Supplemental Data. Short Article. PPARγ Activation Primes Human Monocytes. into Alternative M2 Macrophages. with Anti-inflammatory Properties

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1 Cell Metabolism, Volume 6 Supplemental Data Short Article PPARγ Activation Primes Human Monocytes into Alternative M2 Macrophages with Anti-inflammatory Properties M. Amine Bouhlel, Bruno Derudas, Elena Rigamonti, Rébecca Dièvart, John Brozek, Stéphan Haulon, Christophe Zawadzki, Brigitte Jude, Gérard Torpier, Nikolaus Marx, Bart Staels, and Giulia Chinetti-Gbaguidi Supplemental Experimental Procedures Endarterectomy Tissue Samples Human atherosclerotic plaques were removed from 27 patients eligible for surgical carotid endarterectomy according to the European Carotid Trialists Collaborative Groupe (99), recruited at the Cardiovascular Surgery Department, Hospital of Lille, France (Zawadzki et al., 26). Carotid plaques were obtained from 5 non-diabetic patients with symptomatic carotid artery stenosis scheduled for carotid endarterectomy, recruited at the Department of Thoracic and Vascular Surgery, University of Ulm, Germany (Meisner et al., 26). Before surgery, patients were randomized to weeks placebo or rosiglitazone ( mg BID) after written informed consent was obtained. Immunohistochemical Analysis Immediately after endarterectomy, representative parts of the specimen were fixed in % paraformaldehyde and.2% picric acid in phosphate buffer (ph 7.2), equilibrated in 3% sucrose, embedded in Tissue-Tek OCT compound and frozen. Serial 7-µm cryosections were stained with Oil Red O for detection of lipid deposition. For immunohistochemical analysis, endogenous peroxidase activity was quenched and sections were stained with a mouse monoclonal antibody anti-human CD68 (DakoCytomation, Denmark) and a goat polyclonal antibody anti-human MR (C-2, Santa Cruz Biotechnology, California) or with a goat polyclonal antibody anti-human MCP- (C-7, Santa Cruz Biotechnology, California) after heat-induced epitope retrieval in citrate buffer, followed by detection with appropriate biotinylated secondary antibodies and streptavidin-horseradish peroxidase. Immunostains were visualized using the DAB substrate-chromogen system (Dako Corporation, USA). Cell Culture Human PBMC were isolated from healthy donors by Ficoll density gradient centrifugation (Chinetti et al., 998). Monocyte differentiation in occurred after 7 days of culture in RPMI 6 medium (Gibco, Invitrogen, France) supplemented with gentamicin ( µg/ml), L-glutamine (2 mm) (Sigma-Aldrich, France) and % pooled human serum (Abcys, France). Alternatively differentiated macrophages (M2) were obtained by stimulating freshly isolated

