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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

38573173
202309-1565OC
10.1164/rccm.202309-1565OC
Original Articles
Cystic Fibrosis
Pulmonary Ionocytes Regulate Airway Surface Liquid pH in Primary Human Bronchial Epithelial Cells
Luan Xiaojie 1 2
Henao Romero Nicolas 1 2
Campanucci Veronica A. 1 2
Le Yen 1 2
Mustofa Jannatul 1 2
Tam Julian S. 2 3 *
https://orcid.org/0000-0003-4650-8531
Ianowski Juan P. 1 2 *
1 Department of Anatomy, Physiology, and Pharmacology,
2 Respiratory Research Centre, and
3 Division of Respirology, Critical Care, and Sleep Medicine, Department of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
Correspondence and requests for reprints should be addressed to Juan Ianowski, Ph.D., Department of Anatomy, Physiology, and Pharmacology, College of Medicine, University of Saskatchewan, 2D30.4 Health Science Building, 107 Wiggins Road, Saskatoon, SK, S7N 5E5 Canada. E-mail: juan.ianowski@usask.ca.
* These authors contributed equally to this work.

4 4 2024
15 9 2024
4 4 2024
210 6 788800
8 9 2023
4 4 2024
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).

Rationale

Pulmonary ionocytes are a newly discovered airway epithelial cell type proposed to be a major contributor to cystic fibrosis (CF) lung disease based on observations they express the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel at a higher level than any other cell type in the airway epithelia. Moreover, genetically manipulated experimental models that lack ionocytes develop NaCl transport abnormalities and airway surface liquid (ASL) dehydration consistent with CF. However, no direct evidence indicates ionocytes engage in NaCl transport or contribute to ASL formation, questioning the relevance of ionocytes to CF lung disease.

Objectives

To determine the ion transport properties of pulmonary ionocytes and club cells in genetically intact healthy and CF airway epithelia.

Methods

We measured ion transport at the single-cell level using a self-referencing ion-selective microelectrode technique in primary human bronchial epithelial cell culture.

Measurements and Main Results

cAMP-stimulated non-CF ionocytes do not secrete Na+ or Cl− into the ASL, but rather modulate its pH by secreting bicarbonate via CFTR-linked Cl−/bicarbonate exchange. Non-CF club cells secrete Na+ and Cl− to the lumen side after cAMP stimulation. CF ionocytes and club cells do not transport ions in response to cAMP stimulation, but incubation with CFTR modulators elexacaftor/tezacaftor/ivacaftor restores transport properties.

Conclusions

We conclude that ionocytes do not contribute to ASL formation but regulate ASL pH. Club cells secrete the bulk of airway fluid. In CF, abnormal ionocyte and club cell function results in acidic and dehydrated ASL, causing reduced antimicrobial properties and mucociliary clearance.

Keywords

cystic fibrosis
ionocytes
club cells
elexacaftor
tezacaftor
ivacaftor
Cystic Fibrosis Foundation 10.13039/100000897 002367G221 Cystic Fibrosis Canada 10.13039/100000897 1034245 555681 Institute of Circulatory and Respiratory Health 10.13039/100000897 463882 470114
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pmcAt a Glance Commentary

Scientific Knowledge on the Subject

The cellular basis of cystic fibrosis (CF) lung disease is not fully understood, and there is controversy on the cell types responsible for the ion transport abnormalities that result in defective airway surface liquid (ASL) and mucociliary clearance. Some evidence indicates that ionocytes are involved in cystic fibrosis transmembrane conductance regulator (CFTR)-mediated ion transport that influence ASL volume, pH, and viscosity. Contradicting evidence suggests that club cells, rather than ionocytes, are the main site of CFTR-mediated ion transport. This controversy hinders the development of next-generation molecular strategies to treat CF lung disease, such as gene therapy, that require precise identification of cellular targets for treatment.

What This Study Adds to the Field

We determine the ion transport properties of pulmonary ionocytes and club cells at the single-cell level using a self-referencing ion-selective microelectrode technique on primary human bronchial epithelia cell cultures from donors with and without CF. We conclude that ionocytes do not contribute to ASL formation but regulate ASL pH by secreting bicarbonate via CFTR-linked Cl−/bicarbonate exchange. Club cells secrete the bulk of airway fluid by transporting Na+ and Cl− into the lumen. Incubation with the CFTR modulators elexacaftor/tezacaftor/ivacaftor corrects the ion transport abnormalities of CF ionocytes and club cells.

Cystic fibrosis (CF) lung disease is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel expressed on the apical membrane of airway epithelial cells. CFTR function is required to produce healthy airway surface liquid (ASL) necessary for mucociliary clearance of inhaled pathogens (1). In recent years, interest has renewed in developing molecular strategies to treat CF lung disease (e.g., RNA-based treatments [2], gene editing [3, 4], and gene therapy [5]). All these approaches have the potential to treat patients who cannot benefit from current CFTR modulator treatments, but they require precise identification of cellular targets for treatment. However, the cellular bases of CF lung disease are not well understood, and controversy remains regarding which cell types within the human lung contribute to CF pathology.

