Research ArticlePulmonology Open Access | 10.1172/JCI181995

Guillermo S. Romano Ibarra,1 Lei Lei,2 Wenjie Yu,1 Andrew L. Thurman,1 Nicholas D. Gansemer,1 David K. Meyerholz,3 Alejandro A. Pezzulo,1,4 Paul B. McCray,1,2,4 Ian M. Thornell,1,4 and David A. Stoltz1,4,5,6

1Department of Internal Medicine,

2Department of Pediatrics,

3Department of Pathology,

4Pappajohn Biomedical Institute, and

5Department of Molecular Physiology and Biophysics, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

6Department of Biomedical Engineering, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: David A. Stoltz, Department of Internal Medicine, University of Iowa, 6322 PBDB, 169 Newton Road, Iowa City, Iowa 52242, USA. Phone: 319.356.4419; Email: david-stoltz@uiowa.edu.

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1Department of Internal Medicine,

2Department of Pediatrics,

3Department of Pathology,

4Pappajohn Biomedical Institute, and

5Department of Molecular Physiology and Biophysics, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

6Department of Biomedical Engineering, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: David A. Stoltz, Department of Internal Medicine, University of Iowa, 6322 PBDB, 169 Newton Road, Iowa City, Iowa 52242, USA. Phone: 319.356.4419; Email: david-stoltz@uiowa.edu.

Find articles by Lei, L. in: JCI | PubMed | Google Scholar |

1Department of Internal Medicine,

2Department of Pediatrics,

3Department of Pathology,

4Pappajohn Biomedical Institute, and

5Department of Molecular Physiology and Biophysics, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

6Department of Biomedical Engineering, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: David A. Stoltz, Department of Internal Medicine, University of Iowa, 6322 PBDB, 169 Newton Road, Iowa City, Iowa 52242, USA. Phone: 319.356.4419; Email: david-stoltz@uiowa.edu.

Find articles by Yu, W. in: JCI | PubMed | Google Scholar |

1Department of Internal Medicine,

2Department of Pediatrics,

3Department of Pathology,

4Pappajohn Biomedical Institute, and

5Department of Molecular Physiology and Biophysics, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

6Department of Biomedical Engineering, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: David A. Stoltz, Department of Internal Medicine, University of Iowa, 6322 PBDB, 169 Newton Road, Iowa City, Iowa 52242, USA. Phone: 319.356.4419; Email: david-stoltz@uiowa.edu.

Find articles by Thurman, A. in: JCI | PubMed | Google Scholar |

1Department of Internal Medicine,

2Department of Pediatrics,

3Department of Pathology,

4Pappajohn Biomedical Institute, and

5Department of Molecular Physiology and Biophysics, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

6Department of Biomedical Engineering, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: David A. Stoltz, Department of Internal Medicine, University of Iowa, 6322 PBDB, 169 Newton Road, Iowa City, Iowa 52242, USA. Phone: 319.356.4419; Email: david-stoltz@uiowa.edu.

Find articles by Gansemer, N. in: JCI | PubMed | Google Scholar |

1Department of Internal Medicine,

2Department of Pediatrics,

3Department of Pathology,

4Pappajohn Biomedical Institute, and

5Department of Molecular Physiology and Biophysics, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

6Department of Biomedical Engineering, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: David A. Stoltz, Department of Internal Medicine, University of Iowa, 6322 PBDB, 169 Newton Road, Iowa City, Iowa 52242, USA. Phone: 319.356.4419; Email: david-stoltz@uiowa.edu.

Find articles by Meyerholz, D. in: JCI | PubMed | Google Scholar |

1Department of Internal Medicine,

2Department of Pediatrics,

3Department of Pathology,

4Pappajohn Biomedical Institute, and

5Department of Molecular Physiology and Biophysics, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

6Department of Biomedical Engineering, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: David A. Stoltz, Department of Internal Medicine, University of Iowa, 6322 PBDB, 169 Newton Road, Iowa City, Iowa 52242, USA. Phone: 319.356.4419; Email: david-stoltz@uiowa.edu.