2 monocytes with recombinant human IL- or IL-3 (5 ng/ml) or their combination ( ng/ml of each) added to RPMI medium containing % human serum for 7 days. The PPARγ agonists 929 (, 6 nm) or rosiglitazone (, nm) were added the first and third day of M2 differentiation without replacing the medium (M2γ). Where indicated, the PPARγ antagonist 9662 (µm) was added simultaneously with the PPARγ agonists. were activated in M macrophages by the addition of IL-β (5 ng/ml), TNFα (5 ng/ml) or LPS ( ng/ml). PPARγ agonists were added to 7 day-old 2h before M activation. To induce foam cell formation, acetylated LDL (AcLDL, 5 µg/ml) was added for 2h to together with PPARγ ligands. PBMC were isolated from subjects enrolled in the PIUS study (Balmforth et al., 27) before and after 2 months pioglitazone administration (3 mg/d; n=5 individuals). Indirect M/M2 Coculture Experiments M2 macrophages were cultured for 7 days in the presence (M2γ) or absence (M2) of 929 (6 nm). At the end, medium was removed and cells were cultured for an additional 2h with fresh medium. The resulting M2 or M2γ culture medium was collected and added (2% of M2 or M2γ supernatant in fresh medium) to 7 day-old 2h before activation to M macrophages by incubation with LPS ( ng/ml). After h, M were washed and cultured for an additional 2h in fresh medium. The resulting supernatants were assayed for proinflammatory cytokine and chemokine (TNFα, MCP- and CCL-3 (MIP-α)) by enzymelinked immunosorbent assays (ELISA) according to the manufacturer s instructions (Peprotech, France). RNA Extraction and Analysis Total cellular RNA isolated from human carotid plaques and macrophages using Trizol (Invitrogen) or from PBMC using the PAXgene system, was reverse transcribed. cdnas were quantified by quantitative polymerase chain reaction (qpcr) on an MX apparatus (Stratagene) using specific primers indicated in table. mrna levels were subsequently normalized to those of cyclophilin. Ct is the difference between qpcr Ct of target gene and those of cyclophilin. Flow Cytometry Analysis Protein expression analysis was performed using a FACStar plus with PE-Cy5-conjugated anti- MR or PE-conjugated anti-cd63 human antibodies (BD Bioscience Pharmingen). Cells were incubated h at C with the appropriate antibody and harvested by gentle scrapping. Non-specific staining was avoided by incubating macrophages with PBS-human serum % for 2 minutes at C. Statistical Analysis Pearson correlation coefficients (r) and multiple regression analysis were calculated from qpcr Ct data (expressed as the difference between Ct of target gene and those of cyclophilin) using the R statistical software ( Statistical differences between groups were analyzed by Student t-test and Mann Whitney-test and were considered significant when P<.5.

3 Supplemental References Balmforth, A. J., Grant, P. J., Scott, E. M., Wheatcroft, S. B., Kearney, M. T., Staels, B., and Marx, N. (27). Inter-subject differences in constitutive expression levels of the clock gene in man. Diab Vasc Dis Res, Chinetti, G., Griglio, S., Antonucci, M., Pineda Torra, I., Delerive, P., Majd, Z., Fruchart, J. C., Chapman, J., Najib, J., and Staels, B. (998). Activation of peroxisome proliferatoractivated receptors a and g induces apoptosis of human monocyte-derived macrophages. J. Biol. Chem. 273, European Carotid Trialists Collaborative Groupe (99). MRC European Carotid Surgery Trial: interim results for symptomatic patients with severe (7-99%) or with mild (-29%) carotid stenosis. Lancet 337, Meisner, F., Walcher, D., Gizard, F., Kapfer, X., Huber, R., Noak, A., Sunder-Plassmann, L., Bach, H., Haug, C., Bachem, M., et al. (26). Effect of rosiglitazone treatment on plaque inflammation and collagen content in nondiabetic patients: data from a randomized placebocontrolled trial. Arterioscler Thromb Vasc Biol 26, Zawadzki, C., Susen, S., Richard, F., Haulon, S., Corseaux, D., Jeanpierre, E., Vincentelli, A., Lucas, C., Torpier, G., Martin, A., et al. (26). Dyslipidemia shifts the tissue factor/tissue factor pathway inhibitor balance toward increased thrombogenicity in atherosclerotic plaques Evidence for a corrective effect of statins. Atherosclerosis, in press. Table S. Sequences of Primers Used for qpcr Analysis Gene Forward Reverse MR 5'-CGA GGA AGA GGT TCG GTT CAC C-3' 5'-GCA ATC CCG GTT CTC ATG GC-3' CD63 5'-TTG CCA GCA GT TAA ATG TG-3' 5'-AGG ACA GTG TTT GGG ACT GG-3' AMAC 5'-AGC TCT GCT GCC TCG TCT AT-3' 5'-CCC ACT TCT TAT TGG GGT CA-3' IL- 5'-GAT CCA GTT TTA CCT GGA GGA G-3' 5'-CCT GAG GGT CTT CAG GTT CTC -3' PPARγ 5'-AGT CCT CAC AGC TGT TTG CCA AGC-3' 5'-GAG CGG GTG AAG ACT CAT GTC TGT C-3' IL-6 5'-AGT GCC TCT TTG CTG CTT TCA C-3' 5'-TGA CAA ACA AAT TCG GTA CAT CCT-3' IL-β 5'-GGG CCT CAA GGA AAA GAA TC-3' 5'-TTC TGC TTG AGA GGT GCT GA-3' MCP- 5'-CCC CAG TCA CCT GCT GTT AT-3' 5'-TGG AAT CCT GAA CCC ACT TC-3' TNF-α 5'-CAG AGG GCC TGT ACC TCA TC-3' 5'-GGA AGA CCC CTC CCA GAT AG-3' CD36 5'-TCA GCA AAT GCA AAG AAG GGA GAC-3' 5'-GGT TGA CCT GCA GCC GTT TTG-3' Cyclophilin 5'-GCA TAC GGG TCC TGG CAT CTT GTC C-3' 5'-ATG GTG ATC TTC TTG CTG GTC TTG C-3'

