Oxidative stress induces club cell proliferation and pulmonary fibrosis in Atp8b1 mutant mice

Abstract

Atp8b1 (ATPase, aminophospholipid transporter, class I, type 8B, member 1) is a cardiolipin transporter in the apical membrane of lung epithelial cells. While the role of Atp8b1 in pneumonia-induced acute lung injury (ALI) has been well studied, its potential role in oxidative stress-induced ALI is poorly understood. We herein show that Atp8b1^G308V/G308V mice under hyperoxic conditions display exacerbated cell apoptosis at alveolar epithelium and aberrant proliferation of club cells at bronchiolar epithelium. This hyperoxia-induced ambivalent response in Atp8b1^G308V/G308V lungs was followed by patchy distribution of non-uniform interstitial fibrosis at late recovery phase under normoxia. Since this club cell abnormality is commonly observed between Atp8b1^G308V/G308V lungs under hyperoxic conditions and IPF lungs, we characterized this mouse fibrosis model focusing on club cells. Intriguingly, subcellular morphological analysis of IPF lungs, using transmission electron microscopy (TEM), revealed that metaplastic bronchiolar epithelial cells in fibrotic lesions and deformed type II alveolar epithelial cells (AECs) in alveoli with mild fibrosis, have common morphological features including cytoplasmic vacuolation and dysmorphic lamellar bodies. In conclusion, the combination of Atp8b1 mutation and hyperoxic insult serves as a novel platform to study unfocused role of club cells in IPF.

Keywords: Atp8b1; club cells; hyperoxia; idiopathic pulmonary fibrosis (IPF); oxidative stress.

Figure 1

Atp8b1^G308V/G308V mice under hyperoxic conditions display increased cell death in alveoli, but not in bronchiolar epithelium. Wild-type (WT) and Atp8b1^G308V/G308V mice were exposed to 100% O₂ for 48 hrs. Mice were euthanized and formaldehyde fixed paraffin-embedded lung sections were stained with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). TUNEL-positive cells are denoted by arrowheads. (A & B) Representative photomicrographs focusing on bronchovascular bundles with surrounding alveoli. (C) Quantitative comparison between hyperoxic WT and Atp8b1^G308V/G308V mice (n=3 for each) regarding the total number of TUNEL positive cells per 100x field in the lung. The numbers of TUNEL-positive cells were determined in 7-8 randomly chosen 100x fields for each section. Means ± SE for each group is shown. *p < 0.05. (D & E) Representative photomicrographs of peripheral part of the lung with relatively small bronchioles. Basement membranes of bronchiolar epithelium are highlighted by blue lines. (F) Quantitative comparison between hyperoxic WT and Atp8b1^G308V/G308V mice (n=3 for each) regarding the number of TUNEL positive cells in bronchiolar epithelium. The number of TUNEL-positive cells in bronchiolar epithelium were determined in 7-8 randomly chosen 100x fields. Means ± SE is shown. *p < 0.05. Br: Bronchiolar lumen. Magnifications: (A & B) 200X; (D & E) 100X

Figure 2

Atp8b1^G308V/G308V mice under hyperoxic conditions display increased number of total cells in airspace compared to WT controls. Representative photomicrographs of bronchoalveolar lavage fluid (BAL) cells retrieved from WT (A & C) and Atp8b1^G308V/G308V mice (n=3 for each) (B, D & E-G) following exposure to 100% O₂ for 48 hrs. BAL fluid (BALF) cells were stained with Diff-Quik. Infiltrating neutrophils are indicated by arrows in Panel C & D. Highly vacuolated cells with weakly stained nucleus are encountered in airspace of hyperoxic Atp8b1^G308V/G308V mice (arrowheads in Panel E & F), which are morphologically distinct from surrounding cells that are considered to be macrophages (arrows in Panel E & F). Cells with eccentric nucleus and numerous cytoplasmic granules are occasionally encountered in hyperoxic Atp8b1^G308V/G308V mice, which are not morphologically similar to any immune cell types that are normally encountered in lung airspace (cell designated by arrowheads in Panel G). (H & I) Levels of IL-6 and total protein in BALF from WT and Atp8b1^G308V/G308V mice exposed to normoxia or 100% O₂ for 48 hrs. IL-6 levels in BALF were measured by ELISA (n=3 for each group). Results are presented as Means ± SE. *p < 0.05. Magnifications: (A & B) 200X; (C & D) 400X; (E-G) 1000X. Data presented are representative of two independent experiments.

