The activin A-peroxisome proliferator-activated receptor gamma axis contributes to the transcriptome of GM-CSF-conditioned human macrophages

January 2018 | Volume 9 | Article 311 Original research published: 29 January 2018 doi: 10.3389/fimmu.2018.00031 Frontiers in Immunology | www.frontiersin.org Edited by: Alexandre Corthay, Oslo University Hospital, Norway Reviewed by: Ana Rosa Pérez, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina *Correspondence: Concha Nieto [email protected] †These authors have contributed equally to this work. Specialty section: This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology Received: 26July2017 Accepted: 04January2018 Published: 29January2018 Citation: NietoC, BragadoR, MunicioC, Sierra-FilardiE, AlonsoB, EscribeseMM, Domínguez-AndrésJ, ArdavínC, CastrilloA, VegaMA, Puig-KrögerA and CorbíAL (2018) The Activin A-Peroxisome Proliferator-Activated Receptor Gamma Axis Contributes to the Transcriptome of GM-CSFConditioned Human Macrophages. Front. Immunol. 9:31. doi: 10.3389/fimmu.2018.00031 The activin a-Peroxisome Proliferator-activated receptor gamma axis contributes to the Transcriptome of gM-csFconditioned human Macrophages Concha Nieto1*, Rafael Bragado2, Cristina Municio3, Elena Sierra-Filardi1, Bárbara Alonso1, María M. Escribese1, Jorge Domínguez-Andrés4, Carlos Ardavín4, Antonio Castrillo5,6, Miguel A. Vega1, Amaya Puig-Kröger3† and Angel L. Corbí1† 1 Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain, 2 Instituto de Investigación Sanitaria, Fundación Jiménez Díaz, Madrid, Spain, 3 Instituto de Investigación Sanitaria Gregorio Marañón, Hospital General Universitario Gregorio Marañón, Madrid, Spain, 4 Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain, 5 Instituto Investigaciones Biomédicas “Alberto Sols” (IIBM), and Centro Mixto Consejo Superior de Investigaciones Científicas y Universidad Autónoma de Madrid (ICSIC-UAM), Madrid, Spain, 6 Unidad de Biomedicina (Unidad Asociada al CSIC), IIBM-Universidad Las Palmas de Gran Canaria (ULPGC), and Instituto Universitario de Investigaciones Biomédicas y Sanitarias (IUIBS), Universidad Las Palmas de Gran Canaria (ULPGC), Las Palmas de Gran Canaria, Spain GM-CSF promotes the functional maturation of lung alveolar macrophages (A-MØ), whose differentiation is dependent on the peroxisome proliferator-activated receptor gamma (PPARγ) transcription factor. In fact, blockade of GM-CSF-initiated signaling or deletion of the PPARγ-encoding gene PPARG leads to functionally defective A-MØ and the onset of pulmonary alveolar proteinosis. In vitro, macrophages generated in the presence of GM-CSF display potent proinflammatory, immunogenic and tumor growth-limiting activities. Since GM-CSF upregulates PPARγ expression, we hypothesized that PPARγ might contribute to the gene signature and functional profile of human GM-CSF-conditioned macrophages. To verify this hypothesis, PPARγ expression and activity was assessed in human monocyte-derived macrophages generated in the presence of GM-CSF [proinflammatory GM-CSF-conditioned human monocytederived macrophages (GM-MØ)] or M-CSF (anti-inflammatory M-MØ), as well as in ex vivo isolated human A-MØ. GM-MØ showed higher PPARγ expression than M-MØ, and the expression of PPARγ in GM-MØ was found to largely depend on activin A. Ligand-induced activation of PPARγ also resulted in distinct transcriptional and functional outcomes in GM-MØ and M-MØ. Moreover, and in the absence of exogenous activating ligands, PPARγ knockdown significantly altered the GM-MØ transcriptome, causing a global upregulation of proinflammatory genes and significantly modulating the expression of genes involved in cell proliferation and migration. Similar effects were observed in ex vivo isolated human A-MØ, where PPARγ silencing led to enhanced expression of genes coding for growth factors and chemokines and downregulation of cell surface pathogen receptors. Therefore, PPARγ shapes the transcriptome of GM-CSF-dependent human macrophages (invitro derived GM-MØ and ex vivo isolated A-MØ) in the absence of exogenous activating ligands, and its expression is primarily 2 Nieto et al. PPARγ Transcriptome in GM-CSF-Conditioned Human Macrophages Frontiers in Immunology | www.frontiersin.org January 2018 | Volume 9 | Article 31 regulated by activin A. These results suggest that activin A, through enhancement of PPARγ expression, help macrophages to switch from a proinflammatory to an antiinflammatory polarization state, thus contributing to limit tissue damage and restore