A pneumocyte-macrophage paracrine lipid axis drives the lung. toward fibrosis

A pneumocyte-macrophage paracrine lipid axis drives the lung toward fibrosis Freddy Romero, Dilip Shah, Michelle Duong, Raymond B. Penn, Michael B. Fessler, Jennifer Madenspacher, William Stafstrom, Mani Kavuru, Bo Lu, Caleb B. Kallen, Kenneth Walsh, and Ross Summer ONLINE DATA SUPPLEMENT

Supplementary Methods Mouse BAL recovery and fractionation Bronchoalveolar lavage was performed as described previously (1). The lungs were lavaged three times with the same 1 ml of PBS. Total cell counts were measured in BAL fluid using a TC20 automated cell counter (Bio-Rad Laboratories, Inc). The BAL samples were centrifuged at 200g for 10 min at 4 C to separate the supernatant and cells. The pelleted cells were plated on glass slides using a cytospin centrifuge and stained with hematoxylin and eosin for differential counting, or with Oil Red O for lipid analysis. Supernatants were transferred to microcentrifuge tubes for further analysis. For some experiments, BAL cells were lysed and RNA and lipids were extracted for later analysis. Oil Red O staining Frozen lung tissue and cells isolated from BAL fluid were incubated for 15 min at room temperature in Oil Red O solution. After staining, slides were immersed in 85% propylene glycol, rinsed twice in distilled water, and counterstained with Harris hematoxylin. Isolation of primary alveolar macrophages and ATII cells AM and ATII cells were isolated from BAL lavage as described previously (2). Briefly, following anesthesia with isoflurane, mice were exsanguinated and the pulmonary vasculature was perfused with ice-cold saline. The trachea was cannulated and the lungs were filled with 3 ml dispase (BD Bioscience, San Jose, CA). Low melting agarose (Bio-Rad; 1%, 0.5 ml) was then infused into the airways via the trachea and lungs were placed in ice for 2 min to harden the agarose. The lung tissue was removed and incubated in 1ml of dispase x 6 min at 37 C. Lung

parenchymal tissue was gently minced in DMEM containing 0.01 % DNase I (Sigma) using a Gentle Macs Dissociator (Miltenyi Biotec). Purification of ATII cells was performed using negative depletion with CD45 Microbeads (Miltenyi Biotec) and positive selection for the epithelial cell adhesion molecule (Ep-CAM) using magnetic labeling with Streptavidin Microbeads in MACS LS columns (Miltenyi Biotec). The purity of the cells was determined by flow cytometry as described previously (3) and the phenotype was confirmed by immunohistochemical staining of cytospun cells for surfactant protein C (prosp-c) and ABCA3 (Abcam) staining. Lipid extraction and analysis Total lipids were extracted from BAL fluid, macrophages, lung tissue, and conditioned media using a modified method of Bligh and Dyer in chloroform/methanol (2:1) (4). The organic phase was obtained by centrifugation at 200 g. Lipids were dried under a gentle stream of nitrogen gas. The lipid extracts were assayed for triglyceride, total cholesterol and phospholipids content using a triglyceride and cholesterol kit (BioVision, Mountain View, CA) or a Phospholipids C kit, both according to the manufacturers' protocols (Wako Pure Chemicals, Osaka, Japan). In other experiments, extracts were dissolved in chloroform (200 µl) and aliquots, together with standards, were separated by TLC on silica gel G-60 plates (Sigma-Aldrich) as described previously (5) The neutral lipids components were separated using a mixture of n-hexane: diethyl ether: acetic acid (70:30:1) and polar lipid components were separated using a mixture of chloroform: methanol: acetic acid: water (85:15:10:3.5). After TLC plates were run, plates were removed, air dried, sprayed with primuline (Sigma-Aldrich, St. Louis, MO) to label lipid spots, and viewed under UV light. Spots were identified and quantitated by comparison with lipid standards.