Figure 3

Atp8b1^G308V/G308V mice under hyperoxic conditions display patchy thickening of bronchiolar epithelium with epithelial hypercellularity. (A & B) Photomicrographs of H&E stained lung sections from WT and Atp8b1^G308V/G308V mice exposed to 100% O₂ for 48 hrs (n=3 for each). (C & D) Photomicrographs of TUNEL stained lung sections from WT and Atp8b1^G308V/G308V mice exposed to 100% O₂ for 48 hrs. Arrowheads denote thickened bronchiolar epithelium of hyperoxic Atp8b1^G308V/G308V lungs. TUNEL positive cells are denoted by arrows. Br: Bronchiolar lumen. Magnifications: (A & B) 100X; (C & D) 200X. Data presented are representative of two independent experiments.

Figure 4

Atp8b1^G308V/G308V mice under hyperoxic conditions display proliferation of claudin-10-positive club cells in thickened bronchiolar epithelium. Paraffin-embedded lung sections from WT and Atp8b1^{G308V/G308V mice} exposed to 100% O₂ for 48 hrs were subjected to immunohistochemical staining for club cell secretory protein (CCSP). (A & B), Ki-67 (C & D), and claudin-10 (E-J) (n=3 for each of WT and Atp8b1 mutant mice). Photomicrographs show representative images from either parabronchiolar (A-F) or alveolar regions (G-J) Arrowheads in Figure A-H designate positive cells for the respective markers. (K) claudin-10 positive cells per 100x field in alveoli were quantified in 10 randomized independent fields of 4 mice per each group. Means ± SE of the total number of claudin-10 positive cells for each group is shown. Br: Bronchiolar lumen. Magnifications: (A-F) 100X (G-J) 200X. Data presented are representative of two independent experiments.

Figure 5

Atp8b1^G308V/G308V mice exposed to hyperoxia and returned subsequently to normoxia for recovery develops late-onset interstitial fibrosis. Representative photomicrographs of H&E-stained lung sections from 7-9-wk-old WT (A-D) and Atp8b1^G308V/G308V mice (E-H) that were exposed to 100% O₂ for 48 hrs and allowed to recover under normoxia for 12 days (n=3 for each). WT mice show marked recovery with some hypercellularity remaining in bronchiolar regions. Atp8b1^G308V/G308V lungs display juxtaposition of normal lung with collapsed alveoli beneath the pleura (arrows in Panel E), distinct hyperplastic epithelium (arrowheads in F), thick-walled cystic air space (arrowheads in Panel G), and hypercellularity in bronchovascular interstitium (arrowheads in Panel H). Magnifications: (A-H) 100X. Data presented are representative of two independent experiments.

Figure 6

Atp8b1^G308V/G308V mice exposed to hyperoxia and returned subsequently to normoxia for recovery display abnormal fibrotic reactions in the lung. Representative photomicrographs of Masson's Trichrome-stained lung sections from 7-9-wk-old WT (A-D) and Atp8b1^G308V/G308V mice (E-H) that were exposed to 100% O₂ for 48 hrs and allowed to recover under normoxia for 12 days (n=3 for each). WT mice show minimal collagen deposition mainly at peribronchiolar and perivascular areas. Atp8b1^G308V/G308V mice show patchy distribution of aberrant collagen deposition (areas circled by dashed lines in Panel E, F, G & H): (Panel E) perivascular region, (Panel F) alveoli, (Panel G) alveoli located at subpleura and alveoli adjacent to alveolar duct, and (Panel H) peribronchiolar region. Magnification: (A-H) 100X. Data presented are representative of two independent experiments.

