Alternative Activation Comparison Between Innate, Classic, And

Substrate Fate in Activated Macrophages: A Comparison Between Innate, Classic, and Alternative Activation

This information is current as Juan-Carlos Rodríguez-Prados, Paqui G. Través, Jimena of September 29, 2021. Cuenca, Daniel Rico, Julián Aragonés, Paloma Martín-Sanz, Marta Cascante and Lisardo Boscá J Immunol published online 24 May 2010 http://www.jimmunol.org/content/early/2010/05/24/jimmun

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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 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 24, 2010, doi:10.4049/jimmunol.0901698 The Journal of Immunology

Substrate Fate in Activated Macrophages: A Comparison Between Innate, Classic, and Alternative Activation

Juan-Carlos Rodrı´guez-Prados,*,1 Paqui G. Trave´s,†,‡,1 Jimena Cuenca,† Daniel Rico,x Julia´n Aragone´s,{ Paloma Martı´n-Sanz,†,‡ Marta Cascante,* and Lisardo Bosca´†,‡

Macrophages play a relevant role in innate and adaptive immunity depending on the balance of the stimuli received. From an analytical and functional point of view, macrophage stimulation can be segregated into three main modes, as follows: innate, classic, and alternative pathways.Thesedifferentialactivationsresultintheexpressionofspecificsetsofgenesinvolvedinthereleaseofpro-oranti-inflammatorystimuli. 13 In the present work, we have analyzed whether specific metabolic patterns depend on the signaling pathway activated. A [1,2- C2]glucose tracer-based metabolomics approach has been used to characterize the metabolic flux distributions in macrophages stimulated through the classic, innate, and alternative pathways. Using this methodology combined with mass isotopomer distribution analysis of the new formed Downloaded from metabolites, the data show that activated macrophages are essentially glycolytic cells, and a clear cutoff between the classic/innate activation and the alternative pathway exists. Interestingly, macrophage activation through LPS/IFN-g or TLR-2, -3, -4, and -9 results in similar flux distribution patterns regardless of the pathway activated. However, stimulation through the alternative pathway has minor metabolic effects. The molecular basis of the differences between these two types of behavior involves a switch in the expression of 6-phosphofructo-2- kinase/fructose-2,6-bisphosphatase (PFK2) from the liver type-PFK2 to the more active ubiquitous PFK2 isoenzyme, which responds to Hif-1a activation and increases fructose-2,6-bisphosphate concentration and the glycolytic flux. However, using macrophages targeted for http://www.jimmunol.org/ Hif-1a, the switch of PFK2 isoenzymes still occurs in LPS/IFN-g–activated macrophages, suggesting that this pathway regulates ubiquitous PFK2 expression through Hif-1a-independent mechanisms. The Journal of Immunology, 2010, 185: 000–000. he innate immune system acts as the first line of defense receptors called pattern-recognition receptors. TLRs, a class of pat- and functions by recognizing highly conserved sets of tern-recognition receptors, have the ability to recognize pathogens T molecular patterns (pathogen-associated molecular pat- or pathogen-derived products and initiate signaling events leading to terns [PAMPs]) through a limited number of germline-encoded activation of innate host defense (1, 2). Macrophages play an essential role in the immune response and normal tissue development by guest on September 29, 2021 by producing proinflammatory mediators and by phagocytic clear- *Department of Biochemistry and Molecular Biology, Institute of Biomedicine, Uni- versity of Barcelona; ‡Centro de Investigacioń Biome´dica en Red de Enfermedades ance of pathogens and apoptotic cells (3, 4). It has been described Hepa´ticas y Digestivas, Barcelona; †Instituto de Investigaciones Biome´dicas ‘Alberto that macrophages could undergo different activation processes Sols’ (Consejo Superior de Investigaciones Cientı´ficas-Universidad Autońoma de x depending on the stimuli received (5, 6). The classic activation, Madrid); Centro Nacional de Investigaciones Oncolo´gicas, Melchor Fernańdez de Almagro; and {Servicio de Inmunologıá, Hospital Universitario de la Princesa, Uni- which can be induced by in vitro culture of macrophages with versidad Autońoma de Madrid, Madrid, Spain IFN-g and LPS (inducing TNF-a production), is associated with 1J.-C.R.-P. and P.G.T., in alphabetical order, contributed equally to this work. high microbicidal activity, proinflammatory cytokine, and reactive Received for publication May 28, 2009. Accepted for publication April 10, 2010. oxygen species production and cellular immunity; the innate This work was supported by European Commission (FP7) Etherpath KBBE-Grant activation, which is mediated in culture by ligation of receptors, Agreement no. 222639, Grants SAF2005-01627, SAF2008-00164, and BFU2008- such as TLRs, is associated with microbicidal activity and 02161 from Ministerio de Ciencia e Innovacioń of the Spanish Government, 2005SGR00204 from the Generalitat de Catalunya, S-BIO-0283/2006 from Comu- proinflammatory cytokine production; the alternative activation, nidad de Madrid, Red Tema´tica de Investigacioń Sobre Cańcer (RETICC RD6/0020/ which can be mimicked in vitro after culture with IL-4, IL-13, 0046) and Red Tema´tica de Investigacioń Sobre Enfermedades Cardiovasculares (RECAVA RD6/0014/006), and FIS-RECAVA RD06/0014/0025. RECAVA and glucocorticoids, immune complexes, or IL-10, is associated with Ciberehd are funded by Instituto de Salud Carlos III. J.-C.R.-P. is supported by tissue repair, tumor progression, and humoral immunity (7). Some a fellowship from the University of Barcelona. authors also distinguish macrophage deactivation, which is induced Address correspondence and reprint requests to Dr. Lisardo Bosca´ and Dr. Marta Cas- by cytokines, such as IL-10 or TGF-b, or by ligation of inhibitory cante, Instituto de Investigaciones Biome´dicas ‘Alberto Sols’ (Consejo Superior de Inves- tigaciones Cientı´ficas-Universidad Autońoma de Madrid), Arturo Duperier 4, 28029 receptors, such as CD200 receptor or CD172a, and is related to Madrid, Spain (L.B.) and Department of Biochemistry and Molecular Biology, Faculty anti-inflammatory cytokine production and reduced MHC class II of Biology (edifici Nou) Planta-2.Avinguda Diagonal 645, 08028 Barcelona, Spain (M.C.). E-mail addresses: [email protected] (L.B.) and [email protected] (M.C.) expression (8, 9). Under normal conditions, macrophages are recruited and phago- The online version of this article contains supplemental material. cyte at sites of infection. In addition to playing a crucial role in im- Abbreviations used in this paper: COX-2, cyclooxygenase-2; FDR, false discovery rate; munity, some of the mammalian TLRs have been described to FI, fold induction; Fru-2,6-P2, fructose-2,6-bisphosphate; GCMS, gas chromatography/mass spectrometry; GSEA, gene set enrichment analysis; IP-10, IFN-g–inducible regulate bodily energy metabolism, mostlythrough acting on adipose protein-10; L-PFK2, liver type-PFK2; NOS-2, nitric oxide synthase-2; PAMP, pathogen- tissue. This has recently opened new avenues of research on the role associated molecular pattern; PFK2, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; PI, propidium iodide; poly(I:C), polyriboinosinic:polyribocytidylic acid; PPP, pentose of TLRs in pathologies related to metabolism, such as obesity, in- phosphate pathway; scRNA, scrambled RNA; siRNA, small interfering RNA; uPFK2, sulin resistance, metabolic syndrome, or atherosclerosis (10). The ubiquitous PFK2. accumulation of fatty acids, above all cholesterol in low density Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 lipoprotein form, is the main reason that lipid metabolism has been

