Targeted PPAR{gamma} deficiency in alveolar macrophages disrupts surfactant catabolism.

Surfactant accumulates in alveolar macrophages of granulocyte-macrophage colony-stimulating factor (GM-CSF) knockout (KO) mice and pulmonary alveolar proteinosis (PAP) patients with a functional loss of GM-CSF resulting from neutralizing anti–GM-CSF antibody. Alveolar macrophages from PAP patients and GM-CSF KO mice are deficient in peroxisome proliferator-activated receptor-γ (PPARγ) and ATP-binding cassette (ABC) lipid transporter ABCG1. Previous studies have demonstrated that GM-CSF induces PPARγ. We therefore hypothesized that PPARγ promotes surfactant catabolism through regulation of ABCG1. To address this hypothesis, macrophage-specific PPARγ (MacPPARγ) knockout mice were utilized. MacPPARγ KO mice develop foamy, lipid-engorged Oil Red O positive alveolar macrophages. Lipid analyses revealed significant increases in the cholesterol and phospholipid contents of MacPPARγ KO alveolar macrophages and extracellular bronchoalveolar lavage (BAL)–derived fluids. MacPPARγ KO alveolar macrophages showed decreased expression of ABCG1 and a deficiency in ABCG1-mediated cholesterol efflux to HDL. Lipid metabolism may also be regulated by liver X receptor (LXR)–ABCA1 pathways. Interestingly, ABCA1 and LXRβ expression were elevated, indicating that this pathway is not sufficient to prevent surfactant accumulation in alveolar macrophages. These results suggest that PPARγ mediates a critical role in surfactant homeostasis through the regulation of ABCG1.

System (TaqMan; Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. RNA specimens were analyzed in duplicate using primer sets for mouse LXR ␣ (Mm00443454), LXR ␤ (Mm00437262), ABCA1 (Mm00442646), ABCG1 (Mm00437390), CYP27A1 (Mm00470430), and APOE (Mm00437573) (Applied Biosystems). Relative gene expression was quantifi ed as described ( 26 ). Briefl y, the control group (C57Bl/6) values were calculated by subtracting the raw cycle (CT) data for the housekeeping gene (GAPDH, 4352339E) from the cycle data for the gene of interest. The ensuing values ( ⌬ CT) were averaged and normalized to 1.0. Data from MacPPAR ␥ KO were quantifi ed in a similar fashion and expressed as relative mRNA expression compared with wild-type. For these experiments, BAL cells were isolated from individual MacPPAR ␥ KO mice and compared with pooled samples of C57Bl/6 BAL cells.

Cholesterol effl ux assay
Pooled BAL cells (3.5 × 10 5 /well) were plated in 48-well cell culture plates in complete DMEM media (Invitrogen, Carlsbad, CA) and maintained at 37°C and 5% CO 2 . Nonadherent cells were removed after 1 h. Cells were incubated for 24 h in 2 Ci/ ml of [1,[2][3] H(N)]cholesterol (NEN, Perkin Elmer, Waltham, MA), equilibrated in serum-free media for 24 h, and incubated in the presence of 10% fetal bovine serum (FBS), apolipoprotein A-I (ApoA-I) (25 g/ml) (Sigma) or HDL (25 g/ml) (Intracel, Frederick, MD) for 24 h. Supernatant fl uids were harvested and centrifuged at 1800 rpm for 5 min to remove cellular debris. Cells were washed with PBS and lysed in a 0.2 M sodium hydroxide (NaOH) and 0.1% SDS solution for 1 h at room temperature. Supernatant and cell-associated radioactivity was measured by liquid scintillation. Cholesterol effl ux was expressed as the percentage of radioactivity in the supernatant divided by the total radioactivity of the cells and supernatant. Each assay was performed in duplicate, and results from three independent assays were used to calculate percentage effl ux.

Immunoblotting
For analysis of BAL cell protein, samples were loaded based on equal total protein determined using a modifi ed Lowry assay (Dc Protein Assay, Bio-Rad Laboratories, Hercules, CA). Gels were eletrophoresed under reducing conditions using a 10% Bis-Tris gel (Bio-Rad) with MOPS buffer (Invitrogen). The following primary antibodies and dilutions were used: 1:500 ABCG1 (sc-11150) and 1:500 ABCA1 (sc-5491, Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Bands corresponding to ABCG1 were normalized to ␤ -actin as the loading control, and intensity of the protein bands were quantifi ed using ImageQuant TL (GE Healthcare, Little Chalfont, UK). Bands corresponding to ABCA1 were analyzed in the same manner using ImageJ.

