The common ABCA3E292V variant disrupts AT2 cell quality control and increases susceptibility to lung injury and aberrant remodeling

Abstract

ATP-binding cassette class A3 (ABCA3) is a lipid transporter that plays a critical role in pulmonary surfactant function. The substitution of valine for glutamic acid at codon 292 (E292V) produces a hypomorphic variant that accounts for a significant portion of ABCA3 mutations associated with lung disorders spanning from neonatal respiratory distress syndrome and childhood interstitial lung disease to diffuse parenchymal lung disease (DPLD) in adults including pulmonary fibrosis. The mechanisms by which this and similar ABCA3 mutations disrupt alveolar type 2 (AT2) cell homeostasis and cause DPLD are largely unclear. The present study, informed by a patient homozygous for the E292V variant, used an in vitro and a preclinical murine model to evaluate the mechanisms by which E292V expression promotes aberrant lung injury and parenchymal remodeling. Cell lines stably expressing enhanced green fluorescent protein (EGFP)-tagged ABCA3 isoforms show a functional deficiency of the ABCA3^E292V variant as a lipid transporter. AT2 cells isolated from mice constitutively homozygous for ABCA3^E292V demonstrate the presence of small electron-dense lamellar bodies, time-dependent alterations in macroautophagy, and induction of apoptosis. These changes in AT2 cell homeostasis are accompanied by a spontaneous lung phenotype consisting of both age-dependent inflammation and fibrillary collagen deposition in alveolar septa. Older ABCA3^E292V mice exhibit increased vulnerability to exogenous lung injury by bleomycin. Collectively, these findings support the hypothesis that the ABCA3^E292V variant is a susceptibility factor for lung injury through effects on surfactant deficiency and impaired AT2 cell autophagy.

Keywords: ABCA3; autophagy; diffuse parenchymal lung disease; lung epithelium; pulmonary fibrosis.

Figure 1.

Pretransplant radiography and explant histology from a 47-yr-old patient with DPLD harboring the homozygous E292V variant. A: chest radiograph before transplant showing a diffuse reticular pattern throughout the bilateral lungs. B: chest CT before transplant demonstrating DPLD characterized by architectural distortion with peribronchial thickening with consolidation, diffuse reticular changes, and traction bronchiectasis and bronchiolectasis. C: lung explant histology: H&E staining at low (left) and high (right) magnifications showing end-stage lung disease with diffuse parenchymal remodeling characterized by dilated airspaces with thickened septa replacing the normal distal lung alveolar architecture (left) and extension of smooth muscle into the alveolar septa (right). CT, computed tomography; DLPD, diffuse parenchymal lung disease; H&E, hematoxylin-eosin.

Figure 2.

In vitro-expressed ABCA3^E292V variant is functionally deficient. A: representative confocal images of A549 (top) and human alveolar type 2 (AT2) cells (bottom) 48 h following plasmid introduction of EGFP-tagged WT and the E292V mutant isoform of ABCA3 immunostained with Texas red-conjugated CD63 (top) and LAMP1 (bottom) antibodies. Bar, 5 µm. B: transmission electron microscope (TEM) images from HEK293 cell lines: parental (left) or stably expressing EGFP-tagged with either ABCA3^WT (middle) or ABCA3^E292V (right). LB-like organelles are apparent only in ABCA3^WT-expressing cells. Solid boxes are magnified underneath each image. Bar, 500 nm. C: tubes from sucrose gradient fractionation showing isolated LB band from mouse lung homogenate (2nd from left) and LB-like organelles from HEK293 cell homogenates stably expressing ABCA3^WT (3rd from left) or ABCA3^E292V (right). Strong and faint bands between 0.35 and 0.50 M sucrose fractions (arrowheads) where LBs are known to segregate are apparent in the mouse and ABCA3^WT samples, respectively, but not in parental or ABCA3^E292V samples. D: anti-EGFP immunoblot of ABCA3 primary translation (top bands) and processed (bottom bands) products from sucrose gradient fractionated samples taken from the two right tubes in C that included the upper (U), lower (L), or pellet (P) fractions showing the presence and absence of ABCA3^E292V protein band corresponding to the lower and upper fractions, respectively. ABCA3, ATP-binding cassette subfamily A, member 3; EGFP, enhanced green fluorescent protein; LBs, lamellar bodies; WT, wild type.

