Pulmonary Inflammation Loss of ABCG1 Results in Chronic

Loss of ABCG1 Results in Chronic Pulmonary Inflammation Ángel Baldán, Aldrin V. Gomes, Peipei Ping and Peter A. Edwards This information is current as of September 27, 2021. J Immunol 2008; 180:3560-3568; ; doi: 10.4049/jimmunol.180.5.3560 http://www.jimmunol.org/content/180/5/3560 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Loss of ABCG1 Results in Chronic Pulmonary Inﬂammation1

A´ ngel Balda´n,*† Aldrin V. Gomes,†‡ Peipei Ping,†‡ and Peter A. Edwards2*†§

ABCG1, a member of the ATP-binding cassette transporter superfamily, is highly expressed in multiple cells of the lung. Loss of ABCG1 results in severe pulmonary lipidosis in mice, with massive deposition of cholesterol in both alveolar macrophages and type 2 cells and the accumulation of excessive surfactant phospholipids. These observations are consistent with ABCG1 controlling cellular sterol metabolism. Herein, we report on the progressive and chronic inflammatory process that accompanies the lipidosis in the lungs of Abcg1؊/؊ mice. Compared with wild-type animals, the lungs of aged chow-fed mice deficient in ABCG1 show distinctive signs of inflammation that include macrophage accumulation, lymphocytic infiltration, hemorrhage, eosinophilic crystals, and elevated levels of numerous cytokines and cytokine receptors. Analysis of bronchoalveolar lavages obtained from -Abcg1؊/؊ mice revealed elevated numbers of foamy macrophages and leukocytes and the presence of multiple markers of in flammation including crystals of chitinase-3-like proteins. These data suggest that cholesterol and/or cholesterol metabolites that ؊/؊ accumulate in Abcg1 lungs can trigger inflammatory signaling pathways. Consistent with this hypothesis, the expression of a Downloaded from number of cytokines was found to be significantly increased following increased cholesterol delivery to either primary peritoneal macrophages or Raw264.7 cells. Finally, cholesterol loading of primary mouse macrophages induced cytokine mRNAs to higher levels in Abcg1؊/؊, as compared with wild-type cells. These results demonstrate that ABCG1 plays critical roles in pulmonary homeostasis, balancing both lipid/cholesterol metabolism and inflammatory responses. The Journal of Immunology, 2008, 180: 3560–3568. http://www.jimmunol.org/ nflammation is a complex biological response to tissue injury tral lipid content of the lungs can also modulate pulmonary in- and/or infection that facilitates tissue repair and the removal flammatory responses. These authors demonstrated that disruption I of the causal stimuli. Whereas acute inflammation is gener- of triglyceride and cholesteryl ester metabolism in alveolar mac- ally considered a physiological process that resolves in a relative rophages, as a result of lysosomal acid lipase deficiency, results in short period of time, chronic inflammation represents a prolonged respiratory inflammation, tissue remodeling, and emphysema (11, pathological condition in which simultaneous destruction and heal- 12). Together, these results indicate that factors governing surfac- ing of tissues eventually results in further tissue damage (1, 2). tant composition and neutral lipid metabolism in alveolar cells Chronic inflammation is also characterized by the presence of in- might be associated with chronic pulmonary inflammation result- filtrated mononuclear cells that include macrophages, lympho- ing in asthma, allergy, chronic obstructive pulmonary disease by guest on September 27, 2021 cytes, and plasma cells (1, 2). (COPD),3 or lung cancer. The continuous exposure of pulmonary alveolar spaces to in- ABCG1 is a member of the ATP-binding cassette (ABC) family haled foreign Ags (bacteria, viruses, fungi, and particles of varying of transmembrane transporters (reviewed in Ref. 13). Earlier stud- size) could potentially result in an excessive and violent inflam- ies demonstrated that Abcg1 mRNA levels are highly induced fol- matory response. Thus, precise mechanisms that balance pro- and lowing incubation of human and murine macrophages with either anti-inflammatory responses exist in the lung to ensure an appro- oxidized or acetylated low density lipoprotein (LDL) or with ago- priate response to environmental agents. Anti-inflammatory strat- nists that activate the nuclear liver X receptor (LXR) (14, 15). More egies that suppress the immune response in the lung include an- recent studies have shown that ABCG1 protein facilitates cholesterol ergization of T cells by type 2 pneumocytes (3), disruption of efflux from cells to a variety of exogenous lipid acceptors that include LPS-stimulated cytokine secretion in alveolar macrophages by mature high density lipoprotein (HDL) particles, phospholipid ves- specific surfactant phospholipids or proteins (4–6), and neutral- icles, and phospholipid/apoprotein complexes but not lipid-poor ization of different pathogens by surfactant collectins SP-A and apoA1 (15–21). Studies using both Abcg1Ϫ/Ϫ and ABCG1 transgenic SP-D (7–10). Recently, Lian et