Research ArticleCell biologyPulmonology Open Access | 10.1172/jci.insight.187904

Katrina M. Kudrna,1 Luis F. Vilches,1 Evan M. Eilers,1 Shailendra K. Maurya,1 Steven L. Brody,2 Amjad Horani,3 Kristina L. Bailey,1,4 Todd A. Wyatt,1,4,5 and John D. Dickinson1

1Department of Internal Medicine, Division of Pulmonary, Critical Care, & Sleep, University of Nebraska Medical Center, Omaha, Nebraska, USA.

2Department of Medicine, Division of Pulmonary and Critical Care, Washington University, St. Louis, Missouri, USA.

3Washington University School of Medicine, Division of Pediatric Allergy, Immunology, and Pulmonary Medicine, St. Louis, Missouri, USA.

4Department of Veterans Affairs Nebraska-Western Iowa Health Care System, Omaha, Nebraska, USA.

5Department of Environmental, Agricultural and Occupational Health, College of Public Health, University of Nebraska Medical Center, Omaha, Nebraska, USA.

Address correspondence to: John D. Dickinson, MD, PhD., Division of Pulmonary, Critical Care, & Sleep Medicine, University of Nebraska Medical Center, 985910 Nebraska Medical Center, Omaha, Nebraska, 68198, USA. Phone: 402.559.8442; Email: jdickins@unmc.edu.

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1Department of Internal Medicine, Division of Pulmonary, Critical Care, & Sleep, University of Nebraska Medical Center, Omaha, Nebraska, USA.

2Department of Medicine, Division of Pulmonary and Critical Care, Washington University, St. Louis, Missouri, USA.

3Washington University School of Medicine, Division of Pediatric Allergy, Immunology, and Pulmonary Medicine, St. Louis, Missouri, USA.

4Department of Veterans Affairs Nebraska-Western Iowa Health Care System, Omaha, Nebraska, USA.

5Department of Environmental, Agricultural and Occupational Health, College of Public Health, University of Nebraska Medical Center, Omaha, Nebraska, USA.

Address correspondence to: John D. Dickinson, MD, PhD., Division of Pulmonary, Critical Care, & Sleep Medicine, University of Nebraska Medical Center, 985910 Nebraska Medical Center, Omaha, Nebraska, 68198, USA. Phone: 402.559.8442; Email: jdickins@unmc.edu.

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1Department of Internal Medicine, Division of Pulmonary, Critical Care, & Sleep, University of Nebraska Medical Center, Omaha, Nebraska, USA.

2Department of Medicine, Division of Pulmonary and Critical Care, Washington University, St. Louis, Missouri, USA.

3Washington University School of Medicine, Division of Pediatric Allergy, Immunology, and Pulmonary Medicine, St. Louis, Missouri, USA.

4Department of Veterans Affairs Nebraska-Western Iowa Health Care System, Omaha, Nebraska, USA.

5Department of Environmental, Agricultural and Occupational Health, College of Public Health, University of Nebraska Medical Center, Omaha, Nebraska, USA.

Address correspondence to: John D. Dickinson, MD, PhD., Division of Pulmonary, Critical Care, & Sleep Medicine, University of Nebraska Medical Center, 985910 Nebraska Medical Center, Omaha, Nebraska, 68198, USA. Phone: 402.559.8442; Email: jdickins@unmc.edu.

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1Department of Internal Medicine, Division of Pulmonary, Critical Care, & Sleep, University of Nebraska Medical Center, Omaha, Nebraska, USA.

2Department of Medicine, Division of Pulmonary and Critical Care, Washington University, St. Louis, Missouri, USA.

3Washington University School of Medicine, Division of Pediatric Allergy, Immunology, and Pulmonary Medicine, St. Louis, Missouri, USA.

4Department of Veterans Affairs Nebraska-Western Iowa Health Care System, Omaha, Nebraska, USA.

5Department of Environmental, Agricultural and Occupational Health, College of Public Health, University of Nebraska Medical Center, Omaha, Nebraska, USA.

Address correspondence to: John D. Dickinson, MD, PhD., Division of Pulmonary, Critical Care, & Sleep Medicine, University of Nebraska Medical Center, 985910 Nebraska Medical Center, Omaha, Nebraska, 68198, USA. Phone: 402.559.8442; Email: jdickins@unmc.edu.

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1Department of Internal Medicine, Division of Pulmonary, Critical Care, & Sleep, University of Nebraska Medical Center, Omaha, Nebraska, USA.

2Department of Medicine, Division of Pulmonary and Critical Care, Washington University, St. Louis, Missouri, USA.

3Washington University School of Medicine, Division of Pediatric Allergy, Immunology, and Pulmonary Medicine, St. Louis, Missouri, USA.

4Department of Veterans Affairs Nebraska-Western Iowa Health Care System, Omaha, Nebraska, USA.