homeostasis. Keywords: transcription factor, macrophage, peroxisome proliferator-activated receptor, inflammation, innate immunity inTrODUcTiOn Tissue-resident macrophages in homeostasis, as well as monocytederived macrophages within inflamed tissues, exhibit a huge functional diversity which derives from their exquisite sensitivity to extracellular cues (1, 2). GM-CSF and M-CSF drive macrophage differentiation and survival (3). However, M-CSF is required for the generation of most tissue macrophages (4, 5) while GM-CSF is needed for development and maintenance of pulmonary alveolar macrophages (A-MØ) (6). Besides its role in myeloid cell differentiation, GM-CSF is a central mediator of tissue inflammation (7) and its neutralization has been proposed as a therapeutic strategy for inflammatory disorders (8). As a consequence, both colony-stimulating factors promote the generation of functionally distinct macrophages (9): GM-CSF-conditioned human monocyte-derived macrophages (GM-MØ) produce large amounts of proinflammatory cytokines in response to stimulation, whereas M-CSF-dependent monocyte-derived macrophages (M-MØ) primarily produce anti-inflammatory factors upon activation (9–11). At the transcriptional level, while GM-MØ are characterized by the expression of a “Proinflammatory gene set” (11–13) also detected in macrophages under inflammatory conditions in vivo, M-MØ specifically express an “Anti-inflammatory gene set” and resemble macrophages from homeostatic/anti-inflammatory settings (14, 15). Interestingly, the GM-MØ-specific gene signature is critically determined by activin A both invivo (15) and invitro (11). In this regard, we have previously demonstrated that GM-MØ produce large amounts of activin A, a member of the TGFβ family (16, 17) that regulates inflammatory responses (18), modulates cytokine release (19, 20) and myeloid cell differentiation (21), and whose functional blockade in GM-MØ skews cells toward the acquisition of an anti-inflammatory signature (11). The contribution of GM-CSF to differentiation of lung macrophages relies on the GM-CSF-dependent expression of peroxisome proliferator-activated receptor gamma (PPARγ) (22–24), a nuclear receptor that regulates gene transcription through ligand binding (25–28), antagonism of other transcription factors (e.g., NFκB, AP-1) (29, 30) and recruitment of repressor complexes in the absence of ligands (31). As a critical regulator of inflammatory processes (32–34), PPARγ inhibits human and murine macrophage responses to proinflammatory stimuli (35, 36), contributes to IL-4-driven polarization of human and murine macrophages (37, 38) and determines the acquisition of the metabolic disease-specific phenotype of human macrophages (39). In human cells, the ubiquitously expressed PPARγ1 derives from the PPARG1 and PPARG3 mRNA splicing isoforms, while the PPARG2 mRNA isoform codes for PPARγ2, whose expression is restricted to adipocytes (40). Mouse A-MØ exhibit much higher expression of PPARγ than other macrophages in the steady-state (34), and its GM-CSF-dependent expression is essential for their differentiation and maturation from fetal monocytes (24). In fact, PPARγ expression in A-MØ is lost in GM-CSF-deficient mice and in patients with pulmonary alveolar proteinosis (PAP), a pathology derived from a defective expression or activity of GM-CSF (23, 41) and associated with suppressed activin A expression (42). However, it is currently unknown whether PPARγ is required for maintenance of A-MØ throughout adult life (43). Upon tissue injury, monocyte-derived macrophages modulate inflammation and also promote tissue repair. In the specific case of lung inflammation, monocyte-derived mouse A-MØ are the major drivers of fibrosis and become similar to tissue-resident A-MØ over time (44). Since GM-CSF-conditioned monocytederived human macrophages exhibit potent proinflammatory functions upon stimulation (9, 11), and in spite of the intrinsic anti-inflammatory functions of PPARγ, we hypothesized that PPARγ might contribute to the gene signature and functional profile of human GM-CSF-conditioned macrophages. To address this hypothesis, we evaluated the extent of the PPARγ contribution to the gene signature and functional profile of human GM-CSFdependent macrophages. We now report the activin A-dependent expression and activity of PPARγ in GM-CSF-conditioned human macrophages, and demonstrate that PPARγ displays polarizationdependent activities and significantly shapes the gene signature of proinflammatory monocyte-derived