ORIGINAL RESEARCH published: 27 April 2018 doi: 10.3389/fimmu.2018.00852 Extracellular Purine Metabolism Is the Switchboard of Immunosuppressive Edited by: Martin Herrmann, Macrophages and a Novel Target to Universitätsklinikum Erlangen, Germany Treat Diseases With Macrophage Reviewed by: Robert Adam Harris, Imbalances Karolinska Institute (KI), Sweden Anna Ohradanova-Repic1*, Christian Machacek 1, Celine Charvet 2,3,4†, Franck Lager 2,3,4, Amiram Ariel, Delphine Le Roux 2,3,4†, René Platzer 1, Vladimir Leksa1,5, Goran Mitulovic6, University of Haifa, Israel 7 8 9,10 2,3,4 Birgit Strobl, Thomas R. Burkard , Gerhard J. Zlabinger , Michael B. Fischer , Vincent Feuillet , 2,3,4 11 12 12 Veterinärmedizinische Gilles Renault , Stephan Blüml , Miroslav Benko , Miloslav Suchanek , Universität Wien, Austria Johannes B. Huppa1, Takami Matsuyama13, Artur Cavaco-Paulo14, Georges Bismuth2,3,4 1 *Correspondence: and Hannes Stockinger * Anna Ohradanova-Repic 1 Molecular Immunology Unit, Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infectiology and [email protected]; Immunology, Medical University of Vienna, Vienna, Austria, 2 Institut National de la Santé et de la Recherche Médicale, Hannes Stockinger INSERM U1016, Institut Cochin, Paris, France, 3 Université Paris Descartes, Paris, France, 4 Centre National de la Recherche hannes.stockinger@ Scientifique (CNRS), UMR 8104, Paris, France, 5 Laboratory of Molecular Immunology, Institute of Molecular Biology, Slovak meduniwien.ac.at Academy of Sciences, Bratislava, Slovakia, 6 Clinical Department of Medical and Chemical Laboratory Diagnostics, Medical †Present address: University of Vienna, Vienna, Austria, 7 Bioinformatics Department of the Research Institute of Molecular Pathology and the Celine Charvet, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria, 8 Institute of Immunology, Center IGBMC – CNRS UMR 7104 – for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria, 9 Department of Transfusion INSERM U964, Illkirch, France; Medicine, Medical University of Vienna, Vienna, Austria, 10 Center for Biomedical Technology, Danube University Krems, Delphine Le Roux, Krems, Austria, 11 Division of Rheumatology, Internal Medicine III, Medical University of Vienna, Vienna, Austria, 12 EXBIO Ecole Nationale Vétérinaire d’Alfort, Praha, Vestec, Czechia, 13 The Center for Advanced Biomedical Sciences and Swine Research, Kagoshima University, UMR BIPAR, Université Paris-Est, Kagoshima, Japan, 14 Centre of Biological Engineering, University of Minho, Campus of Gualtar, Braga, Portugal Maisons-Alfort, France Specialty section: If misregulated, macrophage (Mϕ)–T cell interactions can drive chronic inflammation thereby This article was submitted to causing diseases, such as rheumatoid arthritis (RA). We report that in a proinflammatory Molecular Innate Immunity, environment, granulocyte-M (GM-CSF)- and M colony-stimulating factor (M-CSF)- a section of the journal ϕ ϕ Frontiers in Immunology dependent Mϕs have dichotomous effects on T cell activity. While GM-CSF-dependent Received: 03 February 2018 Mϕs show a highly stimulatory activity typical for M1 Mϕs, M-CSF-dependent Mϕs, Accepted: 06 April 2018 marked by folate receptor β (FRβ), adopt an immunosuppressive M2 phenotype. We Published: 27 April 2018 find the latter to be caused by the purinergic pathway that directs release of extracellular Citation: ATP and its conversion to immunosuppressive adenosine by co-expressed CD39 Ohradanova-Repic A, Machacek C, Charvet C, Lager F, Le Roux D, and CD73. Since we observed a misbalance between immunosuppressive and immu- Platzer R, Leksa V, Mitulovic G, nostimulatory Mϕs in human and murine arthritic joints, we devised a new strategy for Burkard TR, Zlabinger GJ, Fischer MB, Feuillet V, Renault G, RA treatment based on targeted delivery of a novel methotrexate (MTX) formulation to Blüml S, Benko M, Suchanek M, the immunosuppressive FRβ+CD39+CD73+ Mϕs, which boosts adenosine production Huppa JB, Matsuyama T, and curtails the dominance of proinflammatory M s. In contrast to untargeted MTX, Cavaco-Paulo A, Bismuth G and ϕ Stockinger H (2018) Extracellular this approach leads to potent alleviation of inflammation in the murine arthritis model. Purine Metabolism Is the In conclusion, we define the ϕM extracellular purine metabolism as a novel checkpoint Switchboard of Immunosuppressive Macrophages and a Novel in Mϕ cell fate decision-making and an attractive target to control pathological Mϕs in Target to Treat Diseases With immune-mediated diseases. Macrophage Imbalances. Front. Immunol. 