al. (11, 12) suggested that the neu- mice demonstrated that ABCG1 plays a critical role in controlling pulmonary and hepatic lipid homeostasis in response to a high fat, high cholesterol diet challenge (20). Subsequently, we reported that *Department of Biological Chemistry, Center for Health Sciences, David Geffen School Ϫ/Ϫ of Medicine, †Department of Medicine, ‡Department of Physiology, and §Molecular Bi- Abcg1 mice exhibit an age-related, progressive pulmonary disease ology Institute, University of California, Los Angeles, CA 90095 that has many of the properties associated with human respiratory Received for publication September 5, 2007. Accepted for publication January distress syndromes (22). Thus, although the lungs of 3-mo-old 2, 2008. Abcg1Ϫ/Ϫ mice appear normal by most criteria, by the age of 8 mo the The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by National Institutes of Health Grants 30568 and 3 Abbreviations used in this paper: COPD, chronic obstructive pulmonary disease; 68445 (to P.A.E.), a grant from the Laubisch Fund (to P.A.E.), and a grant from ´ ABC, ATP-binding cassette; LDL, low density lipoprotein; RT-qPCR, real time quan- Pfizer, Inc. (to P.A.E.). A.B. was partially supported by an American Heart Associ- titative PCR; BAL, bronchoalveolar lavage; HF/HC, high fat/high cholesterol; MMP, ation (Western Affiliate) Postdoctoral Fellowship (0525010Y). matrix metalloproteinases; LXR, liver X receptor; HDL, high density lipoprotein; 2 Address correspondence and reprint requests to Dr. Peter A. Edwards, University of TIMP, tissue inhibitors of matrix metalloproteinases; ACAT, acyl-coenzymeA:cho- California-Los Angeles, Department of Biological Chemistry, 615 Charles E. Young lesterol acyltransferase. Drive South, Box 951737, BSRB, Los Angeles, CA 90095. E-mail address: [email protected] Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 www.jimmunol.org The Journal of Immunology 3561 lungs of chow-fed Abcg1Ϫ/Ϫ mice contain massive numbers of cholesterol- and cholesteryl ester-loaded macrophages and a 5-fold increase in type 2 cells that contain excessive numbers of lamellar bodies (22). Taken together, these studies identified pivotal roles for ABCG1 in controlling pulmonary homeostasis in vivo (20, 22). In- terestingly, based on gene deletion in mice and/or natural mutations in humans, other members of the ABC family of transporters, ABCA1, ABCA3, and ABCC7, have also been shown to play important roles in pulmonary physiology (23–27). In this report, we focus on the progressive, age-dependent, chronic inflammatory process that accompanies the lipidosis in the lungs of Abcg1Ϫ/Ϫ mice. The data are consistent with the proposal that the regulation of lipid/cholesterol metabolism has a critical Ϫ Ϫ role in balancing pulmonary pro- and anti-inflammatory responses FIGURE 1. Histological signs of inflammation in Abcg1 / lungs. and that ABCG1 plays an essential role in these processes. Staining of sections with H&E revealed severe architectural changes in the lungs from Abcg1Ϫ/Ϫ mice, compared with wild-type controls. Alveolar spaces are normal in 8-mo-old, chow-fed wild-type lungs (A). In contrast, Materials and Methods Ϫ Ϫ massive cell accumulation is observed in Abcg1 / lungs, especially in Animals subpleural areas (B). Many of these cells correspond to giant macrophages Downloaded from Male Abcg1Ϫ/ϪLacZ knock-in mice on a C57BL/6 background were gen- (asterisks). White needle-like clefts (arrowheads in B and D) are likely the erated and maintained on a standard rodent diet (Purina 5001) as described result of cholesterol crystals that are lost from the sample during the fix- (20, 22). All protocols involving mice were reviewed and approved by the ation process. D, An enlarged picture of these giant macrophages (aster- University of California-Los Angeles Animal Research Committee. isks) and cholesterol clefts. Massive lymphocytic (L) infiltration and signs Cell culture of hemorrhage (H) were also noted throughout the lung parenchyma in sections from Abcg1Ϫ/Ϫ mice (C). Eosinophilic crystals (arrows in B and Thioglycollate-elicited primary peritoneal macrophages were obtained and E) were present mostly in peri-bronchiolar areas, generally associated to http://www.jimmunol.org/ maintained as described (20). Raw264.7 macrophages (American Type macrophages and/or lymphocytes. Culture Collection) were maintained in DMEM plus 10% FBS. The day before the experiments, cells were plated in 6-well plates in DMEM plus 1% FBS. The next morning the media was switched to DMEM plus 1% ing conditions using NuPAGE 4–20% Bis-Tris gradient gels (Invitrogen FBS plus cyclodextrin Ϯ cholesterol and the cells then incubated for 6 h. Life Technologies). Commassie blue G-250 stained bands of interest were Where indicated, cells were incubated in media containing 50 ng/ml LPS. removed from SDS-PAGE and digested with trypsin as described previ- Cyclodextrin and cyclodextrin-cholesterol (50:1 molar ratio) media was ously (31). Recovered peptide mixtures were dried, resuspended in 