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901698 2 METABOLIC FLUXES IN MACROPHAGE ACTIVATION studied in macrophages in the atherosclerosis framework. Recent phages to the plastic. An aliquot of the cell suspension was used to de- works have built a fatty acid bridge between diet and immune sys- termine the cell density in the peritoneal ﬂuid. The cells were centrifuged at 200 3 g for 10 min at 4˚C, and the pellet was washed twice with 25 ml ice- tem due to the induction of TLR expression by some fatty acids (11). cold PBS. Cells were seeded at 1 3 106/cm2 in RPMI 1640 medium sup- However, few are known on central metabolism patterns in macro- plemented with 10% of heat-inactivated FCS and antibiotics. After incuba- phages since the work of Newsholme and collaborators (12–14) tion for 3 h at 37˚C in a 5% CO2 atmosphere, nonadherent cells were in the 1990s. In this study, we will apply a system biology approach removed by extensive washing with PBS. Experiments were carried out to shed some light in the cross-talk between signal transduction in phenol-red free RPMI 1640 medium and 1% of heat-inactivated FCS plus antibiotics (26). When glucose concentration was changed, this was and central metabolism in macrophages. We studied whether the added to glucose-free RPMI 1640 medium. Prior to stimulation, the me- distinct stimulation pathways required different energy demands or dium was aspirated and replaced by warm medium containing the indicated have different metabolic patterns. To achieve these goals, tracer- TLR ligand or cytokine. When the murine macrophage RAW 264.7 cells based metabolomics experiments have been used and combined with were used, they were processed as indicated for the peritoneal macrophages. analysis of the expression of markers of activation. Following Flow cytometry the normal activation process of the macrophages, cells were fed 13 Cells were harvested and washed in cold PBS. After centrifugation at 4˚C with [1,2- C2]glucose, a nonradioactive isotope of glucose that for 5 min and 1000 3 g, cells were resuspended in annexin V-binding 13 will incorporate [ C] carbons into metabolite end products (i.e., buffer (10 mM HEPES [pH 7.4], 140 mM NaCl, 2.5 mM CaCl2). Cells lactate, glutamate) and the ribose of RNA. This tracer has been were labeled with annexin V FITC solution (BD Pharmingen, San Jose, broadly used before (15). Our data show that activated macro- CA) and/or propidium iodide (PI; 100 mg/ml) for 15 min at room temper- ature in the dark. PI is impermeable to living and apoptotic cells, but stains phages are essentially glycolytic cells, and a clear cutoff between necrotic and apoptotic dying cells with impaired membrane integrity, in the classic/innate activation and the alternative pathway exists. In- contrast to annexin V, which stains early apoptotic cells. Downloaded from terestingly, activation through TLR-2, -3, -4, and -9 results in similar Assay of PFK2 activity patterns of metabolic activation, despite the use of at least in part distinct signaling pathways and expression of different sets of genes. Cultured macrophages (6-cm dishes) were homogenized in 1 ml medium In the classic and innate activation we observe a shift in the expres- containing 20 mM potassium phosphate (pH 7.4, 4˚C), 1 mM DTT, 50 mM NaF, 0.5 mM PMSF, 10 mM leupeptin, and 5% poly(ethylene) sion of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase glycol. After centrifugation (7000 3 g for 15 min), poly(ethylene)glycol (PFK2) isoforms, from the liver type-PFK2 (L-PFK2), which has was added to the supernatant up to 15% (mass/v) to fully precipitate the http://www.jimmunol.org/ a low net activity, to the more active ubiquitous PFK2 (uPFK2) PFK2. After resuspension of the pellet in the extraction medium, PFK2 activity was assayed at pH 8.5 with 5 mM MgATP, 5 mM Fru-6-P, and that maintains higher fructose-2,6-bisphosphate (Fru-2,6-P2) concentrations due to a minor bisphosphatase activity and, there- 15 mM Glc-6-P. One unit of PFK2 activity is the amount of enzyme that catalyzes the formation of 1 pmol Fru-2,6-P per minute (17). fore, potentiates the glycolytic ﬂux (16–18). This isoenzyme is 2 expressed in response to hypoxia through Hif-1a activation (16). Metabolite assays