Figure 3.

Aberrant LB formation in mice carrying the E292V^+/+ variant. A: schematic illustration of the pgk-Neo⁺ (mABCA3^E292V-Neo⁺) mice showing the FRT-pgk-gb2-Neo/km-FRT cassette insert site within intron 8 in the mAbca3 locus together with the E292V point mutation in exon 9 (top) and the removal of the cassette by breeding mice with constitutively active flippase (Flp) deleter mice to produce pgk-Neo free (mABCA3^E292V) mice (bottom). B: total bronchoalveolar lavage (BAL) phospholipid (PL) content of 16-wk-old homozygous mABCA3^E292V (E292V^+/+) and WT littermates were measured; WT, n = 7; E292V^+/+, n = 6. C: mRNA levels from E292V^+/+ mice relative to WT littermates as measured by real-time quantitative PCR from at least five mice per group. D: representative electron micrographs of AT2 cells of 32-wk-old WT and E292V^+/+ mice. AT2 cell profiles in E292V^+/+ mice appear to comprise smaller lamellar bodies compared with WT controls. Boxed regions are enlarged below each image to provide enhanced resolution. E: unbiased stereological measure of LB size using volume-to-surface ratio of LB as a surrogate parameter for number-weighted mean volume of LB (V-to-S ratio) (left) and volume-weighted mean volume of LB [νV (lb) µm³] (right). *P < 0.05 vs. WT using unpaired 2-tailed t test. WT, n = 5; E292V^+/+, n = 6. F: tubes from sucrose gradient fractionation to isolate LBs from mouse lung homogenates of WT (left) and E292V^+/+ (right) mice. A band between 0.42 and 0.47 M sucrose fractions (left arrow) where LBs are known to segregate is apparent in the WT mouse sample, whereas the band from the E292V^+/+ mouse homogenate segregates below the expected level from that of the WT band (right arrow). AT2, alveolar type 2; LBs, lamellar bodies; WT, wild type.

Figure 4.

A: BALF total cell counts from 4-, 8-, 16-, and 32-wk-old E292V^−/+, E292V^+/+, and WT control littermates. *P < 0.05 vs. WT using one-way ANOVA with post hoc Tukey’s test. 4, 8, 16, 32 wk: WT, n = 10, 7, 8, 11; E292V^-/+, n = 9, 10, 9, 5; E292V^+/+; n = 10, 8, 9, 10, respectively. B: representative cytospin samples collected from 8-, 16-, and 32-wk-old E292V^+/+ mice and WT control littermates. Inset: a higher-resolution image of monocytes and macrophages. C: picrosirius red-stained lung section from 16- and 32-wk-old WT and E292V^+/+ mice (left) and dot plots of % area of alveolar collagen from transferred images to ImageJ Data software (right), each dot representing percentage of at least 10 randomly selected fields per mouse lung section (right). Bar, 50 µm. *P < 0.01 using one-way ANOVA with post hoc Tukey’s test. 16 wk, n = 5 per group; 32 wk, WT, n = 5; E292V^+/+, n =  6. BALF, bronchoalveolar lavage fluid; WT, wild type.

Figure 5.