30% prepared as described (28). Ϫ CH3CN containing 1% trifluoroacetic acid, and stored at 20°C until use. Histopathologic analysis LC-MS/MS experiments were performed on a Proteomex LTQ tandem mass spectrometry instrument (Thermo Electron) with a surveyor pump H&E staining of paraffin-embedded lung sections was performed as de- system using a reversed phase column (75 ␮m i.d. 10 cm, BioBasic C18 5 by guest on September 27, 2021 scribed (20). ␮m particle size, New Objective). The flow rate was 5 ␮l/min for sample loading and 250 nL/min for separation. Buffer A was 0.1% formic acid Immunofluorescence studies with 2% acetonitrile in water, and Buffer B was 0.1% formic acid with 20% Preparation of frozen lung sections were as described (20). For immuno- water in acetonitrile. Analyses were performed using a shallow gradient fluorescence studies, FITC-conjugated anti-CD45R/B220 (1/100 dilution, from 5% B to 40% B over 70 min, then from 40% B to 100% B over 20 BD Biosciences no. 553087) and PE-conjugated anti-CD3␧ (1/50 dilution, min, and finally using 100% B for 9 min. The ion transfer tube of the linear BD Biosciences no. 553063) were used following standard protocols. ion trap was held at 200°C; the normalized collision energy was 35% for MS/MS; and the spray voltage was set at 1.9 kV. Briefly, the mass spec- RNA isolation and analysis trometer was operated in the data-dependent mode to switch automatically between MS and MS/MS acquisition. Survey full-scan MS spectra with 1 RNA was isolated and analyzed by real time quantitative PCR (RT-qPCR) microscans (m/z 400-2000) were acquired, followed by five sequential scan as described (20). Each qPCR assay was performed in duplicate using events of MS/MS. Each subsequent MS/MS collision induced dissociation cDNA samples isolated from individual mice (n ϭ 4–6/genotype) or from fragmentation (at a target value of 10,000 ions) was performed on a pre- duplicate dishes of cells. Primer sets are available upon request. Values cursor ion, which was isolated using the data-dependent acquisition mode were normalized to GAPDH and calculated using the comparative Ct to automatically select ions with sequentially top five highest intensities method. Gene expression profile of inflammation markers was determined from the survey scan, with a 3.0 m/z isolation width. In the acquisition using the GEArray mouse inflammatory cytokines and receptors microar- mode, dynamic exclusion was used with two repeat counts within 10 s, and ray system from Superarray Bioscience Corporation, following the manu- with an exclusion duration of 40 s. The spectra were searched with facturer recommendations. SEQUEST against the murine IPI database with cardamidomethyl and methionine oxidation as modifications and the following criteria used Protein isolation and analysis for peptide identification: Xcorr, Ն2.2 (ϩ1), Ն2.5 (ϩ2), Ն3.8 (ϩ3); Protein extracts were obtained from the lungs of wild-type and Abcg1Ϫ/Ϫ DeltaCN Ͼ 0.1; and a peptide probability of 0.001. All proteins were mice, as described (29). Fifty ␮g of protein were resolved in NuPAGE 12% identified on the basis of Ն2 peptides. Bis-Tris gels (Invitrogen Life Technologies) and transferred to polyvinyli- dene difluoride membranes. The expression of MCP-1, TNF-␣, and ␤-actin Statistical analysis was detected using specific Abs (PeproTech P113, Santa Cruz Technology Differences between samples were analyzed by Student’s t test. sc-1350, and Abcam ab6276, respectively) diluted in TBS containing 0.1% Tween 20 and 4% nonfat dried milk. Immune complexes were detected with HRP-conjugated secondary Abs (Bio-Rad) diluted 1/5000. Results Basal inflammation in the lungs of Abcg1Ϫ/Ϫ mice Analysis of bronchoalveolar lavage (BAL) We previously reported that chow-fed mice lacking ABCG1 ex- BAL were performed as described (22). Recovered cells were pelleted by hibit a progressive and severe pulmonary lipidosis; at 3 mo of age gentle centrifugation (500 ϫ g, 5 min). Crystals present in the initial pellet were dissolved in PBS-Isolymph (Gallard-Schlesinger) and purified by the lungs appear normal, but by 6 mo they appear white as a result successive centrifugations as described (30). Cell-free supernatants from of massive lipid deposition (22). Interestingly, this phenotype was Ϫ Ϫ BAL and purified crystals were resolved in SDS-PAGE gels under reduc- greatly accelerated when young Abcg1 / mice were fed a diet 3562 ABCG1 AND LUNG INFLAMMATION Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 2. Abnormal cellular and protein recovery in BAL from Abcg1Ϫ/Ϫ mice. Lavages (n ϭ 4/group) were pooled and gently centrifuged. Pelleted cells were resuspended in the same volume of complete DMEM media, and equivalent aliquots were either plated for 45 min or used to count macrophages (MØ) and lymphocytes (A–C). No crystals were observed in lavages from wild-type mice. Crystals were purified from the BAL, as detailed in Materials and Methods. Aliquots from the supernatants (lanes 1 and 2) and the purified crystals (lane 4) were resolved in SDS-PAGE gels (D). Lane 3 corresponds to a wild-type sample that was run in parallel during the crystal purification process. Specific bands were excised from the gel and the identity of the proteins determined by LC-MS/MS (E). The number of total peptides identified for each protein is noted in brackets. Chitinase 3-like variants 3 and 4 (†) were the only proteins identified in purified crystals (D, lane 4). MS/MS spectra from two peptides identifying variants 3 and 4 (left and right, respectively) (F). .(p Յ 0.01 ,ء ;mRNA levels in total lung for L-plastin, cathepsin B, and chitinase-like proteins were determined by RT-qPCR (G)(n ϭ 4 mice/group

