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. 2020 Apr 24;11(1):2012.
doi: 10.1038/s41467-020-15743-6.

Conditional Deletion of Nedd4-2 in Lung Epithelial Cells Causes Progressive Pulmonary Fibrosis in Adult Mice

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Free PMC article

Conditional Deletion of Nedd4-2 in Lung Epithelial Cells Causes Progressive Pulmonary Fibrosis in Adult Mice

Julia Duerr et al. Nat Commun. .
Free PMC article

Abstract

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease characterized by patchy scarring of the distal lung with limited therapeutic options and poor prognosis. Here, we show that conditional deletion of the ubiquitin ligase Nedd4-2 (Nedd4l) in lung epithelial cells in adult mice produces chronic lung disease sharing key features with IPF including progressive fibrosis and bronchiolization with increased expression of Muc5b in peripheral airways, honeycombing and characteristic alterations in the lung proteome. NEDD4-2 is implicated in the regulation of the epithelial Na+ channel critical for proper airway surface hydration and mucus clearance and the regulation of TGFβ signaling, which promotes fibrotic remodeling. Our data support a role of mucociliary dysfunction and aberrant epithelial pro-fibrotic response in the multifactorial disease pathogenesis. Further, treatment with the anti-fibrotic drug pirfenidone reduced pulmonary fibrosis in this model. This model may therefore aid studies of the pathogenesis and therapy of IPF.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. NEDD4-2 expression is reduced in lung tissue biopsies from patients with IPF.
a Micrographs of lung sections from patients with IPF and age-matched controls stained with anti-NEDD4-2 antibody (representative of n = 9/group). Scale bars, 100 and 10 µm (insets). b, c Levels of NEDD4-2 protein as determined by parallel reaction monitoring mass spectrometry (n = 11/group) (b) and NEDD4-2 mRNA (control, n = 10; IPF, n = 9) (c) in lung biopsies from IPF patients and controls. *P < 0.05, **P < 0.01 compared to controls. Statistical analysis was performed with unpaired two-tailed t test. Data are shown as mean ± S.E.M. Source data are provided in the Source Data file.
Fig. 2
Fig. 2. Conditional deletion of Nedd4-2 in adult lung epithelial cells causes pulmonary fibrosis.
a Survival curves of conditional Nedd4-2−/− mice and littermate controls (n = 26/group). b, c Summary of body weight (n = 8/group) (b) and oxygen saturation (n = 10/group) (c) of conditional Nedd4-2−/− and control mice induced with doxycycline for 3–4 month recorded at day of endpoint study. d Lung compliance was assessed by pulmonary function testing after 0.5 (control, n = 6 mice; conditional Nedd4-2−/−, n = 5 mice), 2 (control, n = 5 mice; conditional Nedd4-2−/−, n = 7 mice), 3 (control, n = 10 mice; conditional Nedd4-2−/−, n = 13 mice), and 4 months (control, n = 9 mice; conditional Nedd4-2/−, n = 6 mice) of doxycycline induction. eg Micro-CT imaging studies were performed after an average of 4 month of doxycycline induction when conditional Nedd4-2−/− mice developed clinical symptoms (n = 7/group). e Representative micro-CT images showing subpleural patchy consolidation with traction bronchiectasis (white arrows), subpleural cystic honeycombing-like destruction of lung parenchyma adjacent to consolidation (black arrows) and subpleural reticulation with parenchymal lines (black arrowheads) in conditional Nedd4-2/− mice. Scale bars, 3 mm. f, g Quantification of pulmonary fibrosis (f) and honeycombing-like cysts (g) by micro-CT scoring. hl Histomorphological evaluation of lungs of clinically symptomatic conditional Nedd4-2/− mice after an average of 4 months of doxycycline induction (n = 5/group). h, i Micrographs of representative hematoxylin and eosin (H&E) stained lung sections. Scale bars, 1 mm (low magnification overview) and 200 µm (high