Find articles by Pezzulo, A. in: JCI | PubMed | Google Scholar |

1Department of Internal Medicine,

2Department of Pediatrics,

3Department of Pathology,

4Pappajohn Biomedical Institute, and

5Department of Molecular Physiology and Biophysics, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

6Department of Biomedical Engineering, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: David A. Stoltz, Department of Internal Medicine, University of Iowa, 6322 PBDB, 169 Newton Road, Iowa City, Iowa 52242, USA. Phone: 319.356.4419; Email: david-stoltz@uiowa.edu.

Find articles by McCray, P. in: JCI | PubMed | Google Scholar |

1Department of Internal Medicine,

2Department of Pediatrics,

3Department of Pathology,

4Pappajohn Biomedical Institute, and

5Department of Molecular Physiology and Biophysics, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

6Department of Biomedical Engineering, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: David A. Stoltz, Department of Internal Medicine, University of Iowa, 6322 PBDB, 169 Newton Road, Iowa City, Iowa 52242, USA. Phone: 319.356.4419; Email: david-stoltz@uiowa.edu.

Find articles by Thornell, I. in: JCI | PubMed | Google Scholar |

1Department of Internal Medicine,

2Department of Pediatrics,

3Department of Pathology,

4Pappajohn Biomedical Institute, and

5Department of Molecular Physiology and Biophysics, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA.

6Department of Biomedical Engineering, University of Iowa, Iowa City, Iowa, USA.

Address correspondence to: David A. Stoltz, Department of Internal Medicine, University of Iowa, 6322 PBDB, 169 Newton Road, Iowa City, Iowa 52242, USA. Phone: 319.356.4419; Email: david-stoltz@uiowa.edu.

Find articles by Stoltz, D. in: JCI | PubMed | Google Scholar |

Published September 10, 2024 - More info

Published in Volume 134, Issue 21 on November 1, 2024
J Clin Invest. 2024;134(21):e181995. https://doi.org/10.1172/JCI181995.
© 2024 Ibarra et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published September 10, 2024 - Version history
Received: April 22, 2024; Accepted: September 6, 2024 View PDF Abstract

The airway surface liquid (ASL) plays a crucial role in lung defense mechanisms, and its composition and volume are regulated by the airway epithelium. The cystic fibrosis transmembrane conductance regulator (CFTR) is abundantly expressed in a rare airway epithelial cell type called an ionocyte. Recently, we demonstrated that ionocytes can increase liquid absorption through apical CFTR and basolateral barttin/chloride channels, while airway secretory cells mediate liquid secretion through apical CFTR channels and basolateral NKCC1 transporters. Th2-driven (IL-4/IL-13) airway diseases, such as asthma, cause goblet cell metaplasia, accompanied by increased mucus production and airway secretions. In this study, we investigate the effect of IL-13 on chloride and liquid transport performed by ionocytes. IL-13 treatment of human airway epithelia was associated with reduced epithelial liquid absorption rates and increased ASL volume. Additionally, IL-13 treatment reduced the abundance of CFTR-positive ionocytes and increased the abundance of CFTR-positive secretory cells. Increasing ionocyte abundance attenuated liquid secretion caused by IL-13. Finally, CFTR-positive ionocytes were less common in asthma and chronic obstructive pulmonary disease and were associated with airflow obstruction. Our findings suggest that loss of CFTR in ionocytes contributes to the liquid secretion observed in IL-13–mediated airway diseases.

Introduction

The airway surface liquid (ASL) that overlies the respiratory tract epithelium is an important component of host defense. The composition of this thin layer of liquid is tightly regulated and maintains ciliary function, mucus biophysical properties, and mucociliary transport (1–4). ASL also contains antimicrobial proteins and peptides that are important in the lung’s innate defense system (5, 6). Changes in ASL pH and volume have been implicated in airway diseases including cystic fibrosis (CF), asthma, and chronic obstructive pulmonary disease (COPD) (2, 5, 7–10).

