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 ol.0901698 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2010/05/24/jimmunol.090169 Material 8.DC1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 29, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 path- ways.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 es- sential 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 Investigacio´n 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 Auto´noma de x depending on the stimuli received (5, 6). The classic activation, Madrid); Centro Nacional de Investigaciones Oncolo´gicas, Melchor Ferna´ndez de Almagro; and {Servicio de Inmunologı´a, Hospital Universitario de la Princesa, Uni- which can be induced by in vitro culture of macrophages with versidad Auto´noma 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 Innovacio´n 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 Investigacio´n Sobre Ca´ncer (RETICC RD6/0020/ which can be mimicked in vitro after culture with IL-4, IL-13, 0046) and Red Tema´tica de Investigacio´n 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 Auto´noma 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 chromatogra- phy/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