5Department of Environmental, Agricultural and Occupational Health, College of Public Health, University of Nebraska Medical Center, Omaha, Nebraska, USA.

Address correspondence to: John D. Dickinson, MD, PhD., Division of Pulmonary, Critical Care, & Sleep Medicine, University of Nebraska Medical Center, 985910 Nebraska Medical Center, Omaha, Nebraska, 68198, USA. Phone: 402.559.8442; Email: jdickins@unmc.edu.

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1Department of Internal Medicine, Division of Pulmonary, Critical Care, & Sleep, University of Nebraska Medical Center, Omaha, Nebraska, USA.

2Department of Medicine, Division of Pulmonary and Critical Care, Washington University, St. Louis, Missouri, USA.

3Washington University School of Medicine, Division of Pediatric Allergy, Immunology, and Pulmonary Medicine, St. Louis, Missouri, USA.

4Department of Veterans Affairs Nebraska-Western Iowa Health Care System, Omaha, Nebraska, USA.

5Department of Environmental, Agricultural and Occupational Health, College of Public Health, University of Nebraska Medical Center, Omaha, Nebraska, USA.

Address correspondence to: John D. Dickinson, MD, PhD., Division of Pulmonary, Critical Care, & Sleep Medicine, University of Nebraska Medical Center, 985910 Nebraska Medical Center, Omaha, Nebraska, 68198, USA. Phone: 402.559.8442; Email: jdickins@unmc.edu.

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

1Department of Internal Medicine, Division of Pulmonary, Critical Care, & Sleep, University of Nebraska Medical Center, Omaha, Nebraska, USA.

2Department of Medicine, Division of Pulmonary and Critical Care, Washington University, St. Louis, Missouri, USA.

3Washington University School of Medicine, Division of Pediatric Allergy, Immunology, and Pulmonary Medicine, St. Louis, Missouri, USA.

4Department of Veterans Affairs Nebraska-Western Iowa Health Care System, Omaha, Nebraska, USA.

5Department of Environmental, Agricultural and Occupational Health, College of Public Health, University of Nebraska Medical Center, Omaha, Nebraska, USA.

Address correspondence to: John D. Dickinson, MD, PhD., Division of Pulmonary, Critical Care, & Sleep Medicine, University of Nebraska Medical Center, 985910 Nebraska Medical Center, Omaha, Nebraska, 68198, USA. Phone: 402.559.8442; Email: jdickins@unmc.edu.

Find articles by Bailey, K. in: PubMed | Google Scholar

1Department of Internal Medicine, Division of Pulmonary, Critical Care, & Sleep, University of Nebraska Medical Center, Omaha, Nebraska, USA.

2Department of Medicine, Division of Pulmonary and Critical Care, Washington University, St. Louis, Missouri, USA.

3Washington University School of Medicine, Division of Pediatric Allergy, Immunology, and Pulmonary Medicine, St. Louis, Missouri, USA.

4Department of Veterans Affairs Nebraska-Western Iowa Health Care System, Omaha, Nebraska, USA.

5Department of Environmental, Agricultural and Occupational Health, College of Public Health, University of Nebraska Medical Center, Omaha, Nebraska, USA.

Address correspondence to: John D. Dickinson, MD, PhD., Division of Pulmonary, Critical Care, & Sleep Medicine, University of Nebraska Medical Center, 985910 Nebraska Medical Center, Omaha, Nebraska, 68198, USA. Phone: 402.559.8442; Email: jdickins@unmc.edu.

Find articles by Wyatt, T. in: PubMed | Google Scholar

1Department of Internal Medicine, Division of Pulmonary, Critical Care, & Sleep, University of Nebraska Medical Center, Omaha, Nebraska, USA.

2Department of Medicine, Division of Pulmonary and Critical Care, Washington University, St. Louis, Missouri, USA.

3Washington University School of Medicine, Division of Pediatric Allergy, Immunology, and Pulmonary Medicine, St. Louis, Missouri, USA.

4Department of Veterans Affairs Nebraska-Western Iowa Health Care System, Omaha, Nebraska, USA.

5Department of Environmental, Agricultural and Occupational Health, College of Public Health, University of Nebraska Medical Center, Omaha, Nebraska, USA.

Address correspondence to: John D. Dickinson, MD, PhD., Division of Pulmonary, Critical Care, & Sleep Medicine, University of Nebraska Medical Center, 985910 Nebraska Medical Center, Omaha, Nebraska, 68198, USA. Phone: 402.559.8442; Email: jdickins@unmc.edu.