ATP and Lactic acid measurement Extracellular ATP and lactic acid in BAL fluid was quantified using Colorimetry kits (Bio Vision, Mountain View, CA) according to the manufacturer s instructions. Measurement of oxidized phospholipids The level of oxpc in BAL fluid was measured by ELISA using E06 antibody (Avanti Polar lipids) as described (6). OxPc intratracheal administration 1-palmitoyl-2(5 -oxi-valeroyl)-sn-glycerol-3-phosphocholine (POVPC, Avanti Polar Lipids) was administered by intratracheal instillation at a dose of 200 µg/mouse. Mice were sacrificed on day 5 and 10 after oxpc installation. Lung collagen measurement Whole lung collagen was determined using a Sircol Collagen Assay kit (Biocolor Ltd., Belfast, Northern Ireland, UK) according to the manufacturer s instructions. Lung histology The trachea was cannulated, and the lungs were inflated with 4% paraformaldehyde in PBS. Lungs were removed en bloc and immersed in the same fixative at 4 C for 18 h and then embedded in either paraffin or OCT. Hematoxylin and eosin, Masson s trichrome stain, and Oil Red O staining were performed on four-µm-thick sections. Immunohistochemistry

Immunostaining was performed on lung sections after antigen retrieval using Retrievagen A (Target Retrieval Solution; Dako, CA) at 100 C for 20 min and quenching endogenous peroxidases with 3% H 2 O 2. Sections were subsequently blocked with 2% BSA in PBS followed by staining with primary anti-abca3 (1:200 dilution, Abcam) overnight at 4 C. After washing slides, secondary antibody was applied, and then sections were exposed to Vectastain ABC (Vector Laboratories, Burlingame, CA) followed by 3,39-diaminobenzidine (Vector Laboratories). Electron microscopy Lungs were inflated with 2% Gluteraldehyde and 0.05M Sodium Cacodylate and fixed in this solution overnight at 4 C. They were then dissected into approximately 5 cubic mm pieces and washed in 0.05M Sodium Cacodylate. Tissue was placed in 2% OsO4 and 0.05M Sodium Cacodylate followed by an H 2 O wash. Tissues were then placed in 1% uranyl acetate for 15 minutes and then washed with H 2 O. Finally, the samples went through an acetone dehydration series (25%, 50%, 75%, 90%, and 100%). Samples were then infiltrated with Spurr's embedding media and polymerized in a silicone mold at 65 C. The resulting blocks were then sectioned to 65nm on a Leica Ultracut UCT ultra microtome and mounted on copper grids. Sections were examined in an FEI Tecnai 12 TEM and electron micrographs were captured with an AMT XR111 CCD camera. RNA isolation and analysis Gene transcript levels were quantified by real-time PCR. Total RNA was isolated using RNeasy Mini-Kit (QIAGEN, Valencia, CA), according to the manufacturer s instructions and quantified with the Nano-Drop spectrophotometer. One microgram of RNA was used to make cdna using