Interleukin-13 (IL-13) is an inflammatory cytokine that plays a role in allergic inflammation often seen in Th2 inflammatory responses (11–14). Individuals with asthma and COPD are more likely to have elevated levels of IL-13 (11, 12), and these diseases are typically characterized by mucus hypersecretion and goblet cell hyperplasia (15–18). Previous studies using human tracheobronchial epithelial cells cultured at the air-liquid interface have shown that Th2 cytokines, IL-4 and IL-13, induce goblet cell hyperplasia and mucus hypersecretion (14, 19–26). Further, inhibition of IL-13–mediated signaling can reverse goblet cell hyperplasia (27, 28), suggesting that IL-13 may contribute to the pathophysiology of airway diseases such as asthma and COPD.

Despite accounting for approximately 1% of the cells in the airway, ionocytes express very high levels of cystic fibrosis transmembrane conductance regulator (CFTR) channels compared with secretory cells (29–32). We recently reported that whereas CFTR channels in human airway epithelial secretory cells increase transepithelial liquid secretion, CFTR channels in ionocytes increase liquid absorption across healthy airways (33), suggesting that cellular segregation of CFTR channels allows for control of ASL volume. Th2 inflammatory responses associated with subsets of asthma and COPD increase both Cl– and mucus secretion (9, 13, 34, 35). How Th2 inflammatory responses, which increase Cl– and mucus secretion through secretory cells, affect ionocytes is unknown. In this study, we investigated how IL-13 impacts airway liquid handling and whether IL-13 treatment affects ionocyte number and/or function.

Results

IL-13 exposure increases goblet cell abundance and Cl– secretion. Treating airway epithelia with IL-13 increased the number of MUC5AC-positive goblet cells (Figure 1, A and B). We evaluated the effect of IL-13 on CFTR-mediated Cl– secretion using the short-circuit current (Isc) technique (Figure 1C). Adding the epithelial Na+ channel (ENaC) inhibitor amiloride induced a larger fall in Isc for control epithelia but only a minor decrease for IL-13 conditions (Figure 1, C and D). The change in Isc (ΔIsc) with forskolin and IBMX was greater for IL-13–treated epithelia compared with controls (Figure 1, C and D). Similarly, TMEM16A inhibition, with Ani9, and CFTR inhibition, with CFTRinh-172, caused a greater ΔIsc for IL-13–treated compared with untreated epithelia (Figure 1, C and D). Inhibition of the basolateral NKCC1 transporter with bumetanide dissipated the remaining Isc (Figure 1, C and D). Therefore, IL-13 treatment increased transepithelial Cl– secretion. The large residual Isc after amiloride treatment produced by IL-13–treated epithelia (Figure 1, C and D) indicated that IL-13 altered the driving force for Na+ across ENaC.

Figure 1

IL-13 treatment increases airway epithelial goblet cell number and bumetanide-sensitive Isc. (A) MUC5AC immunostaining of human airway epithelial cells treated with vehicle control or IL-13 for 6 or 28 days. Scale bar: 100 μm. (B) Quantification of goblet cell abundance (MUC5AC-positive cells) by flow cytometry at various time points following vehicle control or IL-13 treatment. Data are from 8 independent donors. Each symbol represents an individual donor at a given time point. Not all donors are represented at each time point. (C) Representative short-circuit current (Isc) traces of human airway epithelial cultures exposed to vehicle control or IL-13. The following agents were added sequentially: apical amiloride, apical forskolin/IBMX, apical Ani9, apical CFTRinh-172, and basolateral bumetanide. n = 5 donor epithelia per treatment group. (D) Summary Isc values for donor epithelia treated with vehicle control or IL-13 for 3–4 weeks. Ctrl, control.

The post-amiloride increase in Isc with IL-13 depended on CFTR, since IL-13 failed to increase post-amiloride Isc values when CF epithelia were studied (Supplemental Figure 1, A and B; supplemental material available online with this article; https://doi.org/10.1172/JCI181995DS1). Furthermore, in these experiments and additional experiments performed on non-CF epithelia bathed in nominally Cl–-free solutions, IL-13 did not affect amiloride-sensitive Isc values, suggesting that the large increase in Cl– channel function produced the difference in amiloride-sensitive Isc observed after treatment of non-CF epithelia with IL-13 (Supplemental Figure 1, A–D).