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Published May 30, 2025 - More info

Published in Volume 10, Issue 13 on July 8, 2025
JCI Insight. 2025;10(13):e187904. https://doi.org/10.1172/jci.insight.187904.
© 2025 Kudrna 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 May 30, 2025 - Version history
Received: October 28, 2024; Accepted: May 21, 2025 View PDF Abstract

In asthma, airway epithelial remodeling is characterized by aberrant goblet cell metaplastic differentiation accompanied by epithelial cell hyperplasia and hypertrophy. These pathologic features in severe asthma indicate a loss of control of proliferation, cell size, differentiation, and migration. MTOR is a highly conserved pathway that regulates protein synthesis, cell size, and proliferation. We hypothesized that the balance between MTOR and autophagy regulates mucous cell metaplasia. Airways from individuals with severe asthma showed increased MTOR signaling by RPS6 phosphorylation, which was reproduced using an IL-13–activated model of primary human airway epithelial cells (hAEC). MTOR inhibition by rapamycin led to a decrease of IL-13–mediated cell hypertrophy, hyperplasia, and MUC5AC mucous metaplasia. BrdU labeling during IL-13–induced mucous metaplasia confirmed that MTOR was associated with increased basal-to-apical hAEC migration. MTOR activation by genetic deletion of Tsc2 in cultured mouse AECs increased IL-13–mediated hyperplasia, hypertrophy, and mucous metaplasia. Transcriptomic analysis of IL-13–stimulated hAEC identified MTOR-dependent expression of genes associated with epithelial migration and cytoskeletal organization. In summary, these findings point to IL-13–dependent and –independent roles of MTOR signaling in the development of pathogenic epithelial changes contributing to airway obstruction in severe asthma.

Graphical Abstract Introduction

Cells tightly regulate proliferation, size, and differentiation in response to nutrient availability, and inflammatory or infectious cues. Consequently, airway epithelial cells must adapt to external signals for specialized functions. Much is known about the control of the identity, localization, and function of individual airway epithelial cells in the healthy airway (1–3). However, during inflammation, injury, and repair, the maintenance of cell classification and function is perturbed. In response to type 2 airway inflammation, basal cells and secretory cells differentiate to large mucous-secreting cells that are tightly packed with mucin granules that are morphologically termed goblet cells (GC). Histologic studies from autopsy series (4) from fatal asthma attacks and endobronchial biopsies from mild and severe asthmatics (5, 6) have revealed loss of control of epithelial cell size, migration, and proliferation along with GC metaplasia. Increased airway wall thickness collectively is associated with mucous plugging, contributing to airway obstruction and loss of lung function (7). Type 2 cytokines, such as IL-13, play an important role in the development of airway obstruction driving epithelial hyperplasia, GC hypertrophy, mucous hypersecretion, and loss of polarity with aberrant migration (8–12). However, airway epithelial cellular migration in the context of type 2 inflammation remains unresolved. Traditional wounding assays have demonstrated conflicting results on the role of IL-13 on migration (8, 9). At a cellular level, the loss of control of epithelial cell size, proliferation, and migration in the context of dynamic exogenous inflammatory signaling associated with severe asthma is incompletely understood.

Mammalian target of rapamycin (MTOR) is a nutrient-sensing signaling protein complex that regulates cell size, metabolism, migration, and proliferation while inhibiting autophagy (13, 14). The MTOR pathway is a highly conserved serine-threonine kinase signaling complex located at the hub of a network. Two distinct signaling protein complexes (MTORC1 and MTORC2) exist with differing cofactors and phosphorylation targets. MTORC1 phosphorylates S6K1 and ribosomal S6 (RPS6) leading to increased protein synthesis and anabolic metabolism required for cell growth and proliferation (15–17). MTOR also inhibits autophagy through phosphorylation of unc-51 like autophagy activating kinase 1 (ULK1) and autophagy-related protein 13 (ATG13) (18–21). Macroautophagy (referred to here as autophagy) provides a counterbalance to these metabolic demands through bulk protein degradation-recycling proteins to amino acids for new synthesis (22–24).

We and others have reported that autophagy proteins are increased in the airway epithelium during cytokine-mediated mucous metaplasia in vitro (12, 25) and in diseased human airway samples (26). However, we have not been able to reconcile the profound morphologic differences driven by mitogenic effects of cytokines such as IL-13 on the epithelial hyperplasia, hypertrophy, and mucous cell metaplasia with increased autophagy signaling, which is catabolic in nature. Although autophagy is commonly thought to be activated in airway epithelial mucous cell metaplasia, the profound morphologic changes and increased protein production point to a more complex situation in which the balance of MTOR and autophagy may regulate key biologic processes critical to the development, persistence, and resolution of airway disease. Here, we propose a potentially new conceptual framework of mucous cell metaplasia in which the temporal balance of MTOR and autophagy control features of pathogenic mucous cell metaplasia. IL-13–mediated MTOR activation drives loss of control of airway epithelial cell proliferation, hypertrophy, and migration, contributing to airway wall thickening and formation of eGC (eGC), which are key features of mucous cell metaplasia found in severe asthma.