supplemented with high levels of fat and cholesterol (20). Under stained extracellular eosinophilic structures (Fig. 1E), and extra- these latter dietary conditions, the lungs of 3-mo-old Abcg1Ϫ/Ϫ cellular clefts (Fig. 1, B and D). None of these features, with the mice were white, likely as a result of the excess lipids that included exception of very sporadic, very small perivascular lymphocytic cholesterol esters, cholesterol crystals, and phospholipids. In ad- foci, were observed in lung sections from wild-type mice (Fig. 1A; dition, the lungs of these Abcg1Ϫ/Ϫ mice showed evidence of lym- data not shown). Interestingly, giant cells present in the Abcg1Ϫ/Ϫ phocytic infiltration, excess foamy lipid-loaded macrophages and lungs showed a scattered pattern of nuclei distribution (Fig. 1, B proliferating type 2 cells (22). and D, and data not shown) similar to foreign body granuloma Herein, we report that the lungs of Abcg1Ϫ/Ϫ mice also show cells, and in contrast to Langhans-type granuloma cells that typi- signs of severe basal inflammation. H&E-stained sections of the cally contain a circular peripheral arrangement of the numerous lungs of 8-mo-old chow-fed Abcg1Ϫ/Ϫ mice indicate they contain nuclei (32). No signs of fibrosis, as measured by Masson’s large numbers of subpleural macrophages and lymphocytes (Fig. trichrome staining, were noted in the lungs of Abcg1Ϫ/Ϫ or wild- 1, B and C), perivascular lymphocytes (Fig. 1D), large orange- type mice (data not shown). When a semiquantitative method, The Journal of Immunology 3563

tify them as chitinase 3-like variants 3 and 4, respectively. Based on the MS/MS analysis of proteins present in the four major bands (Fig. 2, D and E), we subsequently determined the mRNA levels of L-plastin, cathepsin B, and chitinase 3-like protein. The data show that the expression of all three genes was increased 3- to 6-fold in the lungs of Abcg1Ϫ/Ϫ mice (Fig. 2G), consistent with increased protein recovery from lavages (Fig. 2E). Lymphocytic infiltration in the lungs of Abcg1Ϫ/Ϫ mice As shown above in Fig. 1D, the lungs of chow-fed 8-mo-old Abcg1Ϫ/Ϫ mice contain large numbers of lymphocytic foci, especially in the subpleural areas, the internal regions of the pulmonary parenchyma, and in areas adjacent to bronchioles and larger blood vessels. Direct immunofluorescence, using primary Abs to CD45R or CD3␧, indicates that such lymphocytic infiltrates contain B cells, with few, if any, T cells (Fig. 3). Staining of spleen sections FIGURE 3. Lymphocyte infiltrates in Abcg1Ϫ/Ϫ lungs are predomi- Ϫ/Ϫ obtained from wild-type mice confirmed the specificity of the Abs nantly B cells. Frozen sections from Abcg1 lungs were processed as ␧ described in Materials and Methods. A,4Ј,6-diamidino-2-phenylindole to CD45R and CD3 (data not shown). As expected, lymphocytic (DAPI) (nuclear) staining; (B) FITC-conjugated CD45R/B220 (B cell foci were absent in the lungs of either wild-type litter mates or Downloaded from Ϫ/Ϫ marker); (C) PE-conjugated CD3␧ (T cell marker); and (D) merged image. young (3-mo-old) chow-fed Abcg1 mice (data not shown). In- Original magnification: ϫ1000. Sections from the lungs of wild-type mice terestingly, the B cells that accumulate in the lungs of the 8-mo-old Ϫ Ϫ show no evidence of lymphocytic infiltration (data not shown). Abcg1 / mice stain highly positive for ␤-galactosidase activity (22) (and data not shown), suggesting that ABCG1 is normally expressed at a relatively high level in these B cells. Whether de-

adapted from Card et al. (33), was used to compute different in- letion of ABCG1 affects B cell function remains to be established. http://www.jimmunol.org/ flammatory parameters, Abcg1Ϫ/Ϫ mice showed an 8-fold increase in their basal pulmonary inflammation status, compared with their Increased expression of cytokines and cytokine receptors in the Ϫ/Ϫ wild-type litter mates (data not shown). lungs of Abcg1 mice The presence of macrophage giant cells, lymphocytic infiltration, Markers of inflammation are induced in BAL from pneumocyte type 2 proliferation, accumulation of chitinase 3-like Abcg1Ϫ/Ϫ mice protein, and induction of cathepsin B and L-plastin mRNAs in the Analysis of the cells and proteins recovered after BAL revealed lung are all signs of inflammation. Consequently, we studied the dramatic differences between 8-mo-old chow-fed wild-type and pattern of expression of a panel of different