Aberrant lung remodeling in a mouse model of surfactant dysregulation induced by modulation of the Abca3 gene

Abstract

The lipid transporter, ATP binding cassette class A3 (ABCA3), plays a critical role in the biogenesis of alveolar type 2 (AT2) cell lamellar bodies (LBs). A relatively large number of mutations in the ABCA3 gene have been identified in association with diffuse parenchymal lung disease (DPLD), the most common of which is a missense mutation (valine substitution for lysine at residue 292 (ABCA3^E292V)) that leads to functional impairment of the transporter in vitro. The consequences of ABCA3^E292V gene expression in vivo are unknown. To address this question, we developed mouse models expressing ABCA3^E292V knocked-in to the endogenous mouse locus. The parental (F1) mouse line (mAbca3^E292V) that retained an intronic pgk-Neo selection cassette (inserted in reverse orientation) (mAbca3^E292V-rNeo) demonstrated an allele dependent extracellular surfactant phospholipid (PL) deficiency. We hypothesize that this PL deficiency leads to aberrant parenchymal remodeling contributing to the pathophysiology of the DPLD phenotype. Compared to wild type littermates, baseline studies of mice homozygous for the pgk-Neo insert (mAbca3^E292V-rNeo^+/+) revealed nearly 50% reduction in bronchoalveolar lavage (BAL) PL content that was accompanied by quantitative reduction in AT2 LB size with a compensatory increase in LB number. The phenotypic alteration in surfactant lipid homeostasis resulted in an early macrophage predominant alveolitis which peaked at 8 weeks of age. This was followed by age-dependent development of histological DPLD characterized initially by peribronchial inflammatory cell infiltration and culminating in both an emphysema-like phenotype (which included stereologically quantifiable reductions in both alveolar septal surface area and volume of septal wall tissue) plus foci of trichrome-positive collagen deposition together with substantial proliferation of hyperplastic AT2 cells. In addition to spontaneous lung remodeling, mABCA3^E292V-rNeo mice were rendered more vulnerable to exogenous injury. Three weeks following intratracheal bleomycin challenge, mAbca3-rNeo mice demonstrated allele-dependent susceptibility to bleomycin including enhanced weight loss, augmented airspace destruction, and increased fibrosis. Removal of the rNeo cassette from mAbca3 alleles resulted in restoration of BAL PL content to wild-type levels and an absence of changes in lung histology up to 32 weeks of age. These results support the importance of surfactant PL homeostasis as a susceptibility factor for both intrinsic and exogenously induced lung injury/remodeling.

Keywords: ABCA3; Lamellar bodies; Lung disease; Pathogenesis; Phospholipids; Pulmonary; Surfactant.

Fig. 1

(A) Schematic illustration of the mAbca3^E292V–rNeo⁺ mouse model showing the FRT-pgk-gb2-Neo/km-FRT cassette insert site within intron 8 in the Abca3 locus together with the E292V point mutation in Exon 9. (B) Total bronchoalveolar lavage (BAL) phospholipid (PL) content of 16 wk old mAbca3^{E292 V}–rNeo+^/−, mAbca3^E292V–rNeo^+/+ mice and WT littermates were measured; mAbca3^E292V–rNeo^+/−, N = 9; for mAbca3^E292V–rNeo^+/+, N=10; WT, N = 5. ^*P<0.05, ^**P<0.001. (C, top) Representative immunoblot bands of anti-mature SP-B from BAL of mAbca3^E292V–rNeo^+/+ and WT control mice. (C, bottom) Quantitation of relative intensity of mature SP-B content (as determined by densitometric analysis) from at least 3 mice per group. (D) mRNA levels from mAbca3^E292V–rNeo^+/−, and mAbca3^E292V–rNeo^+/+ mice relative to WT mAbca3 as measured by real time quantitative PCR with at least 5 mice per group. ^*P<0.05, **P<0.01.

Fig. 2

(A) Representative electron micrographs of AT2 cells of 32 wk old WT, mAbca3^E292V–rNeo^+/−, and mAbca3^E292V–rNeo^+/+ mice. The profiles of AT2 cells in mAbca3^E292V–rNeo^+/+ mice appear to comprise smaller lamellar bodies compared to WT or mAbca3^E292V–rNeo^+/− mice. (B and C) LB size measured as number-weighted mean volume of LB (vN (LB) [μm³]) (B) and number of LBs per AT2 cells (N(LB, AT2)) (B). ^*P<0.05 vs WT. (D and E) AT2 cell size measured as number-weighted mean volume of AT (vN (AT2) μm³) (D) and number of AT2 cells (N(AT2, lung)) 10⁶ (E).

Fig. 3

BAL total cell counts from 4,8, 16, and 32 wk old mAbca3^E292V–rNeo^+/−, mAbca3^E292V–rNeo^+/+ mice and WT control littermates. At least 7 mice per group were used. ^*P<0.05, ^**P< 0.005.

Fig. 4

(A–C) Representative H&E staining of histological lung sections from 16 (A) and 32 (B and C) wk old mAbca3^E292V–rNeo^+/+ mice. (A) Moderate inflammatory cell infiltration (arrow) at 16 wk but otherwise normal alveolar structure is exhibited compared to WT littermates. (B) AT 32 wk, common features of the mAbca3^E292V–rNeo^+/+ mouse lung include patchy but significant perinuclear inflammatory cell infiltration (arrow), thinning of the septal wall, and marked enlargement of the alveolar space. (C) Profound distraction of alveolar architecture detected in 32 wk old mAbca3^E292V–rNeo^+/+ mice comprising areas of dense collagen deposition, medial thickening populated by an abundance of abnormally large macrophages (arrows). Boxed areas are magnified on the right of each image for better resolution. (D) Representative lung sections from two 32 wk old mAbca3^E292V–rNeo^{+/ +} mice and wild type littermates immunostained for Texas Red-conjugated proSP-C antibody (anti-NPRO). Bar, 50 μm.

Fig. 5

Light microscopy-based stereological measurement of lung structure showing decreased surface area of alveolar epithelium (S(salvepi, lung) [cm² ]) (A) and decreased volume of septal wall tissue (V(sep, lung) [mm³ ])(B)in mAbca3^E292V–rNeo^+/+ mice compared to mAbca3^E292V–rNeo^+/− mice and WT littermates. ^*P< 0.05, ^**P<0.001 vs WT.

Fig. 6

(A) Weight-loss in mAbca3^E292V–rNeo^+/−, mAbca3^E292V–rNeo^+/+, and WT control mice following IT bleomycin (2U/kg). At least 7 mice per group were used. (B) Representative trichrome staining of histological lung sections from 16 wk old mAbca3^E292V–rNeo^+/−, mAbca3^E292V–rNeo^+/+, mice and WT littermates 21 days post intratracheal (IT) bleomycin (1U/kg). Patchy areas of fibrosis were noted in WT and mAbca3^E292V–rNeo^+/− mice. In contrast, trichrome stain was more prominent in mAbca3^E292V–rNeo^+/+ mice displaying severe alveolar destruction and subpleural fibrosis enveloping several lobes. Boxed areas are magnified on the right of each image for better resolution.