CFTR was long believed to be primarily expressed in airway epithelial ciliated cells and submucosal gland serous cells (6). However, this hypothesis has been challenged by reports that used single-cell RNA-seq profiling to demonstrate the airway surface epithelium consists of a complex ecosystem of various cell types that express CFTR (7–14). The studies described a previously unknown cell type, the pulmonary ionocyte (7, 8). Ionocytes constitute just ∼1% of all epithelial cells but carry 60% of the CFTR-dependent ion flux; blocking ionocyte function is sufficient to generate phenotypes consistent with CF in ferrets, mice, and cultured monolayers of human cells (7, 8, 10, 11). Genetically manipulating human bronchial epithelial cell culture to enhance ionocyte expression, by interfering with the FOXI1 (forkhead box I1) transcription factor required for progenitor specification of ionocytes, triggers an increase in transepithelial Na+ and Cl− reabsorption from the ASL (10). In another study, FOXI1 knockout ferrets showed abnormalities in Na+, Cl−, and bicarbonate transport, resulting in abnormal ASL volume, pH, viscosity, and mucociliary clearance, implicating ionocyte dysfunction as a major cause of airway physiologic abnormalities encountered in CF lung disease (11). However, the genetic manipulation of this experimental model not only affects ionocytes but also alters the ion transporter expression of basal, ciliated, and club cells (11). Thus, the phenotypes described in FOXI1-modified experimental models could result from the ionocyte manipulation alone or reflect a contribution from other airway cellular populations. Significantly, other publications confirm the existence of CFTR-rich ionocytes, but they also describe club cells that dominate CFTR expression (14); these studies suggest club cells, not ionocytes, are responsible for normal secretion of the ASL and the development of lung disease in CF (14).

Thus, we set out to study ionocyte function in genetically unaltered primary human bronchial epithelial (pHBE) cell cultures. To definitively determine the function of ionocytes and club cells, we used a single-cell physiological assay to study the ion transport properties of individual cells and distinguish their contribution to transepithelial ion transport from that of the other airway epithelial cells. We optimized a noninvasive self-referencing ion-selective microelectrode (SRISM) method with sufficiently high temporal and spatial resolution to measure Na+, Cl−, and H+ flux across single epithelial cells (Figure 1). Using SRISM, we studied the ion transport properties of ionocyte and club cells in pHBE cell cultures obtained from donors without CF and with CF (homozygous F508del-CFTR) grown to confluence at the airway–liquid interface (ALI). The results show ionocytes regulate ASL pH via CFTR-linked bicarbonate secretion through an anion exchanger but do not transport Na+. In contrast, club cells produce ASL by driving Na+ and Cl− transport into the lumen through CFTR. Both cell types fail to transport ions in CF preparations, but their activity is recovered after incubation with CFTR modulators elexacaftor/tezacaftor/ivacaftor (ETI). Some of the results of these studies have been reported in the form of abstracts (15–17).

Figure 1. Self-referencing ion-selective microelectrode (SRISM) system setup. (A) Schematic representation of the SRISM system for ion flux measurements using double electrodes in primary human bronchial epithelial (pHBE) cell culture. A video camera connected to a microscope facilitates placement of the microelectrodes. Movement of the microelectrodes is computer controlled. The electrodes are connected to an amplifier and data acquisition system for offline analysis. (B) We tested the spatial resolution of our SRISM experimental protocol by measuring the ability to resolve two NaCl point sources consisting of borosilicate micropipettes with a ∼5-μm tip diameter, filled with 1 M NaCl in 1% agarose at pH 6.5 and separated by 10 μm. Our SRISM system resolved the Na+, H+, and Cl− fluxes emanating from the two artificial sources. MitoTracker Deep Red FM (100 nM) was used to locate mitochondria-rich (C) ionocytes and (D) club cells in pHBE live cell culture. We marked the position of the cell of interest by scraping the preparation surface with two perpendicular linear markings (located 100 μm away from the cell), the projections of which intersected at the position of the cell. The markings were produced with a ∼5-μm tipped tool dragged along the cell culture using a computer-controlled micromanipulator. The cell identity was verified by staining with ionocyte-specific marker FOXI1 and club cell–specific marker SCGB1A1. (E) FOXI1-, BSND-, and MitoTracker-labeled ionocyte. Scale bars in B–E, 10 μm.

Methods

Preparation of pHBE Cells

First-passage non-CF and CF (F508del homozygous) pHBE cells were purchased from the Primary Airway Cell Biobank at McGill University. pHBE cells were cultured until 90% confluence at ALI in ALI medium for 21 to 28 days. The transepithelial resistance of pHBE cultures reached a minimum of 500 Ω.cm2, as measured with a Millicell-ERS voltmeter-ohmmeter (Merck Millipore). To test the effect of CFTR modulators, CF pHBE cell cultures were incubated with VX445 (elexacaftor, 3 μM), VX661 (tezacaftor, 3 μM), and VX770 (ivacaftor, 1 μM) in the basolateral media for 24 hours before the experiments. For a detailed description, see the online supplement.