the GoScript Reverse Transcription System (Promega, Madison, WI). The primer sets for the PCR reaction contained 1 µm forward primer and 1 µm reverse primer with SYBR Green I GoTaq qpcr Master Mix (Promega, Madison, WI). The primer set was amplified using cycles of 94 C for 1 min, 58 C for 0.5 min, 72 C for 1 min. Q-PCR analyses were performed by using the IQ5 Multicolor Real-Time PCR Detection System (Bio-Rad, Hercules, CA.), and values were determined using IQ5 Multicolor Real-Time Detection System software (Bio-Rad, Hercules, CA), normalized to Gapdh. The following gene specific primers (sense and anti-sense) were used in the study (IDT, USA): Srebp1 (forward) 5'- GGA ACC ATG GAT TGC ACA TT -3' and (reverse) 5'- AGG AAG GCT TCC AGA GAG GA -3'; Chrebp (forward) 5'- GTC CGA TAT CTC CGA CAC ACT CTT -3' and (reverse) 5'- CAT TGT CAA CAT AAG CAT CTT CTG -3'; Acc (forward) 5'- CCC AGC AGA ATA AAG CTA CTT TGG -3' and (reverse) 5'- TCC TTT TGT GCA ACT AGG AAC GT -3'; Fasn (forward) 5'- CCT GGA TAG CAT TCC GAA CCT - 3' and (reverse) 5'- AGC ACA TCT CGA AGG CTA CAC A -3'; Hmgcr (forward) 5'- CTT GTG GAA TGC CTT GTG ATT -3' and (reverse) 5'- AGC CGA AGC AGC ACA TGA T -3'; Lxr-a (forward) 5'- TCC ATC AAC CAC CCC CAC GAC -3' and (reverse) 5'- CAG CCA GAA AAC ACC CAA CCT -3'; Ppar-γ (forward) 5'- CAC AAT GCC ATC AGG TTT GG -3' and (reverse) 5'- GCT GGT CGA TAT CAC TGG AGA TC -3'; Abcg1 (forward) 5 - TTC CCC TGG AGA TGA GTG TC-3 and (reverse) 5 - CAG TAG GCC ACA GGG AAC AT-3, Abca1 (forward) 5 - CGT TTC CGG GAA GTG TCC TA -3, (reverse) 5 - GCT AGA GAT GAC AAG GAG GAT GGA - 3 ; Cd36 (forward) 5 -CCT TAA AGG AAT CCC CGT GT -3, (reverse) 5 -TGC ATT TGC CAA TGT CTA GC -3 ; Sra-1 (forward) 5 - CAT GAA CGA GAG GAT GCT GAC T-3, (reverse) 5 - GGA AGG GAT GCT GTC GTC ATT GAA-3 ; Tgf-β1 (forward) 5 - TTG CCC TCT ACA ACC AAC ACA A-3, (reverse) 5 -GGC TTG CGA CCC ACG TAG TA -3 ; Tnf-

α (forward) 5 - TCG TAG CAA ACC ACC AAG TG -3, (reverse) 5 - AGA TAG CAA ATC GGC TGA CG -3 ; Il-1-α (forward) 5 - CGC CAA TGA CTC AGA GGA AGA -3, (reverse) 5 - AGG GCG TCA TTC AGG ATG AA -3 ; Arg1 (forward) 5 -GAA CAC GGC AGT GGC TTT AAC -3, (reverse) 5 -TGC TTA GCT CTG TCT GCT TTG C -3 ; Cd86 (forward) 5 -TTG TGT GTG TTC TGG AAA CGG AG -3 (reverse) 5 -AAC TTA GAG GCT GTG TTG CTG GG -3 ; Cd163 (forward) 5 -TCC ACA CGT CCA GAA CAG TC -3 (reverse) 5 -CCT TGG AAA CAG AGA CAG GC -3 ; Ym1 (forward) 5 -GGG CAT ACC TTT ATC CTG AG -3 (reverse) 5 - CCA CTG AAG TCA TCC ATG TC -3 ; Ccr7 (forward) 5 -AAA GCA CAG CCT TCC TGT GT -3 (reverse) 5 -AGT CCA CCG TGG TAT TCT CG -3 ; Gapdh (forward) 5'- TGC ACC ACC AAC TGC TTA GC -3' and (reverse) 5'- GGC ATG GAC TGT GGT CAT GAG -3'; Hprt1 (forward) 5 -GTT GGA TAC AGG CCA GAC TTT GT-3, (reverse) 5 -CAC AGG ACT AGA ACA CCT GC-3 ; Cd36 (Human, forward) 5 -TCA GCA AAT GCA AAG AAG GGA GAC -3, (Human, reverse) 5 -GGT TGA CCT GCA GCC GTT TTG -3 ; Cd163 (Human, forward) 5 -TTG CCA GCA GTT AAA TGT G -3 (Human, reverse) 5 -AGG ACA GTG TTT GGG ACT GG -3 ; Tnf-α (Human, forward) 5 - CAG AGG GCC TGT ACC TCA TC -3, (Human, reverse) 5 - GGA AGA CCC CTC CCA GAT AG -3 ; Ym1 (Human, forward) 5 -ACT GGT CTG CTA CTA CAC CA -3 (Human, reverse) 5 -CTT GGC AAT GTC ATA GCT GC - 3 ; Hprt1 (Human, forward) 5 -CTT CCT CCT CCT GAG GAG TC-3, (Human, reverse) 5 - CCT GAC CAA GGA AAG CAA AG-3 Western blot analysis Lung tissues were homogenized in ice cold lysis buffer (PBS, 0.05% Triton X-100, ph 7.4) containing protease inhibitors (Roche Complete mini) and phosphatase inhibitor. After centrifugation (14,000g, 15 min, 4 C), the supernatant was collected. Lung nuclear fraction was