cytokines and their Abcg1Ϫ/Ϫ mice (Fig. 2). As compared with wild-type mice, the receptors in 8-mo-old chow-fed animals. The microarray data sug- by guest on September 27, 2021 BAL recovered from Abcg1Ϫ/Ϫ mice contained increased numbers gest that many genes associated with inflammation are highly in- of total macrophages, lipid-loaded foamy macrophages, and leu- duced in the lungs of these older chow-fed Abcg1Ϫ/Ϫ mice (Fig. 4, kocytes (22) (Fig. 2, A–C), in addition to crystals of unknown A–C; complete microarray data is accessible from the Gene Ex- composition (Fig. 2B). We also used SDS-PAGE to analyze pro- pression Omnibus (GEO) repository). RT-qPCR analysis of a se- teins present in cell-free extracts recovered after BAL. Based on lective subset of these genes confirmed that mRNA levels for Coomassie blue staining of the gels, we identified four bands that Mcp-1, Tnf-␣, Mip-1␤, Il-1␤, Ccr-5, and iNos are all significantly are increased in the cell-free supernatants recovered from elevated (2.5- to 35-fold) in the lungs of Abcg1Ϫ/Ϫ mice (Fig. 4D). Abcg1Ϫ/Ϫ mice (Fig. 2D, lanes 2 vs 1; bands a–d). Individual The induction of inflammatory cytokines is specific because the bands were subsequently excised from the acrylamide gel, trypsin- levels of many other mRNAs (e.g., Vcam-1) are unchanged (Fig. 4D). digested, and analyzed by LC-MS/MS (see Materials and Meth- We next studied the expression of two representative cytokines, ods). Proteins shown to be highly enriched in, or present exclu- MCP-1 and TNF-␣, in crude extracts of lung tissue. As shown in Fig. sively in, the lavages from Abcg1Ϫ/Ϫ mice included moesin, ezrin, 4E, protein levels of both cytokines were markedly increased in the Ϫ/Ϫ radixin, L-plastin, cathepsin B precursor, and chitinase 3-like lungs of Abcg1 mice, compared with wild-type lungs. protein variants 3 and 4 (also known as Ym-1 and Ym-2) (Fig. We next asked whether increased expression of inflammatory 2E). Interestingly, the presence of some of these proteins in cytokines could be observed in the lungs of 3-mo-old chow-fed sputum or bronchial lavages has been correlated with pulmo- Abcg1Ϫ/Ϫ mice, at a time when there is no evidence of lipid nary inflammation (34–37). Only trace amounts of chitinase accumulation. The data of Fig. 4F show that of those cytokines 3-like variant 3 (and no variant 4) were detected in lavages tested, only Mip-1␤ mRNA levels were significantly increased obtained from wild-type mice (Fig. 2E). Finally, based on the in the lungs of the young chow-fed Abcg1Ϫ/Ϫ mice as compared number of peptides identified using LC-MS/MS, we estimate with wild-type mice. However, administration of a high fat/high that transferrin and albumin levels were increased ϳ2-fold in cholesterol (HF/HC) diet resulted in increased expression of a lavages from Abcg1Ϫ/Ϫ mice (Fig. 2E). number of mRNAs, including those encoding for Mip-1␤, The crystals present in the BAL obtained from Abcg1Ϫ/Ϫ mice Mcp-1, Ccr-5, and Il1-␤ (Fig. 4F). Interestingly, the expression (Fig. 2B) were purified by repeated centrifugation on PBS- of these same mRNAs was significantly higher in the lungs of Isolymph solutions and shown to migrate as a single band on SDS- HF/HC-fed Abcg1Ϫ/Ϫ mice as compared with their wild-type PAGE (Fig. 2D, lane 4). Analysis of this band by LC-MS/MS litter mates (Fig. 4F). These differences in cytokine expression revealed that the crystals were a mixture of chitinase 3-like vari- between Abcg1Ϫ/Ϫ and wild-type mice occurred even though ants 3 and 4, and that together these two proteins comprised Ͼ90% plasma lipid levels were not significantly different between the of the protein recovered from the gel (Fig. 2E). The data of Fig. 3F two genotypes (data not shown). These results strongly suggest show representative MS/MS spectra from two peptides that iden- that pulmonary lipid content plays a crucial role in eliciting 3564 ABCG1 AND LUNG INFLAMMATION

FIGURE 4. Abcg1Ϫ/Ϫ lungs display signs of inﬂam- mation. RNA from the lungs of wild-type and Abcg1Ϫ/Ϫ mice (n ϭ 4/genotype) were used to synthesize biotin- ylated cRNA. GEArray microarray membranes were Downloaded from probed, washed, and exposed to x-ray ﬁlms following the manufacturer’s recommendations (A). Films were scanned and analyzed using the manufacturer’s software (B and C). Dotted lines in B represent 2.5-fold increase/ decrease in gene expression. Relative gene expression was plotted as a gradient of colors from light green (low expression) to bright red (high expression) for wild-type http://www.jimmunol.org/ (w) and Abcg1Ϫ/Ϫ (k) samples (C). The expression of selected genes in the lungs of 8-mo-old chow-fed mice was determined by RT-qPCR using appropriate primer sets in D and by Western blot (n ϭ 3/genotype) in E. Samples from 3-mo-old chow- or HF/HC-fed mice were analyzed by RT-qPCR in F. Wild-type (Ⅺ) and Abcg1Ϫ/Ϫ (f) mRNAs (n ϭ 4 mice/group) were each analyzed in duplicate. Data is expressed as mean Ϯ Ϫ/Ϫ p Յ 0.05 Abcg1 vs wild-type; §, p Յ 0.05 by guest on September 27, 2021 ,ء ;SEM diet vs chow.