Identifying Pulmonary Ionocytes and Club Cells in pHBE Live Cell Culture

Ion transport is a metabolically demanding function of epithelial cells. Thus, epithelial cell types that actively transport ions express high levels of mitochondria. Indeed, ionocytes are also known as mitochondria-rich cells in other vertebrates because of their high mitochondrial expression. Thus, we used MitoTracker before SRISM ion flux measurement to screen for mitochondria-rich cells in pHBE cell cultures and select them for experimentation. The identities of ionocytes and club cells were later assessed using the ionocyte-specific marker FOXI1 (Figure 1C) and the club cell–specific marker SCGB1A1 (Figure 1D). Experiments showed 53% (60/114 cells) of the cells screened using MitoTracker in cell culture were ionocytes, 43% (49/114 cells) were club cells, and 4% (5/114) were unidentifiable and removed from the data pool (Figure 1E). Studies suggest more than one type of ionocyte (10, 11, 18) may exist, including some that may be FOXI1+ and/or BSND+ but do not express CFTR. Thus, we surveyed the expression of FOXI1, BSND, and CFTR in CF and non-CF pHBE cell cultures. We identified and counted all FOXI1+ and BSND+ cells. The majority of FOXI1+ cells were also BSND+. Approximately 18% of FOXI+ cells were BSND−; these cells tended to be located more on the serosal side of the tissue culture and frequently did not express CFTR but costained with “tuft-like” cell marker POU2F3, consistent with the hypothesis that they constitute a FOXI+/POU2F3+ progenitor cell population (see Figure E6 in the online supplement) (19). We did not identify any cells that were FOXI1−/BSND+. After the SRISM experiments, we marked the position of the cell of interest by scraping the preparation surface with two perpendicular linear markings ∼100 μm away from the cell; the projections of these markings intersected at the position of the cell. For a detailed description, see the online supplement.

Ion-Selective Microelectrode Construction

Ion-selective microelectrodes were fabricated from borosilicate nonfilamented capillary glass (TW150-4; WPI). Capillaries were pulled into microelectrodes with ∼3-μm diameter tips using a vertical micropipette puller (PC-10 Narishige). To make the glass hydrophobic, the pulled capillaries were silanized with dimethyldichlorosilane and baked overnight at 250°C. Finally, a short column (∼50 μm) of liquid ion-selective cocktail was introduced into the microelectrode tip and backfilled with the appropriate solution. For a detailed description, see the online supplement.

SRISM Technique

The SRISM technique is a noninvasive method capable of measuring transepithelial ion flux at high spatial and temporal resolutions. The cells of an ion-transporting epithelia generate an ion concentration gradient in an unstirred layer adjacent to the tissue. The concentration gradient within the unstirred layer is proportional to the rate of epithelial transport. We mapped and measured patterns of Na+, Cl−, and H+ flow across the epithelial surfaces by measuring the ion concentration gradients in the unstirred layer. A micromanipulator positioned an ion-selective microelectrode at two sites within this unstirred layer to measure the small ion concentration gradient generated by transepithelial transport (Figure 1A). Fick’s law of diffusion was then used to calculate the rate of diffusion of ions within the unstirred layer, which in the steady state matches the rate of net epithelial ion transport regardless of the transport pathway (transcellular or paracellular) or whether it is passive or active. We tested the spatial resolution of our SRISM system using artificial sources of Na+, Cl−, and H+ placed 10 μm apart (i.e., two glass electrodes filled with 1% agar containing 1 M NaCl at pH 6.5) (Figure 1B). The SRISM system was able to clearly resolve the Na+, Cl−, and H+ flux signals from sources 10 μm apart (Figure 1B). For a detailed description, see the online supplement.

Immunofluorescence Imaging

pHBE cell ALI cultures were fixed by submerging them in freshly made 4% paraformaldehyde for 30 minutes at room temperature; then they were conserved in Dulbecco’s phosphate-buffered saline (DPBS) at 4°C. For immunostaining, cells were permeabilized with 0.3% Triton X-100 diluted in DPBS for 15 minutes and then blocked with 5% donkey serum for 1 hour at room temperature. After three DPBS washes, cells were incubated with primary antibodies overnight at 4°C (see online supplement for detailed description of antibodies used). Finally, pHBE cells were incubated with appropriate secondary antibodies at room temperature for 1 hour and then covered with antifading medium before imaging.

The preparations were imaged with a Nikon Ti2E spinning disk confocal microscope with an ×40 water objective (Nikon Canada Inc.). High-resolution three-dimensional (3D) images and Z-stack images were acquired with a 0.4-μm step size through the entire depth of the sample. Z sections and 3D images were reconstructed and analyzed using Nikon NIS-Elements Viewer 5.21 software. Each experiment was repeated at least five times on at least two different cell cultures.

Chemicals

Chemicals were purchased from Sigma-Aldrich unless otherwise indicated. Stock solutions of pharmacological agents were prepared in DMSO to make 1,000-fold concentrated stock solutions.