Lipid extraction
Total lipids were extracted from BAL cells and fl uid using a modifi ed method of Bligh and Dyer in HPLC-grade chloroform/methanol/1M sodium chloride (NaCl) (2/1/1.25, v/v/v) (Sigma) ( 27 ). The organic phase was obtained by centrifugation at 1500 rpm. Lipids were dried under a gentle stream of nitrogen gas. The Phospholipids C kit (Wako Pure Chemicals, Osaka, Japan) was used according to manufacturer's instructions. Phospholipid content was expressed as mg phospholipid per mg protein.

Cholesterol content analysis
The total cholesterol content from BAL cells and fl uid was analyzed using the Amplex Red Cholesterol Assay (Invitrogen) according to the manufacturer's protocol. Samples were assayed stitutively expressed in the alveolar macrophages of healthy controls and is upregulated by GM-CSF ( 12,13 ). Our previous studies have shown that the alveolar macrophages of PAP patients and the GM-CSF knockout (GM-CSF KO) mouse model of PAP are defi cient in PPAR ␥ ( 12,14 ).
While the role of PPAR ␥ in surfactant catabolism in the lung remains unclear, PPAR ␥ is known to directly and indirectly regulate many genes involved in cholesterol metabolism and transport, including the nuclear transcription factor liver X receptor ␣ (LXR ␣ ) and ATP-binding cassette (ABC) lipid transporters ABCG1 and ABCA1 (14)(15)(16)(17). Studies have suggested that PPAR ␥ defi ciencies result in decreased expression of ABCG1 ( 14,16 ). The deletion of ABCG1 in mice (ABCG1 KO) results in severe pulmonary lipidosis ( 18 ). Cholesterol and phospholipid accumulate and foam-cell formation occurs in the macrophages of ABCG1 KO (18)(19)(20). Moreover, ABCG1 KO macrophages display reduced capacities to effl ux cholesterol and phospholipid ( 19,(21)(22)(23). We therefore hypothesized that PPAR ␥ may promote surfactant catabolism through regulation of the lipid transporter ABCG1. To test this hypothesis, we investigated the alveolar macrophages from macrophage-specifi c PPAR ␥ knockout (MacPPAR ␥ KO) mice.

Mice
Animal studies were conducted in conformity with Public Health Service policy on the humane care and use of laboratory animals and were approved by the Institutional Animal Care Committee. C57Bl/6 wild-type (WT) mice were obtained from Jackson Laboratory (Bar Harbor, ME). Macrophage-specifi c PPAR ␥ knockout (MacPPAR ␥ KO) mice have been previously described ( 24 ). Bronchoalveolar lavage (BAL) cells were obtained as described earlier from 8-to 12-week-old MacPPAR ␥ KO mice and age-and gender-matched wild-type C57Bl/6 controls ( 24 ). Briefl y, the thoracic cavity was opened and the lungs were exposed. After cannulating the trachea, a tube was inserted, and BAL was carried out with warmed (37°C) PBS in 1 ml aliquots × 5. Except where indicated, the sample number (n) refers to sets of BAL cells pooled from 3-5 mice, whereas BAL fl uid was analyzed from individual mice. Following previously established guidelines for analysis of acellular components of BAL fl uid ( 25 )

RNA purifi cation and analysis
Total RNA was extracted from BAL cells by the RNeasy protocol (Qiagen, Valencia, CA). Expression of mRNA was determined by real-time RT-PCR analysis using the ABI Prism 7300 Detection type (4.5 ± 0.3%), and the overall cholesterol effl ux to media supplemented with FBS was decreased in the MacPPAR ␥ KO (59.5 ± 1.7%) relative to wild-type (70.5 ± 3.5%) ( Fig. 3 ). We next measured the effl ux of cholesterol to acceptor molecules HDL and ApoA-I. Cholesterol effl ux to ApoA-I in MacPPAR ␥ KO (25.7 ± 1.7%) was signifi cantly increased over wild-type (17.3 ± 1.5%), and effl ux to HDL was signifi cantly decreased in MacPPAR ␥ KO (46.2 ± 1.5%) compared with wild-type (56.7 ± 3.6%). These results suggest impairment of ABCG1-mediated cholesterol effl ux. in serial dilution in 96-well plates. Cholesterol content was expressed as g cholesterol per mg protein.