magnification). i Quantification of honeycombing-like cysts determined from H&E stained lung sections. j Micrograph of representative adjacent lung sections stained with H&E and anti-α-smooth muscle actin (αSMA) antibody depicting fibroblast foci-like alterations (arrowheads). Scale bars, 50 µm. k Quantification of fibroblast foci-like alterations as determined from H&E stained lung sections. l Masson–Goldner–Trichrome staining of lung sections depicts increased collagen deposition in the lung parenchyma of conditional Nedd4-2/− mice. Scale bars, 200 µm. m Collagen content (Sircol) in lungs of conditional Nedd4-2−/− mice and littermate controls (n = 5/group). n Mean septal wall thickness in conditional Nedd4-2−/− mice and littermate controls after 0.5, 2, and 3 months of doxycycline induction (n = 6/group). o Transmission electron microscopy reveals ultrastructural abnormalities in alveolar type 2 cells in conditional Nedd4-2−/− mice (n = 6/group). Scale bars, 2 µm. *P < 0.05, **P < 0.01, ***P < 0.001 compared to littermate controls of the same age. Survival in a was analyzed with log rank test. Statistical analysis was performed with unpaired two-tailed t test in (b, d, n), and with two-tailed Mann–Whitney test in (c, f, g, i, k, m). Data are shown as mean ± S.E.M. Source data are provided in the Source Data file.
Fig. 3
Fig. 3. Conditional deletion of Nedd4-2 in lung epithelial cells causes peripheral airway remodeling with increased Muc5b production.
ad Representative micrographs and high magnification insets illustrating the airway cell population of terminal bronchioles from conditional Nedd4-2−/− mice and littermate controls after 3 months of doxycycline induction stained with Alcian blue and periodic acid-Schiff (AB-PAS) (control, n = 5 mice; Nedd4-2−/−, n = 9 mice) (a), or co-stained with anti-Muc5b and anti-acetylated α-tubulin antibodies (control, n = 5 mice; Nedd4-2−/−, n = 6) (b, c), or with anti-Muc5ac and anti-CCSP antibodies (n = 4/group) (d). Scale bars, 60 and 15 µm (insets). ei Summary of numeric densities of club cells (n = 5 mice/group) (e), ciliated cells (n = 6/group and proximal, conditional Nedd4-2−/−, n = 5 mice) (f), goblet cells (proximal, control, n = 5 mice; conditional Nedd4-2−/−, n = 6 mice; distal, control, n = 5 mice; conditional Nedd4-2−/−, n = 7 mice, terminal, n = 9/group) (g), Muc5b expressing cells (proximal, n = 5/group; distal, control, n = 5 mice; conditional Nedd4-2−/−, n = 6 mice, terminal, n = 6/group) (h), and Muc5ac expressing cells (n = 4/group) (i) in proximal, distal and terminal conducting airways of conditional Nedd4-2−/− mice and littermate controls after 3 months of doxycycline induction (BM basement membrane). j, k mRNA expression levels of Muc5b (j) and Muc5ac (control, n = 15 mice; conditional Nedd4-2−/−, n = 6 mice) (k) in whole lungs from 3-month induced conditional Nedd4-2−/− and control mice. *P < 0.05, **P < 0.01, ***P < 0.001. Statistical analysis in e, g, h, i, k was performed with two-tailed Mann–Whitney test, and in f, and j with unpaired two-tailed t test. Data are shown as mean ± S.E.M. Source data are provided in the Source Data file.
Fig. 4
Fig. 4. Conditional deletion of Nedd4-2 in airway epithelial cells causes increased ENaC activity, reduced airway surface liquid (ASL) height and impaired mucociliary transport.