inflammatory responses, and that small increases in cellular ste- diate extra-cellular matrix degradation in a variety of physiological rol levels might be sufficient to promote cytokine expression. processes such as development, growth, and wound repair (reviewed in Refs. 38, 39). Deregulation of the activity of MMPs and TIMPs in Altered expression of matrix metalloproteinases in the lungs of the lungs has been shown to result in a variety of pathological effects, Ϫ/Ϫ Abcg1 mice including asthma, COPD, and respiratory distress syndrome (38). Figs. 1 and 4 show that the lungs of Abcg1Ϫ/Ϫ mice undergo: i) Consistent with the altered histology and increased cytokine a profound tissue remodeling, with massive accumulation of expression, we found elevated expression of Mmp-8 and Mmp-12 macrophages and lymphocytes, and ii) a chronic inflammatory mRNAs in the lungs of Abcg1Ϫ/Ϫ mice, compared with wild-type process. Consequently, we next studied the pulmonary expres- controls (Fig. 5). The expression of Mmp-9 mRNA, however, was not sion of several matrix metalloproteinases (MMPs) and their in- significantly different between the two genotypes (Fig. 5). We also hibitors (tissue inhibitors of matrix metalloproteinases; TIMPs). noted a small but significant increase in Timp-1, but not Timp-2 and Both families of proteins are a diverse group of molecules that me- Timp-3, mRNA levels in the lungs of Abcg1Ϫ/Ϫ mice (Fig. 5). The Journal of Immunology 3565

sence of cholesterol resulted in decreased Abcg1 expression (Fig. 6A), presumably as a result of a decrease in LXR activation following the efflux of cellular sterols to exogenous cyclodextrin (that functions as a sterol sink). Interestingly, the basal mRNA expression of Tnf-␣ and Il-1␤, but not Mcp-1, was elevated ϳ4-fold in primary peritoneal macrophages derived from Abcg1Ϫ/Ϫ mice, as compared with wild-type controls (Fig. 6B). Incubation of these primary macrophages with exogenous cholesterol/cyclodextrin resulted in a marked induction of all three cytokines (Fig. 6B). However, it is notable that cytokine Ϫ/Ϫ Ϫ Ϫ mRNA levels were always significantly higher in Abcg1 ,as FIGURE 5. Altered expression of MMPs in the lungs of Abcg1 / mice. RNA from the lungs of wild-type and Abcg1Ϫ/Ϫ mice were used to compared with wild-type macrophages, following treatment determine the expression of a subset of MMPs and their inhibitors (TIMPs) with either cholesterol/cyclodextrin or LPS (Fig. 6B). by RT-qPCR. Samples from wild-type (Ⅺ) and Abcg1Ϫ/Ϫ (f) mice (n ϭ 4/group) were each analyzed in duplicate. Data is expressed as mean Ϯ Discussion p Յ 0.01 Abcg1Ϫ/Ϫ vs wild-type. ABCG1 is broadly expressed in many tissues and cell types where ,ء ;SEM it is thought to promote the efflux of cellular cholesterol to specific exogenous acceptors, such as HDL or phospholipid/apoA1 com-

Taken together, the data from Figs. 1–5 suggest that the lungs of plexes (15–21). In the current report, we show that loss of ABCG1 Downloaded from Ϫ Ϫ Abcg1 / mice undergo a dramatic increase in the inflammatory results in an age-dependent chronic inflammation that is limited process that is initiated at a time when there is no measurable to the lungs. Thus, although the lungs of 3-mo-old chow-fed change in tissue lipids. However, inflammation is progressive and Abcg1Ϫ/Ϫ mice appear normal, by the age of 6–8 mo the lungs parallels the subsequent accumulation of lipids that occurs in contain massive infiltrates of lymphocytes and macrophages, crys- Ϫ Ϫ chow-fed Abcg1 / mice. In additon, we also show that the in- tals of chitinase 3-like protein and cholesterol, and increased ex- flammatory process in the lung is greatly accelerated following pression of multiple cytokines and cytokine receptors. The lungs of http://www.jimmunol.org/ administration of a diet enriched in fat and cholesterol. Abcg1Ϫ/Ϫ mice also show evidence of tissue hemorrhage. In contrast, no signs of inflammation or lipid deposition are observed in Cholesterol loading stimulates cytokine production wild-type litter mates. in macrophages Histological studies show that numerous “giant cells” contain- The data of Fig. 4F indicate that dietary fat and cholesterol is ing an enlarged cytoplasm, lipid droplets, and multiple centrally sufficient to increase pulmonary inflammation in both wild-type located nuclei are present in the subpleural region of Abcg1Ϫ/Ϫ and Abcg1Ϫ/Ϫ mice. To test the hypothesis that increased in- lungs (Fig. 1). Such giant cells have been observed in several tracellular cholesterol promotes the expression of inflammatory chronic inflammatory conditions and during osteoclastic remod- mediators, we analyzed the expression of three representative eling (reviewed in Ref. 40). Although the formation of these by guest on September 27, 2021 cytokines, Tnf-␣, Il-1␤ and Mcp-1, in the murine macrophage cells is poorly understood, they are believed to be active in cell line Raw264.7 after loading the cells with excess choles- phagocytosis of large extracellular components including for- terol. As shown in Fig. 6A, compared with cells incubated for eign bodies (32, 40, 41). Whether the giant cells accumulate in 6 h with cyclodextrin alone, cyclodextrin/cholesterol resulted in response to the extracellular crystals of cholesterol and chiti- a significant increase in the expression of all three cytokines. As nase 3-like in the Abcg1Ϫ/Ϫ lungs is unknown. Although the expected, incubation of the cells with cyclodextrin in the ab- molecular and cellular mechanisms of macrophage fusion are