Figure 7

Atp8b1^G308V/G308V mice exposed to hyperoxia and returned subsequently to normoxia for recovery display aberrant deposition of collagen in the lung. Photomicrographs of lung sections immunohistochemically labeled for type I collagen. WT and Atp8b1^G308V/G308V mice at 7-9 weeks of age were exposed to room air or 100% O₂ for 48 hours, and then allowed to recover under normoxia for 12 days (n=3 for each). Arrowheads indicate areas showing strong signals for type I collagen. Atp8b1^G308V/G308V mice display aberrant collagen deposition in both perivascular and alveolar regions. Br: Bronchiolar lumen; V: Vessel (bronchiolar artery). Magnification: (A-D) 100X. Data presented are representative of two independent experiments.

Figure 8

Atp8b1^G308V/G308V mice exposed to hyperoxia and returned subsequently to normoxia for recovery show impaired bronchiolar regeneration. WT and Atp8b1^G308V/G308V mice at 7-9 weeks of age were exposed to room air or 100% O₂ for 48 hours, and then allowed to recover under normoxia for 12 days (n=3 for each). Representative photomicrographs of lung sections immunohistochemically labeled for claudin-10 are shown. Arrows denote relatively intact bronchiolar lumens. (A) WT lungs recovered from hyperoxia display normal regeneration of bronchioalveolar structures wherein organized arrangements of bronchioles surrounded by intact alveoli are noted (area designated by one asterisk). (B) Atp8b1^G308V/G308V lungs recovered from hyperoxia display impaired regeneration of bronchioalveolar structures wherein incomplete bronchiolar structures (two asterisks) are juxtaposed to highly remodeled lesions (areas circled by dashed lines). Br: Bronchiolar lumen. Magnifications: (A and B) 200X. Data presented are representative of two independent experiments.

Figure 9

IPF lungs show aberrant CCSP expression at bronchiolar epithelium with hypercellularity. Immunohistochemical staining for CCSP was performed on lung tissue sections from patients with IPF and patients with COPD (control for IPF) (n=4 for each). Labeled boxes correspond to their respective enlarged images. (A, B, C & D) Representative photomicrographs of lung sections from COPD patients display relatively organized arrangement of CCSP expression in apical side of bronchiolar epithelium. (E, F, G & H) Representative photomicrographs of lung sections from IPF patients. Hyperplastic bronchiolar epithelium randomly displays accumulation of cells with varying degrees of CCSP expression. Magnifications: (A, C, E, G) 40X; (H) 100x; (B, D, F) 200X. Data presented are representative of one experiments.

Figure 10

IPF lungs show increased claudin-10 expression. (A) Western Blot analysis was performed using whole protein lysates from IPF and control lung samples (n=3 for each), to determine relative abundance of claudin-10. Equal amounts of protein (50μg) were loaded per lane. Data presented are representative of two independent experiments. (B) Expression of claudin-10 in A was normalized to β-actin and presented in arbitrary units.