a Amiloride-sensitive short circuit current (Isc) in freshly excised airway tissues of conditional Nedd4-2−/− and control mice (control, n = 20 mice; conditional Nedd4-2−/−, n = 27 mice) induced with doxycycline for 2 weeks. b, c Confocal images of ASL height (b) and summary of measurements (c) at baseline and 2, 4, 8, and 24 h after application of rhodamine dextran to primary murine airway epithelial cultures from conditional Nedd4-2−/− mice and littermate controls induced with doxycycline for 2 weeks (n = 3 independent experiments performed in triplicates). Scale bars, 15 µm. d, e Mucociliary transport (MCT) velocity was determined from transport rates of fluorescent beads added on the surface of primary murine airway epithelial cultures. Representative bead tracks that visualize MCT velocity (d) and summary of measurements (e) in primary cultures from conditional Nedd4-2−/− mice and littermate controls (n = 3 independent experiments). Scale bars, 50 µm. **P < 0.01, ***P < 0.001. Statistical analysis in a was performed with two-tailed Mann–Whitney test and in c, e with unpaired two-tailed t test. Data are shown as mean ± S.E.M. Source data are provided in the Source Data file.
Fig. 5
Fig. 5. Lack of Nedd4-2 causes misprocessing of proSP-C in alveolar type 2 (AT2) cells, but genetic deletion of Sftpc does not prevent pulmonary fibrosis in conditional Nedd4-2−/− mice.
a Representative immunofluorescence images of lungs from conditional Nedd4-2−/− mice and littermate controls stained with anti-proSP-C antibodies. The subcellular distribution of proSP-C in control lungs represents predominantly large subplasma membrane organelles consistent with lamellar bodies (white arrowheads) while expression of proSP-C in conditional Nedd4-2−/− lungs also occurs in smaller cytosolic vesicles (n = 3/group). Scale bars, 50 and 10 µm (insets). b Western blot of isolated AT2 cells demonstrating aberrations in the proSP-C processing profile in conditional Nedd4-2/− compared to control mice (n = 3/group). c, d Representative Western blot (c) and densitometric analysis (d) of mature SP-C in bronchoalveolar lavage fluid from conditional Nedd4-2−/− mice and littermate controls (n = 3/group). eg Micrographs of representative hematoxylin and eosin (H&E) stained lung sections (scale bars, 1 mm) (e) and measurements of pressure–volume curves (n = 13/group) (f) and static lung compliance (g) from conditional Nedd4-2−/− (n = 7 mice), conditional Nedd4-2−/−/Sftpc−/− (n = 10 mice), littermate Sftpc−/− (n = 11 mice), and control mice (n = 7 mice) induced with doxycycline for an average of 4 months. *P < 0.05, **P < 0.01, ***P < 0.001. Statistical analysis was performed with unpaired two-tailed t test in d and ANOVA with Tukey’s post hoc test in g. Data are shown as mean ± S.E.M. Source data are provided in the Source Data file.
Fig. 6
Fig. 6. Conditional deletion of Nedd4-2 in lung epithelial cells causes increased levels of TGFβ and exaggerated Smad2/3 signaling.
a Levels of active TGFβ in whole lung of conditional Nedd4-2−/− mice and littermate controls after 0.5 (n = 4/group), 2 (n = 4/group), 3 (n = 5/group), and 4 months (n = 6/group) of doxycycline induction. b, c mRNA expression levels of the mesenchymal marker genes Vim and Fn1 (n = 9/group) (b) and alveolar type 2 (AT2) cell markers Sftpc (n = 9/group) and Sftpd (n = 6/group) (c) of whole lungs from conditional Nedd4-2/− and control mice after 3 months of doxycycline induction. di AT2 cells isolated from conditional Nedd4-2−/− and control mice induced with doxycycline for 2 weeks were pretreated with 1 ng/ml TGFβ and analyzed for phosphorylated Smad2/3 (pSmad2/3) and transcript levels of TGFβ-inducible genes. df Representative Western blot (d) and densitometric analysis of pSmad2 (e) and pSmad3 (f) at baseline and after 1–3 h of TGFβ stimulation. gi mRNA levels of Serpine1 (g), Smad7 (h), and Skil (i) after the indicated time of TGFβ stimulation. All in vitro data are derived from 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared to controls. Statistical analysis was performed using two-tailed Mann–Whitney test in a, b, two-tailed unpaired t test in c, and two-way ANOVA with Sidac’s post hoc test in ei. Data are shown as mean ± S.E.M. Source data are provided in the Source Data file.
Fig. 7
Fig. 7. Comparison of proteomic signatures of pulmonary fibrosis in conditional Nedd4-2−/− mice and patients with IPF.