Interestingly, LPS challenge results in an increase in Hif-1a levels Fru-2,6-P2 was extracted from cells (24-well dishes) after homogenization that might contribute to this uPFK2 expression (19–21). Using in 100 ml 50 mM NaOH, followed by heating at 80˚C for 10 min. The macrophages targeted for this transcription factor, we have in- metabolite was measured by the activation of the pyrophosphate-dependent vestigated the contribution of Hif-1a to the enhancement of the phosphofructo-1-kinase (17). Lactate and glucose were measured enzymi- by guest on September 29, 2021 cally in the culture medium. NO release was determined spectrophotomet- glycolytic flux and to the expression of inflammatory molecules in rically by the accumulation of nitrite and nitrate in the medium (phenol red response to classic and alternative activation (22, 23). free). Nitrate was reduced to nitrite, and this was determined after reduction of nitrate to nitrite and the latter with Griess reagent (26) by adding 1 mM sulfanilic acid and 100 mM HCl (final concentration). Materials and Methods Cytokine assay Chemicals The accumulation of TNF-a, IL-6, and IL-10 in the culture medium was Reagents were from Sigma-Aldrich (St. Louis, MO), Roche (Basel, Switzer- measured per triplicate using commercial kits (Biotrak, GE Healthcare, land), Invitrogen (Carlsbad, CA), or Merck (Darmstadt, Germany). TLR Barcelona, Spain, [TNF-a and IL-6] and eBioscience, San Diego, CA ligands were from InvivoGen (San Diego, CA). Cytokines were from Pepro- [IL-10]), following the indications of the supplier. Tech (Rocky Hill, NJ). Commercial Abs were from Santa Cruz Biotechnol- ogy (Santa Cruz, CA), Cell Signaling Technology (Danvers, MA), Abcam Preparation of cell extracts (Cambridge, U.K.), R&D Systems (Minneapolis, MN), Sigma-Aldrich, or The macrophage cultures (six-well dishes) were washed twice with ice-cold PeproTech. Serum and media were from BioWhittaker (Walkersville, PBS, and the cells were homogenized in 0.2 ml buffer containing 10 mM MD). Commercial Pfkfb3 small interfering RNAs (siRNAs) were from Tris-HCl (pH 7.5), 1 mM MgCl2, 1 mM EGTA, 10% glycerol, 0.5% Ambion (Austin, TX), and Pfkfb3 TaqMan gene expression assays were CHAPS, 1 mM 2-ME, and 0.1 mM PMSF and a protease inhibitor mixture 13 . from Applied Biosystems (Carlsbad, CA). [1,2- C2]glucose ( 99% (Sigma-Aldrich). The extracts were vortexed for 30 min at 4˚C and centri- enriched) was purchased from Isotec (Miamisburg, OH). fuged for 15 min at 13,000 3 g. The supernatants were stored at 220˚C. Proteins levels were determined using the Bio-Rad (Richmond, CA) Treatment of animals and preparation of peritoneal detergent-compatible protein reagent. All steps were carried out at 4˚C. macrophages Western blot analysis Mice were housed and bred in our pathogen-free facility. C57BL/6 micewere Samples of cell extracts containing equal amounts of protein (30 mg/lane) used for macrophage isolation. Mice with myeloid lineage-specific HIF1a were boiled in 250 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, and 2% floxed-UBC-cre/ERT2 mice for conditional HIF1a inactivation were used 2-ME, and size separated in 10–15% SDS-PAGE. The gels were blotted (24, 25) and were a generous gift of M. Landa´zuri and J. Aragone´s (Hospital onto a polyvinylidene difluoride membrane (GE Healthcare) and processed La Princesa, Madrid, Spain). Experimental procedures were approved by as recommended by the supplier of the Abs against the murine Ags: NO the Committee for Research Ethics of the Universidad Autonoma de Madrid synthase-2 (NOS-2; sc-7271), cyclooxygenase-2 (COX-2; sc-1999), in accordance with Spanish and European guidelines. Animals were used MHC-II (sc-59322), hemeoxygenase-1 (AB-1284), Arg-1 (sc-20150), sup- aged 8–12 wk, as follows: 4 d prior to the assay, mice were i.p. injected pressor of cytokine signaling-3 (2923s), KC/CXCL1 (AF-453-NA), IFN- 2.5 ml 3% (w/v) of thioglycolate broth (26). Elicited peritoneal macro- g–inducible protein-10 (IP-10)/CXCL10 (500-p129), L-PFK2 (sc-10096), phages were prepared from light-ether anesthetized mice (four to six ani- Hif-1a (MAB1536), and b-actin (A-5441). For uPFK2, specific peptides of mals per condition), killed by cervical dislocation, and injected i.p. 10 ml the isoenzyme were used to generate polyclonal Abs by immunization of sterile RPMI 1640 medium. The peritoneal fluid was carefully aspirated, rabbits (New Zealand White) with multiple intradermal injections with avoiding hemorrhage, and kept at 4˚C to prevent the adhesion of the macro- 300 mg Ag in 1 ml CFA, followed by boosters with 100 mgAginIFA The Journal of Immunology 3

(a gift of R. Barrtrons, University of Barcelona, Barcelona, Spain). The 200˚C. A Varian VF-5 capillary column (30-m length, 250-mm diameter, blots were developed by ECL protocol (GE Healthcare), and different 0.25-mm film thickness) was used to analyze all of the compounds studied. exposition times were performed for each blot with a charged coupling device camera in a luminescent image analyzer (Molecular Imager, Bio- Statistical analysis Rad) to ensure the linearity of the band intensities. The data shown are the means 6 SD of three or five experiments. Statistical significance was estimated with Student t test for unpaired observations. A RNA isolation and RT-PCR analysis p value of less than 0.05 was considered significant. A total of 1 mg total RNA, extracted with TRIzol reagent (Invitrogen) according to the manufacturer’s instructions, was reverse transcribed using Transcriptor First Strand cDNA Synthesis Kit for RT-PCR following the Results indications of the manufacturer (Roche). Real-time PCR was conducted Distribution of substrate fluxes in activated macrophages with SYBR Green on a MyiQ Real-Time PCR System (Bio-Rad) using the Elicited peritoneal macrophages were isolated, maintained in SYBR Green method. PCR thermocycling parameters were 95˚C for 10 min, 40 cycles of 95˚C for 15 s, and 60˚C for 1 min. All samples were culture overnight, and activated for the indicated periods of time analyzed for 36B4 expression in parallel. Each sample was run in duplicate through TLRs involved in innate immunity (TLR4, LPS; TLR2, and was normalized to 36B4. The replicates were then averaged, and fold lipoteichoic acid; TLR3, polyriboinosinic:polyribocytidylic acid; induction was determined in DDCt-based fold-change calculations. Primer and TLR9, CpG), classic activation (LPS/IFN-g), and alternative sequences are available on request. activation (IL-4/IL-13; IL-10) (5, 6). Fig. 1A shows the expression Transient transfections of a panel of representative markers of macrophage activation. NOS-2 and COX-2 were expressed in cells activated by the in- For transient transfections, macrophages were seeded at 80% confluence and transfected using the Cell Line Nucleofector Kit V, following the manu- nate and classic response, except after CpG stimulation. MHC-II Downloaded from facturer’s instructions (Amaxa, Cologne, Germany). Forty-eight hours after upregulation was very sensitive to LPS signaling and to the classic nucleofection with pfkfb3 Silencer Predesigned siRNA or a scrambled RNA activation pathway, and Arg-1 was notably upregulated in re- (scRNA), macrophages were stimulated for 12 h and mRNA isolated. Trans- sponse to IL-4/IL-13. Hemeoxygenase-1 was moderately induced fections were performed in triplicate, and expression of a GFP vector was by innate and alternative pathways of activation. Suppressor used as a control for transfection efficiency. of cytokine signaling-3 was upregulated in response to LPS or Microarray analysis