Autophagy is disrupted in AT2 cells of E292V^+/+ mice. A: representative immunoblots from whole cell lysates of freshly isolated mouse AT2 cells showing baseline levels of autophagy-associated species (left) and band intensity of densitometry quantitation from Western blots of at least three separate experiments (right) of 16- and 32-wk-old E292V^+/+ mice and WT littermates. Each dot represents AT2 cell samples pooled from at least six mice. *P < 0.01 vs. WT using one-way ANOVA with post hoc Tukey’s test. LC3, p62, and Tom20, n = 7 per group; LAMP1, n = 6 per group. B: representative immunoblots of freshly isolated AT2 cells treated with bafilomycin A1 (Baf. A1) for 18 h showing increased levels of LC3 and p62 species at baseline and reaching optimum level at lower doses of Baf. A1 treatment in E292V^+/+ mice (bottom) compared with WT control littermates (top). C: representative immunoblots of whole cell lysates from AT2 cells treated with an autophagy inducer, Rapamycin (Rapa), alone or together with an inhibitor of autophagy, Baf. A1. D: representative immunoblots (top) and band intensity densitometer quantitation (bottom) showing levels of the apoptosis marker, activated/cleaved caspase 3, in AT2 cells of 16- and 32-wk-old E292V^+/+ mice. Each dot represents cleaved bands from AT2 cell samples pooled from at least six mice. *P < 0.05 vs. WT using unpaired 2-tailed t test. WT, n = 5; E292V^+/+, n = 6. ns, not significant. E: representative epifluorescence images of lung sections from 32-wk-old E292V^+/+ mice and WT control stained for SP-C (red) and apoptotic cells using TUNEL assay (green). Nuclei are stained with DAPI (blue). Solid boxes are magnified beneath each image to illustrate double positive (TUNEL⁺ + SP-C⁺) AT2 cells (arrows). Areas of patchy distribution of clustered TUNEL-labeled AT2 cells were noted in the lungs of 32-wk-old E292V^+/+ mice. Bar, 50 µm. Bottom: dot-plots representing % of double-positive cells per total SP-C⁺ cells per mouse from at least 10 fields (each at ×20 magnification). *P < 0.05 vs. WT using unpaired 2-tailed t test. WT, n = 5; E292V^+/+, n = 6. AT2, alveolar type 2; SP-C, surfactant protein C; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Figure 6.

A: weight loss in E292V^+/+ and WT control mice following intratracheal (IT) bleomycin (0.5 U/kg) instillation. *P < 0.05 vs. WT using unpaired 2-tailed t test. WT, n = 8; E292V^+/+, n = 7. B, top: representative baseline H&E (rows 1 and 3) and trichrome (rows 2 and 4) staining of histological lung sections from 32-wk-old WT and E292V^+/+ mice. Bar, 4 mm. Bottom: fold change (compared with WT average) in collagen genes messages from lung tissues measured by RT-qPCR. *P < 0.01 vs. WT by unpaired 2-tailed t test. WT, n = 5; E292V^+/+, n = 8. C: representative H&E (left column) and trichrome (right three columns) staining of histological lung sections from 32-wk-old E292V^+/+ mice and WT littermates 21 days after IT bleomycin. Patchy trichrome-stained regions were noted in WT mice. More prominent trichrome staining was observed in E292V^+/+ mice displaying severe alveolar destruction and subpleural fibrosis enveloping several lobes. Boxed and numbered regions (left column) are enlarged on the right to provide enhanced resolution. Bar, 3 mm. D: representative distribution of picrosirius red-stained lung section from 32-wk-old WT and E292V^+/+ mice (left) and dot plots of % area of alveolar collagen from transferred images to ImageJ Data software (right), with each dot representing percentage of at least 10 randomly selected fields per mouse lung section (right). *P < 0.05 vs. WT using unpaired 2-tailed t test. n = 6 per group. E, top: representative epifluorescence images of lung sections from 32-wk-old E292V^+/+ mice and WT littermates (untreated or 21 days after IT bleomycin) immunostained for proSP-C. Nuclei are stained with DAPI (blue). Bar, 50 µm; Inset bar, 10 µm. Bottom: dot-plots of average cell number of SP-C⁺ cells per mouse lung obtained from at least 10 fields (each at ×20 magnification field). Alveolar regions absent of large airways were randomly selected for cell count using ImageJ, version 1.8.0, multipoint tool. *P < 0.01 vs. WT Bleo, **P < 0.001 vs. untreated E292V^+/+ by one-way ANOVA with post hoc Tukey’s test. n = 5 per group. F: cell counts (top left) and pulmonary function tests that included static compliance (top right), inspiratory capacity (bottom left), and elastance (bottom right) of E292V^+/+ mice and WT littermates 21 days after IT bleomycin. *P < 0.05 vs. WT using unpaired 2-tailed t test. n = 8 per group. G: Kaplan–Meier survival curve of 42-wk-old E292V^+/+ mice and WT littermates following IT bleomycin treatment (0.75 U/kg). n ≥ 12. End points were defined as death or body weight <75% of starting weight on 2 consecutive days. *P < 0.05 vs. WT by log-rank (Mantel–Cox) test. WT, n = 6; E292V^+/+, n = 8. Bleo, bleomycin; H&E, hematoxylin-eosin; SP-C, surfactant protein; WT, wild type.

