IL13 activates autophagy to regulate secretion in airway epithelial cells

Abstract

Cytokine modulation of autophagy is increasingly recognized in disease pathogenesis, and current concepts suggest that type 1 cytokines activate autophagy, whereas type 2 cytokines are inhibitory. However, this paradigm derives primarily from studies of immune cells and is poorly characterized in tissue cells, including sentinel epithelial cells that regulate the immune response. In particular, the type 2 cytokine IL13 (interleukin 13) drives the formation of airway goblet cells that secrete excess mucus as a characteristic feature of airway disease, but whether this process is influenced by autophagy was undefined. Here we use a mouse model of airway disease in which IL33 (interleukin 33) stimulation leads to IL13-dependent formation of airway goblet cells as tracked by levels of mucin MUC5AC (mucin 5AC, oligomeric mucus/gel forming), and we show that these cells manifest a block in mucus secretion in autophagy gene Atg16l1-deficient mice compared to wild-type control mice. Similarly, primary-culture human tracheal epithelial cells treated with IL13 to stimulate mucus formation also exhibit a block in MUC5AC secretion in cells depleted of autophagy gene ATG5 (autophagy-related 5) or ATG14 (autophagy-related 14) compared to nondepleted control cells. Our findings indicate that autophagy is essential for airway mucus secretion in a type 2, IL13-dependent immune disease process and thereby provide a novel therapeutic strategy for attenuating airway obstruction in hypersecretory inflammatory diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis lung disease. Taken together, these observations suggest that the regulation of autophagy by Th2 cytokines is cell-context dependent.

Keywords: IL13; MUC5AC; airway epithelial cells; autophagy; reactive oxygen species; secretion.

Figure 1.

Goblet cell hypertrophy in autophagy-deficient mice. WT and Atg16l1 hypomorphic (Atg16l1^HM/HM) mice were administered vehicle or IL33 intranasally. Lungs were evaluated on d 7 post-challenge. (A) Representative photomicrographs of lung sections stained with PAS. Scale bars, top and middle panels: 100 μm; lower panel: 10 μm. (B) Quantification of area per goblet cell (n = 6 mice per group, (C) percentage of PAS staining (on histological sections) of total airway epithelium area (n = 6 mice per group) of IL33-treated WT and Atg16l1^HM/HM, and (D) levels of MUC5AC in bronchoalveolar lavage (BAL) fluid from lungs of IL33-treated mice (n = 3 mice per group). (B–D) are graphs of the means ±SEM. Means with different letters are significantly different by the unpaired 2-tailed Student's t-test.

Figure 2.

IL13 increases MUC5AC expression and secretion. (A) In vitro protocol for IL13 treatment of human tracheal/bronchial epithelial cells (hTEC) differentiated using air-liquid interface conditions (ALI). Cells were assayed at the indicated times. (B) Representative images of hTEC treated with vehicle or IL13 for the indicated periods, then immunostained for MUC5AC and cilia marker acetylated tubulin (acetylated-TUB). Nuclei were stained with DAPI. MC, goblet cell; arrows, secreted MUC5AC on the cell surface. Scale bars: 10 μm. (C) IL13-induced MUC5AC mRNA levels (+) relative to vehicle (−) (n = 6 preparations/condition). (D) ROS production in hTEC cultured with or without IL13 during ALI d 14–21 d. Three h prior to loading the ROS probe DCF (CM-H2DCFDA), cells were treated with combinations of IL13 and the NOX inhibitor DPI (5 μM), or vehicle controls and the resulting mean fluorescent intensity signal was measured from 3 random fields. AU, arbitrary units. (E) MUC5AC secretion in hTEC preparations cultured with or without IL13 for 21 d as in (A) and pre-treated with DPI (5 μM) or DMSO vehicle prior to the MUC5AC secretion assay. Levels of secreted MUC5AC were measured by ELISA after performing a series of washes (to obtain baseline levels) followed by a 1 h incubation with IL13. Values were normalized to the baseline amount of MUC5AC for each condition and reported as fold change (n = 7 for DMSO vehicle pretreatment, n = 4 for DPI pretreatment). Measures in (C–E) were obtained from hTEC preparations obtained from at least 3 unique donors and independent experiments, displayed as the graph of the means ±SEM. In (C), means from each time point were compared to vehicle alone using the paired Student t test. In (D, E), means with different letters are significantly different by ANOVA with Bonferroni's correction.

Figure 3.