LPS/IFN-g. The chemokine CXCL1/KC was expressed in cells http://www.jimmunol.org/ Normalized expression data were obtained from National Center for Bio- activated through classic activation pathway, and innate immunity technology Information GEO dataset GDS2429 (27) using GEOquery mediated by TLR2 and TLR4 and CXCL10/IP-10 was upreg- package from Bioconductor (Berkeley, CA) (28). Differential expression for ulated through the classic and the innate response. In addition to the following comparisons was tested using limma Bioconductor package (29): 1) naive mature macrophage versus classic activated macrophage, and these parameters related to macrophage activation, cell viability 2) alternatively activated macrophage. Two gene lists were generated after was determined; apoptosis was evaluated by annexin V exposure each comparison, and they were ranked according to the test statistic for of the cells and by positivity for PI labeling, the later characteristic subsequent gene set enrichment analysis (GSEA). Enrichment of gene sets of dying cells. As Fig. 1B shows, an increase of annexin V was of interest in each list was accomplished using the GSEA method, as de- observed in cells treated with LPS/IFN-g, and to a lesser extent scribed by Mootha et al. (30). We used “Which genes?” (www.whichgenes. org/) to retrieve the REACTOME (31) pathways as gene sets. The genes with LPS; however, the percentage of PI-positive cells at this time by guest on September 29, 2021 from the two lists were also mapped into canonical pathways using In- (12 h) was modest, suggesting that the integrity of the plasma genuity Pathway Analysis software (Ingenuity Systems, Redwood, CA; see membrane remained preserved. These data were reminiscent of Supplemental Material). the expression of NOS-2, indicating that NO plays an important Gas chromatography/mass spectrometry sample preparation role in the induction of apoptosis in these cells (38). Staurosporine and procedure was used as a reference for apoptosis induction in macrophages The macrophage cultures (10-cm dishes) were washed twice with ice-cold (39). Moreover, the accumulation in the culture medium of nitrite/ 13 PBS, and culture media were replaced by 50% enriched [1,2- C2]glucose nitrate, IL-6, TNF-a, and IL-10 contributed to establish the profile containing the indicated TLR ligand or cytokine. After incubation, cells of activation elicited by the different stimuli used; for example, were centrifuged (200 3 g for 5 min), and incubation medium and cell CpG failed to promote NOS-2 expression, but notably increased 2 pellets were obtained and stored at 80˚C until processing. Glucose, IL-6 and TNF-a synthesis (Fig. 1C). lactate, glutamine, and glutamate incubation medium concentrations were determined, as previously described (32, 33), using a Cobas Mira Plus Previous data suggested the relevance of carbohydrate metab- chemistry analyzer (HORIBA ABX, Montpelier, France). Lactate from olism in the commitment for activation of macrophages (40–43). In the cell culture media was extracted by ethyl acetate after acidification this regard, we investigated the glucose consumption and fate in with HCl. Lactate was derivatized to its propylamide-heptafluorobutyric macrophages activated through the innate, classic, and alternative form, and the m/z 328 (carbons 1–3 of lactate, chemical ionization) was monitored for the detection of m1 (lactate with a [13C] in one position) and pathways. As Fig. 2A shows, two clear profiles were observed in m2 (double-labeled lactate) for the estimation of pentose cycle activity terms of glucose consumption and lactate release. TLR activation versus anaerobic glycolysis (34). RNA/Ribose was isolated by acid hydro- promoted an enhancement in the glycolytic flux to lactate that can lysis of cellular RNA after TRIzol purification of cell extracts. Ribose be divided into two time-dependent steps: one from 0 to 4 h, and isolated from RNA was derivatized to its aldonitrile acetate form using a second flux rate from 4 to 12 h. Indeed, when the same exper- hydroxylamine in pyridine and acetic anhydride. We monitored the ion 13 cluster around the m/z 256 (carbons 1–5 of ribose, chemical ionization) to iment was performed with [1,2- C2]-glucose, the incorporation of find the molar enrichment of [13C] labels in ribose (34). Glutamate was [13C] atoms from glucose in different metabolites, according to the separated from the medium using ion-exchange chromatography (35). Glu- scheme depicted in Fig. 2B, allowed the measurement of the pre- tamate was converted to its n-trifluoroacetyl-n-butyl derivative, and the ion cise contribution of these time-dependent fluxes in activated mac- clusters m/z 198 (carbons 2–5 of glutamate, electron impact ionization) and m/z 152 (carbons 2–4 of glutamate, electron impact ionization) were mon- rophages. From 0 to 4 h, the glycolytic activity was much lower itored. Isotopomeric analysis of C2–C5 and C2–C4 fragments of glutamate than from 4 to 12 h (Fig. 2C). Interestingly, alternative stimulation in the medium was done to estimate the relative contributions of pyruvate exhibited a metabolic profile that essentially matched that of con- carboxylase and pyruvate dehydrogenase to the Krebs cycle (36, 37). Mass trol cells, despite the influence of the expression of a specific set of spectral data were obtained on the QP2010 Shimadzu mass selective de- tector connected to a GC-2010 gas chromatograph. The settings were as genes (vide infra). Also, macrophages displayed a metabolic flux follows: gas chromatography inlet for glucose and ribose 250˚C and 200˚C that involved the conversion of glucose into lactate by more than for lactate and glutamate, transfer line 250˚C, mass spectrometry source 95%, regardless of the activation pathway considered (Fig. 3A). 4 METABOLIC FLUXES IN MACROPHAGE ACTIVATION