extracted using a commercially available kit (Active motif, Carlsbad, CA) according to the manufacturer s instructions. Protein concentration was determined by Pierce TM BCA assay kit (Thermo Scientific, Rockford, IL). Protein samples (20 µg) were solubilized in 4 Laemmli sample buffer, heated at 95 C for 10 min, centrifuged at 3,000 g for 1 min, loaded on a 10% Tris-HCl-SDS-polyacrylamide gel and run for 1 h at 120 V. Protein was transferred to a nitrocellulose membrane (Bio-Rad) and then blocked with Odyssey Blocking Buffer (Li-Cor Biosciences, Lincoln, NE) for 1 h at room temperature followed by incubation overnight at 4 C with a specific polyclonal rabbit primary antibody to AMPK, pampk, ACC, pacc, FASN, ABCG1, ABCA1, GAPDH (Cell Signaling, Danvers, MA), SREBP-1, HMGCR (Abcam, Cambridge, MA) at a dilution of 1:1,000 in blocking buffer with 0.1% Tween-20, followed by incubation with Donkey anti-rabbit or anti-mouse secondary antibody (Li-Cor Biosciences, Lincoln, NE) at a dilution of 1:5,000 in blocking buffer. After three washings with PBS, all immunoblots were visualized using the Odyssey infrared imaging system (Li-Cor Biosciences, Lincoln, NE). In vitro exposure to bleomycin MLE-12 murine lung epithelial cells and MH-S AM were obtained from ATCC (Manassas, VA). MLE-12 cells were maintained in HITES medium supplemented with 5% fetal calf serum. Cells were plated in 6-well plates cultured with or without bleomycin (250 µg/ml). After 48 h, supernatant was collected, centrifuged to remove cell debris, and immediately frozen at -80 C. Samples were later assayed for TGF-β1, lactic acid, and lipids. Whole cell lysates were used for mrna expression by Real-Time PCR. In vitro production of foam cells

To generate foam cells, MH-S cells were cultured in RPMI medium (Invitrogen, Life technology, Grand Island, NY) supplemented with 10% fetal bovine serum (Invitrogen) and maintained at 37 C in a humidified atmosphere with 5% CO 2. The cells were seeded in a 6-well plate and incubated in serum-free medium containing lipid extracts from BAL fluid. For some studies, cells were cultured with either 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) or POVPC (50µg/ml) Avanti Polar Lipids (Alabaster, Alabama) at 37 C for 24 h. The culture media was harvested and stored at -80 C and cells were lysed for RNA isolation and protein extraction. Liposome preparation Liposomes were prepared as described previously (7). In brief, each lipid (Cholesterol, Phosphatidylcholine) (Sigma-Aldrich) was dissolved in chloroform at a concentration of 10 mg/ml. Phosphatidylethanolamine (PE)-Bodipy was dissolved in methanol at a concentration of 0.3 mg/ml. The lipids were mixed in a weight ratio: 86:8:6 (Phosphatidylcholine: Cholesterol:PE-bodipy). The mixture of lipids was dried under a stream of nitrogen gas. Liposomes were re-suspended in PBS at a concentration of 0.25 mg/ml and sonicated for 2 min on ice. Measurement of liposome uptake by ATII cells The uptake of liposomes was carried out as described previously (8). Briefly, MLE-12 were plated at a density of 2 x 10 6 cells/well onto six well plate for 24h at 37 C in HITES medium supplemented with 5% fetal calf serum. MLE-12 cells were washed twice with PBS and scraped from the plate and resuspended in solution 1 (140 mm NaCl, 5 mm KCl, 20 mm Hepes, 6 mm glucose, 1 mm CaCl 2 and 1 mm MgCl 2 ) to a concentration of 2 x 10 6 cells/ml. MLE-12 cells (5 x