IL13 increases autophagy activity. (A) hTEC were cultured in the presence of vehicle or IL13 for 21 d of ALI conditions. Cell lysates were prepared at the indicated time points of ALI culture for immunoblot analysis for ATG5 and LC3. Representative blots are shown. (B) Quantification of immunoblot density of the LC3-II to actin ratio as the mean ±SEM (n = 3 independent experiments each from a preparation of a unique cell donor). (C) Autophagy flux assay to determine the influence of IL13 on autophagy activity in hTEC. (D) Representative LC3-immunoblots of hTEC incubated with vehicle (−) or IL13 (+) for 7 d then treated with chloroquine (Chloro) as shown in (C). Lysates were harvested at ALI 21. (E) Immunoblot image densities of LC3-II to actin ratios shown as the mean ±SEM (n = 5 independent experiments). Mean LC3 to actin band densities with different letters are significantly different by ANOVA with Bonferroni's correction. (F) Representative images of LC3 puncta in hTEC (E) in the presence or absence of IL13 for 7 d, on ALI d 14–21, then treated with chloroquine as shown in (C). Scale bar: 10 μm. (G) Quantification of the number of LC3 puncta as mean ±SEM from triplicate values per high-powered field (hpf) obtained from photomicrographs for each experiment (n = 3 independent experiments). Means with different letters are significantly different by unpaired 2-tailed Student t test.

Figure 4.

ATG5 depletion reduces autophagy activity in hTEC preparations. hTEC were transduced with nontargeted (NT) or ATG5-specific shRNA sequences. (A) ATG5 levels in representative immunoblots for NT or 2 different ATG5 shRNA sequences (#1 and #2) at ALI d 21 with corresponding protein levels of SQSTM1 and LC3. (B) Representative images of LC3 (red) and SQSTM1 (green) puncta in hTEC treated with vehicle or IL13 in shRNA-transduced cells NT and ATG5 (sequence #2) shRNA. Scale bars: 10 μm. (C and D) Quantification of LC3 and SQSTM1 puncta in transduced cells treated with vehicle (−) or IL13 (+) for 7 d. (C and D) are graphs of number of puncta as the mean ±SEM of triplicate samples from high-powered field images obtained from n = 4 different cell preparations. Means with different letters are significantly different by ANOVA with Bonferroni's multiple comparison tests.

Figure 5.

Depletion of ATG5 is associated with airway goblet cell hypertrophy. hTEC were transduced with nontargeted (NT) or ATG5-specific shRNA and treated with vehicle or IL13 for 7 d (ALI days 14–21). (A) Representative confocal photomicrographs of hTEC in the x,y plane, stained for MUC5AC and actin using phalloidin. Scale bars: 10 μm. (B) Representative x,z images demonstrating the optical level selected in (A) relative to the apical membrane (dashed line). (C) Quantification of (A) as percent (%) goblet cells of total cell number on the apical surface, (D) MUC5AC-staining area per goblet cell and (E) MUC5AC signal intensity per goblet cell. Graphs in (C–E) are from are the mean ±SEM from triplicate values per high-powered field (hpf) obtained from photomicrographs for each experiment (n = 3–4 independent experiments, each from a unique donor). Means in (C–E) with different letters are significantly different by ANOVA with Bonferroni's multiple comparison test. (F) Representative transmission EM images of goblet cells treated with IL13. Red lines mark cell borders. Scale bars: 2 μm. (G) Quantification of number of mucin granules per cell was performed with 8 goblet cells evaluated from each group. Shown are the means ±SEM with different letters indicating a significant difference by the unpaired 2-tailed Student t test.

Figure 6.

Depletion of ATG5 and ATG14 reduces IL13-mediated MUC5AC secretion. (A) Scheme of in vitro IL13-mediated MUC5AC secretion protocol in hTEC. Secreted MUC5AC was measured 1 h following the application of fresh IL13 on cells transduced with NT or autophagy gene shRNA. (B) hTEC transduced with ATG5 (sequence #2) shRNA. (C) hTEC transduced with ATG14 shRNA. Graphs in (B and C) are the means ±SEM from cells derived from 4 unique preparations. Means with different letters are significantly different by the unpaired 2-tailed Student t test.

Figure 7.

Depletion of ATG5 and ATG14 reduces IL13-mediated ROS activity. hTEC transduced with NT, ATG5 or ATG14 shRNA, treated with vehicle (−) or IL13 (+) for 7 d, ALI d 14–21 as in Figure 5. (A) Representative images of DCF (CM-H2DCFDA) probe fluorescent signal; Scale bar: 100 μm. (B and C) Quantification of ROS signal intensity is shown. Graphs in (B and C) are the means ±SEM of values obtained from images of 3 random fields from 4 unique preparations. Means with different letters are significantly different by ANOVA with Bonferroni's multiple comparison test (B) or the unpaired 2-tailed Student t test (C).