FIGURE 1. Characterization of macrophages stimulated through the classic, innate, and alternative pathways. Peritoneal macrophages were maintained in culture and stimulated for 12 h with the indicated stimuli: LPS/IFN-g (250 and 20 ng/ml, respectively); LPS (100 ng/ml); IL-13, IL-4, and IL-10 (20 ng/ml each); lipoteichoic acid (5 mg/ml); polyriboinosinic: polyribocytidylic acid (25 mg/ml); CpG (3 mg/ml); and staurosporine (100 ng/ml). The levels of the indicated proteins were determined by Western blot (A). The extent of apoptosis/necrosis was determined by the staining with annexin V and PI, respectively (B). The release of nitrite plus nitrate, TNF-a, IL-6, and IL-10 to the culture medium was determined, as described in Materials and Methods (C). Results show the mean 6 SD of ﬁve experiments. pp , 0.01 versus

the none condition. Downloaded from

The metabolic features revealed by glucose consumption and lac- that classic activation has a stronger effect on the expression of genes tate release are followed by a basal consumption of glutamine and related with the energetic metabolism, favoring the upregulation of http://www.jimmunol.org/ a release of glutamate (Figs. 2A,3A). Besides, low enrichment in genes from the glycolytic pathway and the repression of genes encod- glutamate and RNA (Fig. 3A) evidenced the high glycolytic rate in ing for proteins that participate in oxidative phosphorylation. peritoneal macrophages. Peritoneal macrophages are quiescent PFK2 isoenzyme changes in activated macrophages cells; however, when the macrophage cell line RAW 264.7 was The aforementioned differences in transcription of the glycolysis used and kept overnight with 1% FCS, stimulation for 12 h under gene set between classic and alternative activation of macrophages these conditions revealed that the basal proliferation rate of these allowed us to hypothesize that PFK2, one of the key enzymes of cells did not influence the metabolic profile associated with the the pathway, could be regulated at the protein and/or enzymatic macrophage activation process. As Fig. 3B shows, RAW 264.7 cells level. Accordingly, the expression of the isoforms of PFK2 and the by guest on September 29, 2021 followed a similar m0 (nonlabeled lactate) and m2 (double-labeled levels of Fru-2,6-P were determined under these conditions as an lactate produced from [1,2-13C ]glucose through glycolysis) mass 2 2 indication of the capacity of these cells to metabolize glucose (17, isotope distribution than peritoneal macrophages, without 44). As Fig. 4B shows, resting macrophages expressed the L-type differences in the distribution among the different pathways of isoenzyme of PFK2, but not the uPFK2 isoenzyme, resulting in stimulation, and with ~80% of the glucose label converted into low steady state levels of Fru-2,6-P . However, as result of the lactate. However, proliferation induces higher metabolic fluxes 2 activation through the classic and innate pathway, but not the and [13C] enrichment in ribose and glutamate (enrichment is the alternative pathway, a robust expression of the uPFK2 iso- weighted sum of all the labeled species; Fig. 3C). enzyme occurred, concomitant with a 9-fold rise in the PFK2 Gene profiling in stimulated macrophages activity and a 5-fold increase in the levels of Fru-2,6-P2. In- The previous experiments showed that the carbohydrate metabolism terestingly, the levels of expression of uPFK2, and more impor- in macrophages is fundamentally glycolytic, and that the rate of tantly, the enzyme activity and intracellular concentration of Fru- glucoseconsumptionislowerinalternativelyactivatedmacrophages 2,6-P2 exhibited parallel profiles for each activation condition. than in those activated through the classic pathway. Therefore, the Moreover, neither IL-4/IL-13 nor IL-10 was able to change the expression levels of genes involved in energetic metabolism expression pattern of PFK2 or to increase Fru-2,6-P2 levels. To may differ between the two types of macrophage activation. To test evaluate the capacity of these ILs to influence the response to LPS this hypothesis, we compared the functional enrichment (in up- or costimulation, studies were done. As Fig. 4C shows, when com- downregulated genes) of the following REACTOME (31) pathways: bining IL-10 and IL-4/IL-13 with LPS, the intracellular levels of glycolysis, pyruvate metabolism and TCA cycle, and electron trans- Fru-2,6-P2 were only minimally influenced with respect to the port chain. Glycolysis was significantly enriched in upregulated genes LPS condition, and an important expression of uPFK2 occurred, after classic activation (false discovery rate [FDR] 0.032; Fig. 4A); despite maintaining a certain level of L-PFK2 expression. The however, the enrichment trend of glycolysis upon alternative activa- time course of the changes in Fru-2,6-P2 (Fig. 5A) was com- tion was not significant (FDR 0.547; Fig. 4A, Supplemental Fig. 1). pared with that of uPFK2 (Fig. 5B) and exhibited a parallel pat- Moreover, pyruvate/TCA cycle pathways were most clearly enriched tern. Because uPFK2 expression has been described to be regu- in upregulated genes after IL-4/IL-13 activation (FDR 0.097), lated by Hif-1a, the levels of this protein were determined and whereas the opposite trend was observed for LPS/IFN-g activation, only the classic pathway was able to promote its expression (Fig. although statistically not significant (FDR 0.455) (Fig. 4A,Supple- 5B); COX-2 and NOS-2 levels exhibited the expected profile. In mental Fig. 2). Noteworthily, the most significant enrichment (FDR addition to these protein levels, the time course of the expression 0.000) was found in electron-transport chain genes that were down- of Hif-1a, uPFK2, IL-10, and Glut-1 and Glut-4 was determined, regulated during classic activation, an effect not observed for the al- showing a kinetics compatible with the changes in Fru-2,6-P2 ternative activation (Fig. 4A, Supplemental Fig. 3). These data suggest concentration (Fig. 5C). Interestingly, when the mRNA levels The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 2. Metabolic fluxes in macrophages stimulated through the classic, innate, and alternative pathways. The time course of glucose and glutamine consumption and lactate and glutamate release was determined enzymically by sampling the culture medium at periods of 2 h (A). Macrophages were 13 13 loaded with [1,2- C2]-glucose, and the [ C] trace was followed by GCMS to establish unambiguously the fate of glucose in cells stimulated by the classic, 13 13 innate, and alternative pathways. [1,2- C2]-glucose could follow the oxidative branch of PPP to provide [1- C1]-ribose (═ line) or the upper part of 13 glycolysis driving to the [1,2- C2]-glyceraldehyde-3-phosphate formation (▬ line). Recycling through the nonoxidative branch of PPP allows the formation 13 13 of [1- C1]-glyceraldehyde-3-phosphate and [1,2- C2]-ribose, respectively. Label pattern does not change in the lower part of glycolysis, and the lactate formed could drive into the four depicted isotopomers of glutamate, depending on whether pyruvate dehydrogenase or pyruvate carboxykinase is used to 13 enter in the Krebs cycle (B). The fluxes of glucose consumption and lactate release using [1,2- C2]-glucose were quantified from 0 to 4 h and from 4 to 12 h (C). Results show the mean of five experiments (A), and the mean 6 SD of four experiments (C). pp , 0.01 versus the none condition. GCMS, gas chromatography/mass spectrometry. of the four PFK2 isoenzymes were determined at 12 h in LPS/ set between classic and alternative activation of macrophages might be IFN-g–treated macrophages, minimal differences in L-PFK2 ex- explained through the changes in Hif-1a promoted after LPS pression were observed, whereas uPFK2 and testis-PFK2 were activation (23). However, as Fig. 6A shows, targeting of Hif-1a in upregulated by classic activation and downregulated in alterna- macrophages failed to induce changes in the levels of Fru-2,6-P2 tively activated cells (Fig. 5D). (cells treated with IL-10 exhibited a similar behavior as with IL-4/ IL-13), or in the glycolytic flux measured as lactate release (Fig. 6B), a Macrophage activation in the absence of Hif-1 or in the expression of uPFK2 in these cells (Fig. 6C). Moreover, uPFK2 has been described to be a gene regulated by Hif-1a (18, 45) the mRNA levels of the Hif-1a– and the Hif-1a–targets PHD3, and, therefore, the differences in transcription of the glycolysis gene Glut-1, and, to a lesser extent, Glut-4 exhibited the expected drop in 6 METABOLIC FLUXES IN MACROPHAGE ACTIVATION

