Pulmonary Ionocytes at the Crossroads of Absorption and Secretion: Clues from the Ferret

Ionocytes were first identified in the gill epithelium of eels in the early 1930s, when they were proposed to play a central role in osmoregulation by controlling salt and water balance in aquatic environments (1). Since then, ionocytes have been found in various mammalian epithelia, where they contribute to ion and fluid regulation. The analysis of airway epithelia by single-cell sequencing has identified various types of airway epithelial cells, including a small subpopulation (∼1%) of ionocytes that highly expresses cystic fibrosis transmembrane conductance regulator (CFTR) (2, 3). Airway fluid absorption, alongside secretion, is crucial for maintaining optimal airway surface liquid (ASL) volume and properly hydrated mucus. Continuous, balanced absorption and secretion maintain ASL volume within a narrow healthy range. In cystic fibrosis (CF), ASL is dehydrated and acidified because of defects in the CFTR protein abrogating Cl⁻ and HCO₃⁻ secretion, causing excessive Na⁺ and fluid absorption, and thick, concentrated mucus that impairs clearance and increases infection risk. Ionocytes display uniquely high CFTR expression and activity (4, 5) and may regulate ASL pH (6). Studies in transgenic ferret models showed that ionocyte depletion resulted in decreased CFTR-mediated Cl⁻ secretion and mucociliary clearance, resembling defects seen in human CF airways (7). However, others have suggested that ionocytes mainly mediate ASL absorption, whereas secretory cells are responsible for fluid secretion (8). Thus, the cell type–specific contributions to CFTR-mediated ASL volume regulation remain unclear (7, 8). In addition, a considerable debate persists over whether rare ionocytes or the far more abundant secretory club cells are the main mediators of CFTR-dependent ASL regulation in human airways (9).

In this issue of the Journal, Yuan and colleagues (pp. 1935–1950) used transgenic ferret models with ionocyte-specific CFTR deletion and reactivation to define the role of ionocytes in airway fluid regulation (10). Electrophysiological assays in Ussing chambers demonstrated that ionocyte CFTR supports bidirectional Cl⁻ transport. Cl⁻ gradients across the apical and basolateral buffers were established to enhance the driving force for Cl⁻ movement. Under low apical Cl⁻ conditions, Cl⁻ secretion was observed (Figure 1A), whereas low basolateral Cl⁻ conditions promoted Cl⁻ absorption (Figure 1B). Loss of ionocyte CFTR caused ∼70% reduction in secretion and complete loss of absorption, which resembles the effects seen in FOXI1-knockout–driven ionocyte-depleted epithelia (7). Reintroduction of ionocyte CFTR restored secretion and absorption, including substantial functional recovery upon monoallelic rescue.

Figure 1.

Ferret airway ionocytes are capable of secretion and absorption. (A) Ussing chamber assays to evaluate the secretory capacity of ionocytes. A low Cl⁻ buffer on the apical/mucosal side (low Cl⁻; ∼5 mM Cl⁻) and a high Cl⁻ buffer (high Cl⁻; ∼140 mM) on the basolateral side led to transepithelial Cl⁻ secretion via the basolateral NKCC1 and the apical cystic fibrosis transmembrane conductance regulator (CFTR) channels. (B) Ussing chamber assays to evaluate the absorptive capacity of ionocytes. A high Cl⁻ buffer on the apical side and a low Cl⁻ buffer on the basolateral side led to transepithelial Cl⁻ absorption via the apical CFTR channel and the basolateral barttin Cl⁻ channel. Under both conditions (A, B), Cl⁻ is moving along the electrochemical gradient in response to CFTR activation by forskolin and 3-isobutyl-1-methylxanthine. Whether secretion and absorption are controlled within the same cell or by independent ionocyte subtypes remains to be determined. (C) Ferret airway epithelial organoids with lumen-facing apical membranes swell in response to forskolin activation of CFTR and amiloride inhibition of ENaC, reflecting CFTR-dependent Cl⁻ and fluid secretion. Created with BioRender.com. ENaC = epithelial sodium channel; NKCC1 = Na⁺/K⁺/2Cl⁻ cotransporter.