IL-13 treatment reduces barttin-positive ionocyte abundance. In healthy human airway epithelia, Lei et al. found that CFTR channels on secretory cells participate in Cl– secretion while ionocytes absorb Cl– through apical CFTR channels and ionocyte-specific basolateral barttin/Cl– channels (33). To evaluate how these barttin-containing ionocytes respond to IL-13, we optimized a multicolor flow cytometry approach to determine the cellular composition of airway epithelia (Figure 2, A–C, and Supplemental Figure 2). IL-13 treatment increased the number of goblet cells and reduced ciliated cell abundance (Figure 2, D and E). Surprisingly, we detected about half as many barttin-positive ionocytes in IL-13–treated epithelia compared with controls (Figure 2E). At the transcript level, IL-13 increased epithelial CFTR expression, yet decreased epithelial BSND, which encodes ionocyte-specific barttin (Figure 2, F and G). These data suggest that IL-13 increases CFTR in secretory cells while simultaneously decreasing the absorption function of ionocytes.

Figure 2

IL-13 reduces barttin-positive ionocyte abundance in airway epithelia. Human airway epithelial cultures were dissociated and analyzed by multicolor flow cytometry. (A–C) Top row represents vehicle control epithelia, and lower row represents epithelia treated with IL-13 for 3 weeks. (A) Panels are gated on live singlets and show NGFR detection levels by barttin (BSND) levels to identify basal cells (teal) and ionocytes (pink). (B) Panels are gated on gray gate in A (NGFR-negative, barttin-negative) and depict CD66c detection by MUC5AC detection. Goblet cells are indicated by dark blue gate. (C) Panels are gated on non-goblet cells (MUC5AC-negative) gray gate in B and show cells by detection of CD66c versus α-tubulin. Ciliated cells are α-tubulin–positive. (D) User-defined gates from flow cytometric data graphed in A–C displayed as a t-distributed stochastic neighbor embedding (t-SNE) plot. (E) Cell type abundance quantified from flow cytometry data in vehicle control (gray symbols) or IL-13–treated (red symbols) epithelia. (F and G) Total epithelial transcript levels for CFTR (F) and BSND (gene encoding barttin) (G). Data points connected by a line represent paired experiments from a single human donor. Data are shown as mean ± SD. P values were obtained using a 2-way ANOVA with multiple comparisons or paired, 2-sided Student’s t test. Ctrl, control. *P < 0.05; ***P < 0.001; ****P < 0.0001.

IL-13 increases the number of CFTR-positive goblet cells, but decreases the number of CFTR-positive ionocytes. Finding an increase in CFTR transcripts and a decrease in BSND transcripts and barttin-positive ionocytes suggested that secretory cells drove the increase in CFTR. To test this hypothesis, we performed immunofluorescence confocal microscopy after a time course of IL-13 exposures to investigate which cell types express CFTR. IL-13 exposure increased the total number of cells and cells with CFTR within 6 days (Figure 3, A and B). CFTR was nearly always observed on goblet cells, and IL-13 increased the number of goblet cells as early as day 4 (Figure 3, C and D). Accompanying the increase in goblet cells was a decrease in ciliated cells (Figure 3E). We rarely detected CFTR in ciliated cells (Figure 3, B and E). IL-13 treatment decreased the number of barttin-positive ionocytes and CFTR detection in remaining ionocytes as early as 4 days, and CFTR was rarely detected in ionocytes by day 12 (Figure 3, B, F, and G). Using fluorescence in situ hybridization, we found that IL-13 treatment reduced CFTR mRNA expression on ionocytes (Supplemental Figure 3). These results and our flow cytometry data indicate that an increase in CFTR-expressing goblet cells drives the increase in CFTR during IL-13 exposure, and any remaining barttin-positive ionocytes lack CFTR.