Data Analysis

To quantify the ion flux, we report the maximum ion flux, calculated as the largest flux measured after forskolin plus 3-isobutyl-1-methylxanthine (IBMX) stimulation, and the total ion transport, calculated by integrating all ion fluxes within 120 seconds after forskolin plus IBMX stimulation minus the baseline ion transport within 60 seconds before stimulation.

Statistical analyses were performed using GraphPad Prism (GraphPad Software) and unpaired ANOVA and Tukey or Holm-Sidak post hoc tests. Differences were considered statistically significant at P < 0.05. Data are presented as the mean ± SEM.

Results

Ionocytes Regulate ASL pH

Simultaneous SRISM measurement of Na+ and H+ or Cl− and H+ fluxes under unstimulated conditions showed ionocytes from non-CF pHBE ALI culture display minimal transport of Na+, H+, and Cl− (Figures 2A–2C). To stimulate CFTR-mediated ion transport, we treated the preparations with forskolin (adenylate cyclase activator, 10 μM) plus IBMX (phosphodiesterase inhibitor, 100 μM) on the apical side (20). Forskolin plus IBMX treatment triggered H+ and Cl− transport at similar rates from the apical to basolateral side across ionocytes (i.e., generating a negative value for ion flux measurements); however, Na+ transport remained unchanged (Figures 2A–2C). All ionocytes investigated were later confirmed to express FOXI1, BSND, and CFTR. In contrast, forskolin plus IBMX treatment of CF pHBE cell cultures showed minimal ion transport across ionocytes (Figures 2D–2F). Forskolin plus IBMX triggered significantly higher total H+ and Cl− transport to the basolateral side via ionocytes in non-CF than CF culture (Figures 2K and 2L). Moreover, the maximum H+ and Cl− flux triggered by forskolin plus IBMX stimulation was significantly higher in non-CF versus CF ionocytes (Figures 2N and 2O). Treating CF pHBE cell cultures with the CFTR modulators elexacaftor (3 μM)/tezacaftor (3 μM)/ivacaftor (1 μM) (ETI), to model the effect of current treatments, for 24 hours (21) resulted in a recovery of forskolin plus IBMX–triggered H+ and Cl− transport across ionocytes (Figures 2G–2I). The total (Figures 2J–2L) and maximum (Figures 2M–2O) H+ and Cl− fluxes of ETI-treated preparations were not different than those of non-CF preparations.

Figure 2. Ionocytes regulate airway surface liquid pH. Effect of forskolin (10 μM) plus 3-isobutyl-1-methylxanthine (IBMX) (100 μM) stimulation on non–cystic fibrosis (non-CF) pulmonary ionocyte transport of (A) Na+ (n = 9 from four donors), (B) H+ (n = 16 from five donors), and (C) Cl− (n = 16 from five donors). Effect of forskolin plus IBMX stimulation on CF ionocyte transport of (D) Na+ (n = 10 from four donors), (E) H+ (n = 14 from six donors), and (F) Cl− (n = 4 from three donors). Effect of 24-hour incubation of CF ionocytes with elexacaftor (3 μM), tezacaftor (3 μM), and ivacaftor (1 μM) (ETI) on forskolin plus IBMX–stimulated transport of (G) Na+ (n = 11 from four donors), (H) H+ (n = 13 from six donors), and (I) Cl− (n = 10 from four donors). Total transport of (J) Na+, (K) H+, and (L) Cl− and maximum transport rate of (M) Na+, (N) H+, and (O) Cl− by ionocytes from non-CF, CF, and CF treated with ETI for 24 hours (CF + ETI) preparations. Positive flux values indicate ion movement from the basolateral into the apical side of the airway epithelial cells, and negative values indicate transport in the opposite direction. Data are presented as mean ± SEM and were subjected to unpaired ANOVA and Holm-Sidak post hoc tests. Differences were considered statistically significant at P < 0.05. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. ns = no significant difference.

CFTR-linked Bicarbonate Transport through an Anion Exchanger by Ionocytes

Our results suggest ionocytes are involved in CFTR-dependent pH modulation of the ASL, and we thus progressed to study the ion transporter mechanisms involved. Ionocytes express at high levels a vacuolar type H+ ATPase (7, 8). Ionocytes may pump H+ from the apical to basolateral side mediated by the V-type H+ ATPase. We tested the effect of treating ionocytes with the V-type H+ ATPase inhibitor bafilomycin A1 (100 nM) (22). Blocking the V-type H+ ATPase had no effect on the forskolin plus IBMX–triggered ion transport by the ionocytes, even though all ionocytes tested expressed V-type H+ ATPase (ATP6V0C subunit), as well as FOXI1 and BSND (Figures 3A–3C and E1). Immunohistochemistry analysis showed the V-type H+ ATPase (ATP6V0C) is expressed on the apical side of pulmonary ionocytes (Figure 3D); this suggests ionocyte V-type H+ ATPase would transport H+ into the ASL, inconsistent with the hypothesis that basic ASL is produced after forskolin plus IBMX–triggered H+ reabsorption (23, 24). Thus, the V-type H+ ATPase expressed in ionocytes is not involved in CFTR-mediated ASL alkalinization. An alternative possibility is that ASL alkalinization may be the result of bicarbonate secretion into the ASL rather than reabsorption of H+.