Results

Mucous cell metaplasia in severe asthma is characterized by the emergence of eGC associated with regions of epithelial hyperplasia and hypertrophy. In severe asthma, the airway epithelium undergoes remodeling characterized by increased epithelial thickness and mucous cell metaplasia with increased size and number of GC, collectively contributing to airway obstruction (4–6, 27, 28). To identify key pathologic features of the asthmatic airway epithelia, we examined segmental bronchi cross sections that contained a defined lamina propria layer tangential to the epithelium. Airway sections from nonasthma controls contained a pseudostratified epithelial layer with irregularly localized MUC5AC+ GC along the luminal surface (Figure 1A). In contrast, airway sections from patients with severe asthma demonstrated significantly increased epithelial thickness, increased numbers of MUC5AC+ GC (Figure 1, A–C), and occasional luminal occlusion by a mucous plug in the small airways (<2 mm in diameter) (Figure 1A). Furthermore, the epithelial layer was fundamentally disorganized, characterized by multiple layers of MUC5AC+ GC. In both large and small airways, many of the GC were localized deep within the epithelial layer without an outlet to the luminal surface. We termed these morphologically abnormally localized GC “eGC.” To better characterize eGC, we utilized E-cadherin to mark the cell borders of individual cells in the airway cross sections. The fraction of eGC relative to all GC, and total number of eGC per airway segment, were significantly increased in eGC from asthma airways (Figure 1, D and E). eGC were not unique to asthmatic airway sections. Some nonasthma control airway sections also contained scattered eGC, though at a lower frequency. Furthermore, we identified hypertrophic areas of increased epithelial thickness in individuals with severe asthma. These hypertrophied areas contained a higher density of eGC. The fraction of eGC was related to the epithelial thickness of the corresponding airway epithelial segment (Figure 1F). These epithelial hypertrophied areas may reflect regions of high aberrant metaplastic differentiation and migration. We therefore sought to understand the factors that led to these regions of hypertrophy, MUC5AC mucous cell metaplasia, and aberrant migration.

Figure 1

Airway epithelium from severe asthmatics is characterized by mucous metaplasia with ectopic goblet cell distribution. (A) Representative airway immunostaining for MUC5AC and E-Cadherin: Large airway section from nonasthmatic control (upper left panel), large airway section from #1 asthmatic sample (upper right panel), large airway section from #2 asthma sample (lower left panel), small airway section from #3 asthmatic sample (lower right panel). White asterisk marks airway luminal surface goblet cells (GC), golden asterisk marks airway ectopic goblet cells (eGC). DAPI for nuclear counter stain. Scale bar: 50 μm. (B) Quantification of MUC5AC volume density per airway. Each color represents a unique nonasthma or asthma patient sample. (C) The total epithelial area μm2 per airway basement membrane segment was determined for nonasthma controls and asthmatic airways. (D and E) The fraction of ectopic goblet cells (eGC) relative to total goblet cells and total cells along the linear baseline membrane was calculated with n = 5 asthmatic airway and 5 normal airway donors with n = 8 to 15 distinct images per donor airway. Unpaired t-test (2-tailed) for statistical difference with *P < 0.05. (F) Simple linear regression comparison of airway epithelial area and fraction eGC relative to total GC. Upper panel is nonasthma controls, and lower panel is asthmatic airways. Mean slope and 95% CI are shown.

MTOR signaling is increased in severe asthma airways with mucous cell metaplasia. Metaplastic airway epithelium in asthma is characterized by many of the central features of MTOR activation: increased epithelial cell hyperplasia and cellular hypertrophy. We examined the airway sections from nonasthma controls and individuals with severe asthma for MTOR activation using phosphorylated serine 240/244 (phospho-RPS6), which regulates key MTOR-dependent signaling pathways in cell proliferation, cell size, and protein synthesis (29). We found a significant increase in phospho-RPS6 staining in asthma epithelial sections compared with controls (Figure 2, A–C). In contrast, the phospho-RPS6 staining was scattered in individual cells in the nonasthma airways. This finding indicates that MTOR signaling is activated in the airway epithelium of severe asthmatics. We further subclassified severe asthma airways into 2 categories by the morphologic identification of GC. Airway sections with < 50% GC were classified as low GC airways. Sections with estimated GC > 50% were classified as GC rich. Phospho-RBS6 staining was significantly higher in the GC-rich asthma airway sections than the GC-low asthma airways (Figure 2, D and E). This suggests that MTOR signaling may be associated with mucous cell metaplasia.