10 5 cells/ml) were incubated with 25 µg/ml of liposome solution for 1 h at 37 C. Uptake was stopped by addition of 2 ml of ice-cold-pbs. The cell suspension was centrifuged at 100 g for 10 min at 4 C. Subsequently, epithelial cells were washed twice with cold-pbs and centrifuged again. Finally, the pellet was resuspended in 200 µl of PBS and fluorescence was determined (Biotek Instrument, Inc.). To assess the effect of bleomycin on lipid reuptake, MLE-12 cells were pre-incubated with 125 µg/ml of bleomycin for 24 h prior to measuring liposome uptake. Additionally, to assess the effects of metabolic stress on lipid reuptake, MLE-12 cells were ATP depleted as described previously (9). Briefly, cells were washed twice with solution 1 containing 50 mm 2-DG (ATP depleted) or 50 mm Glucose (control). Subsequently, the MLE-12 cells were incubated for 30 min at 37 C either with solution 1 containing 50 mm 2-DG or with 50 mm glucose prior to measuring liposome uptake. Measured of TGF-β1 by enzyme-linked immunosorbent assay (ELISA) TGF-β1 was measured using a commercially available DuoSet ELISA development kits (R&D System Inc., Minneapolis, MN). Briefly, Nalgene Nunc Maxisorp plates were coated overnight with mouse anti TGF-β1 antibody (4 µg/ml). The following morning, plates were washed and blocking was performed with 5% Tween 20 in PBS for 2 h. Immunoreactive TGF-β1 was generated from latent (non-immunoreactive) TGF-β1 by acidifying and then neutralizing conditioned media according to the manufacturer s instructions. Samples were incubated for 2 h, followed by washing and incubating with biotinylated chicken anti TGF- β1 antibody (75 ng/ml) for 2 h. This step was followed by the addition of streptavidin-hrp conjugate (1:200 dilutions) for 20 min, and incubation with a substrate solution (1:1 volume of substrate A and B). The

reaction was subsequently halted with stop solution and plates were read at 450 nm (Biotek Instrument, Inc.). References: 1. Govender P, Romero F, Shah D, Paez J, Ding SY, Liu L, Gower A, Baez E, Aly SS, Pilch P, et al. Cavin1; a regulator of lung function and macrophage phenotype. PLoS One 2013;8(4):e62045. 2. Lasbury ME, Durant PJ, Bartlett MS, Smith JW, Lee CH. Correlation of organism burden and alveolar macrophage counts during infection with pneumocystis carinii and recovery. Clin Diagn Lab Immunol 2003;10(2):293-302. 3. Gereke M, Autengruber A, Grobe L, Jeron A, Bruder D, Stegemann-Koniszewski S. Flow cytometric isolation of primary murine type ii alveolar epithelial cells for functional and molecular studies. J Vis Exp 2010(70). 4. Iverson SJ, Lang SL, Cooper MH. Comparison of the bligh and dyer and folch methods for total lipid determination in a broad range of marine tissue. Lipids 2001;36(11):1283-1287. 5. Matyash V, Liebisch G, Kurzchalia TV, Shevchenko A, Schwudke D. Lipid extraction by methyltert-butyl ether for high-throughput lipidomics. J Lipid Res 2008;49(5):1137-1146. 6. Horkko S, Miller E, Dudl E, Reaven P, Curtiss LK, Zvaifler NJ, Terkeltaub R, Pierangeli SS, Branch DW, Palinski W, et al. Antiphospholipid antibodies are directed against epitopes of oxidized phospholipids. Recognition of cardiolipin by monoclonal antibodies to epitopes of oxidized low density lipoprotein. J Clin Invest 1996;98(3):815-825. 7. Poelma DL, Zimmermann LJ, Scholten HH, Lachmann B, van Iwaarden JF. In vivo and in vitro uptake of surfactant lipids by alveolar type ii cells and macrophages. Am J Physiol Lung Cell Mol Physiol 2002;283(3):L648-654. 8. Poelma DL, Ju MR, Bakker SC, Zimmermann LJ, Lachmann BF, van Iwaarden JF. A common pathway for the uptake of surfactant lipids by alveolar cells. Am J Respir Cell Mol Biol 2004;30(5):751-758. 9. Guthmann F, Haupt R, Looman AC, Spener F, Rustow B. Fatty acid translocase/cd36 mediates the uptake of palmitate by type ii pneumocytes. Am J Physiol 1999;277(1 Pt 1):L191-196.