Figure 3. Bicarbonate transport by ionocytes in non–cystic fibrosis (non-CF) primary human bronchial epithelial cell culture. Effect of 15-minute incubation of non-CF ionocytes with bafilomycin A1 (100 μM) on forskolin (10 μM) plus 3-isobutyl-1-methylxanthine (IBMX) (100 μM)-stimulated (A) transport of H+ (black trace, n = 8 from five donors). (B) Total H+ transport and (C) maximum transport rate of H+ by ionocytes from non-CF, non-CF treated with bafilomycin A1 (non-CF + BafA1), and CF preparations. (D) Confocal immunofluorescence of BSND (ionocyte marker), FOXI1 (ionocyte marker), ATP6V0C (V-type ATPase marker), and merged image of non-CF ionocytes. Scale bars, 5 μm. Effect of 15-minute incubation with acetazolamide (ACTZ; 100 μM) on forskolin plus IBMX–stimulated non-CF ionocytes on (E) H+ flux (black trace, n = 14 from six donors). (F) Total transport of H+ and (G) maximum transport rate of H+ by ionocytes from non-CF, non-CF treated with ACTZ (non-CF + ACTZ), and CF preparations. (H) Effect of HCO3−-free bathing saline solution on forskolin plus IBMX–stimulated non-CF ionocytes on H+ (black trace, n = 7 from four donors). (I) Total transport and (J) maximum transport rate of H+ by ionocytes from non-CF, non-CF incubated in HCO3−-free bathing saline (non-CF + HCO3−-free), and CF preparations. (K) Effect of combined treatment with ACTZ and HCO3−-free bathing saline on forskolin plus IBMX–stimulated non-CF pulmonary ionocytes on H+ (black trace, n = 7 from four donors). (L) Total transport and (M) maximum transport rate of H+ by non-CF ionocytes. Red trace represents the H+ measured under control conditions and shown in Figure 2B. Data are presented as mean ± SEM and were subjected to unpaired ANOVA and Holm-Sidak post hoc tests. Differences were considered statistically significant at P < 0.05. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. ns = no significant difference.

To study whether ionocytes transport bicarbonate, we tested the effects of bicarbonate-free Krebs bathing saline solution and acetazolamide (ACTZ), a carbonic anhydrase inhibitor that blocks cellular bicarbonate production. ACTZ treatment (100 μM, on the basolateral side) (25) reduced the forskolin plus IBMX–triggered ASL pH gradient (Figures 3E and 3F) and maximum H+ flux by ∼50% (Figures 3G and E2A–E2F) in non-CF ionocytes. Similarly, bicarbonate-free Krebs bathing saline solution reduced the forskolin plus IBMX–triggered pH gradient (Figures 3H and 3I) and maximum H+ flux by ∼50% (Figures 3J and E2G–E2I) in non-CF ionocytes. Furthermore, simultaneous treatment with ACTZ and bicarbonate-free Krebs saline completely blocked the development of the forskolin plus IBMX–triggered pH gradient (Figures 3K and 3L) and resulted in a maximum H+ flux similar to that of CF ionocytes (Figures 3M and E2J–E2L). These results suggest ionocytes engage in a CFTR-dependent ion transport process that results in bicarbonate secretion to the ASL and, at the same time, Cl− transport in the opposite direction from the apical (i.e., lumen) to the basolateral side. However, anion movement through the CFTR usually drives Cl− and bicarbonate secretion into the ASL, not Cl− reabsorption as we observed (26–28). Thus, we progressed to testing the ion transport machinery underlying the CFTR-dependent Cl− and bicarbonate transport across ionocytes.