GM-MØ and human A-MØ in the absence of exogenous ligands. The activin A-dependent expression of PPARγ in GM-MØ and in A-MØ also suggests a role for activin A in promoting inflammation resolution. eXPeriMenTal PrOceDUres generation of human Monocyte-Derived Macrophages InVitro and Ex Vivo isolation of a-MØ Buffy coats were obtained from healthy blood donors, as anonymously provided by the Comunidad de Madrid blood Bank. Ethical approvals for all blood sources and processes used in this study were approved by the Centro de Investigaciones Biológicas Ethics Committee. All experiments were carried out in accordance with the approved guidelines and regulations. Human PBMCs were isolated from buffy coats over a Lymphoprep™ gradient (#1114545, Axis-Shield PoC AS) according to standard procedures. Monocytes were purified from PBMCs by magnetic cell sorting using human CD14 microbeads (#130-050-201, Miltenyi Biotech). Monocytes (95% CD14+ cells) were cultured 3 Nieto et al. PPARγ Transcriptome in GM-CSF-Conditioned Human Macrophages Frontiers in Immunology | www.frontiersin.org January 2018 | Volume 9 | Article 31 at 0.5 × 106 cells/ml for 7 days in RPMI 1640 (#21875-034, Gibco) supplemented with 10% inactivated fetal calf serum (FCS) (#S1810-500, Biowest) (complete medium), at 37°C in a humidified atmosphere with 5% CO2, and containing 1,000U/ml human GM-CSF (#11343125, Immunotools GmbH) or 10ng/ml human M-CSF (#11343115, Immunotools GmbH), to generate GM-MØ or M-MØ, respectively. Cytokines were added every 2 days. Blocking anti-activin A Ab (100 ng/ml) (#MAB3381, clone 69403, R&D Systems) or the inhibitors of ALK4, ALK5, and ALK7, SB431542 (10µM) (#S4317, Sigma-Aldrich) or A-83 (1µM) (#2039, Tocris) were added every 24h. Finally, polarized macrophages were treated with ultrapure Escherichia coli 0111:B4 strain LPS (10 ng/ml) (#tlrl-3pelps, Invivogen) for 14–16 h. Exposure to recombinant human activin A (25ng/ml) (#120-14P, Preprotech) was done for 24h (monocytes and THP-1 cells) or 48h (M-MØ). The acute monocytic leukemia cell line THP-1, obtained from ATCC® (#TIB-202™), was cultured in complete medium at 37°C in a humidified atmosphere with 5% CO2. A-MØ were obtained from patients undergoing bronchoalveolar lavage (BAL) following the Fundación Jiménez Díaz Medical Ethics committee procedures and after written informed consent from all subjects, in accordance with the Declaration of Helsinki. BAL procedure was performed with a flexible bronchoscope with a total volume of 200ml of sterile isotonic saline solution at 37°C. BAL fluid fractions were maintained at 4°C and cellular debris removed using a 40µm cell strainer (45). BAL cells were washed with PBS, centrifuged and resuspended in complete medium containing 100 U/mL penicillin and 100µg/mL streptomycin (#15140-122, Gibco), 50 µg/ml gentamicin (#G1397, Sigma-Aldrich), and 2.5 µg/ml amphotericin B (#A2942, Sigma-Aldrich). The cells were seeded at 6–8×105 cells per well in 12-well plates for 1h and washed extensively to remove non-adherent cells. Finally, 2ml of complete medium with antibiotics was added to each well and the adherent cells incubated for 16–18h before transfection. More than 95% of adherent BAL cells were identified as macrophages according to morphology and phenotypic analysis. generation of Murine Bone Marrow-Derived Macrophages InVitro All experiments on mice were conducted according to the Spanish and European regulations on care and protection of laboratory animals and were approved by the Centro de Investigaciones Biológicas animal facility and the Consejo Superior de Investigaciones Científicas Ethics Committee. Bone marrow-derived GM-MØ or M-MØ were obtained by flushing the femurs of 6–10-week-old C57BL/6 mice (provided by the Animal facility at the Centro de Investigaciones Biológicas), and culturing cells during 7days in DMEM (#41966-029, Gibco) supplemented with 10% FCS and 50mM 2-ME, containing either murine GM-CSF (1,000U/ml) (#315-03, PreProtech) or human M-CSF (25ng/ml) (#11343115, Immunotools GmbH), respectively (46, 47). Cytokines were added every 2days. Flow cytometry Mouse monoclonal antibodies specific for human CD14 (Alexa Fluor-647-labeled antihuman CD14, #301818, clone M5E2, Biolegend) and human CD163 (PerCP-labeled antihuman CD163, #333625, clone GHI/61, Biolegend) were used. Isotype-matched PerCP-labeled Mouse IgG1 (κ Isotype Ctrl Antibody, #400147, clone MOPC-21, Biolegend) and Alexa Fluor-647 Mouse IgG2a (κ Isotype Ctrl Antibody, #400234, clone MOPC-173, Biolegend) were