Supplementary Figures Figure S1 BALF Cell Counts 2.5 10 6 Neutrophils Lymphocytes 2.0 10 6 Macrophages 1.5 10 6 1.0 10 6 5.0 10 5 0 # ns ns # $ BSL 3d 7d 14d 21d Days after Bleomycin injection * ns $ * ns ns Total and differential cell counts in BAL fluid at baseline, 3, 7, 14 and 21 days after bleomycin (n=6, at each time point). The statistical significance was assessed by one-way ANOVA. The symbols#,* and $ correspond to p<0.05 versus BSL group for neutrophils, lymphocytes and macrophages, respectively.

Figure S2 Silica and radiation injury induce foam cell formation and lung fibrosis. (A) Oil red O staining for neutral lipids in AM at baseline (BSL), 3 and 14 days after silica administration (n=6).(b) Oil red O staining for neutral lipids in AM at baseline, 4 and 8 weeks after radiation injury (16 Gy) (n=5). (C) TGF-β1in BAL fluid at BSL, 3, 7 and 14 days after silica (n=6). (D) Collagen content in the lung at BSL, 3, 7 and 14 days after silica (n=6). (E) Trichrome staining of lung at BSL, 7 and 14 days after silica. Data are expressed as mean ± SE. The statistical significance was assessed with a one-way ANOVA test. *p.0.05, ** p< 0.01,*** p< 0.001 versus BSL group.

Figure S3 Pro-fibrotic injury induces expression of lipid receptors and transporters in AM. (A)Quantitative mrna expression for Cd36, Sra- 1and Lxr-α in AM isolated at baseline (BSL), 3 and 14 days after bleomycin (n=6). (B) Transcript levels for Abca1 and Abcg1 in AM isolated at BSL, 3 and 14 days after bleomycin (n=6). (C) Western blot analysis for ABCA1 and ABCG1 on protein lysates from AM isolated at BSL and 14 days after bleomycin. (D) Quantitative mrna analysis for Cd36, Sra-1and Lxr-α in AM isolated at BSL and 14 days after silica (n=6). Data are expressed as mean ± SE. In (A) and (B), the statistical significance was assessed with a one-way ANOVA test, whereas Student s t test was employed in (C) and (D). **, p< 0.01, and ***, p< 0.001 versus BSL group.

Figure S4 Silica promotes surfactant lipid accumulation in BAL fluid. (A) Total cholesterol in BAL fluid at baseline (BSL), 3, 7 and 14 days after silica (n=6). (B) Triglyceride concentration in BAL fluid at BSL, 3, 7, 14 days after silica (n=6). (C) Thin layer chromatography (TLC) of phospholipids in BAL fluid from mice at BSL, 3 and 14 days after silica. Phosphatidylcholine (PC) and phosphatidylserine (PS) are increased at 3 and 14 days (densitometry data). The image is representative of at least two independent experiments. Data are expressed as mean ± SE. The statistical significance was assessed with a one-way ANOVA test. *p.0.05, **, p< 0.01, and ***, p< 0.001 versus BSL group.

Figure S5 Radiation injury promotes surfactant lipid accumulation in BAL fluid. (A) Thin layer chromatography (TLC) for neutral lipids (N) and phospholipids in BAL fluid at baseline (BSL) and 4 weeks after radiation injury (16 Gy). Phosphatidylcholine (PC) is increased at 4 weeks (p<0.05) after radiation (by densitometry). The image is representative of two independent experiments. (B) Triglyceride concentration in BAL fluid at BSL, 1, 4 and 8 weeks after radiation (n=5). (C) Total cholesterol in BAL fluid at BSL, 1, 4 and 8 weeks after radiation (n=5). (D) TGF-β1in BAL fluid at BSL, 1, 4 and 8 weeks after radiation (n=5). Data are expressed as mean ± SE. The statistical significance was assessed with a one-way ANOVA test. *p< 0.05 and **p< 0.01 versus BSL group.