a, b Heatmap of differentially regulated matrisome annotated proteins in lungs from conditional Nedd4-2−/− mice (n = 11) and control mice (n = 13) induced with doxycycline for 3 months (a), and in lung tissues from IPF patients and controls (n = 11/group) (b). c Venn-diagram showing proportion of unique and common differentially regulated proteins in lungs of conditional Nedd4-2/− mice and IPF patients. d Summary of differentially regulated matrisome proteins found in conditional Nedd4-2/− mice and human IPF samples. e Micrographs of representative lung sections from conditional Nedd4-2/− mice and littermate controls stained with anti-Tnc, anti-Col14a1, and anti-Serpinh1 antibodies (n = 6/group). Scale bars, 100 µm.
Fig. 8
Fig. 8. Pirfenidone treatment reduces pulmonary fibrosis and inflammation in conditional Nedd4-2−/− mice.
a Representative micro-CT images of paraffin embedded lungs of 3-months doxycycline-induced conditional Nedd4-2−/− and control mice treated with pirfenidone (pirf) or vehicle alone for one month. Fibrotic areas show patches of increased tissue density (white arrowheads) (control, n = 8; conditional Nedd4-2−/−, n = 8; conditional Nedd4-2−/− +pirf, n = 10). b Volumetric analysis of fibrotic regions in the three experimental groups (control, n = 8; conditional Nedd4-2−/−, n = 8; conditional Nedd4-2−/− +pirf, n = 10). c, d Effect of pirfenidone treatment on static compliance (control, n = 30; conditional Nedd4-2−/−, n = 28; conditional Nedd4-2/− +pirf, n = 27) (c) and on concentrations of active TGFβ in lung homogenates (control, n = 27; conditional Nedd4-2−/−, n = 24; conditional Nedd4-2−/ +pirf, n = 24) (d). eg Number of neutrophils (control, n = 29; conditional Nedd4-2−/−, n = 25; conditional Nedd4-2−/− +pirf, n = 25) (e) and concentration of IL-13 in bronchoalveolar lavage fluid (control, n = 21; conditional Nedd4-2−/−, n = 24; conditional Nedd4-2−/− +pirf, n = 22) (f), and concentration of IL-1β in lung homogenates (control, n = 15; conditional Nedd4-2−/−, n = 15; conditional Nedd4-2−/− +pirf, n = 16) (g) of conditional Nedd4-2−/− and control mice treated with pirfenidone or vehicle alone. *P < 0.05, **P < 0.01, ***P < 0.001. hk Effect of pirfenidone on TGFβ signaling in alveolar type 2 cells isolated from conditional Nedd4-2−/− and control mice induced with doxycycline for 2 weeks that were pretreated with 1 ng/ml TGFβ and analyzed for phosphorylated Smad2 (pSmad2) and transcript levels of TGFβ-regulated genes. h Densitometric analysis of pSmad2 at baseline and after 1–3 h of TGFβ stimulation. ik mRNA levels of Serpine1 (i), Smad7 (j), and Skil (k) at indicated times after TGFβ stimulation in the presence or absence of pirfenidone. (n = 3 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001 compared to control mice. #P < 0.05, ##P < 0.01, ###P < 0.001 compared to pirfenidone treated conditional Nedd4-2−/− mice. Statistical analysis was performed with Kruskal wallis test and Mann–Whitney test corrected for multiple comparisons with Bonferroni method as post hoc test in b, dg, one-way ANOVA with Tukey’s post hoc test in c and two-way ANOVA with Tukey’s post hoc test in hk. Data are shown as mean ± S.E.M. Source data are provided in the Source Data file.
Fig. 9
Fig. 9. Pirfenidone treatment reverts proteomic changes associated with pulmonary fibrosis in conditional Nedd4-2−/− mice.
a Heatmap of differentially regulated matrisome annotated proteins in lungs of 3-months doxycycline-induced conditional Nedd4-2−/− and control mice treated with pirfenidone (pirf) or vehicle alone (n = 9/group and Nedd4-2−/− vehicle, n = 8). b Principal component analysis of all significantly regulated proteins. c, d Ingenuity Pathway Analysis of significantly regulated proteins in the four experimental groups. Top enriched diseases and functions (c) and canonical pathways (d) based on the predicted activation z-score are shown.

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