society
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0. For commercial usage and reprints, please e-mail Diane Gern (dgern@thoracic.org).

National Heart, Lung, and Blood Institute 10.13039/100000050 R01HL163602 Cystic Fibrosis Foundation 10.13039/100000897 BOUCHE19R0 National Institute of Diabetes and Digestive and Kidney Diseases 10.13039/100000062 P30DK065988
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pmcAirway mucociliary clearance is a key innate defense mechanism for lung health. Mucus concentration and biophysiochemical properties are tightly regulated by airway epithelial cellular activities to provide effective host innate defense, including mucus transport and airway surface liquid (ASL) pH regulation. Quantitative and qualitative mucus abnormalities are common features of muco-obstructive lung diseases, including cystic fibrosis (CF) (1, 2). CF is characterized by the presence of hyperconcentrated mucus, reflecting loss-of-function mutations in the CF transmembrane conductance regulator (CFTR) gene and consequent defective control of ASL volume by CF airway epithelia. It has been suggested that CFTR dysfunction also leads to acidification of mucus in model systems that is associated with reduced bacterial killing in CF airways (3, 4). However, a study of children with CF reported that the pH of their ASL in vivo was the same as that in children without CF (5). Therefore, it remains critically important to understand how airway epithelial cells regulate CFTR-dependent mucus properties to maintain mucus homeostasis and how it fails in CF.

Recent technical advances in single cell–based omics approaches have revealed substantial diversity among airway epithelial cells, including the identification of pulmonary ionocytes that account for <1% of airway epithelial cells (6, 7). Accumulating single cell–based transcriptional data have revealed widespread cellular CFTR expression among airway epithelial cells, with specific emphasis on pulmonary ionocytes and club (i.e., secretory) cells (8, 9). Notably, rare ionocytes exhibit the highest levels of CFTR expression, whereas club cells comprise the largest proportion of CFTR-expressing cells, albeit with lower levels per individual cell. Given our lack of understanding regarding the contributions of ionocytes and club cells to CF pathophysiology, there is a critical need to comprehensively relate single cell–based airway epithelial cell biology to physiology.

Ionocytes were initially discovered in fish and amphibians, in which they play a role in maintaining ion and fluid homeostasis. Since their discovery in airways, the role of CFTR-rich ionocytes has been extensively investigated. Several electrophysiological studies demonstrated positive correlations between the number of ionocytes and CFTR-mediated Cl− secretion in human bronchial epithelial cells, emphasizing the capacity of ionocytes to mediate transepithelial Cl− secretion despite their rarity (10, 11). Furthermore, a significant contribution of ionocytes to tracheal CFTR function was recently reported in studies of transgenic ferret models, in which approximately 70% reductions of CFTR-mediated Cl− secretion and mucociliary transport rates were observed in ferret tracheal epithelia genetically depleted of ionocytes (12). However, the study of Lei and coworkers reported that human airway ionocytes mediate Cl−/fluid absorption rather than secretion (13). This study also demonstrated that ionocyte-mediated Cl−/fluid absorption requires epithelial sodium channels and barttin (BSND)/Cl− channels (13). The juxtaposition of these findings indicates that there is a need for more evidence to elucidate the physiological roles of ionocytes in airway epithelial homeostasis and CF pathogenesis.

In this issue of the Journal, Luan and colleagues (pp. 788–800) have now added additional data describing cell type–specific ion transport function in human bronchial epithelial cells, with a focus on ionocytes and club cells (14). They used a noninvasive self-referencing ion-selective microelectrode technique to measure transepithelial ion fluxes at single-cell levels. They observed that forskolin plus 3-isobutyl-1-methylxanthine triggered cAMP-stimulated H+ and Cl− transport from the apical to the basolateral surfaces of ionocytes, whereas no Na+ transport was detected. They hypothesized that the ionocyte-mediated reduction of apical H+ concentrations, i.e., alkalization, reflected bicarbonate (HCO3−) secretion into the apical compartment. Based on a series of pharmacological inhibitor studies of relevant ion channels and transporters, Luan and colleagues identified an ionocyte role in generating bicarbonate flow to the apical surface, linking CFTR-mediated Cl− secretion with an anion exchange (AE2 [anion exchanger protein 2], SLC4A2) activity that exchanges Cl− for bicarbonate (14). This finding aligns with a significant decrease in HCO3− currents reported in ferret tracheal epithelial cells with depleted ionocytes (12). In contrast to the minimal contributions of ionocytes to Na+ and Cl− secretion into ASL, Luan and colleagues proposed that cAMP-mediated CFTR activation resulted in a robust increase in club cell–mediated Cl− and Na+ concentrations in ASL that was suppressed by CFTR inhibitor (14), suggesting that CFTR in club cells mediates transcellular secretion of Cl− accompanied by paracellular permeation of Na+ into the airway lumen. These findings are consistent with a selective role for club cells in maintaining purinergically regulated Cl− secretion (8), but leave unanswered the role of club cells for epithelial sodium channel–mediated fluid absorption. Collectively, Luan and colleagues propose distinct electrophysiological functions for ionocytes and club cells, the former regulating ASL pH and the latter regulating ASL volumes and hydration (14), both important for maintaining airway mucus functions. Importantly the CFTR modulators elexacaftor/tezacaftor/ivacaftor rescued ionocyte- and club cell–mediated ion fluxes in CF airway epithelial cells, indicating that both cell types may need to be targeted for CFTR molecular therapies.