We tested the role of CFTR on ionocyte bicarbonate transport by treating non-CF pHBE cell cultures with the CFTR blocker CFTRinh172 (10 μM) (29). Blocking CFTR inhibited the ionocytes from generating the pH gradient in the ASL after forskolin plus IBMX treatment (Figures 4A–4C and E3). These results suggest CFTR is indeed involved in bicarbonate secretion by ionocytes. As ion flux through the CFTR channel unlikely allows simultaneous bicarbonate transport into the ASL and Cl− movement in the opposite direction (Figure 2C), CFTR could be a component of a more complex Cl−-linked bicarbonate transport. Thus, we tested whether an anion exchanger (AE) (i.e., a Cl−/bicarbonate exchanger; AE2, SLC4A2) reported to be expressed in ionocytes (13) may play a role in Cl−-linked bicarbonate transport. Inhibiting AE function by incubating with 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS, 500 μM) (30) blocked the forskolin plus IBMX–triggered pH gradient generated by ionocytes (Figures 4E–4G), suggesting AE2 may be involved in bicarbonate secretion. Interestingly, DIDS had no effect on forskolin plus IBMX–triggered NaCl transport in club cells, suggesting DIDS is not toxic to our cell cultures (Figure E4). Using immunohistochemistry, we detected the expression of both CFTR and AE2 (SLC4A2) on the apical membrane of ionocytes (Figures 4D and 4H); this finding is consistent with the hypothesis that the apical membrane of the ionocyte expresses CFTR and AE2 and that these transporters work together to drive bicarbonate into the ASL. Apical secretion of intracellularly produced bicarbonate requires the basolateral extrusion of excess H+ produced during carbonic acid dissociation. Basolateral extrusion of H+ may involve a Na+/H+ exchanger (NHE), as seen in pancreatic duct cells (31). Treatment with amiloride (10 μM) (32) on the apical (Figures 4I–4K) or basolateral side (Figures 4L–4N) to block NHE partially reduced forskolin plus IBMX–triggered H+ flux. Moreover, treatment with a more specific NHE blocker, 5-(N-Ethyl-N-isopropyl)-Amiloride (EIPA, 5 μM, basolateral and apical) (33), completely blocked forskolin plus IBMX–triggered H+ flux (Figures 4O–4Q). Consistently, immunohistochemistry shows the basolateral expression of NHE (Figure 4R). Amiloride may also block epithelial Na+ channels (ENaC); however, it had no effect on Na+ flux (Figures 4S–4U), and immunofluorescence imaging failed to detect the expression of ENaC in ionocytes (Figure 4V). We hypothesize the bicarbonate generated intracellularly in ionocytes by carbonic anhydrase is secreted into the ASL via apical AE2 in exchange for Cl−. The Cl− ions are recycled through CFTR to avoid Cl− depletion, thus maintaining the driving force for AE2 function. Blocking CFTR function inhibits AE2 bicarbonate secretion because it reduces Cl− availability for bicarbonate exchange. The H+ generated by the dissociation of the carbonic acid produced by intracellular carbonic anhydrase may be extruded across the basolateral membrane through NHE. Transepithelial Cl− flux from the lumen to the basolateral side of the ionocyte would occur through paracellular pathways and act as a counterion to H+ reabsorption, which results in net HCl transport from the lumen to basolateral side.

Figure 4. Bicarbonate secretion involves the coordinated function of apical cystic fibrosis transmembrane conductance regulator (CFTR) and AE2 (anion exchanger 2) with basolateral NHE2 (Na+/H+ exchanger 2). Effect of 15-minute incubation with CFTRinh172 (10 μM) on forskolin plus 3-isobutyl-1-methylxanthine (IBMX)-stimulated non–cystic fibrosis (non-CF) ionocytes on (A) H+ flux (black trace), (B) total transport of H+, and (C) maximal H+ transport rate (n = 9 from four donors). Effect of 15-minute incubation with DIDS (500 μM) on forskolin plus IBMX–stimulated non-CF ionocytes on (E) H+ flux (black trace), (F) total transport of H+, and (G) maximal H+ transport rate (n = 8 from two donors). Effect of 15-minute incubation with apical amiloride (10 μM) treatment on forskolin (10 μM) plus IBMX (100 μM) stimulation of non-CF primary human bronchial epithelial (pHBE) ionocytes on (I) H+ flux (black trace, n = 6 from three donors), (J) total transport of H+, and (K) maximal transport rate of H+. Effect of 15-minute incubation with basolateral amiloride (10 μM) treatment on forskolin (10 μM) plus IBMX (100 μM) stimulation of non-CF pHBE ionocytes on (L) H+ flux (black trace, n = 7 from three donors), (M) total transport of H+, and (N) maximal transport rate of H+. Effect of 15-minute incubation with EIPA (5 μM basolateral and apical) treatment on forskolin (10 μM) plus IBMX (100 μM) stimulation of non-CF pHBE ionocytes on (O) H+ flux (black trace, n = 8 from three donors), (P) total transport of H+, and (Q) maximal transport rate of H+. Red trace represents the H+ measured under control conditions and shown in Figure 2B. Effect of 15-minute incubation with apical amiloride (10 μM) treatment on forskolin (10 μM) plus IBMX (100 μM) stimulation of non-CF pHBE ionocytes on (S) Na+ flux (black trace, n = 5 from three donors), (T) total transport of Na+, and (U) maximal transport rate of Na+. Red trace represents control Na+ flux (Figure 2A). Positive flux values indicate ion movement from the basolateral side into the apical side of the airway epithelial cells, and negative values indicate transport in the opposite direction. Red trace represents control Na+ flux (Figure 2A). Data are presented as mean ± SEM and were subjected to unpaired ANOVA and Tukey post hoc tests. Differences were considered statistically significant at P < 0.05. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Confocal immunofluorescence of non-CF ionocytes of (D) BSND (ionocyte marker), FOXI1 (ionocyte marker), CFTR, and merged image; (H) BSND (ionocyte marker), FOXI1 (ionocyte marker), SLC4A2 (AE2 channels), and merged image; (R) BSND (ionocyte marker), FOXI1 (ionocyte marker), SLC9A2 (NHE2 marker), and merged image; and (V) BSND (ionocyte marker), FOXI1 (ionocyte marker), SCNN1B (epithelial Na+ channel marker), and merged image. Scale bars in D, H, R, and V, 10 μm. DIDS = 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid; EIPA = 5-(N-Ethyl-N-isopropyl)-amiloride; ns = no significant difference.