Figure 3

IL-13 rapidly reduces CFTR detection on ionocytes and increases goblet cell abundance. Immunofluorescence quantification and representative confocal microscopy images of different cell types and percentage of CFTR-positive cells over time in vehicle control and IL-13–treated epithelia. (A) Quantification of all cell types (DAPI-positive) and CFTR detection. (B) Epithelia were defined by DAPI (gray, all nuclei), CFTR (yellow), barttin (magenta, ionocytes), and α-tubulin (cyan, ciliated cells). (C) Quantification of goblet cells (MUC5AC-positive) and CFTR detection. (D) Epithelia were defined by DAPI (gray, all nuclei), CFTR (yellow), barttin (magenta, ionocytes), and MUC5AC (cyan, goblet cells). (E) Quantification of ciliated cells (α-tubulin–positive) and CFTR detection. (F) Quantification of ionocytes (barttin-positive) and CFTR detection. (G) Epithelia were defined for CFTR (yellow) and barttin (magenta, to identify ionocytes). Yellow arrows denote CFTR-positive ionocytes. Data are shown as mean ± SD. For percentage CFTR graphs, each symbol represents an independent donor. P values were obtained using an ordinary 1-way ANOVA with Dunnett’s multiple-comparison test between treatment time points versus day 0 controls. n = 3–7 independent human donors. (B and D, top panels, and G): XY projection. (B and D, bottom panels): Z projections taken at the dashed line and shown below the XY image. Scale bars: 15 μm. *P < 0.05; ****P < 0.0001.

IL-13 treatment reduces transepithelial liquid absorption. Since IL-13 decreased CFTR and barttin in ionocytes, we hypothesized that IL-13 would decrease liquid absorption. We tested this hypothesis using 2 approaches. First, we measured ASL volume and found that IL-13 treatment significantly increased ASL volume in cultured epithelium, a finding consistent with reduced transepithelial liquid absorption (Figure 4A). Second, we directly assayed the epithelial liquid absorption rate by adding saline to the apical epithelial surface and 4 hours later measuring the recovered liquid on the apical surface (33, 36–38). Under control conditions, human airway epithelia are absorptive (36–38). Therefore, we predicted that IL-13 treatment would reduce liquid absorption. IL-13–treated epithelia had a decreased liquid absorption rate, and we occasionally observed liquid secretion (Figure 4B). This shift toward a liquid-secretion phenotype, as indicated by a decrease in absorption, began as early as 3 days (Figure 4B). Thus, IL-13 created a less absorptive epithelium, consistent with our observation that after IL-13 exposure most epithelial CFTR channels exist in secretory cells where CFTR mediates Cl– secretion.

Figure 4

IL-13 treatment decreases airway epithelial liquid absorption. (A) ASL volume of control and IL-13–treated human airway epithelial cultures. (B) Liquid flux (Jv) in epithelial cultures at various time points after IL-13 exposure and their respective controls. n = 5–14 independent human donors. Data are shown as mean ± SD. P values were obtained by a paired 2-tailed t test or mixed-effects analysis using Dunnett’s multiple-comparison test comparing treatment time points with controls (day 0). Ctrl, control. *P < 0.05; **P < 0.01; ***P < 0.001.

Increasing ionocyte abundance limits IL-13–induced liquid secretion. We hypothesized that reintroducing ionocytes to IL-13–treated epithelia would increase ASL absorption. Forkhead box I1 (FOXI1) overexpression increases ionocyte number and liquid absorption in airway epithelial cultures (29, 30, 33, 39). Using viral vectors, we expressed GFP or FOXI1-GFP, then treated airway epithelia with IL-13 (Figure 5A). We found that FOXI1 overexpression increased the number of barttin-positive, NGFR-positive ionocytes about 100-fold in both control and IL-13–treated epithelia (Figure 5, B–D). Using CF epithelia, we previously reported that ionocytes provide a CFTR-dependent pathway for passive Cl– absorption across the apical membrane (33). We performed a similar series of experiments after increasing the number of ionocytes and treating the epithelia with vehicle or IL-13. Under transepithelial voltage-clamp conditions (Vclamp = 0), epithelia were treated with apical amiloride and apical 4,4′-dilsothiocyano-2,2′-stilbenedifulonic acid (DIDS) to isolate CFTR-dependent ion flow. Then we bathed the basolateral surface of epithelia in low-[Cl–] solutions to drive Cl– movement from the apical to the basolateral chamber. Finally, we revealed CFTR-mediated apical-to-basolateral Cl– flow by treating epithelia with forskolin/IBMX and the CFTR inhibitor CFTRinh-172 (Figure 6, A–C) (33). Compared with control epithelia, CFTRinh-172 blocked less transepithelial current (It) in IL-13–treated epithelia (Figure 6, A–D), consistent with our earlier findings that IL-13 treatment reduced ionocyte number and ionocyte CFTR expression. In epithelia overexpressing FOXI1, we saw a significant increase in the CFTR-dependent It in the absence or presence of IL-13 (Figure 6D). Finally, increasing the number of ionocytes, in both control and IL-13–treated epithelia, significantly decreased ASL volume and increased the liquid absorption rate (Figure 6, E and F). These results indicate that a decrease in ionocyte-mediated ASL absorption contributes to the IL-13–mediated increase in ASL volume.