Figure 2

Increased MTOR signaling in asthmatic airway epithelium. (A) Representative IHC image of MTOR substrate ribosomal S6 phosphorylated at serine 240/244 (phospho-RPS6). Images from nonasthma controls on the left and asthmatic airway sections on the right. Vertical scale bar: 200 μm. (B) Dashed box indicates area of higher magnification. Scale bar: 25 μm. (C) Quantification of phospho-RPS6 levels normalized to volume density per airway. (D) Representative IHC image of phospho-RPS6 from severe asthma airways classified based on morphologic identification of goblet cells (GC) as either GC low or GC rich (estimated ≥ 50% GC). Yellow asterisks mark GC. Scale bar: 25 μm. (E) Quantification of phospho-RPS6 staining in GC low versus GC rich regions. N = 5 nonasthma controls and 5 asthmatic airway donors. N = 10–15 images per donor. Unpaired t-test (2-tailed) for statistical difference with *P < 0.05.

Given the complexities of MTOR signaling, we next sought to characterize the role of MTOR using an in vitro model of de novo IL-13–induced mucous cell metaplasia. IL-13 stimulation recapitulates many of the morphologic features of the diseased epithelium in severe asthma, including increased cell size, epithelial cell hyperplasia, MUC5AC staining, and formation of eGC (11, 30–35) (Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.187904DS1). Primary human airway epithelial cells (hAEC) were fully differentiated under air-liquid interface (ALI) conditions (ALI ≥ 21 days) and examined for MTOR signaling by evaluation of multiple downstream substrates during IL-13 activation (Figure 3A). Starting at ALI day 21, hAEC were evaluated after treatment with IL-13 for +2 or +7 days or 3 days after withdrawal of IL-13 (Figure 3B). We found that MTOR protein levels themselves did not change during IL-13–mediated mucous metaplasia development and there was variability in MTOR protein levels by immunoblot across different hAEC donors (Figure 3, C and D). IL-13 increased phosphorylation of S6K1 and RPS6 at both 2 and 7 days of IL-13 days (Figure 3, C, E, and F). MTOR also negatively regulates autophagy by phosphorylation of serine residue S757 of ULK1 (20, 21). We (12, 25) and others (36, 37) previously have shown that autophagosome-related protein LC3 II levels are increased during the development of mucous cell metaplasia in hAEC treated with IL-13 in the context of lysosomal inhibition with bafilomycin A1 or chloroquine. These autophagy flux assays revealed concurrent signals of autophagy activation and inhibition of lysosome-autophagosome fusion both driving increased LC3 II levels. We hypothesized that the inhibition of autophagosome-lysosome fusion was temporally mediated by MTOR signaling (19, 38). Consistent with increased MTOR signaling during early IL-13–activation, we found a significant increase in phosphorylation of ULK1 at serine 757 in hAEC at +2 day of IL-13 treatment relative to untreated controls (Figure 3, C and G). However, during prolonged IL-13 activation and after IL-13 withdrawal, MTOR signaling is reduced and autophagy activation predominates. We next tested the role of MTOR using this time-dependent IL-13 model in the development and maintenance of airway mucous cell metaplasia associated with severe asthma.

Figure 3

IL-13 increases MTOR substrate phosphorylation. (A) MTOR signaling regulates both MTOR and autophagy by protein phosphorylation. (B) Schematic for IL-13–mediated mucous metaplasia (IL-13, 10 ng/mL) in human airway epithelial cells (hAEC) and resolution according to time points after withdrawal of IL-13. (C) Representative immunoblots for MTOR levels and total and phosphorylated RPS6 (S240/244), S6K1 (T389), and ULK1 (S757) during IL-13–mediated mucous metaplasia and resolution 3 days after IL-13 withdrawal. (D–G) Corresponding quantification of protein levels normalized to total protein for MTOR or nonphosphorylated protein levels (S6K1, RPS6, and ULK1). n = 8–10 per group from n = 4 unique donors. Each experiment and hAEC donor is denoted with a different color. ANOVA with mixed-effect test (1-tailed) for statistical difference comparing each time point to untreated vehicle was used. *P < 0.05.