Figure 6.

A: weight loss in E292V^+/+ and WT control mice following intratracheal (IT) bleomycin (0.5 U/kg) instillation. *P < 0.05 vs. WT using unpaired 2-tailed t test. WT, n = 8; E292V^+/+, n = 7. B, top: representative baseline H&E (rows 1 and 3) and trichrome (rows 2 and 4) staining of histological lung sections from 32-wk-old WT and E292V^+/+ mice. Bar, 4 mm. Bottom: fold change (compared with WT average) in collagen genes messages from lung tissues measured by RT-qPCR. *P < 0.01 vs. WT by unpaired 2-tailed t test. WT, n = 5; E292V^+/+, n = 8. C: representative H&E (left column) and trichrome (right three columns) staining of histological lung sections from 32-wk-old E292V^+/+ mice and WT littermates 21 days after IT bleomycin. Patchy trichrome-stained regions were noted in WT mice. More prominent trichrome staining was observed in E292V^+/+ mice displaying severe alveolar destruction and subpleural fibrosis enveloping several lobes. Boxed and numbered regions (left column) are enlarged on the right to provide enhanced resolution. Bar, 3 mm. D: representative distribution of picrosirius red-stained lung section from 32-wk-old WT and E292V^+/+ mice (left) and dot plots of % area of alveolar collagen from transferred images to ImageJ Data software (right), with each dot representing percentage of at least 10 randomly selected fields per mouse lung section (right). *P < 0.05 vs. WT using unpaired 2-tailed t test. n = 6 per group. E, top: representative epifluorescence images of lung sections from 32-wk-old E292V^+/+ mice and WT littermates (untreated or 21 days after IT bleomycin) immunostained for proSP-C. Nuclei are stained with DAPI (blue). Bar, 50 µm; Inset bar, 10 µm. Bottom: dot-plots of average cell number of SP-C⁺ cells per mouse lung obtained from at least 10 fields (each at ×20 magnification field). Alveolar regions absent of large airways were randomly selected for cell count using ImageJ, version 1.8.0, multipoint tool. *P < 0.01 vs. WT Bleo, **P < 0.001 vs. untreated E292V^+/+ by one-way ANOVA with post hoc Tukey’s test. n = 5 per group. F: cell counts (top left) and pulmonary function tests that included static compliance (top right), inspiratory capacity (bottom left), and elastance (bottom right) of E292V^+/+ mice and WT littermates 21 days after IT bleomycin. *P < 0.05 vs. WT using unpaired 2-tailed t test. n = 8 per group. G: Kaplan–Meier survival curve of 42-wk-old E292V^+/+ mice and WT littermates following IT bleomycin treatment (0.75 U/kg). n ≥ 12. End points were defined as death or body weight <75% of starting weight on 2 consecutive days. *P < 0.05 vs. WT by log-rank (Mantel–Cox) test. WT, n = 6; E292V^+/+, n = 8. Bleo, bleomycin; H&E, hematoxylin-eosin; SP-C, surfactant protein; WT, wild type.