Figure 5

Overexpressing FOXI1 increases ionocyte abundance in IL-13–treated epithelia. Human airway epithelia were transduced with a lentivirus expressing GFP or FOXI1 and GFP, to generate and label ionocytes, respectively. Epithelia were then treated with vehicle control or IL-13 for 24 days. (A) Representative flow cytometry plots showing live, single cells. Cells are plotted by NGFR and barttin detection. (B) The relative abundance (percent) of ionocytes (NGFR-positive, barttin-positive events) within live single cells using flow cytometry. Data points connected by a line represent paired experiments from a single human donor. Data are shown as mean ± SD. (C) Confocal images of airway epithelia stained with DAPI (gray, all nuclei), CFTR (yellow), and barttin (magenta, ionocytes). Scale bar: 15 μm. (D) XZ projections of the confocal images in C taken at the dashed lines showing DAPI (gray, all nuclei), CFTR (yellow), barttin (magenta, ionocytes), and GFP (green). Scale bar: 15 μm. Ctrl, control; GFP, green fluorescent protein. *P < 0.05; ****P < 0.0001.

Figure 6

Increasing ionocyte abundance limits IL-13–induced liquid secretion. Human airway epithelia were transduced with a lentivirus expressing GFP or FOXI1 and GFP, to generate and label ionocytes. Epithelia were then treated with vehicle control or IL-13 for 24 days. (A–C) Representative transepithelial current (It) tracings. To drive passive Cl– current into cells through apical CFTR channels and out through basolateral barttin/Cl– channels, transepithelial voltage was held at 0 mV and the basolateral [Cl–] was reduced (depicted in inset). (A) No IL-13 treatment (control). (B) IL-13 treatment. (C) Focus on CFTRinh-172–sensitive It generated by GFP controls from A and B. (D) CFTRinh-172–sensitive It summary data. (E) ASL volume of human airway epithelial cultures. (F) Liquid flux (Jv) in epithelial cultures. Data points connected by a line represent paired experiments from a single human donor. Data are shown as mean ± SD. P values were obtained by a 2-way ANOVA with Šidák’s multiple-comparison test. Baso, basolateral; Ctrl, control; DIDS, 4,4′-dilsothiocyano-2,2′-stilbenedifulonic acid; F&I, forskolin and 3-isobutyl-1-methylxanthine; GFP, green fluorescent protein; Inh-172, CFTRinh-172; It, transepithelial current. *P < 0.05; **P < 0.01; ****P < 0.0001.

Ionocyte CFTR expression is reduced in IL-13–associated human airway diseases. We found that IL-13 treatment caused significant changes in ionocyte number and CFTR expression. Thus, we hypothesized that ionocytes in human airway diseases with elevated IL-13 would also exhibit altered CFTR detection. Asthma and COPD are both heterogeneous diseases, with their phenotypes associated with IL-13 (11, 12, 40, 41). To test our hypothesis, we performed immunofluorescence microscopy on formalin-fixed lung sections from deceased donors. We obtained lung sections from 1 control without lung disease, 2 COPD donors, a donor with asthma, and a donor who died from an asthma exacerbation (status asthmaticus). We stained serial sections for MUC5AC and tubulin, to look for evidence of goblet cell metaplasia, or for barttin and CFTR. In the control donor, we consistently observed strong CFTR labeling at the apical surface of barttin-positive ionocytes (Figure 7A). In COPD and asthma samples, we found an increased number of MUC5AC-positive cells (goblet cell metaplasia), consistent with elevated exposure to IL-13 (Supplemental Figure 4). However, we did not detect apical CFTR on most ionocytes but detected CFTR staining on other cells (Figure 7, B and C, and Supplemental Figure 5). These findings are consistent with our results from IL-13–treated human airway epithelia.