included as negative controls. Quantitative real Time rT-Pcr Total RNA was extracted using the total RNA and protein isolation kit (Macherey-Nagel). RNA samples were retrotranscribed with the High-Capacity cDNA Reverse Transcription kit (AB), and individually amplified cDNA was quantified using the Universal Human Probe Roche library (Roche Diagnostics). Oligonucleotides for selected genes were designed according to the Roche software for quantitative real-time PCR (qRT-PCR), and their sequence is indicated in Table S1 in Supplementary Material. qRT-PCR was performed on a LightCycler® 480 (Roche Diagnostics). Assays were made in triplicates, and results were normalized according to the expression levels of TBP mRNA or/ and GAPDH mRNA (for qRT-PCR) or to the mean of the expression level of endogenous reference genes HPRT1, TBP and RPLP0 (for microfluidic gene cards). Results were expressed using the ΔΔCT (cycle threshold) method for quantification. elisa Macrophage supernatants were tested for the presence of cytokines using commercially available ELISA sets for human TNFα (BD OptEIA Human TNF ELISA set, #555212, BD Biosciences), CCL2 (BD OptEIA Human MCP-1 ELISA set, #555179, BD Biosciences), IL-10 (ELISA MAX Standard set, #430601, BioLegend), IL-6 (ELISA MAX Standard set, #430501, BioLegend), and activin A (DuoSet, #DY338, R&D Systems), following the protocols supplied by the manufacturers. cell Transfection and reporter gene assays HEK293-Tcells, provided by the Cell culture facility at the Centro de Investigaciones Biológicas, were transfected with an expression vector for PPARγ2 (pBABE-PPARγ2, Addgene) or an empty vector using Superfect transfection reagent (#301305, Qiagen). Human GM-MØ or M-MØ (1 × 106 cells) were transfected using the Human Macrophage Nucleofector® Kit (#VPA-1008, Lonza) with 1µg of PPAR reporter DNA mixture (#CCS-3026L, Cignal PPAR Reporter assay kit, Qiagen). This mixture contains a PPAR-responsive firefly luciferase construct and a constitutively expressing Renilla luciferase (40:1) The PPAR-dependent construct encodes the firefly luciferase gene under the control of a minimal CMV promoter and tandem repeats of the PPAR responsive element (PPRE). Firefly and Renilla luciferase activities were determined by using the Dual-Luciferase® Reporter Assay System (#E1910, Promega). Western Blot assay Cell lysates (40µg) and nuclear extracts (30µg) were subjected to SDS-PAGE and transferred onto an Immobilon polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). After 4 Nieto et al. PPARγ Transcriptome in GM-CSF-Conditioned Human Macrophages Frontiers in Immunology | www.frontiersin.org January 2018 | Volume 9 | Article 31 blocking the unoccupied sites with 5% nonfat dry milk, protein detection was carried out with a goat polyclonal against PPARγ2 (G-18, #sc-22020, Santa Cruz Biotechnology), a goat affinity purified polyclonal antibody against Sp1 (PEP2, #sc-59-G, Santa Cruz Biotechnology), or a monoclonal antibody against GAPDH (6C5, #sc-32233, Santa Cruz Biotechnology), and using the SuperSignal West Pico Chemiluminescent system (#34081, Thermo Fisher Scientific). small interfering ribonucleic acid (sirna) Transfection To silence PPARG gene expression, human GM-MØ, M-MØ (1×106 cells) or A-MØ (6–8×105 cells) were transfected with a PPARG-specific siRNA (siPPARG) (50nM) (#s10888, Thermo Fisher Scientific), using HiPerFect transfection reagent (#301705, Qiagen). A negative control siRNA from the same company was used as a transfection control (siControl) (#4390843, Thermo Fisher Scientific). After 6h of transfection, cells were allowed to recover from transfection in RPMI 1640 medium with 10% FCS and the cells were treated with GW7845 (1µM) (kindly provided by Jon Collins, Glaxo SmithKline, USA) or DMSO for 18–24h before assessing for PPARγ markers. Microarray analysis Global gene expression analysis was performed on RNA obtained from three independent samples of GM-MØ that had been transfected with siPPARG or siControl for 48 h, and using a whole human genome microarray from Agilent Technologies (Palo Alto, CA, USA). Only probes with signal values >60% quantile in at least one condition were considered for the differential expression and statistical analysis. Statistical analysis for differential gene expression was carried out using empirical Bayes moderated t test implemented in the limma package1 and using paired t-test. All the above procedures were coded in R.2 Microarray data were deposited in the Gene Expression Omnibus3 under accession