Thus, we are presented with three candidate roles for pulmonary ionocytes in ASL physiology emanating from a spectrum of experimental techniques and models: 1) Cl−/fluid secretion, 2) Cl−/fluid absorption, and 3) Cl−/HCO3−–dependent pH regulation (Figures 1A–1C). How do these functions relate to normal lung physiology and CF disease pathogenesis? And how do we move forward? What is clear is that the ion transport/pH processes in airway epithelia are dynamically regulated in vivo by the concentrations of ion transport regulatory molecules in ASL, e.g., extracellular nucleotides/nucleosides (15). One approach is to study the secretory, absorptive, and pH-regulating roles of ionocytes and club cells under dynamic phasic motion conditions that reprise breathing conditions on the thin films of ASL that manifest in vivo (16). In parallel, ionocytes may have regional differences in prevalence within the respiratory tract. For example, what is the function of ionocytes in the high-prevalence submucosal gland ducts (7) and olfactory epithelia (17) versus the low-prevalence small airways (8, 12)? Another approach is to integrate the in vitro data with in vivo observations. For example, why is pH in CF airways seemingly no different than in controls in vivo but different in vitro? Why do human subjects with mutations in a transcription factor for ionocytes or putative ionocyte ion transport channels, i.e., FOXI1 and barttin, respectively, not have a pulmonary mucoinfective phenotype? Understanding the physiological role of pulmonary ionocytes and club cells (Figure 1) could guide the development of therapeutic approaches for mucus-obstructive conditions such as CF. The studies of Luan and coworkers (14) help spur this forward movement.

Figure 1. Models proposed for pulmonary ionocyte and club cell functions. (A) Ionocytes participate in Cl−/fluid secretion, facilitated by cystic fibrosis transmembrane conductance regulator (CFTR)- and NKCC1-mediated ion transport processes (12). (B) Ionocytes mediate Cl−/fluid absorption, a process involving ion movement facilitated by CFTR, epithelial sodium channel (ENaC), and barttin/Cl− channels (13). (C) Ionocytes control the airway surface liquid pH through a Cl−/HCO3− exchange mechanism (14). CFTR contributes indirectly to HCO3− movement in ionocytes, which is mediated by anion exchanger protein 2. (D) Cl−/fluid secretion in club cells as described by Luan and colleagues (14). (E) Regulated Cl−/fluid secretion under physiological conditions. Mechanical stretch associated with normal tidal breathing promotes ATP release from airway epithelia, which activates P2Y2 signaling that inhibits ENaC and promotes CFTR-mediated Cl− secretion in club cells that is associated with paracellular Na+ movement (8). (F) In a nonregulated state (i.e., when the ATP concentration is low as a result of increased airway surface liquid volume), ENaC is active and club cells absorb Na+/fluid, which is accompanied by paracellular Cl− movement. Created with BioRender.com. AE2 = anion exchanger 2, Cl−HCO3− exchanger; NaK pump = Na+/K+ ATPase; NHE2 = Na+/H+ exchanger 2; NKCC1 = Na+-K+-2Cl− cotransporter 1.

Supported by National Heart, Lung, and Blood Institute grant R01HL163602, Cystic Fibrosis Foundation grant BOUCHE19R0, and National Institute of Diabetes and Digestive and Kidney Diseases grant P30DK065988.

Originally Published in Press as DOI: 10.1164/rccm.202404-0727ED on May 3, 2024

Author disclosures are available with the text of this article at www.atsjournals.org.
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