Statistical analysis
Data were analyzed by Student's t -test using Prism software (GraphPad, Inc., San Diego, CA). Signifi cance was defi ned as P р 0.05.

PPAR ␥ defi ciency results in lipid accumulation and dysregulation of lipid transporters in alveolar macrophages
Wright-Giemsa staining revealed large foamy alveolar macrophages and Oil Red O staining showed that 88.8 ± 1.7% of MacPPAR ␥ KO alveolar macrophages stained positive, compared with 2.4 ± 1.0% of wild type, indicating neutral lipid accumulation in the MacPPAR ␥ KO ( P < 0.0001) ( Fig. 1A ). Because of the lipid accumulation, we evaluated mRNA expression of the lipid transporters ABCG1 and ABCA1, which are known to be involved in lipid metabolism in macrophages and are downstream targets of PPAR ␥ ( 28 ). ABCG1 mRNA was decreased by 30%; in contrast, ABCA1 was increased 5.9-fold ( Fig. 1B ). Decreased ABCG1 and increased ABCA1 protein expression were confi rmed by immunoblotting ( Fig. 1C-D ).

Surfactant lipids accumulate in the lungs of MacPPAR ␥ KO mice
The composition of the lipid accumulating in the lungs of the MacPPAR ␥ KO was determined by measuring both extracellular and intracellular cholesterol and phospholipid levels in BAL fl uids and alveolar macrophages. Compared with wild-type mice, cellular content of free cholesterol was signifi cantly increased in MacPPAR ␥ KO mice (0.39 ± 0.07 versus 5.80 ± 1.69 g/mg protein) while the cholesteryl ester content was not signifi cantly different (0.12 ± 0.01 versus 0.58 ± 0.29 g/mg protein) ( Fig. 2A ). Free cholesterol was also elevated in the BAL fl uid of MacPPAR ␥ KO mice (59.6 ± 5.7 g/mg protein) compared with the wild-type mice (17.8 ± 1.3 g/mg protein) ( Fig. 2B ). Cholesteryl esters were not detected in the BAL fl uid of wild-type or MacPPAR ␥ KO mice. The cellular phospholipid content in MacPPAR ␥ KO alveolar macrophages was signifi cantly increased over wild-type (0.03 ± 0.01 versus 0.26 ± 0.07 mg/mg protein) ( Fig. 2C ). Extracellular phospholipids were elevated in the BAL fl uid of MacPPAR ␥ KO mice (257.5 ± 28.9 mg/mg protein) compared with wild-type (174.2 ± 16.0 mg/mg protein) ( Fig.  2D ).

PPAR ␥ defi ciency results in decreased cholesterol effl ux to HDL from alveolar macrophages
The accumulation of cholesterol in the lungs and alveolar macrophages of the MacPPAR ␥ KO and decreased expression of key cholesterol effl ux mediators led us to evaluate the cholesterol effl ux system. Baseline cholesterol effl ux (no acceptor) was increased in the MacPPAR ␥ KO alveolar macrophages (8.3 ± 0.8%) compared with wild- alveolar macrophages, indicating that the LXR pathway is enhanced ( Fig. 4B ).

DISCUSSION
In the present study we show that the targeted knockout of PPAR ␥ in macrophages results in the accumulation of surfactant-like material in the alveolar spaces of the lung and within the alveolar macrophages. This is the fi rst report directly linking the defi ciency of PPAR ␥ to lipid accumulation in the lung. MacPPAR ␥ KO alveolar macrophages phenotypically resemble those of PAP patients in that they are foamy and Oil Red O-positive for neutral lipid accumulation ( 11 ). Additionally, surfactant lipid components (phospholipid and cholesterol) are increased within extracellular BAL fl uids. Finally, the alveolar macrophages of MacPPAR ␥ KO mice are ABCG1-defi cient and exhibit reduced ABCG1-mediated cholesterol effl ux to HDL. Our results support the hypothesis that PPAR ␥mediated regulation of ABCG1 expression is critical for surfactant catabolism in alveolar macrophages.
Previous studies have suggested that PPAR ␥ is a key mediator of surfactant clearance and catabolism by alveolar macrophages ( 12,14 ). Surfactant accumulates in alveolar macrophages of PAP patients. PPAR ␥ is defi cient in the alveolar macrophages of these patients and is associated with the presence of neutralizing auto-antibodies against the hematopoietic growth factor GM-CSF ( 12 ). PPAR ␥ defi ciencies were also demonstrated in the GM-CSF KO mouse model of the disease ( 14 ).
GM-CSF also promotes cell survival, proliferation, and differentiation of alveolar macrophages and promotes the transcription of PPAR ␥ in macrophages ( 13,30,31 ). The biological loss of GM-CSF has been reported to impair the differentiation of alveolar macrophages through dysregulation of the transcription factor PU.1 ( 4 ). It was further demonstrated that PU.1 is defi cient in the alveolar macrophages of PAP patients and GM-CSF KO mice ( 32,33 ); however, no defi ciency in GM-CSF or PU.1 mRNA expression was observed in the alveolar macrophages of the MacPPAR ␥ KO mice. GM-CSF was up-