Figure 7

CFTR loss in ionocytes is associated with worsening airflow obstruction in asthmatic subjects. (A–C) Lung histology sections from deceased human donors stained with CFTR (yellow), barttin (magenta, ionocytes), and β-tubulin (cyan, ciliated cells). Sections were obtained from a control without known lung disease (A) and subjects with a history of asthma (B) and status asthmaticus (C). Scale bar: 15 μm. (D–J) scRNA-Seq and pulmonary function data are from Vieira Braga et al. (42). Data are from 6 asthmatic subjects and 6 age-matched controls. scRNA-Seq data were obtained from tracheal biopsies. The authors’ cluster definitions were preserved for this analysis. (D and E) t-SNE plots with cell type clusters for control and asthmatic subjects. (F) Ionocyte cluster. (G) Relative contributions of cell types per subject (individual symbols) by cell cluster identity. (H) Normalized ionocyte CFTR expression for each subject. Within a subject, individual ionocytes are each represented by a symbol. The percentage of ionocytes with CFTR was evaluated for subjects with more than 3 ionocytes. (I) Percentage of ionocytes with CFTR. Each symbol represents a different subject. (J) The percentage of CFTR-positive ionocytes was plotted versus FEV1% predicted for control and asthmatic subjects. The black line represents a nonlinear best-fit model. Data are shown as mean ± SD. P values were obtained by 2-tailed Mann-Whitney ranking test comparing asthmatic donors with control donors. Asth, asthma; Ctrl, control; FEV1, forced expiratory volume in 1 second. *P < 0.05.

To further investigate ionocyte CFTR expression in asthma, we analyzed previously published single-cell RNA sequencing (scRNA-Seq) data obtained from lower-airway biopsies of asthma patients or controls (Figure 7, D–F) (42). We preserved the cluster classifications designated by the original authors. The clustering revealed more goblet cells in people with asthma compared with controls; however, ionocytes occurred at a similar frequency (Figure 7, E–G). We next determined the mean expression of CFTR per ionocyte for each participant. We restricted our analysis to samples with more than 3 ionocytes, which excluded 2 controls and 1 person with asthma. Nearly all control ionocytes contained CFTR (Figure 7H). In contrast, ionocytes from people with asthma appeared as 2 populations: one population expressed CFTR mRNA at levels comparable to controls, while the other population of ionocytes lacked CFTR mRNA (Figure 7H). Overall, the percentage of ionocytes with CFTR was reduced in asthmatic samples (Figure 7I). Interestingly, when we compared the number of ionocytes with detectable CFTR in a participant with their spirometry, we found that the CFTR-positive ionocyte number fell with worsening airflow obstruction (Figure 7J). These data suggest that in asthma and other IL-13–driven diseases the absorption property of ionocytes involving CFTR and barttin/Cl– channels could be affected, while the total number of ionocytes might remain unchanged.

Discussion

We found that IL-13 has divergent effects on CFTR expression/function in different types of airway epithelial cells, secretory cells versus ionocytes. We and others have shown that IL-13– and/or IL-4–mediated Th2 airway inflammation increases goblet cell number, CFTR expression, and Cl– secretion and is accompanied by increased ASL volume (19–21, 25, 27, 43–49). The ionocyte’s role in this response has been unknown. Unexpectedly, we discovered that IL-13 decreases the number of CFTR-rich barttin-positive ionocytes and eliminates apical CFTR detection in the remaining ionocytes. Thus, IL-13 increases ASL volume by both increasing Cl– secretion, through secretory cells, and inhibiting Cl– absorption, through ionocytes.