no. GSE88768. The differentially expressed genes were analyzed for annotated gene sets enrichment using the online tool ENRICHR4 (48, 49). Enrichment terms were considered significant when they had a Benjamini-Hochberg-adjusted p value<0.05. For gene set enrichment analysis (GSEA) (50), the previously defined “Proinflammatory gene set” and “Anti-inflammatory gene set” (12), which contain the top and bottom 150 probes from the GM-MØ versus M-MØ limma analysis of the microarray data in GSE68061 (ranked on the basis of the value of the t statistic), were used. statistical analysis Statistical analysis was performed using paired Student’s t-test, and p<0.05 was considered significant (*p<0.05, **p<0.01, and ***p<0.001). 1 http://www.bioconductor.org 2 http://www.r-project.org 3 http://www.ncbi.nlm.nih.gov/geo 4 http://amp.pharm.mssm.edu/Enrichr/ resUlTs PParγ activation has Different Transcriptional and Functional Outcomes in human gM-MØ and M-MØ To initially assess the PPARγ activation-dependent transcriptional profile of GM-MØ and M-MØ, both human macrophage subtypes were exposed for 24h to the PPARγ agonist GW7845 and the expression of the GM-MØ-specific “Proinflammatory gene set” and M-MØ-specific “Anti-inflammatory gene set” (derived from the data contained in the Gene Expression Omnibus GSE68061) (11, 12) was determined. PPARγ activation upregulated the paradigmatic PPARγ target genes CD36 and FABP4, and downregulated FLT1 and CSF1 expression, in both macrophage subtypes (Figure1A). However, GW7845 downregulated IL6, IL10, CCL2, HAMP, and CCR2 and enhanced THBS1, exclusively in GM-MØ (Figure1A). These GW7845-triggered gene expression changes were dependent on PPARγ activation as they were significantly impaired upon siRNA-mediated knockdown of PPARG mRNA (Figures1B,C). Specifically, PPARG mRNA knockdown inhibited the GW7845-mediated modulation of CD36 and CSF1 expression in M-MØ (Figure1B) and significantly impaired the GW7845mediated modulation of CD36, CSF1, FLT1, CCL2, CCR2, IL10, and HAMP in GM-MØ (Figure1C). Analogous findings were observed in murine bone marrow-derived macrophages, where Pparγ activation modified the expression of a common set of genes in both macrophage subtypes but significantly diminished the expression of Csf1 and Ccr2 only in GM-MØ (Figure 2). Therefore, although PPARγ activation alters the expression of known PPARγ targets in both GM-MØ and M-MØ, it also promotes human macrophage subtype-dependent transcriptional changes because the expression of CCR2, IL10, CCL2, and HAMP is downregulated by GW7845 only in proinflammatory GM-MØ. To determine whether the distinct transcriptional effects of PPARγ activation in GM-MØ and M-MØ had a functional correlate, the LPS-induced cytokine-producing ability of both macrophage subtypes was evaluated in the presence of GW7845. As expected, LPS stimulation of GM-MØ caused the preferential production of the proinflammatory cytokines TNFα and IL-6, whereas LPSstimulated M-MØ primarily released IL-10 (9–11) (Figure1D). In line with the transcriptional results, GW7845 significantly reduced the LPS-induced production of TNFα and IL6 from GM-MØ, but had no effect on the LPS-induced cytokine release from M-MØ (Figure1D). Importantly, the inhibitory effect of GW7845 on the LPS-induced TNFα production of GM-MØ was PPARγ-dependent, as it was reduced upon PPARγ knockdown (Figure1E). Therefore, agonist-mediated activation of PPARγ exclusively modulates the LPS-induced cytokine production from proinflammatory human monocyte-derived GM-MØ, further arguing for a polarizationdependent effect of PPARγ in human macrophages. PParγ is Preferentially expressed by Proinflammatory gM-csF-Dependent human Macrophages Given the different effect of PPARγ on GM-MØ and M-MØ, we next determined PPARγ expression and function in both FigUre 1 | Peroxisome proliferator-activated receptor gamma (PPARγ) mediates the differential effect of GW7845 on the gene and cytokine profile of human GM-CSF-conditioned monocyte-derived macrophages (GM-MØ) and M-CSF-dependent monocyte-derived macrophages (M-MØ). (a) Expression of the indicated genes in GM-MØ and M-MØ exposed for 24h to either GW7845 (GW, 1µM) or vehicle (DMSO), as determined by quantitative real-time PCR assay using microfluidic gene cards. Results are indicated as the expression of each gene after GW7845 treatment relative to its expression in the presence of DMSO. Each experiment was performed in triplicate, and mean and SEM of three independent experiments is shown (*p<0.05; **p<0.01; ***p<0.001). (B,c) Expression