PPAR ␥ defi ciency results in dysregulated LXR ␣ and LXR ␤ expression
Given the increased expression of ABCA1 in MacPPAR ␥ KO alveolar macrophages, we next investigated the expression of the LXR transcription factors, which may regulate cholesterol metabolism in macrophages in part by mediating transcription of the ABC transporters ( 29 ). RT-PCR analysis revealed LXR ␣ mRNA was downregulated by 40% and LXR ␤ mRNA was upregulated 2.1-fold in MacPPAR ␥ KO alveolar macrophages ( Fig.  4A ). RT-PCR analysis also revealed increased expression in sterol 27-hydroxylase (CYP27A1) (2.3-fold) and apolipoprotein E (ApoE) mRNA (34-fold) in MacPPAR ␥ KO  RT-PCR analysis of the MacPPAR ␥ KO alveolar macrophages revealed similar gene expression patterns of downstream PPAR ␥ direct and indirect targets to those previously reported from PAP patients and GM-CSF KO mice with decreased expression of ABCG1 mRNA and increased expression in ABCA1 mRNA ( 12,14 ). These results, which are consistent with several studies, indicate that defi ciency of one ABC transporter is compensated by the other transporter and is mediated by the sterol-sensing nuclear transcription factor LXR in response to the buildup of oxysterol ligands ( 44 ) or the oxidation of cholesterol metabolites by CYP27A1 in foamy macrophages ( 45 ). The two isoforms of LXR-LXR ␣ and LXR ␤ -have overlapping roles in promoting cellular cholesterol export through regulation of the ABC transporters and ApoE ( 29 ).
In contrast to the increased LXR ␣ expression reported in PAP and GM-CSF KO ( 14 ), LXR ␣ was downregulated in the MacPPAR ␥ KO mice. The differential expression of LXR ␣ , which is regulated in part by PPAR ␥ ( 16 ), may be explained by the varying levels of PPAR ␥ in these systems: PPAR ␥ is defi cient in PAP and GM-CSF KO while it is absent in MacPPAR ␥ KO. This is supported by the fi nding that LXR ␤ expression, which is regulated independently of PPAR ␥ ( 16 ), is increased 2-fold in the MacPPAR ␥ KO alveolar macrophages. The expression of LXR ␤ has not been reported in PAP or GM-CSF KO alveolar macrophages.
While more study is needed to elucidate the possible mechanisms and differential regulation of the LXRs, it has been shown that the function and expression of the LXR isoforms are tissue-dependent ( 29,46,47 ). LXR ␤ is expressed at higher levels than LXR ␣ in macrophages and is more effective than LXR ␣ at upregulating ABCA1 in response to sterol ligands ( 48 ). While the contributions of the individual LXR isoforms are unknown, as specifi c gene targets have yet to be identifi ed, the LXR pathway overall is enhanced in the MacPPAR ␥ KO, as evidenced by increased expression of downstream targets ABCA1 and ApoE. We show that increased expression of the LXR pathway is not suffi cient to maintain surfactant catabolism in the absence of PPAR ␥ .
The accumulation of cholesterol in the lungs and alveolar macrophages of MacPPAR ␥ KO mice and the dysregulation of several cholesterol transport genes led us to investigate the effl ux of cholesterol in MacPPAR ␥ KO alveolar macrophages in vitro. PPAR ␥ promotes lipid infl ux and effl ux in macrophages through transcriptional regulation of ABC transporters and LXRs. ABCG1 mediates transport of cholesterol to extracellular acceptor HDL, and ABCA1 transports cholesterol to lipid-free ApoA-I ( 19,22,23,49,50 ). In the present study, overall cholesterol effl ux to FBS (10% serum) was decreased compared with wild-type, consistent with the high levels of cholesterol in the macrophages in vivo. ABCA1-mediated effl ux to ApoA-I was increased, while ABCG1-mediated cholesterol effl ux to HDL was reduced. These fi ndings indicate that the reduction in cholesterol effl ux in the MacPPAR ␥ KO alveolar macrophages may be because of defi cient regulated 2.7 ± 0.25-fold (n = 5, P = 0.02) while PU.1 expression was not different from wild-type mice (n = 3). These data suggest that maturation of the MacPPAR ␥ KO alveolar macrophages is not disrupted as it is in PAP patients and GM-CSF KO mice ( 4 ). This is consistent with current literature, which suggests that although PPAR ␥ is not necessary for the differentiation of monocytes, a variation in the expression levels of PPAR ␥ may modulate differentiation (34)(35)(36).