Figure S6 A mrna/gapdh 1.2 1.0 0.8 0.6 0.4 0.2 Control D7 *** ** ** B 0.0 Srebp-1 Fasn Acc C D FAS GAPD Bleomycin promotes metabolic changes in the lung. (A) Quantitative mrna expression for sterol-response element binding protein (Srebp-1), fatty acid synthase (Fasn) and acetyl CoA carboxylase (Acc) in freshly isolated ATII cells from the baseline (BSL) and day 7 bleomycin-injured lungs (n=6). (B) Western blot analysis for SREBP-1 at BSL and 7 days after bleomycin (n=6). (C) Western blot analysis for FASN at BSL and 7 days after bleomycin (n=6). (D) Western blot analysis for 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) at BSL and 7 days bleomycin (n=6). Western blot images are representative of at least two independent experiments. Data are expressed as mean ± SE. The statistical significance was assessed with astudent s t test. ** p< 0.01 and *** p< 0.001 versus BSL group.

Figure S7 800 Controls Bleo 125 µg/ml Bleo 250 µg/ml Lactic acid (ηmol/ml) 600 400 *** *** *** *** 200 0 3 h 6h Lactic acid production is increased in MLE-12 cells after culture with bleomycin. Lactic acid in supernatant was measured at 3 and 6 h after incubation with bleomycin. Data are expressed as Mean ± SE of n=3 independent experiments, ***p< 0.05 versus control group.

Figure S8 Extracellular ATP (eatp) is increased in BAL fluid after bleomycin injury. (A) eatp in BAL fluid at baseline, 1, 3, 14 and 21 days after bleomycin. (B) Quantification of surfactant uptake MLE-12 cells cultured in media containing either vehicle or bleomycin (125 µg/ml) (n=6). (C) Quantification of surfactant uptake in MLE-12 cells cultured in media containing 50 mm of either glucose or 2-DG (n=6). Data are expressed as mean ± SE. In (A), the statistical significance was assessed with one-way ANOVA test, whereas Student s t test was employed in (B) and (C) * p<0.05, and *** p< 0.001 versus BSL group.

Figure S9 Uptake of oxpc induces TGF-β1production and promotes an M2-like macrophage phenotype. (A) Lipids from BAL fluid of uninjured mice do not promote MH-S cell TGF-β1production (n=3 independent experiments). (B) TGF-β1concentration in supernatant of MH-S cells after culture in media containing either oxidized or non-oxidized phosphatidylcholine for 24 h (n=3 independent experiments). (C) Quantitative mrna expression for Ym1, Ppar-γ, Cd36, Arg-1,Cd163, Ccr7, Il-1β, Tnf-α, Cd86in MH-S cells cultured in media or in media supplemented with either oxidized or non-oxidized phosphatidylcholine (3 independent experiments). (D) TGFβ1concentration in supernatant and transcript levels for Cd36, Ym1, Cd163 and Tnf-α in human alveolar macrophages that were cultured in media containing either oxidized or non-oxidized phosphatidylcholine for 48 h (n=6). Data are expressed as mean ± SE. The statistical significance was assessed with a one-way ANOVA test and Student s t-testand * p< 0.05 and *** p< 0.001 versus control group.

Figure S10 GM-CSF attenuates metabolic stress and lipid release from MLE-12 in culture. (A) Western blot analysis for total and phosphorylated AMPK (Thr172) in whole lung lysates from bleomycin-injured mice receiving vehicle or daily ip. GM-CSF (10µg) administration. Levels are compared to baseline (BSL) uninjured mice. (B) Quantitative mrna expression for Chrebp, Srebp-1 and Fasn in MLE-12 cells cultured in media or in media supplemented with either vehicle or GM-CSF (n=3 independent experiments). (C) Lactic acid levels in supernatant from MLE-12 cells cultured in media or in media supplemented with either vehicle or GM-CSF (3 independent experiments). (D) Triglyceride concentration in supernatant from MLE-12 cells cultured in media or in media supplemented with either vehicle or GM-CSF (3 independent experiments). Data are expressed as mean ± SE. The statistical significance was assessed with a one-way ANOVA test. *p<0.05 versus BSL group and ## p<0.01 versus bleomycin group.