FIGURE 6. Cholesterol-loading promotes cytokine expression in macrophages. A, Raw264.7 cells were incubated overnight in 1% FBS. Next morning, fresh media (1% FBS) supplemented with cyclodextrin (CD) or cyclodextrin-cholesterol (CD-c) was added to the cells. Total RNA was obtained imme- diately (basal) of after 6 h, and the expression of Tnf-␣, Mcp-1, Il-1␤, and Abcg1 determined by RT-qPCR using appropriate primer sets. B, Thioglycollate- elicited peritoneal macrophages from wild-type (Ⅺ) and Abcg1Ϫ/Ϫ mice (f) were allowed to adhere to culture dishes for 48 h and then treated as described ,p Յ 0.05 ,ء ;in A. Where indicated, cells were incubated in the presence of LPS (50 ng/ml). Data is expressed as mean Ϯ SEM; ¶, p Յ 0.05, CD vs basal CD-c vs CD (A)orAbcg1Ϫ/Ϫ vs wild-type (B). Insets show relative mRNA levels under basal conditions. 3566 ABCG1 AND LUNG INFLAMMATION still largely unknown, several cytokines and plasma membrane context of the lungs of Abcg1Ϫ/Ϫ mice, the chronic exposure of receptors/ligands have been proposed to be involved (42–46). alveolar cells to surfactant-derived cholesterol results in a slow and Surprisingly, the lymphocytic infiltrates observed throughout progressive accumulation of intracellular sterols that, in turn, stim- the lungs of Abcg1Ϫ/Ϫ mice are comprised of B220ϩ B cells with ulates cytokine production and inflammation. According to this few, if any, T cells (Fig. 3). B cells can be subdivided into a model, inflammation is a secondary process that develops in re- number of subtypes including B-1 and B-2 (47). The activity of sponse to the lipid accumulation in the lungs of the Abcg1Ϫ/Ϫ B-2 cells, that comprise the majority of B cell subtypes, is mod- mice. It is not clear, however, whether treating Abcg1Ϫ/Ϫ mice ulated/regulated by T cells in the so-called T cell-dependent B cell with anti-inflammatory drugs (such as glucocorticoids or nonste- activation. However, B-1 cells show a T cell-independent activa- roidal anti-inflammatory drugs) or decreasing the oxidative burden tion and express a unique set of surface markers including CD11b of the lungs (i.e., administering N-acetylcysteine) would amelio- and IgM (48–50). These cells are thought to be essential in innate rate or exacerbate the pulmonary lipidosis. Additional experiments immunity and in the generation of natural Abs (IgM) (51). The will be necessary to test this proposal. finding that the pleural cavity of Abcg1Ϫ/Ϫ mice shows a signifi- Nevertheless, based on the profound changes that occur in the cant enrichment of B220ϩ, CD11bϩ, and IgMϩ cells (data not lungs of Abcg1Ϫ/Ϫ mice, it is tempting to speculate that loss of shown) is consistent with an enrichment of B-1 cells. Whether this ABCG1 might affect pulmonary function in response to a variety enrichment of B-1 cells is a response to a specific lipid that accu- of insults, such as bacterial, viral, or fungal infection. Interestingly, mulates in the lungs of Abcg1Ϫ/Ϫ mice will require extensive ad- two recent papers describe a marked down-regulation of LXR tar- ditional studies. However, previous reports demonstrating that a get genes, including ABCG1, when peritoneal macrophages are number of natural Abs (e.g., E06, E014, and T15) produced by incubated in vitro with influenza A virus or E. coli (58), or when Downloaded from B-1 cells bind to oxidized phosphatidylcholine present in oxi- J774 macrophages are cultured with LPS (59). These later studies dized LDL, to phosphatidylcholine from bacterial capsules, and suggest that certain pathogens might modulate cholesterol ho- to apoptotic cells (reviewed in Ref. 52) would be consistent meostasis affecting the transcriptional activity of LXR and, con- with this proposal. sequently, the expression of some of its targets, including ABCG1. To identify changes in inflammatory mediators, we profiled the Whether modulation of ABCG1 expression plays a critical role in