4 * IL-3 IL- + IL-3 CD63 / Cyclophilin (AU) * CD63 / Cyclophilin (AU).8.2. * * IL-3 IL- + IL-3 Figure S. PPARγ Agonists Enhance the M2 Phenotype in IL-3 or IL-/IL-3 Differentiated Human Macrophages In Vitro Alternatively differentiated macrophages (M2) were obtained by stimulating freshly isolated monocytes with recombinant human IL-3 (5 ng/ml) or combination of IL- and IL-3 (each at ng/ml) added to RPMI medium containing % human serum for 7 days. The PPARγ agonists 929 (, 6 nm) or rosiglitazone (, nm) were added the first and third day of M2 differentiation without replacing the medium. MR and CD63 mrna was measured by qpcr. Each bar is the mean value ± SD of triplicate determinations, representative of 2 independent experiments. Statistically significant differences are indicated (t-test; vs M2 p <., p <. and p <.5; vehicle vs or in M2 *p <., p <. and *p <.5).

5 M2 / M2γ medium 2h h + LPS Wash + Fresh medium 2h Cytokine & chemokine quantification Figure S2. Protocol Description of Indirect Coculture Experiments were incubated with conditioned medium from M2 or M2γ macrophages (2 % v/v) for 2h and thereafter activated with LPS ( ng/ml). Cells were washed and fresh medium added for an additional 2h. Culture supernatants were assayed for cytokine and chemokine secretion.

6 MR / cyclophilin (AU) AMAC / cyclophilin (AU) CD63 / Cyclophilin (AU) A B.6.8. IL-β TNFα LPS IL- M 7 * 7 ns IL- IL-β TNFα LPS M.. * ns AMAC / cyclophilin (AU) CD63 / Cyclophilin (AU) * ns IL-β TNFα LPS IL- M AMAC / Cyclophilin (AU) CD63 / Cyclophilin (AU) C D AMAC / Cyclophilin (AU) CD63 / Cyclophilin (AU) FC FC FC Figure S3. PPARγ Activation Does Not Switch, M, or Foam Cells into a M2 Phenotype (A and B) were treated with 929 (6 nm) or rosiglitazone ( nm) in the absence (A) or in the presence of IL- (5 ng/ml) for 2h (B). (C) were stimulated with IL-β (5 ng/ml), TNFα (5 ng/ml) or LPS ( ng/ml) for h. 929 (6 nm) was added 2h before stimulation. (D) were 2h cholesterol loaded with AcLDL (5 µg/ml) in combination with 929 (6 nm) or rosiglitazone ( nm). mrna levels were measured and each bar is the mean value ± SD of triplicate determinations, representative of 3 independent experiments. Statistically significant differences are indicated (t-test; *p <., *p <.5, (ns) = nonsignificant).