MTOR regulates cell proliferation, size, and the emergence of eGC during mucous metaplasia. To determine the functional effect of MTOR signaling in IL-13–mediated mucous cell metaplasia, we treated fully differentiated hAEC (ALI day 21) with vehicle control, MTOR inhibitor rapamycin, IL-13, or IL-13 plus rapamycin for 7 days (Supplemental Figure 2A). This concurrent model will allow examination of the role of MTOR in an IL-13–dependent and –independent fashion. When we examined morphologic changes, we found IL-13 increased airway epithelial cell size, MUC5AC immunostaining per airway, and number of eGC (Supplemental Figure 1). During IL-13 activation, concurrent rapamycin led to a significant decrease in levels of MTOR phosphorylated substrates RPS6 and S6K1, indicating that MTOR signaling was inhibited (Figure 4, A–D). In addition, rapamycin led to a decrease in levels of sequestosome-1 (SQSMT1), which is an autophagy cargo protein degraded during autophagy in the lysosome (39). This confirmed that MTOR inhibition corresponded to an increase in autophagy. Concurrent rapamycin did not change IL-13–induced signal transducer and activator of transcription 6 (STAT6) phosphorylation nor MUC5AC gene expression (Supplemental Figure 2, B and C). MTOR inhibition with rapamycin reduced cell size and number independently of the presence of IL-13 activation (Figure 4, E–G). However, in the presence of IL-13 activation as a model of severe asthma, MTOR signaling was required for key features of airway mucous cell metaplasia, MUC5AC staining levels, and number of eGC (Figure 4, E and G–I). There was no effect of MTOR inhibition on cilia formation either with or without IL-13 (Supplemental Figure 3). There was no corresponding increase in cleaved caspase 3 (Supplemental Figure 2D), suggesting that cellular apoptosis could not explain the rapamycin-induced decrease in cell size and number. Furthermore, the effect of MTOR inhibition on epithelial hypertrophy and hyperplasia was not due to reduction in the number of TP63+ basal cells (Supplemental Figure 3E). Interestingly, there was a trend toward higher TP63+ basal cells during concurrent IL-13 activation and MTOR inhibition. In summary, MTOR signaling was required for the development of key features of metaplastic changes including cellular hypertrophy, hyperplasia, and eGC formation.

Figure 4

MTOR regulates airway epithelial hypertrophy, hyperplasia, and ectopic goblet cell formation. (A–D) Representative immunoblots for phosphorylated RPS6 (S-240-244), S6K1 (T389) levels, and total SQSTM1 with corresponding quantification. n = 7 per group from 4 hAEC donors. (E) Representative images of MUC5AC immunostaining for goblet cells and Actin immunostaining to define cell borders of hAEC in untreated vehicle, rapamycin alone (250 nM), IL-13 (10 ng/mL) for 7 days, and IL-13 plus concurrent rapamycin. DAPI (blue) for nuclear counterstain. Scale bars: 25 μm. White asterisk marks airway luminal surface goblet cells, golden asterisk marks airway ectopic goblet cells (eGC). (F–I) Quantification of area per cell, total number of cells, MUC5AC volume density immunostaining normalized to airway basement membrane length, eGC fraction per total cells. Images from n = 10–12 microscopic fields from 9 or 10 replicate inserts per condition from n = 6 unique normal airway donors. ANOVA (2-tailed) with Tukey multiple-comparison test for statistical difference. *P < 0.05.

Based on our new data of short-term IL-13 stimulation leading to the development of MTOR-dependent features of mucous cell metaplasia and our prior work showing persistent increase in autophagy and ROS levels during prolonged IL-13 stimulation, including after cytokine withdrawal (12, 25), we hypothesized that epithelial cells would be more responsive to autophagy activation during resolution of mucous metaplasia. We have previously demonstrated that autophagy contributes to the resolution of mucous metaplasia through the degradation of mucin granules in the lysosome (40). We predicted that MTOR inhibition would accelerate the degradation of mucin granules. hAEC were treated with IL-13 for 7 days, and the cytokine was withdrawn to model resolution. hAEC were treated with a single high-dose rapamycin or vehicle control for 48 hours and then harvested at day 7 after cytokine withdrawal (Figure 5A). There was a reduction in phosphorylated-RPS6 by immunoblot, indicating MTOR inhibition (Figure 5B). We found that MTOR inhibition with rapamycin significantly decreased MUC5AC levels by immunoblotting and immunostaining (Figure 5, C–F). These data indicate that activating autophagy by MTOR inhibition hastens resolution and confirms that the temporal balance of MTOR and autophagy is critical in regulating key features of mucous cell metaplasia.

Figure 5

MTOR inhibition hastens resolution of IL-13–mediated mucous metaplasia by reducing MUC5AC protein levels. (A) Schematic for treating fully differentiated hAEC for 7 days with IL-13 (10 ng/mL) and then treated with vehicle or rapamycin (1 μM) for 48 hours following withdrawal of IL-13. (B) Representative immunoblots for total and phosphorylated RPS6. n = 4 inserts from n = 2 independent hAEC donors. (C and D) Representative immunoblot for MUC5AC from IL-13–treated AEC following vehicle or rapamycin treatment with corresponding quantification. n = 8 from 3 independent hAEC donors. (E) Representative immunostaining for mucin MUC5AC and MUC5B with DAPI for nuclear staining in IL-13–treated hAEC following vehicle or rapamycin treatment. Scale bar: 20 μm. (F) Quantification of MUC5AC volume density normalized by basement membrane length in vehicle control and rapamycin-treated hAEC. n = 8–10 microscopic images per hAEC donor. Six inserts from n = 4 unique hAEC donors. Unpaired t test (2-tailed) for statistical difference with *P < 0.05.