of the indicated genes in M-MØ (B) or GM-MØ (c) transfected with either siPPARG or siControl (siC), and treated with GW7845 (1µM) or DMSO for 24h. Relative mRNA expression indicates the expression of each gene in the different conditions and relative to its expression in DMSO-treated siC-transfected cells (arbitrarily set to 1). (Left panels) PPARG mRNA expression in siPPARG-transfected cells relative to the PPARG mRNA level in siC-transfected cells (arbitrarily set to 1). Mean and SEM of four independent experiments are shown (*p<0.05; **p<0.01; ***p<0.001). (D) TNFα, IL-10, and IL-6 production in LPS-treated (24h) GM-MØ and M-MØ that had been preexposed (4h) to DMSO or GW7845 (GW, 1µM). Results indicate the concentration of each cytokine for each condition relative to the cytokine levels detected in cells treated with DMSO and LPS (arbitrarily set to 1). Mean and SEM of three independent experiments are shown (*p<0.05; ***p<0.001). (e) TNFα production in LPS-treated (24h) untransfected (−), siControl-transfected or siPPARG-transfected GM-MØ that had been preexposed (4h) to DMSO or GW7845 (GW, 1µM). Results indicate the concentration of TNFα for each condition relative to the cytokine levels detected in untransfected cells treated with DMSO and LPS (arbitrarily set to 1). Mean and SEM of four independent experiments are shown (*p<0.05). 5 Nieto et al. PPARγ Transcriptome in GM-CSF-Conditioned Human Macrophages Frontiers in Immunology | www.frontiersin.org January 2018 | Volume 9 | Article 31 FigUre 2 | Differential effect of peroxisome proliferator-activated receptor gamma activation on mouse GM-CSF-conditioned bone marrow-derived macrophages (GM-MØ) and M-CSF-dependent bone marrow-derived macrophages (M-MØ). Relative expression of the indicated genes in murine bone marrow-derived GM-MØ (a) and M-MØ (B) exposed to either DMSO or GW7845 (1µM) for 24h, as determined by quantitative real-time PCR. Results are expressed as the expression of each gene in the presence of GW7845 relative to its expression in the presence of vehicle (DMSO). Mean and SD of three independent experiments is shown (*p<0.05). 6 Nieto et al. PPARγ Transcriptome in GM-CSF-Conditioned Human Macrophages Frontiers in Immunology | www.frontiersin.org January 2018 | Volume 9 | Article 31 human macrophage subtypes. Transfection of a PPRE reporter construct in both macrophage subtypes revealed that global PPAR-dependent transcriptional activity is higher in GM-MØ than in M-MØ (Figure3A), thus suggesting that GM-MØ are endowed with a stronger PPARγ-dependent transcriptional activity. Regarding expression, GM-MØ contained higher levels of PPARG1/3 (encoding the ubiquitous PPARγ1 isoform) and PPARG2 (coding for the PPARγ2 isoform) mRNAs than M-MØ (Figure 3B). In fact, the adipocyte-restricted PPARG2 mRNA (40) was barely detectable in M-MØ (Figure3B). The preferential expression of the PPARγ2-encoding mRNA was also observed in murine bone marrow-derived GM-MØ, whereas mouse M-MØ exhibited significantly higher Pparg1 expression than mouse GM-MØ (Figure3C), in agreement with a previous report (51) and in line with the distinct gene profiles of monocyte-derived human M-MØ and bone marrow-derived mouse M-MØ (13, 52). Kinetic analysis revealed that PPARG2 mRNA is upregulated in human monocytes exposed to GM-CSF for 3, 5, and 7days (Figure 3D). Although PPARG1 is expressed at higher levels than PPARG2 mRNA (25-fold approx.), PPARγ2 protein could be detected in whole cell and nuclear extracts from GM-MØ (Figures3E,F). Therefore, GM-CSF-conditioned proinflammatory human macrophages exhibit a higher expression of PPARγ (PPARγ1 and PPARγ2) than M-CSF-conditioned antiinflammatory human macrophages. activin a controls PParγ expression in gM-csF-Dependent Macrophages The GM-CSF-dependent expression of PPARγ is essential for the differentiation of A-MØ (24). Since proinflammatory human GM-MØ polarization is dependent on the autocrine/paracrine action of activin A (11), we next questioned whether activin A contributes to the preferential expression of PPARG mRNAs in GM-MØ. Activin A significantly elevated PPARG1/3 and PPARG2 mRNA levels in M-MØ, monocytes and THP-1 myeloid cells (Figure 4A). Moreover, inhibition of activin A-initiated Smad signaling by either SB431542 (Figure4B) or A-83 (Figure4C), or blockade of activin A with an anti-activin neutralizing antibody (Figure4D), significantly reduced PPARG1/3 and PPARG2 mRNA levels in GM-CSF-dependent proinflammatory GM-MØ. In line with these results, generation