CFTR-mediated Na+ and Cl− Secretion by Club Cells

Experiments showed approximately 40% of the cells highly labeled with MitoTracker were club cells (see Methods). Similar to ionocytes, SRISM measurements from non-CF club cells revealed minimal transport of Na+, H+, and Cl− under unstimulated conditions (Figures 5A–5C). Treatment with forskolin (10 μM) plus IBMX (100 μM) (20) on the apical side to stimulate CFTR-mediated ion transport triggered a large increase in Na+ and Cl− transport into the apical (i.e., luminal) side of the preparation (Figures 5A and 5C). In addition, club cells produced a small level of apical alkalinization that was approximately 10 times smaller than that generated by ionocytes (Figure 5B). CF club cells failed to respond to forskolin plus IBMX treatment (Figures 5D–5F). Both total (Figures 5J and 5L) and maximum (Figures 5M and 5O) Na+ and Cl− transport were significantly higher in non-CF than CF cultures, consistent with the hypothesis that CF airways have reduced ASL formation. Moreover, forskolin plus IBMX stimulation triggered a small but significantly higher maximum H+ flux in non-CF versus CF club cells (Figures 5K and 5N), suggesting a minor contribution of club cells to ASL pH regulation. Treating CF pHBE cell cultures with ETI for 24 hours (21) resulted in a recovery of forskolin plus IBMX–triggered Na+ and Cl− transport across club cells (Figures 5G–5I). The total (Figures 5J–5L) and maximum (Figures 5M–5O) ion fluxes of ETI-treated preparations were not different from those of non-CF preparations.

Figure 5. Club cells secrete Na+ and Cl−. Effect of forskolin (10 μM) plus 3-isobutyl-1-methylxanthine (IBMX) (100 μM) stimulation on non–cystic fibrosis (non-CF) club cell transport of (A) Na+ (n = 15 from five donors), (B) H+ (n = 36 from seven donors), and (C) Cl− (n = 21 from six donors). Effect of forskolin plus IBMX stimulation on CF club cell transport of (D) Na+ (n = 11 from four donors), (E) H+ (n = 17 from five donors), and (F) Cl− (n = 5 from three donors). Effect of 24-hour incubation of CF club cells with elexacaftor (3 μM), tezacaftor (3 μM), and ivacaftor (1 μM) (ETI) on forskolin plus IBMX–stimulated transport of (G) Na+ (n = 7 from three donors), (H) H+ (n = 14 from four donors), and (I) Cl− (n = 7 from four donors). Total transport of (J) Na+, (K) H+, and (L) Cl− and maximum transport rate of (M) Na+, (N) H+, and (O) Cl− by club cells from non-CF, CF, and CF treated with ETI for 24-hour (CF + ETI) preparations. Positive flux values indicate ion movement from the basolateral into the apical side of the airway epithelial cells, and negative values indicate transport in the opposite direction. Data are presented as mean ± SEM and were subjected to unpaired ANOVA and Holm-Sidak post hoc tests. Differences were considered statistically significant at P < 0.05. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. ns = no significant difference.

We tested the role of CFTR on the transport of Na+, H+, and Cl− by club cells by treating non-CF pHBE cell cultures with the CFTR blocker CFTRinh172 (10 μM) (29). Blocking CFTR inhibited Na+ (Figures 6A–6C) and Cl− (Figures 6H–6J) transport by club cells (Figure 6D). The total Na+ and Cl− transport (Figures 6B and 6I) and maximum ion flux (Figures 6C and 6J) were significantly reduced and not different from CF club cells. CFTRinh172 treatment also reduced H+ flux (Figures 6E–6G). The basolateral uptake of Cl− and Na+ has been proposed to be mediated by the Na+:K+:2Cl− cotransporter (NKCC) (28), and, thus, we tested the effect of the NKCC blocker bumetanide (100 μM basolateral) (34). Blocking NKCC inhibited Cl− transport by club cells (Figures 6N–6P). The total Cl− transport (Figure 6O) and maximum ion flux (Figure 6P) were significantly reduced and not different from CF club cells. Bumetanide treatment had no statistically significant effect on H+ flux (Figures 6K–6M). These results indicate club cells engage in CFTR-dependent Na+ and Cl− transport into the airway lumen and have a minor effect on ASL pH (Figure 7B).