Consistent with the fi ndings in PAP patients and GM-CSF KO mice (37)(38)(39)(40)(41), the MacPPAR ␥ KO mice exhibit e levated levels of the major lipid components of surfactant, including cholesterol and phospholipid, in the BAL fl uid and alveolar macrophages. The alveolar macrophages from the MacPPAR ␥ KO mice had signifi cant accumulation of free (unesterifi ed) cholesterol. Cellular deposition of cholesterol is considered to be an initial step in foamcell formation ( 42 ) and is consistent with the foamy phenotype of the MacPPAR ␥ KO alveolar macrophages. The impact that free cholesterol accumulation has on cell signaling, plasma membrane rigidity, and induction of pro-apoptotic cascades warrants further study ( 43 ). The pattern of lipid accumulation both in the alveolar space and alveolar macrophages of the lungs of MacPPAR ␥ KO mice suggests defi cient or incomplete surfactant catabolism by the alveolar macrophages. transporter-mediated cholesterol effl ux pathways, specifically transport mediated by ABCG1.
Interestingly, similar patterns of cholesterol and phospholipid accumulation and altered lipid effl ux have been reported in ABCG1 KO models ( 18,20 ). A reduction in total cholesterol effl ux and a specifi c reduction in effl ux to HDL were noted in ABCG1 KO peritoneal macrophages ( 20 ). Importantly, the authors also noted a signifi cantly increased effl ux to ApoA-I in ABCG1 KO, which indicated compensation by ABCA1. Taken together, defi ciencies in ABCG1 may result in dysregulated or insuffi cient cholesterol effl ux and, therefore, cholesterol accumulation in the lung.
A summary of the differential gene expression of various lipid regulators and transporters in the alveolar macrophages from MacPPAR ␥ KO mice, GM-CSF KO mice, and PAP patients is presented in Table 1 . Comparison of the data supports the hypothesis that PPAR ␥ -mediated regulation of ABCG1 is necessary to prevent the accumulation of surfactant. Additionally, the LXR pathway is enhanced in all of the groups, as evidenced by increased expression in ABCA1. An interesting difference, however, is the expression of LXR ␣ , which is increased in PAP and GM-CSF KO (PPAR ␥ -defi cient) alveolar macrophages and decreased in the MacPPAR ␥ KO model. We speculate that lipid accumulation activates the LXR-ABCA1 pathway as a compensation mechanism and that, in the absence of PPAR ␥ , LXR ␤ is the predominant isoform driving the upregulation of ABCA1 and ApoE.
In the MacPPAR ␥ KO mouse model, the absence of PPAR ␥ results in reduced expression levels of ABCG1 and LXR ␣ . Despite increased expression of LXR ␤ and ABCA1 and increased ABCA1-mediated cholesterol effl ux, surfactant components accumulate in the alveolar macrophages and BAL fl uid of MacPPAR ␥ KO mice. Our results indicate that as part of surfactant catabolism, ABCG1-mediated cholesterol effl ux to HDL may be a pathway for cholesterol effl ux in alveolar macrophages. Thus PPAR ␥mediated regulation of ABCG1 expression may be critical to the maintenance of surfactant homeostasis. This is the fi rst report directly linking PPAR ␥ defi ciency in alveolar macrophages to lipid accumulation in the lungs. Understanding the role of PPAR ␥ in normal surfactant homeo- Abbreviations: ABC, ATP binding cassette; GM-CSF, granulocytemacrophage colony-stimulating factor; KO, knockout; LXR, liver X receptor; MacPPAR ␥ , macrophage-specifi c PPAR ␥ ; PAP, pulmonary alveolar proteinosis. Numbers in parentheses are reference citations. a GM-CSF mRNA is increased ( 52 ). However, protein is functionally reduced due to neutralizing antibodies against GM-CSF ( 1 ).