of GM-MØ in the presence of A-83 resulted in significantly reduced expression of the PPARγ target gene ABCA1, a gene whose expression is responsive to PPARγ-LXR activation in human macrophages (Figure 4E). Further, analysis of ex vivo isolated human A-MØ revealed the constitutive expression of activin A (Figure 4F), and that the expression of PPARG1/3 and PPARG2 mRNA, as well as the expression of the PPARγ target ABCA1 mRNA, were significantly reduced in the presence of the A-83 Smad signaling inhibitor (Figure4G). Altogether, these results indicate that activin A is a positive regulator of PPARγ expression and activity in GM-CSFconditioned macrophages both invitro and invivo. identification of the PParγ-Dependent gene Profile in gM-csF-conditioned Proinflammatory human Macrophages Given the transcriptional effects of PPARγ knockdown (Figure1C), and to more thoroughly address the role of PPARγ in in vitro generated GM-CSF-conditioned macrophages, we determined the PPARγ-dependent transcriptional profile of GM-MØ in the absence of exogenous agonists. siRNA-mediated PPARγ knockdown significantly modified the transcriptome of GM-MØ, altering the expression of 314 probes (283 annotated genes) (p< 0.003, Table S2 in Supplementary Material). Specifically, PPARγ knockdown led to downregulation of 139 genes and upregulation of 144 genes in GM-MØ (Figure5A). Twenty-five percent of the genes downregulated by siPPARG (36 out of 139) had been previously predicted as PPAR targets (53), including 20 genes upregulated by long-term rosiglitazone treatment of human monocyte-derived dendritic cells (54) and two genes whose expression is also diminished in mouse Pparγ−/− macrophages (CD36 and GPD1) (24) (Figure5B). Similarly, the set of genes upregulated upon PPARγ knockdown contained 19 genes FigUre 3 | Expression of peroxisome proliferator-activated receptor gamma (PPARγ) isoforms in human and mouse GM-CSF-conditioned macrophages (GM-MØ) and M-CSF-dependent macrophages (M-MØ). (a) Basal PPAR-dependent transcriptional activity in GM-MØ and M-MØ. Mean and SEM of the relative PPARdependent luciferase activity (compared to Renilla luciferase activity) of seven independent experiments is shown (*p<0.05). (B) PPARG1/3 and PPARG2 mRNA expression levels in GM-MØ and M-MØ, as determined by quantitative real-time PCR (qRT-PCR) and relative to TBP mRNA levels. Mean and SEM of three independent experiments is shown (*p<0.05; ***p<0.001). (c) Pparg1 and Pparg2 mRNA expression in bone marrow-derived murine GM-MØ and M-MØ, as determined by qRT-PCR and relative to Tbp mRNA levels. Mean and SD of three independent samples is shown (*p<0.05). (D) PPARG1/3 and PPARG2 mRNA expression levels along GM-MØ and M-MØ differentiation, as determined by qRT-PCR and relative to TBP mRNA levels. A representative experiment is shown. (e,F) PPARγ2 protein levels in whole cell (e) or nuclear lysates (Nucl. Ext.) (F) from mock-transfected (C) or human PPARγ2-transfected HEK293Tcells (e) and two independent samples of GM-MØ and M-MØ (donor #1 and donor #2). GAPDH (e) and Sp1 (F) protein levels were detected in parallel as protein loading controls. The band corresponding to PPARγ2 protein is indicated by an asterisk. 7 Nieto et al. PPARγ Transcriptome in GM-CSF-Conditioned Human Macrophages Frontiers in Immunology | www.frontiersin.org January 2018 | Volume 9 | Article 31 predicted as PPAR targets (53) (Figure5B), including 5 genes upregulated by rosiglitazone in human dendritic cells (54) and CCL2 and CCL7, whose orthologous genes are overexpressed in murine Pparγ−/− macrophages (24). Conversely, siPPARG downregulated the expression of MSR1, whose mouse ortholog is overexpressed in Pparγ−/− macrophages (24). The PPARγ-regulated gene set also included genes whose expression distinguishes A-MØ from other tissue-resident mouse macrophages (KRT79, BCAR3, MAFF, WWTR1) (55) or have been defined as human A-MØ-enriched genes (EDN1, CXCL1, TNFAIP6, IL7R) (56) (Table S2 in Supplementary Material). Therefore, PPARγ knockdown in GM-CSF-conditioned human macrophages allowed the identification of a large set of genes (Table S2 in Supplementary Material) whose expression is specifically modulated by PPARγ in the absence of an exogenous agonist. Besides, and in agreement with the divergent transcriptional profiles of functionally similar human and mouse macrophages (57), the human macrophage PPARγ-dependent gene set in human macrophages only partially overlaps with the list PPARγ-regulated genes previously identified in mouse