FIGURE 3. Metabolic fluxes using tracer-based distribution of [13C] in stimulated macrophages. Peri- toneal macrophages were maintained in culture and 13 stimulated for 12 h in the presence of [1,2- C2] glucose, as indicated in Fig. 1. Samples of culture medium were collected at 4, 8, and 12 h, and the [13C] label distribution in lactate (m0, nonlabeled 13 13 [ C0]lactate; m1, one label [ C1]lactate; and m2, 13 two labels [ C2]lactate) was determined by GCMS to establish the metabolic fluxes (A). The macrophage cell line RAW 264.7 was stimulated in the presence of 13 [1,2- C2]glucose as indicated for peritoneal macrophages, samples of culture medium were collected at 6 and 12 h, and the distribution of the [13C] label was determined by GCMS to establish the metabolic Downloaded from fluxes (B). The comparison between peritoneal macrophages and the RAW 264.7 cell line of the main metabolic fluxes and [13C] enrichment in ribose and glutamate is shown in C. Results show the mean 6 SD of four experiments. pp , 0.001 versus peritoneal macrophages. http://www.jimmunol.org/

Hif-1a2/2 macrophages (Fig. 6D); however, those of the pro- Hif-1a2/2 macrophages activated with LPS/IFN-g or IL-4/IL-13, inﬂammatory cytokines IL-6 and IP-10, and the inﬂammatory mark- respectively (Fig. 6E). In agreement with these data, the protein ers NOS-2 and Arg-1, showed a similar expression in Hif-1a+/+ and levels of COX-2, NOS-2, KC, IP-10, uPFK2, and L-PFK2 did not by guest on September 29, 2021

FIGURE 4. GSEA of energy metabolism pathways and PFK2 isoenzyme switch in activated macrophages. The normalized enriched score calculated by GSEA was shown in the bars, and numbers indicated the FDR for the enrichment. Positive NES indicates enrichment in upregulated genes, whereas negative normalized enriched score corresponds to enrichment in downregulated genes; see Supplemental Material for more details (A). Peritoneal macrophages were maintained in culture and stimulated for 12 h, as indicated in Fig. 1. The levels of the L-PFK2 and uPFK2 isoenzymes were determined by Western blot using speciﬁc Abs. Cell extracts were prepared, and the activity of PFK2

was determined in vitro. The levels of Fru-2,6-P2 were determined in cell extracts after collection of the cell pellets in 50 mM NaOH at 80˚C (B). Cotreatment of cells with LPS and IL-10 or IL-4 results in a decrease

