MTOR signaling regulates the development of airway mucous cell metaplasia associated with severe asthma

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.

Keywords: Asthma; Autophagy; Cell biology; Cell migration/adhesion; Pulmonology.

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.

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.

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.

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.

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.

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.

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.

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 Tsc2^fl/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 Tsc2^fl/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⁺ Tsc2^fl/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.