Figure 11

Dysmorphic lamellar bodies, cytoplasmic vacuolation, and euchromatic nucleus are widely shared in metaplastic bronchiolar epithelial cells in IPF lung. Transmission electron microscopy was performed on lung samples from patients with IPF (n=5). Data presented are from Patient No. 2 and Patient No. 3. Labeled boxes correspond to their respective enlarged images. (A & I) A small bronchiole (circled by dashed line in Panel A) is occluded by a suspected cell structure featuring multiple granules, lamellar bodies (arrowheads in Panel I), and an enlarged endoplasmic reticulum (arrows in Panel I). (B-G & L) Bronchiolar epithelial cells exhibit morphological variation with frequently encountered features of euchromatic nucleus (asterisks in Panel C, D, E, F & G) and dysmorphic lamellar bodies. (H) A dysmorphic lamellar body next to a trace of mitochondria. (J & K) Lamellar bodies present at the apical surface of a bronchiolar epithelial cell are about to secrete their contents into the airspace. An arrow in Panel K denotes mitochondria. (M) A narrowed bronchiolar lumen lined by highly vacuolated epithelial cells with dysmorphic lamellar structures (arrows). (N) A cell with euchromatic nucleus (asterisk) and morphological feature of club cell (numerous secretory granules in cytoplasm) is located in bronchiolar epithelium. Prominent phagosomes are noted in cytoplasm (arrows). (O & P) Hyperplastic bronchiolar epithelium with collagen deposition in the interstitium. Cells with euchromatic nucleus (asterisks in Panel O) are located across apoptotic cells (arrows in Panel P) from collagen bundle bundles (CB). Cells with numerous secretory granules in cytoplasm, which are considered to be club cells, are noted (arrowhead in Panel O). Seen at luminal side are cells with numerous lamellar structures in cytoplasm (arrows in Panel O). (Q-T) Metaplastic epithelial cell on fibroblastic foci features dysmorphic mitochondria (arrows in Panel S), numerous phagosomes (double asterisks in Panel S & T), and a small number of lamellar bodies. AS: Airspace; FF: Fibroblastic foci; LB: Lamellar body. *(single asterisk) = Euchromatic nucleus, **(double asterisks) = Phagosome. Magnifications: (A) 3000X; (B, C, and R) 8000X; (D & L) 20000X; (E, F, G, I, N & P) 12000X; (H & K) 60000X; (J & S) 30000X; (M) 4000X; (O) 2500X; (Q) 1500X; (T) 80000X.

Figure 12

Morphologically abnormal type II alveolar epithelial cells with similar subcellular structures to metaplastic bronchiolar epithelial cells are encountered in alveoli of IPF lung. TEM was performed on human IPF samples from patient No. 3. Labeled boxes correspond to their respective enlarged images. (A-E) Alveolar regions with slight interstitial fibrosis show random distribution of cells with varying degrees of apoptosis. Cells with highly condensed chromatin, which are considered to be at late stages apoptosis, are designated by asterisks while cells exhibiting narrow cytoplasm and marginal condensation of chromatin, which are considered to be at early stages of apoptosis, are designated by open arrows. (F) Floating in airspace is a highly atypical cell featuring high nucleus-to-cytoplasm (N/C) ratio, dysmorphic lamellar body (arrowheads) and granular structures suspected of either secretory granules or degraded mitochondria (double asterisks). Note the appearance of cytoplasm and cytoplasmic membrane reminds loss of cell viability meanwhile the nucleus does not show the features of apoptosis. (G) A highly atypical cell with a lot of granular structures is barely attached to the alveolar tissue as if it has migrated and just landed on alveolar tissue. (H) A suspected epithelial cell type (circled by dashed line) featuring microvilli and dysmorphic lamellar bodies, quite similar to the highly vacuolated bronchiolar epithelial cells seen in bronchiolar region (arrows in Fig. 11M). (I-L) Metaplastic epithelial cells suspected of either type II AECs or progenitor cells committed to type II AECs (circled by dotted line) featuring dysmorphic lamellar bodies (arrowheads), granular structures, slightly euchromatic nucleus and vacuolated cytoplasm. Note the juxtaposition of collagen producing active fibroblast (Fib) with a metaplastic epithelial cell with type II AEC morphology (Panel J), which suggests epithelial-to-mesenchymal interaction. Type I AEC (arrows in Panel J) across the active fibroblast from metaplastic epithelial cell show relatively intact appearance. AS: Airspace; AEC I: type I alveolar epithelial cell; CB: Collagen Bundle; Fib: Fibroblast. *(single asterisk) = Apoptotic cell, **(double asterisks) = Granular structures suspected of secretory granules or degraded mitochondria. Magnifications: (A and D) 1200X; (B, E, G, H & I) 4000X; (C) 2500X; (F) 20000X; (J and K) 8000X; (L) 12000X.