Enhancing Autophagy with Drugs or Lung-directed Gene Therapy Reverses the Pathological Effects of Respiratory Epithelial Cell Proteinopathy

Abstract

Recent studies have shown that autophagy mitigates the pathological effects of proteinopathies in the liver, heart, and skeletal muscle but this has not been investigated for proteinopathies that affect the lung. This may be due at least in part to the lack of an animal model robust enough for spontaneous pathological effects from proteinopathies even though several rare proteinopathies, surfactant protein A and C deficiencies, cause severe pulmonary fibrosis. In this report we show that the PiZ mouse, transgenic for the common misfolded variant α1-antitrypsin Z, is a model of respiratory epithelial cell proteinopathy with spontaneous pulmonary fibrosis. Intracellular accumulation of misfolded α1-antitrypsin Z in respiratory epithelial cells of the PiZ model resulted in activation of autophagy, leukocyte infiltration, and spontaneous pulmonary fibrosis severe enough to elicit functional restrictive deficits. Treatment with autophagy enhancer drugs or lung-directed gene transfer of TFEB, a master transcriptional activator of the autophagolysosomal system, reversed these proteotoxic consequences. We conclude that this mouse is an excellent model of respiratory epithelial proteinopathy with spontaneous pulmonary fibrosis and that autophagy is an important endogenous proteostasis mechanism and an attractive target for therapy.

Keywords: autophagy; chronic obstructive pulmonary disease (COPD); misfolded α1antitrypsin; protein misfolding; proteostasis; pulmonary fibrosis; α1antitrypsin deficiency.

FIGURE 1.

Expression of ATZ and activation of autophagy in lungs of PiZ mice. A, immunoblot analysis for ATZ, GAPDH, and p62 in alveolar epithelial cells from PiZ (n = 2) and control (n = 2) mice. In each case 50 μg protein is loaded. B, immunoblot analysis for LC3 isoforms in alveolar epithelial cells from control (n = 2) and PiZ mice (n = 3) after incubation in the absence (−) or presence of lysosomal enzyme inhibitors. Quantification of the LC3-II to LC3-I ratio is shown at the bottom of the blot. This ratio in the presence of lysosomal inhibitors was significantly increased (3.57 ± 0.28 versus 2.07 ± 0.30, p = 0.0297). A loading control is not necessary because lanes are compared with each other based on the ratio of two bands in each lane. C, immunostaining for AT (red) and LC3 (green) in the respiratory epithelium of PiZ x GFP-LC3 (upper) compared with control, GFP-LC3 mouse (lower). Panels on the left show both red and green channels, and panels on the right show only the green channel. Nuclei are stained blue. D, immunoblot analysis for ATZ in the lungs of PiZ (n = 3) and PiZ x Bec (n = 3) mice at age 12 months. The negative control is lung tissue from a Bec mouse. In each case 10 μg of protein is loaded. Densitometric analysis indicates that the increase in PiZ x Bec compared with PiZ is significant, p = 0.0397. The migration of partially and fully glycosylated ATZ is indicated at the right margin by an asterisk and arrowhead, respectively.

FIGURE 2.

Collagen deposition and leukocyte infiltration in lungs of PiZ mice. A, histochemical staining with trichrome. PiZ x GFP-LC3 (n = 2) is in the right panels, and control, GFP-LC3 (n = 2), age 6 months, is in the left panels. % area stained was 9.83 ± 0.92 for PiZ-GFP-LC3, n = 61 versus 2.83 ± 0.41 for control, n = 21; p < 0.0001). B, hydroxyproline content. The mean ± S.E. is shown for each group (n = 5–8). *, difference from control, p < 0.005; **, difference from PiZ, p < 0.005. C, TEM imaging. Sections from 2 mice each at 50,000× magnification. The thickness was significantly increased in PiZ (n = 6) versus control (n = 5) at age 6–12 months analyzing 27 and 25 fields, respectively (p < 0.0001). The arrows point to basement membrane. The control in each is GFP-LC3. D, immunostaining of leukocytes in the lungs of PiZ mice using anti-CD45 (pink). Quantitative morphometry was done for PiZ (n = 4) and the control (n = 3) at age 6 months, analyzing 10 and 9 fields, respectively, for % area stained in sections shown as the mean ± S.E. p = 0.0006 at the right. The control was GFP-LC3.

FIGURE 3.

Pulmonary function test in PiZ mice. Lung compliance (A), tissue resistance (B), airways resistance (C), hysteresis (D), and tissue elastance (E) are shown for PiZ (n = 13) and wild type (n = 11) at 9 months of age. Hysteresis is also shown for PiZ (n = 11) and wild type (n = 8) at 15–18 months of age (F), Bars represent the mean ± 1.0 S.E., and p values are shown at the bottom of each panel. Asterisks indicate statistically significant difference from control. In G, pressure-volume loops for three representative PiZ and three control mice are shown.

