ABCG1 regulates pulmonary surfactant metabolism in mice and men

Abstract

Idiopathic pulmonary alveolar proteinosis (PAP) is a rare lung disease characterized by accumulation of surfactant. Surfactant synthesis and secretion are restricted to epithelial type 2 (T2) pneumocytes (also called T2 cells). Clearance of surfactant is dependent upon T2 cells and macrophages. ABCG1 is highly expressed in both T2 cells and macrophages. ABCG1-deficient mice accumulate surfactant, lamellar body-loaded T2 cells, lipid-loaded macrophages, B-1 lymphocytes, and immunoglobulins, clearly demonstrating that ABCG1 has a critical role in pulmonary homeostasis. We identify a variant in the ABCG1 promoter in patients with PAP that results in impaired activation of ABCG1 by the liver X receptor α, suggesting that ABCG1 basal expression and/or induction in response to sterol/lipid loading is essential for normal lung function. We generated mice lacking ABCG1 specifically in either T2 cells or macrophages to determine the relative contribution of these cell types on surfactant lipid homeostasis. These results establish a critical role for T2 cell ABCG1 in controlling surfactant and overall lipid homeostasis in the lung and in the pathogenesis of human lung disease.

Keywords: ATP binding cassette transporter G1; cholesterol; lung; phospholipid; pulmonary alveolar proteinosis.

Fig. 1.

ABCG1 has a critical role in nonhematopoietic cells. A: Schematic of BM transplantation studies. Wild-type and Abcg1^−/− mice were irradiated and received BM from either wild-type or Abcg1^−/− donor animals. After a 4 week recovery period, all mice were fed a HF/HC (21% fat, 0.2% cholesterol) diet for 16 weeks. B–E: Frozen lung sections (10 μM) of BM-transplanted mice [as in (A)] were stained with filipin for the presence of free cholesterol. White arrows mark filipin-positive areas. Images are at 20× magnification. F: Frozen lung sections (10 μM) from Abcg1^EEC-KO mice (Abcg1^−/− mice receiving Abcg1^+/+ BM) were stained with antibodies for T2 cells (pro-SP-C; green arrows) and macrophages (Mac-3; red arrows), followed by staining with filipin (blue arrows) for free cholesterol. White arrows indicate areas of colocalization. Images are at 100× magnification. G–J: Representative electron micrographs (original magnification: 9,900×) from BM-transplanted mice [as in (A)]. K: The relative area of lamellar bodies within each T2 cell was determined in electron micrographs (n = 32) from each group of transplanted mice (G–J). Significance was measured by two-way ANOVA followed by Bonferroni correction. Data are expressed as mean ± SEM. *P < 0.01 wild-type versus Abcg1^−/− donor; ^§P < 0.01 wild-type versus Abcg1^−/− recipient. AM, alveolar macrophage; CC, cholesterol crystal; LB, lamellar body; LD, lipid droplet.

Fig. 2.

Mice with selective deletion of Abcg1 in T2 cells have abnormal surfactant and lamellar body homeostasis. A: The fresh weight of the lungs was increased in Abcg1^T2-KO mice. B: Representative electron micrographs from Abcg1^flox/flox and Abcg1^T2-KO mice (original magnification: 17,400×). Increased T2 cell number (C) and relative area of lamellar bodies within each T2 cell (D). E: Flow cytometry gating strategy to identify T2 cells (defined as EpCAM^hiT1α⁻ cells). Single-cell suspensions of negatively selected CD45⁻ cells were stained with fluorophore-conjugated antibodies and analyzed by flow cytometry. Among single cells, the live cells were selected for further analysis to identify T2 cells (EpCAM^hiT1α⁻). F: Abcg1 expression is significantly reduced in EpCAM^hiT1α⁻ T2 cells. G: ABCG1 protein is absent from EpCAM^hiT1α⁻ T2 cells. H: Abcg1 expression is unchanged in CD45⁺ cells isolated from Abcg1^T2-KO mice. I: Increased Abca3 and Abca1 expression in EpCAM^hiT1α⁻ T2 cells. J: Decreased Srebp-2, Fdps, and Ldlr expression in EpCAM^hiT1α⁻ T2 cells. K: Increased Il1β, Il6, and Tnfα expression in EpCAM^hiT1α⁻ T2 cells. Significance was measured by Student’s t-test. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3.