macrophages. To gain formation on the biological processes significantly affected after PPARγ knockdown in human macrophages, functional enrichment analysis was performed using GSEA (50). Confirming the validity of the results, PPARγ knockdown led to a very significant reduction in the expression of genes associated with the terms “KEGG_PPAR_Signaling_Pathway” and “KEGG_Peroxisome” (Figure5C). In line with its known antiinflammatory function (32, 58), reduction of PPARγ expression FigUre 4 | The activin A/Smad signaling pathway determines the differential expression of PPARG1 and PPARG2 in human GM-CSF-conditioned monocyte-derived macrophages (GM-MØ) and M-CSF-dependent monocyte-derived macrophages (M-MØ). (a) PPARG1 and PPARG2 expression in untreated or activin A-treated M-MØ, monocytes and THP-1 cells. Cells were treated with 25ng/ml recombinant human activin A for 24h (monocytes and THP-1 cells) or 48h (M-MØ). Mean and SEM of three independent experiments is shown (*p<0.05). (B) PPARG1 and PPARG2 gene expression in GM-MØ generated in the presence of either DMSO or the Smad signaling inhibitor SB431542 (10µM). Mean and SEM of three independent experiments is shown (*p<0.05; ***p<0.001). (c) PPARG1 and PPARG2 gene expression in GM-MØ generated in the presence of either DMSO or the Smad signaling inhibitor A-83 (1µM) for 1, 3, or 7days. Mean and SEM of three independents experiments is shown (*p<0.05; **p<0.01; ***p<0.001). (D) PPARG1 and PPARG2 gene expression in GM-MØ generated in the presence of a neutralizing antiactivin A antibody (α-ActA) or an isotype-matched antibody (−). One representative experiment is shown. In (a–D), results are referred to the PPARG1 or PPARG2 mRNA levels detected in untreated cells (arbitrarily set to 1). (e) ABCA1 gene expression in GM-MØ generated in the presence of either DMSO or the Smad signaling inhibitor A-83 (1µM) for seven days. Results are referred to the ABCA1 mRNA level in DMSO-treated cells. Mean and SEM of three independents experiments is shown (*p<0.05). (F) Activin A expression levels in seven independent samples of human alveolar macrophages kept in culture for 24h after isolation. (g) PPARG1, PPARG2, and ABCA1 mRNA levels in human alveolar macrophages cultured for 24h after isolation in the presence of either DMSO or the Smad signaling inhibitor A-83 (1µM). Results are referred to the mRNA levels of each gene in DMSO-treated cells (arbitrarily set to 1). Means and SEM of three independents samples is shown (*p<0.05). 8 Nieto et al. PPARγ Transcriptome in GM-CSF-Conditioned Human Macrophages Frontiers in Immunology | www.frontiersin.org January 2018 | Volume 9 | Article 31 in GM-MØ caused a significant increase in the expression of genes within the “Hallmark_Inflammatory Response,” “Hallmark_TNFA signaling via NFKB,” and “GO_Cellular Response to IL1” gene sets (Figure5D). Also in agreement with the anti-inflammatory activity of PPARγ (32, 58), PPARγ knockdown promoted a significant global upregulation of the GM-MØspecific “Proinflammatory gene set” (12) (Figure5D), and specially of two GM-MØ-specific genes like ECSCR and HSD11B1 (11) FigUre 5 | Peroxisome proliferator-activated receptor gamma (PPARγ) controls the global transcriptional signature of GM-CSF-conditioned human monocytederived macrophages (GM-MØ). (a) Number of annotated genes whose expression is higher or lower in siPPARG-transfected than in siControl-transfected (siC) GM-MØ at the indicated p-values. (B) Venn diagram analysis of the genes differentially expressed in siPPARG-transfected and siControl-transfected GM-MØ compared to experimentally verified PPARγ target genes (PPARγ targets) and computationally predicted PPAR target genes (predicted PPAR targets), as reported in the PPARgene database (53). (c–e) Gene set enrichment analysis on the “t statistic-ranked” list of genes obtained from the siPPARG-GM-MØ versus siControl-GMMØ limma analysis, using the indicated gene set. In (D), the previously defined GM-MØ-specific “Proinflammatory gene set” (12) was also used. (F) Expression of the indicated genes in siPPARG-transfected and siControl-transfected (siC) GM-MØ, as determined by quantitative real-time PCR on three to five independent GM-MØ samples. Results are indicated as the mRNA levels of each gene in siPPARG-transfected relative to the levels in siControl-transfected GM-MØ (n=3–5; *p<0.05; **p<0.01). 9 Nieto et al. PPARγ Transcriptome in GM-CSF-Conditioned Human Macrophages Frontiers in Immunology | www.frontiersin.org January 2018 | Volume 9 | Article 31