Figure 6. Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated transport by club cells. Effect of 15-minute incubation with CFTRinh172 (10 μM) on forskolin plus 3-isobutyl-1-methylxanthine (IBMX)-stimulated non–cystic fibrosis (non-CF) club cells on (A) Na+ flux (black trace, n = 10 from four donors), (B) total transport of Na+, (C) maximal transport rate of Na+, (E) H+ flux (black trace, n = 17 from six donors), (F) total transport of H+, (G) maximal transport rate of H+, (H) Cl− flux (black trace, n = 7 from three donors), (I) total transport of Cl−, and (J) maximal transport rate of Cl−. Effect of 15-minute incubation with bumetanide (100 μM basolateral) on forskolin plus IBMX–stimulated non-CF club cells on (K) H+ flux (black trace, n = 6 from three donors), (L) total transport of H+, (M) maximal transport rate of H+, (N) Cl− flux (black trace, n = 6 from three donors), (O) total transport of Cl−, and (P) maximal transport rate of Cl−. Positive flux values indicate ion movement from the basolateral into the apical side of the airway epithelial cells, and negative values indicate transport in the opposite direction. Red trace represents control ion flux (Figures 2A and 2B). Data are presented as mean ± SEM and were subjected to unpaired ANOVA and Holm-Sidak post hoc tests. Differences were considered statistically significant at P < 0.05. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. (D) Confocal immunofluorescence of SCGB1A1 (club cell marker), CFTR, and merged image of a club cell. CFTR was localized at the apical membrane of ionocytes. Scale bars represents 10 μm. ns = no significant difference.

Figure 7. Models of ion transport. Schematic model of working hypothesis of how (A) pulmonary ionocytes and (B) club cells function in primary human bronchial epithelial cell culture stimulated with forskolin plus 3-isobutyl-1-methylxanthine. AE2 = anion exchanger 2; CFTR = cystic fibrosis transmembrane conductance regulator; NHE2 = Na+/H+ exchanger 2; NKCC = Na+:K+:2Cl− cotransporter.

Discussion

Recent publications suggest ionocytes are a major pathway for Na+ and Cl− transport across the airway epithelia and imply ionocyte malfunction leads to the development of the hallmark signs of CF lung disease, including ASL dehydration, mucus abnormality, and reduced mucociliary clearance (10, 11).

Our results contradict the hypothesis that ionocytes are involved in Na+ and Cl− transport. We detected no Na+ transport across ionocytes after forskolin and IBMX stimulation. Rather, ionocytes produce bicarbonate flow to the apical side at high rates via a CFTR-linked Cl−/bicarbonate exchange that maximizes bicarbonate secretion, as described in pancreatic duct cells (31), suggesting a role in ASL pH regulation. We did not detect any contribution of ENaC to ion transport across ionocytes (Figure 7A). On the other hand, we found club cells engage in Na+ and Cl− transport to the apical side, consistent with the observation proposed by Okuda and colleagues (14) that club cells produce most of the ASL (Figure 7B). We also found that club cells secrete bicarbonate at low rate.

The Na+ transport properties of ionocytes we observed may differ from those assigned to ionocytes by Lei and colleagues (10) and Yuan and colleagues (11) because of differences in experimental techniques and models. Our SRISM single-cell ion flux measurements are ideally suited to isolate ionocyte transport properties from those of the rest of the tissue, which cannot be achieved using techniques, such as Ussing chamber assays, that test the function of an entire epithelium. Moreover, we used genetically unmodified pHBE cells, which avoids the potential complication that genetic manipulation may disturb ion transport in other cell types (11). Because the ion transport of an ionocyte is dependent on the ion electrochemical potentials, transepithelial membrane potential, and paracellular pathway properties of the entire epithelium, some of the observed phenotypes attributed to ionocyte genetic manipulation may be explained based on alterations to the function and transport properties of other cell types. Finally, our data contradict the hypothesis that several functional subtypes of ionocytes exist, including those that secrete NaCl, reabsorb NaCl, and transport bicarbonate (10, 11, 35). We did not detect such a diversity of function, as all ionocytes surveyed (Figure E5) performed the same ion transport function, namely bicarbonate secretion into the airway lumen. Club cells transport bicarbonate at a low rate but may affect ASL pH because of their abundance.

Our results indicate ionocytes and club cells have different and specific functions in maintaining ASL integrity. Treatments aimed at augmenting CFTR function in ionocytes may resolve the acidic pH in the CF airway, which would improve the airway innate immune defense but may not increase airway hydration. Targeting club cells may improve airway hydration but not necessarily pH abnormalities. The function of multiple cell types may need to be restored to achieve healthy ASL.

Acknowledgment

The authors thank Dr. Terry Machen for his contributions to data interpretation and Gillian Larkin for her assistance with manuscript editing.

Supported by Cystic Fibrosis Foundation (USA) grant 002367G221, Cystic Fibrosis Canada grants 1034245 and 555681, Canadian Institutes of Health Research Institute of Circulatory and Respiratory Health grants 463882 and 470114, the Saskatchewan Health Research Foundation, and the Respiratory Research Centre and College of Medicine at the University of Saskatchewan.

Author Contributions: X.L. contributed to developing experimental techniques, conducting experiments, acquiring data, analyzing data, and writing the manuscript. N.H.R., V.A.C., and Y.L. contributed to conducting experiments and acquiring and analyzing data. J.M. contributed to developing experimental techniques. J.S.T. and J.P.I contributed to securing funding, designing of research studies, designing and developing experimental techniques, interpretation of the data, and writing the manuscript.

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Originally Published in Press as DOI: 10.1164/rccm.202309-1565OC on April 4, 2024

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