Disrupted lipid homeostasis and immunity in Abcg1^T2-KO mice. A–E: BAL fluid from Abcg1^flox/flox and Abcg1^T2-KO mice was diluted 1:5 to 1:250 and tested for binding to IgG (A), IgA (B), IgG2c (C), IgG1 (D), and IgM (E). ALP-conjugated antibodies were used for detection. Data are presented as mean antibody titer (ng/ml) ± SEM (n = 5–10 mice/genotype). F–H: Cholesterol, cholesteryl ester, and PC and their derivatives were quantified by ESI-MS/MS in BAL fluid from Abcg1^flox/flox and Abcg1^T2-KO mice. Data are presented as mean lipid level (pmol/μl) ± SEM (n = 3–6 mice/genotype). Significance was measured by Student’s t-test. *P < 0.05, **P < 0.01.

Fig. 4.

ABCG1 is required for the synthesis and secretion of cholesterol and phospholipids from A549 T2 cells. A, B: A549 T2 cells were infected overnight with either control adenovirus or Ad-ABCG1. Cells were incubated with ¹⁴C-acetate in medium containing 10% FBS for 6 h before total cellular lipids were extracted and separated by thin-layer chromatography to determine levels of neutral lipids (A) and phospholipids (B). C: A549 T2 cells were infected as in (A, B). Cells were pulse labeled with ¹⁴C-acetate for 4 h, followed by a 2 h chase in medium containing 10% FBS in the presence or absence of a secretagogue cocktail (100 μM ATP, 0.1 μM phorbol-12-myristate-13-acetate, 20 μM terbutaline). Total secreted lipids were extracted from the medium and separated by thin-layer chromatography to determine the levels of phospholipids. D–F: A549 T2 cells were transfected with a control scrambled siRNA sequence or siRNA sequences directed against ABCG1. D: Reduced ABCG1 expression in A549 cells treated with ABCG1 siRNA. Total cellular (E) and secreted (F) cholesterol and phospholipids were quantified by enzymatic assay according to manufacturer’s instructions. Data are presented as mean ± SEM (n = 6 replicates/condition). Significance was measured by one-way ANOVA followed by Bonferroni correction. *P < 0.01.

Fig. 5.

Abcg1^−/− macrophages signal to wild-type T2 cells. A, B: ABCG1 is absent in alveolar macrophages isolated from Abcg1^MAC-KO mice. A: Abcg1 expression in alveolar macrophages isolated from Abcg1^flox/flox and Abcg1^MAC-KO mice. B: ABCG1 protein in alveolar macrophages isolated from Abcg1^flox/flox and Abcg1^MAC-KO mice. C: Representative electron micrographs from Abcg1^flox/flox and Abcg1^MAC-KO mice (original magnification: 17,400×). D: Electron micrograph of a T2 cell from Abcg1^MAC-KO mice (original magnification: 22,600×). Increased T2 cell number (E) and relative area of lamellar bodies within each T2 cell (F) in Abcg1^MAC-KO mice. Data are expressed as mean ± SEM (n = 4–6 mice/genotype). AM, alveolar macrophage; CC, cholesterol crystal; LB, lamellar body; T2, T2 cell. Significance was measured by Student’s t-test. **P < 0.01, ***P < 0.001.

Fig. 6.

Sequence polymorphisms in human ABCG1 in patients with PAP. A: Genomic location of ABCG1 sequence polymorphisms. B: Sequence trace from control or PAP patient showing sequence polymorphisms. C, D: The 2,000 bp upstream of the ABCG1 transcriptional start site was cloned upstream of the luciferase gene from control and PAP patient genomic DNA. The reporter plasmid was transfected into CHO-K1 cells together with a β-galactosidase expression plasmid and increasing amounts of LXRα and RXRα expression plasmids in the presence or absence of LXRα agonist, GW3965 (1 μM). Promoter activity was normalized to β-galactosidase activity. Data are presented as mean ± SEM. Significance was measured by Student’s t-test. ***P < 0.001.

Fig. 7.

Reduced activation of ABCG1 by LXR in a patient with PAP. A–D: Macrophages isolated from a PAP patient and non-PAP control were plated and treated with a specific LXR agonist (1 μM GW3965) for the indicated time period. ABCG1 (A), ABCA1 (B), IDOL (C), and LPCAT3 (D) activation by LXR. Gene expression was normalized to 36B4 and presented as fold changes. Data are presented as mean ± SEM. Significance was measured by two-way ANOVA followed by Bonferroni correction. *P < 0.001.