MTOR regulates epithelial cell migration during IL-13–mediated mucous cell metaplasia. To explore fundamental MTOR-dependent pathways during IL-13–mediated mucous metaplasia, we undertook RNA-Seq of IL-13–stimulated hAEC in the presence or absence of concurrent MTOR inhibition by rapamycin (Figure 6A). We found a large set of significant differentially expressed genes (DEG) between the 2 treatment groups (Figure 6B and Supplemental Data 1). A comparison among these DEG with the highest difference (≥log2 fold change) revealed that MTOR inhibition by rapamycin was associated with a decrease in expression of genes related to cytoskeletal organization and epithelial migration, including VIL1, RHOJ, KCNN1, MUC13, and ENPP2, among others (Figure 6C). Gene ontology (GO; https://geneontology.org) of biological processes confirmed that genes involved with cytoskeletal organization and cell migration were decreased in the IL-13 plus rapamycin treatment group (Figure 6D and Supplemental Data 2). To further characterize the functional role of MTOR in epithelial cell migration, we assessed epithelial migration in response to mechanical wounding assays using an undifferentiated hAEC model. MTOR inhibition with rapamycin in the presence of IL-13 significantly decreased the percentage of wound closure at both the 6- and 24-hour time points (Supplemental Figure 4, A–D). This suggested that MTOR signaling plays an important role in epithelial migration in an injury repair model.

Figure 6

MTOR inhibition leads to decreased expression of cytoskeletal organization and migration genes during IL-13 stimulation. (A) hAEC under ALI conditions were treated with IL-13 in the presence or absence of concurrent rapamycin (250 nM) for 8 days. (B) Heatmap of hAEC from IL-13 (10 ng/mL) versus IL-13 plus rapamycin condition for differentially expressed genes (DEG) adjusted P < 0.05. n = 5 unique donors with n = 2–3 replicates per donor. (C) Volcano plot showing log2 fold change for DEG identified from B. (D) GO Annotation pathways analysis of 40 genes log2 2-fold decreased DEG from the IL-13 plus rapamycin group versus IL-13 alone from C. Pathways are on the left of the figure with rank order of significance by corresponding P < 0.5.

Previous studies using an injury repair model have demonstrated inconsistent roles of IL-13 on epithelial wound closure (8, 9). To further explore the effect of MTOR on migration during mucous cell metaplasia independently of cellular injury in fully differentiated airway epithelial cells, we utilized BrdU labeling in hAEC under ALI conditions treated with vehicle control, rapamycin, IL-13, or concurrent IL-13 + rapamycin for 7 days (Figure 7A). BrdU was removed from the culture media after two IL-13 treatments. This approach allowed us to focus on the migration of previously labeled cells rather than ongoing proliferation at day 7 of IL-13 stimulation when the cells are harvested. Under control conditions, BrdU-labeled cells were predominately along the basement membrane. Under MTOR inhibition conditions, rapamycin led to a significant decrease in the total number of BrdU-labeled cells independent of IL-13 (Figure 7, B and C). In response to IL-13, we found a significant increase in the number of BrdU-labeled hAEC. These BrdU-labeled cells were also more apically dispersed in the parabasal epithelial layer, rather than only along the basement membrane (Figure 7, B and D). These data suggest that, under conditions of IL-13–mediated mucous cell metaplasia, there is basal-apical migration of labeled cells independent of mechanical injury. Concurrent MTOR inhibition with rapamycin significantly reduced the IL-13–driven basal-apical migration of BrdU-labeled cells. Instead, BrdU-labeled cells were primarily concentrated along the basement membrane comparable to vehicle control conditions (Figure 7, B and D). We next sought to examine the role of proliferation in this model system. There were few costained Ki-67 and BrdU labeled cells. This likely reflects the timing of BrdU withdrawal in our model system (Figure 7A). There was a nonsignificant trend toward a higher number of Ki-67+ cells with IL-13 activation. However, concurrent MTOR inhibition and IL-13 activation did not lead to a reduction in Ki-67 staining (Figure 7, B and E). This trend was confirmed when we examined gene expression from the same conditions in the bulk RNA-Seq data set for MKI-67 and PCNA genes (Figure 7, F and G). These findings indicate that inhibiting MTOR led to a loss of migration of airway epithelial cells during IL-13–mediated mucous cell metaplasia.