FIGURE 4.

Cigarette smoking challenge of PiZ compared with C57B6 mice. Groups of male mice were subjected to cigarette smoking (S) from 2 to 8 months of age and compared with equal numbers of mice that were not smoked (NS). Chord length was significantly increased by cigarette smoking, p = 0.0317 for C57B6 (*) and p = 0.0286 for PiZ (**). There were no significant differences for PiZ NS versus C57B6 NS (p = 0.4857) and PiZ versus C57B6 S (p = 0.4440), two-tailed Mann-Whitney test.

FIGURE 5.

Effect of lung-directed TFEB gene transfer on lung collagen deposition and leukocyte infiltration. A, collagen I immunostaining. B, Sirius Red staining. C, CD45 staining. Morphometric results and p values are shown to the right of the images. The mice were age 24 months, n = 9 for each group. The number of fields analyzed were 15 and 14 for A, 48 and 54 for B, and 14 each for C. D, ATZ levels as determined by densitometric scanning of immunoblots. The mice were age 24 months, n = 4, for TFEB, n = 3 for control. AU, absorbance units.

FIGURE 6.

Effect of autophagy enhancer drugs on lung collagen deposition, lung leukocyte infiltration, and lung function in PiZ mice. A, immunostaining for collagen I in lung. PiZ mice at 3 months of age (n = 19 in each group) were treated for 3 weeks with placebo or both CBZ 200 mg/kg/day and Flu 7.5 mg/kg/day by oral gavage. B, immunostaining for CD45 from same specimens used for A. Quantitative morphometry for 30–37 fields in each case was expressed as mean ± S.E. is shown on the right of A and B. Asterisks indicate a statistically significant difference from placebo. C, pulmonary function tests. PiZ mice at 12–15 months of age were treated with placebo (n = 5) or CBZ 200 mg/kg/day and Flu 7.5 mg/kg/day (n = 7) for 6 weeks. Bars represent the mean ± 1.0 S.E. Asterisks indicate statistically a significant difference from placebo. D, pressure-volume loops for representative PiZ mice treated with placebo (n = 2; dark red, dots and dashes) and for representative PiZ mice treated with CBZ and Flu (n = 2; green) are shown overlaying pressure-volume loops for representative wild type (light blue) and PiZ mice (light red) from the experiments of Fig. 3.

FIGURE 7.

Relative AT RNA and protein levels in human respiratory epithelial cells and alveolar macrophages. Relative expression of AT was determined by RT-qPCR and ELISA in cultures of type II pneumocytes (ATII, AECT2), type I pneumocytes (ATI, AECT1), and alveolar macrophages (AM) isolated from human lung. The negative control was a human skin fibroblast cell line. A, RNA levels. B, protein levels in ng/μg RNA. C, protein levels in ng/ml. D, protein levels in basal versus apical extracellular fluid.

FIGURE 8.

Expression of ATZ and activation of autophagy in BECs from human with ATD. A, BECs from lung of ATD patient (ZZ) and control (MM) were subjected to pulse-radiolabeling for 2 h and a chase period of 2 h. Samples from HTO/Z (Z) and HTO/M (M) cell lines were used as controls for intracellular lysates (IC) and extracellular fluid respectively (HTO). The 52- and 55-kDa forms of AT are indicated by the tick marks at the left. EC, extracellular fluid. B, BECs from the ATD patient (ZZ) and control (MM) were harvested for assay of p62 levels and loading control (β-actin) by immunoblot analysis. C, BECS from the ATD patient (ZZ) and control (MM) were incubated in the absence or presence of lysosomal enzyme inhibitors for 4 h and then harvested for LC3 immunoblot analysis. The LC3-II:LC3-I ratio was as follows in the 4 groups: 1.02 in MM, 1.14 in MM with lysosomal inhibitors (lyso inhib), 1.85 in ZZ, 4.06 in ZZ with lysosomal inhibitors. Loading control is not necessary because the lanes are being compared for ratio of two bands.

FIGURE 9.

TEM imaging of lung epithelial cells in ATD patients. Large arrowheads point to dilated endoplasmic reticulum, and arrows point to autophagosomes. Small arrows point to collagen fibers in basement membrane. Double arrows point to increased thickness of basement membrane. The asterisk points to collagen deposition. A orients to air surface.

FIGURE 10.

TEM imaging of lung interstitium in ATD patients. Arrowheads point to bundles of collagen, and arrow points to collagen fibers within the basement membrane.

FIGURE 11.

Trichrome staining (blue) of human lung specimens. A, sections from the lungs of two normal controls are shown in the upper panels and from three ATD patients are shown in the lower panels. B, scoring from blinded pathological ranking of the degree of trichrome staining for lung tissue specimens from idiopathic pulmonary fibrosis (n = 3), ATD (n = 16), and normal (n = 9). Scores for ATD were significantly greater than normal, p = 0.0118.