expression of inflammatory genes in the lungs of wild-type and pulmonary-pathogen infectivity remains to be established. Testing http://www.jimmunol.org/ Abcg1Ϫ/Ϫ mice. The results show that a large number of cytokines the hypothesis that loss of ABCG1 compromises the ability of the and cytokine receptors, including Tnf␣, Il-1␤, Mcp-1, Ccr-5, iNos, murine lungs to respond to environmental challenges, such as and Mip-1␤, are induced in the lungs of Abcg1Ϫ/Ϫ mice (Fig. acute or chronic bacterial/viral/fungal infection, or allergens will 4, A–E). We suggest that the massive lipid accumulation in the require extensive additional experiments. Interestingly, elevated alveolar spaces of these animals acts as the triggering stimuli for levels of chitinase-3-like proteins (Fig. 2) have been associated in the inflammatory response. Consistent with this hypothesis, we humans and rodents with asthma and pulmonary allergic responses noted that pulmonary cytokine expression was induced in wild- (34, 35, 60, 61). type mice following administration of a diet enriched in fat and Respiratory inflammation and increased expression of cytokines cholesterol for 9 wk (Fig. 4F). This same diet resulted in even have been reported to increase in mice lacking functional lysoso- by guest on September 27, 2021 greater levels of cytokine expression in the lungs of Abcg1Ϫ/Ϫ mal acid lipase, or following administration of a cholesterol-rich mice as compared with wild-type litter mates (Fig. 4F). Collec- diet (11, 12, 62, 63). Conversely, recent studies suggest a protec- tively, these data suggest that increased intracellular lipids/choles- tive role of the cholesterol-lowering drugs statins in smoke-driven terol induce the expression of certain inflammatory mediators by a respiratory inflammation, COPD, and asthma in both mice (64– process that is enhanced in cells lacking ABCG1. The finding that 67) and humans (68, 69). To date, no functional mutations have incubation of either Raw264.7 cells or primary macrophages with been described in human ABCG1. However, Thomassen et al. (70) cyclodextrin-cholesterol resulted in the induction of Tnf-␣, Il-1␤, recently reported the intriguing finding that a subgroup of patients and Mcp-1 (Fig. 6) suggests that uptake of exogenous cholesterol with pulmonary alveolar proteinosis show a marked decrease in is sufficient to induce the expression of certain cytokines. ABCG1 mRNA and protein expression in alveolar macrophages Earlier studies reported that treatment of macrophages with ox- recovered from BALs, compared with samples from healthy vol- idized LDL resulted in induction of Il-8 (53), and that this effect unteers. Taken together, all these data suggest a critical role for could be enhanced by cotreatment of the cells with an acyl-coen- ABCG1 in controlling pulmonary homeostasis and balancing both zymeA:cholesterol acyltransferase (ACAT) inhibitor (thus inhib- lipid/cholesterol metabolism and inflammatory responses. iting cholesterol esterification) (54). A subsequent report showed that induction of Il-6 and Tnf-␣ in response to oxidized or acety- Acknowledgments lated LDL was dependent on the presence of the ACAT inhibitor We thank Drs. Robert Strieter and John Belperio from the Department of (55). These authors proposed that the increased cholesterol content Pathology and Laboratory Medicine at University of California, Los An- in the endoplasmic reticulum, following inhibition of ACAT, re- geles for their help with the cytokine analysis. We thank Dr. Sam Hawgood sulted in activation of NF-␬B, multiple MAP kinase cascades, and from the Cardiovascular Research Institute and the Department of Pediat- rics at University of California, San Francisco and Dr. Steve Bensinger the unfolded protein response pathway (55). Activation of this lat- (University of California, Los Angeles) for helpful discussions. We also ter pathway in cultured cells, in response to elevated levels of thank the members of the Edwards lab for critical reading of the intracellular unesterified cholesterol, can result in accelerated ap- manuscript. optosis (56, 57). Importantly, the lungs of Abcg1Ϫ/Ϫ mice contain high levels of unesterified cholesterol and cholesterol crystals (22), Disclosures together with increased numbers of TUNEL-positive apoptotic The authors have no financial conflict of interest. cells (data not shown) in the absence of exogenous inhibitors of ACAT. Based on these data, we propose that ABCG1 plays a References critical role in controlling intracellular cholesterol redistribution in 1. Feghali, C. A., and T. M. Wright. 1997. Cytokines in acute and chronic inflammation. Front. Biosci. 2: d12–d26. macrophages and other cell types, and that loss of ABCG1 results 2. Lawrence, T., and D. W. Gilroy. 2007. Chronic inflammation: a failure of reso- in abnormal cholesterol metabolism. We hypothesize that, in the lution? Int. J. Exp. Pathol. 88: 85–94. The Journal of Immunology 3567

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