in Fru-2,6-P2 content (C). Results show the mean 6 SD of ﬁve experiments. pp , 0.05; ppp , 0.01; pppp , 0.001 versus resting macrophages (A). pp , 0.01 versus untreated cells; #p , 0.05 versus the LPS condition (C). The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 5. Time course of glycolytic changes in activated macrophages. Peritoneal macrophages were activated through the innate, classic, FIGURE 6. Macrophages lacking Hif-1a fail to show changes in the and alternative pathways, and the time course of the levels of Fru-2,6-P2 expression of uPFK2 and in the glycolytic flux upon activation. Peritoneal was determined (A). The time-dependent changes in uFPK-2/L-PFK2, Hif- macrophages from Hif-1a+/+ and myeloid Hif-1a2/2 mice were activated 1a, COX-2, NOS-2, and b-actin (for normalization of lane charge) were through the innate, classic, and alternative pathways, and the determined by Western blot (B). The changes in mRNA levels as fold concentrations of Fru-2,6-P2 (A), the lactate flux (B), and the FI of induction (FI) of Hif-1a, uPFK2, IL-10, Glut-1, and Glut-4 were deter- uPFK2 mRNA (C) were determined. The response of a series of genes by guest on September 29, 2021 mined during the initial 12 h after activation and using 36B4 mRNA for involved in hypoxia (Hif-1a, PHD3, Glut-1, and Glut-4) and inflammation normalization (C). The changes in the expression of the mRNA isoforms of (IL-6, IL-10, NOS-2, and Arg-1) was determined at different times upon PFK2 (pfkfb) in macrophages activated through the classic and alternative challenge with LPS/IFN-g or IL-4/IL-13 (D, E). The levels of a panel of pathways were compared at 12 h (D). Results show the mean 6 SD of four proteins involved in the activation through the innate (polyriboinosinic: experiments. polyribocytidylic acid [poly(I:C)]), classic, and alternative pathways were determined at 18 h (F). Results show the mean 6 SD of five experiments (B, C), the means of three independent experiments (A, D, E), or a repre- 2 2 exhibit differences between Hif-1a+/+ and Hif-1a / macrophages; sentative experiment of three (F). Arg-1 protein levels were slightly higher in Hif-1a2/2 cells activated with IL-4/IL-13 (Fig. 6F). metabolic aspect is to modulate glucose availability, from 5 to 100 mM, and therefore to modify the glycolytic flux. As Fig. 7E shows, uPFK2 interference attenuates macrophage activation the intracellular levels of Fru-2,6-P2 and the synthesis of lactate and In view of the minimal effects observed on metabolic fluxes after nitrite in LPS/IFN-g–activated macrophages were modulated by Hif-1a targeting, the interference of uPFK2 expression was in- glucose concentration. However, the protein levels of COX-2, vestigated. Using a GFP expression vector as a tracer for the esti- NOS-2, uPFK2, and Arg-1 apparently did not show significant dif- mation of the percentage of transfected cells, the maximal efficiency ferences among the range of glucose concentrations assayed (Fig. was ∼30%, and higher levels of uPFK2 siRNA or scRNA resulted in 7F). At the mRNA level, small, but reproducible differences in a loss of macrophage viability (Fig. 7A). Fig. 7B shows the analysis NOS-2 and Arg-1 at 5 mM glucose versus 25–100 mM glucose were of PI-positive cells upon stimulation with LPS/IFN-g or IL-4/IL-13. observed in macrophages activated for 8 h with LPS/IFN-g or IL-4/ Interference of uPFK2 with 100 nM siRNA increased significantly IL-13, respectively (Fig. 7G). Taken together, these data suggest the apoptosis of LPS/IFN-g–activated cells at 18 h. For this reason, a role for uPFK2 expression and activity in the metabolic activation further analyses were carried out in cells after 10 h of stimulation. As elicited after LPS/IFN-g treatment of macrophages. Fig. 7C and 7D shows, treatment with uPFK2 siRNA decreased the protein levels of uPFK2, NOS-2, and COX-2, as well as those of the Discussion corresponding mRNAs in macrophages stimulated with LPS/IFN-g. Previous work established that peritoneal macrophages are es- These data suggest that impairment of the expression of uPFK2 sentially glycolytic cells (13, 14, 41, 42, 46, 47). Compared with results in attenuation of the classic activation of macrophages. other cell types, macrophages use mainly anaerobic glycolysis in However, the loss of viability observed after transfection with the metabolization of glucose, and the flux in isolated cells is siRNA and the moderate efficiency of the process required further ∼10% of the actual capacity through 6-phosphofructo-1-kinase, approaches to validate the effects due to a reduction in the expres- one of the enzymes that controls the glycolytic pathway (42). sion (or activity) of uPFK2. An additional way to circumvent this In addition to this, glutamine is converted into glutamate and 8 METABOLIC FLUXES IN MACROPHAGE ACTIVATION

FIGURE 7. Attenuation of uPFK2 expression impairs macrophage viability upon activation. Perito- neal macrophages were incubated with different concentrations of a GFP expression vector and siRNA speciﬁc for uPFK2 or the corresponding scrambled se- quence scRNA. The levels of expression of GFP are shown in A. Cells treated with 100 nM scRNA, siRNA of uPFK2 or the transfection reagent (vehicle) were stimulated with LPS/IFN-g or IL-4/IL-13, and viability wasdeterminedafter18h byPI staining (B).The protein levels of uPFK2, COX-2, and NOS-2 (C) and the FI of the mRNA of these genes (D) were determined at 10 h. Alternatively, macrophages were maintained in culture with 5, 25, or 100 mM glucose in RPMI 1640 medium, Downloaded from and the levels of Fru-2,6-P2 and the accumulation of lactate and nitrite in the culture medium were determined (E). The protein levels at 18 h of genes related to inﬂammation and uPFK2 were measured by immunoblot (F). The mRNA fold inductions of these genes were determined at 8 h (G). Results show the http://www.jimmunol.org/ mean 6 SD of four experiments. pp , 0.01 versus the vehicle condition in nonstimulated cells; #p , 0.05 versus the corresponding vehicle condition (B, D); pp , 0.01 versus the corresponding 5 mM glucose condition (E). by guest on September 29, 2021