The discovery of ionocytes has prompted multiple groups to consider how CFTR is differentially regulated in different cells (33, 39, 50, 51). These studies underlie the idea that CFTR has opposing functions depending on the cell type that expresses it. This current study furthers the idea that CFTR performs Cl– secretion in secretory cells and contributes to fluid absorption in ionocytes. By challenging epithelia with IL-13, we demonstrated that the same agonist has opposing effects on CFTR regulation in a cell type–specific manner. IL-13 increases the contribution from secretory cells to epithelial CFTR function, while decreasing the contribution from ionocytes to epithelial CFTR function. These changes were associated with an increase in ASL volume. How specific cell types differentially regulate CFTR expression remains to be determined, but could have therapeutic implications.

The mechanism(s) underlying reduced ionocyte CFTR expression following IL-13 treatment are unknown. Sonic hedgehog (SHH) signaling was recently discovered to play a key role in ionocyte specification (52). Inhibition of SHH signaling reduced both the total number of barttin-positive ionocytes and the fraction of barttin-positive cells that were CFTR-positive; these results are similar to what we found with IL-13 treatment. However, several studies found that IL-4/IL-13 enhances SHH signaling, which would not fit with our findings. Thus, whether changes in SHH signaling account for our results is unknown and could depend on differences in cell culture models, in vitro versus in vivo conditions, and/or acute versus chronic treatments. While we focused our study on the effect of IL-13 on CFTR in ionocytes, it will be important in the future to investigate the effect of IL-13 on other ionocyte-specific proteins, including ATP6v0d2, and the transcription factors FOXI1 and ASCL3 (29, 30, 52).

IL-13 is increased in patients with airway disease, including asthma and COPD (11, 12, 40, 41). To determine the relevance of our in vitro findings for human disease, we used 2 approaches. First, we obtained lung histological samples from patients with COPD, asthma, and status asthmaticus. We hypothesized that ionocytes in Th2-diseased lungs would have reduced CFTR levels. In healthy control lungs, we consistently found that barttin-positive ionocytes had strong CFTR immunofluorescence at the apical surface. In contrast, in all 4 donors with airway disease, barttin-positive ionocytes lacked detectable CFTR immunofluorescence. This was associated with an increase in goblet cell abundance in all donor samples. Second, to further investigate how IL-13 might alter ionocyte morphology in the context of disease, we explored published scRNA-Seq databases. We found that asthmatic patients had higher goblet cell proportions, and that ionocytes from asthmatic donors were more likely to lack CFTR than ionocytes from healthy controls. How losing CFTR expression on ionocytes alters airway physiology or contributes to disease is unknown. However, we found an association between the proportion of ionocytes with CFTR in asthmatic patients and airflow obstruction. Whether the loss of ionocyte CFTR is contributing to worsening pulmonary function or simply reflects a more advanced disease state is unknown and requires further study.

The degree to which ionocytes contribute to ASL volume regulation could vary by region, and upper versus lower airway disease may differentially impact ionocyte function. Scudieri et al. found a “proximal-to-distal” gradient of ionocytes with more ionocytes present in nasal versus bronchial samples (53). In the nasal mucosa of children with chronic rhinosinusitis, Han et al. found that the number of CFTR-positive ionocytes was reduced (54). These data suggest that the changes we observed could occur in both upper and lower airways.

The therapeutic implications of our findings are unknown. However, this work raises several questions. First, in the setting of increased mucus secretion, is it beneficial or harmful to switch from an absorptive to secretory epithelium? Earlier work from Galietta and colleagues demonstrated that bicarbonate secretion is required for effective mucin release in IL-4–treated airway epithelial cells (26), suggesting that a secretory phenotype is likely beneficial in the setting of goblet cell hyperplasia. Second, do biologics that target the IL-4/IL-13 pathway, in asthma or COPD, affect the number of CFTR-rich barttin-positive ionocytes (55, 56)? And if so, are there therapeutic effects? Finally, does this work have implications for CF? In the absence of functional CFTR, other strategies to enhance liquid secretion could be beneficial, such as targeting other chloride channels (57, 58).

Our study has strengths and limitations. Its strengths include: (a) We optimized a multicolor flow cytometry–based approach to study epithelial populations. Other groups have published techniques for using flow cytometry on airway epithelial cells (59, 60). We were able to capture the diverse range of cells in airway epithelia. Our technique has the distinct advantages that it uses markers commonly used in airway biology; results in discrete, mutually exclusive populations; and is scal