Figure 7

MTOR regulates movement of airway epithelial cells during IL-13–mediated mucous cell metaplasia. (A) Schematic showing timing of BrdU labeling and withdrawal during IL-13 mediated mucous metaplasia. (B) Representative Ki-67 and BrdU immunostaining with DAPI for nuclear staining in hAEC cells for 4 conditions: vehicle DSMO control, rapamycin, IL-13 for 7 days (10 ng/mL), and IL-13 plus rapamycin 7 days. Dashed circles indicate migrating BrdU labeled cells. Scale bar: 25 μm. (C) Quantification of fraction of BrdU-labeled cells compared with total cell number per image. (D) Quantification of mean distance (μm) of each BrdU-labeled cell from the basement membrane (represented by a vertical bar). (E) Quantification of fraction of Ki-67+ cells relative to total cells per image. n = 10 or 12 replicates from n = 6 unique hAEC donors with 10–15 microscopic images per airway segment. ANOVA (2-tailed) with Tukey multiple-comparison test for statistical difference. *P < 0.05. (F and G) Gene expression data for MKI67 (F) and PCNA (G) was compared from the bulk RNA-Seq dataset.

MTOR activation is necessary but not sufficient to drive mucous cell metaplasia in mouse airway cells. To complement the pharmacologic loss-of-function studies using rapamycin, we sought an upstream MTOR activator using a genetic model. Tuberous sclerosis complex 2 (Tsc2) encodes the MTOR-suppressing protein tuberlin (41–44). Mice with a conditional deletion of Tsc2 have increased MTOR signaling in the presence of Cre activation (45). Mouse tracheal Tsc2 fl/fl-derived AECs were propagated in culture and found to have the expected increase in phosphorylated RPS6 levels upon AAV2-6-mediated Cre activation (Figure 8, A–C). To determine the effect on mucous cell metaplasia, Tsc2fl/fl mouse AECs that had been transduced with either AAV2-6 control CMV-Empty or AAV2-6 CMV-Cre-EGFP viral particles were treated with mouse recombinant IL-13 or vehicle control. Transduction efficiency was confirmed by EGFP signal at ALI day 28 (Figure 8D). Mouse AAVCre+Tsc2fl/fl AECs had significant increases in airway cell hypertrophy and mucous cell metaplasia as measured by PAS staining (Figure 8, E–H). Comparable with our rapamycin hAEC model, the effect of MTOR activation cell size was independent of IL-13. However, MTOR activation alone was insufficient to increase mucous cell metaplasia independently of IL-13 (Figure 8, E and H). Taken together, these findings indicate the central role of MTOR signaling in perturbing key features of epithelial control of cell size, organization, and differentiation that contribute to airway obstruction in asthma.

Figure 8

MTOR activation by Tsc2 deletion leads to increased mucous metaplasia and epithelial cell hypertrophy. (A) Illustration demonstrating the effect of Tsc2 deletion on MTOR activity. (B) Schematic for AAV transduction of mouse Tsc2fl/fl airway epithelial cells (AECs). (C) Immunoblotting for total and phosphorylated RibS6 and total MTOR level. n = 2 per group. (D) Schematic for repetitive transduction of AAV of Tsc2fl/fl mAEC under ALI conditions and subsequent IL-13 activation (10 ng/mL). Lower panel shows representative EGFP signal in CMV-Empty and CMV-Cre-EGFP AAV transduced cells. Scale bar: 100 μm. (E) Representative PAS images of AAV Empty and AAV Cre+Tsc2fl/fl mAECs with and without IL-13 treatment. Scale bar: 10 μm. (F–H) Quantification of total epithelial area per airway (F), area per cell (G), and PAS staining volume density immunostaining normalized to airway basement membrane length (H). n = 10–12 images per insert, n = 5 inserts derived from 3 Tsc2 flox mice. ANOVA (2-tailed) with Tukey multiple-comparison test for statistical difference. *P < 0.05.

Discussion

In airway disease such as severe asthma, there is an increase in epithelial hyperplasia, hypertrophy, and mucous cell metaplasia, all contributing to airway wall thickening, mucous plugging, and loss of lung function (4, 5, 46). Morphologically, these alterations were observed in the airway sections from those with severe asthma. At a cellular level, these pathogenic changes are the result of a loss of control of size, morphology, migration, and differentiation. These pathogenic changes are initiated by allergens, viruses, and type 2 inflammatory cytokines such as IL-13. Changes in airway epithelial cell fate and differentiation by type 2 inflammatory conditions have been studied (30, 47). However, the maladaptive epithelial response that leads to loss of control of cell size, proliferation, and migration is not well understood. In addition, we (12, 25) and others (36, 48–50) have observed that autophagy proteins are increased in the airway epithelium from asthma or in response to IL-13. However, autophagy is predominately a catabolic process in response to cell stress, including oxidant injury. To date, it has been challenging to reconcile the morphologic and differentiation changes in severe asthma with autophagy activation. Here, we propose what we believe to be a new paradigm: that the balance of MTOR and autophagy determines the development, persistence, and resolution of airway mucous cell metaplasia found in severe asthma.

In airway sections from individuals with severe asthma, we found an increased airway epithelial thickness and m