aspartate, and less than 10% is being oxidized, at the time that the uPFK2 isoenzyme. Interestingly, uPFK2 exhibits a higher ca- fatty acids appear to be the main substrates for oxidative metab- pacity to accumulate Fru-2,6-P2 in macrophages, due to its lower olism (41). In the present work, we have assessed the metabolic bisphosphatase activity compared with L-PFK2, which results in an profiles associated with stimulation of macrophages through three increase in the enzymatic activity determined in vitro, and in the well-defined activation pathways, using a metabolomic approach. glycolytic flux. Moreover, the substitution between the two isoen- The data obtained show that regardless of the stimulation pathway zymes is remarkable, and, in fact, the protein levels of L-PFK2 involved, macrophages remain glycolytic cells and accelerate nota- almost disappear and are substituted by the uPFK2 form in the bly the conversion of glucose into lactate when challenged through course of TLR-2, -3, -4, or -9 activation, despite the use of non- the classic/innate activation pathways; however, the activation redundant signaling pathways (i.e., MyD88-dependent and inde- through the alternative pathway exerted minimal effects on the basal pendent pathways). One likely mechanism to explain this switch consumption of glucose. One of the relevant regulators of glucose between PFK2 isoenzymes after classic activation is the rise in the metabolism is the rise in the levels of Fru-2,6-P2, which in turn levels of Hif-1a because a functional relationship has been activates the flux through 6-phosphofructo-1-kinase (43, 48, 49). described between the hypoxia-responsive element motifs present Fru-2,6-P2 is synthesized/degraded by the bifunctional enzyme. in the uPFK2 promoter and the expression of this isoenzyme (16, 18, Four genes encode the PFK2 in higher mammals. The L type is 19, 22, 23, 53). Accordingly, experiments in macrophages lacking encoded by the PFKFB1 gene and is mainly expressed in liver, Hif-1a were carried out with the idea to impair uPFK2 expression. and a splicing variant is expressed in muscle. This enzyme has However, our data clearly show that classic activation retains almost a balanced kinase/bisphosphatase ratio and, unless the bisphos- intact the ability to promote uPFK2 expression, and, indeed, the phatase activity is inhibited, it maintains low levels of Fru-2,6-P2 glycolytic flux was identical to that of parental macrophages through a futile cycle of synthesis and degradation of the metabolite expressing Hif-1a after LPS/IFN-g challenge. These results were (16, 48–51). The uPFK2 encoded by the PFKFB3 gene has a higher unexpected in view of the decreased glycolysis observed in B cells kinase activity (∼10:1 kinase/bisphosphatase), is induced by lacking Hif-1a (54). Moreover, a cross-talk between Hif-1a and hypoxia, and can be regulated by phosphorylation, playing a role LPS signaling has been reported in macrophages, and it appears to in the high glycolytic rate of various cell types, such as cancer cells be involved in the development of immune tolerance to LPS and in (18, 43, 45, 49, 52). A relevant finding of this work is the observation sepsis (21, 55, 56). More recently, a specific polarization of classic that concomitant to the classic/innate activation, there is a signi- and alternative activation of macrophages has been attributed to ficant change in the expression of the PFK2 from the L type to Hif-1a and Hif-2a, with a predominant role of Hif-1a in classic The Journal of Immunology 9 activation, in particular when comparing bone marrow-derived due to the recycling through the nonoxidative branch of PPP. Fi- macrophages versus thioglycolate-induced peritoneal macrophages nally, the lower glycolytic flux in RAW 264.7 indicates that pro- (22). In this regard, a drop in the expression of NOS-2 in bone mar- liferation promotes the recruitment of other carbon source like row-derived macrophages from Hif-1a knockout mice challenged glutamine in an anaplerotic flux, corroborating the role of gluta- with pathogens was previously described (57); however, this is not mine metabolism in macrophage activation already described (12). the case when peritoneal cells were used, probably because of the In conclusion, the data show that the stimulation of macrophages expression of Hif-2a, as recently reported (22). The question remains through the classic, innate, and alternative pathways exhibits, as on the identification of the regulatory elements inthe uPFK2 promoter expected, a distinct expression of activation markers due to dif- that are responsible for this induction upon classic activation in the ferent signaling events involved in each pathway. However, the absence of Hif-1a. In addition to other mechanisms, the possibility of innate and classic activation show a higher glycolytic rate versus the involvement of Hif-2a in the induction of uPFK2 cannot be alternative activation, due to a switch in the expression of PFK2 disregarded, as described in tumor-associated macrophages (23, isoenzymes that appears to be a common event after LPS/IFN-g or 58). Next, the expression of uPFK2 was interfered with a specific TLR-2, -3, -4, and -9 stimulation. Despite the differences in glu- siRNA. However, primary cultures of macrophages (and also bone cose consumption and lactate release, label distribution analysis marrow-derived macrophages; data not shown) were difficult to trans- shows that there is a common anaerobic pattern followed regard- fect and, using concentrations up to 300 nM siRNA, the maximal less of the activation pathway. Further studies on the molecular efficiency was ∼35% of the cells with a clear toxicity and loss of mechanisms that govern the switch between the PFK2 isoenzymes viability. Using this approach, it was clearly observed that upon clas- might contribute to unravel metabolic aspects related to accom- sic activation interfered macrophages exhibited a high apoptotic rate, modation of macrophages to biological microenvironments, such Downloaded from not observed in the scRNA counterparts nor in the IL-4/IL-13– as sepsis, endotoxin tolerance, or tumor growth. activated cells, suggesting that the siRNA was decreasing uPFK2 expression and promoting an apoptotic response. In this regard, the Acknowledgments levels of expression of some inflammatory markers, such as COX-2 or We thank Dr. Simon Bartlett for discussion, advice, and critical reading of the NOS-2, were significantly attenuated in the absence of uPFK2. Al- manuscript. We thank Dr. Ramoń Bartrons (University of Barcelona) for the ´ ternatively to this approach, attempts to decrease the concentration of generous gift of the uPFK2 Ab and Dr. M. de Landazuri (Hospital La http://www.jimmunol.org/ Princesa, Madrid) and Dr. Luis del Peso (Instituto de Investigaciones Bio- Fru-2,6-P2 via modulation of glucose availability showed that the metabolic activity of enzymes involved in inflammation, such as me´dicas, Madrid) for help on Hif-1. NOS-2, exhibited a decreased activity at 5 mM versus the cells maintained at 25 and 100 mM glucose. Interestingly, a certain modulation Disclosures by glucose of NOS-2 mRNAwas observed in macrophages activated The authors have no financial conflicts of interest. through the classic pathway. Regarding the mass isotopomer distribution in both resting and References activated macrophages, the distribution of the label is essentially 1. Gordon, S., C. Pasare, and R. Medzhitov. 2007. The macrophage: past, present

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