Pulmonary ionocytes display high concentrations of apical CFTR as well as basolateral Na⁺/K⁺/2Cl⁻ cotransporter (NKCC1) and Na⁺/K⁺-ATPase to drive secretion and also basolateral barttin Cl⁻ channels to enable absorption (Figures 1A and 1B). Interestingly, CFTR deletion not only abolished secretion but also reduced NKCC1 expression (10). Notably, under symmetrical Cl⁻ buffers, the dominant short-circuit current was secretory.

To verify that ionocytes mediate salt and fluid secretion under more physiological conditions, organoid swelling assays were conducted (10). Ferret airway organoids, with apical membranes oriented toward the lumen, exhibited pronounced swelling when CFTR was activated with forskolin (Figure 1C). This response reflects CFTR-mediated Cl⁻ secretion by ionocytes into the lumen, driving osmotic fluid movement. Conditional CFTR knockout, specifically in ionocytes, abolished organoid swelling, demonstrating that ionocyte CFTR is both necessary and sufficient for net luminal fluid secretion. These findings highlight the central role of ionocytes in regulating airway epithelial fluid transport in assays beyond short-circuit current measurements. Another key finding enabled by ferret lineage mapping was that CFTR loss in ionocytes triggers rapid regeneration of CFTR-competent ionocytes from FOXI1-lineage–negative progenitors, resulting in more than a sixfold increase across tracheal and nasal epithelia (10). This effective regeneration suggests that basal cells can respond to ionocyte CFTR dysfunction and highlights the potential for CFTR gene editing in progenitor cells to restore functional ionocytes in the airways.

Yuan and colleagues (10) demonstrated that ionocyte CFTR contributes about 70% of secretion in proximal airways, but the contribution of other cell types to the remaining 30% was not investigated. Because ionocyte abundance declines toward distal airways, other CFTR-expressing cells, such as secretory club cells, may play a larger role in maintaining fluid secretion and hydration in these regions where CF pathology is most pronounced (11, 12). These findings focus primarily on proximal ionocytes, leaving the contributions of other cell types and potential differences between proximal and distal CFTR populations to be further explored. Recent studies showed that asthma-related and IL-13–driven inflammation reduces ionocyte numbers while increasing goblet cells, suggesting that other cell types may assume key roles (13, 14). Similarly, viral or bacterial airway inflammation may alter ionocyte function, shifting secretion and absorption to other cells. Thus, it remains to be explored how ionocyte absorptive, secretory, and pH-regulating properties are affected under pathophysiological conditions such as infection, inflammation, and hypoxia resulting from mucus obstruction. Furthermore, breathing-induced shear stress regulates ion transport and fluid secretion through ATP- and adenosine-mediated pathways within the ASL thin film in vivo and may also influence the relative roles of ionocytes and other cell types in fluid regulation (15).

In conclusion, Yuan and colleagues (10) have addressed a long-standing controversy regarding the precise role of pulmonary ionocytes and their highly expressed CFTR channel in regulating ASL volume. Using novel transgenic ferret models, the research demonstrates conclusively that ionocyte-expressed CFTR is essential for both Cl⁻ absorption and secretion in the large airways. Future work should determine whether these opposing roles are performed by distinct ionocyte subtypes or by context-dependent functional shifts within the same ionocyte and how environmental factors affect ionocyte functions. It is important to identify the signals and mechanisms that govern basal cell differentiation into new ionocytes after CFTR deletion and fluid imbalance. In addition, ferret airway tissue can be analyzed directly as a powerful tool for pulmonary ionocyte studies, because culture conditions may alter ionocyte abundance. Finally, extending conditional knockout and knock-in approaches to lung cell types other than ionocytes will deepen our understanding of CFTR’s role in airway physiology and may provide critical insights for strategies aimed at restoring CFTR function in people with CF.