9:852. Keywords: macrophage polarization, chronic inflammation, macrophage-T cell interaction, purine metabolism, doi: 10.3389/fimmu.2018.00852 adenosine, methotrexate, rheumatoid arthritis Frontiers in Immunology | www.frontiersin.org 1 April 2018 | Volume 9 | Article 852 Ohradanova-Repic et al. Adenosine-Mediated Switch of M-CSF-Dependent M1 Mϕs INTRODUCTION To elucidate mechanisms how different ϕM subtypes contri- bute to chronic inflammation in RA and to identify pathways Macrophages (Mϕs) are myeloid immune cells essential for tissue controlling their identity, we aimed to generate variously activated homeostasis and immunity. Their differentiation and main tenance human GM-CSF- or M-CSF-differentiated ϕM s and address their is controlled in a tissue-specific manner mainly by two growth ability to produce inflammatory mediators and influence T cell factors, Mϕ and granulocyte-Mϕ colony-stimulating factor responses. We show that FRβ+ M-CSF-dependent Mϕs respond (M-CSF/CSF-1 and GM-CSF/CSF-2), respectively (1). In addi- to proinflammatory stimuli by modulating gene expression of tion, the Mϕ phenotype is further shaped by local stimuli, to the purinergic pathway as a means to produce and respond to which Mϕs respond with high plasticity (2). During infection or extracellular adenosine. Adenosine skews these cells toward the sterile inflammation, proinflammatory cyto kines, such as IFNγ, M2 state and suppresses autologous T cells. GM-CSF-dependent or toll-like receptor (TLR) ligands activate Mϕs into the proin- Mϕs resist this adenosine-mediated switch, so that only specific flammatory M1 type with microbicidal and tumoricidal activity. enhancement of the purinergic metabolism in the FRβ+ Mϕs Alternatively, Th2 cytokines IL-4/IL-13 promote M2 ϕM s with potently limits inflammation in the arthritis mouse model. a tissue remodeling or immunoregulatory phenotype, while stimulation with IL-10, transforming growth factor-β (TGF-β), or glucocorticoids generates highly immu nosuppressive “M2-like” MATERIALS AND METHODS Mϕs (3–5). The shift in theϕ M polarizing stimuli during the Reagents resolution phase of infection is thought to convert M1 Mϕs to the M2 type, restoring tissue homeos tasis (4, 6). LPS (Escherichia coli serotype O55:B5) and adenosine were pur- Recently, it has become apparent that ligation of TLRs or chased from Sigma-Aldrich (St. Louis, MO, USA). Deuterated cytokine receptors also triggers profound changes in key metabolic adenosine was from CDN Isotopes (Quebec, Canada). Adenosine events in Mϕs, enabling coordinate induction and maintenance 5′-triphosphate disodium salt (ATP) was from Thermo Fisher of Mϕ effector activities (7–9). In M1 Mϕs, aerobic glycolysis is Scientific (Waltham, MA, USA). Recombinant human M-CSF, induced to readily provide cells with energy in a form of adenosine IFNγ, and IL-10 were obtained from Peprotech (Rocky Hill, 5′-triphosphate (ATP). Aerobic glycolysis additionally feeds to NJ, USA). Recombinant human GM-CSF and IL-4 were from the pentose phosphate pathway for production of nucleotides and Novartis AG (Basel, Switzerland). The RPMI 1640 medium, NADPH, the latter being required for generation of microbicidal l-glutamine, streptomycin, penicillin, and heat-inactivated fetal reactive oxygen species (8, 9). Furthermore, the glycolytic switch calf serum (FCS) were obtained from Gibco, Thermo Fisher is associated with an increase in several metabolic intermediates Scientific. CD39 inhibitor POM-1 was from Tocris Bioscience that are incorporated into signaling pathways to support the (Bristol, UK). The cell proliferation dye CFSE and calcium inflammatory phenotype. In contrast, M2 ϕM s rely on oxidative sensor Fluo-4, AM was from Molecular Probes, Thermo Fisher metabolism that enables long-term cell survival and promotes Scientific. Brilliant Violet 421-conjugated streptavidin used M2 functions (7). Another example is l-arginine metabolism, as a second step in flow cytometry analyses was purchased which is a hallmark of differently polarized mouse ϕM s (10). from BioLegend (San Diego, CA, USA). Phorbol 12-myristate In M1 Mϕs, arginine is a substrate to nitric oxide synthase (NOS2) 13-acetate (PMA), ionomycin calcium salt (ionomycin) from induced by proinflammatory stimuli to produce antibacterial S. conglobatus and monensin A sodium salt (monensin) were NO. In M2 Mϕs, arginine is metabolized by the M2 marker argi- purchased from Sigma-Aldrich. nase 1 (Arg1) to urea and l-ornithine, a precursor of polyamines important for wound healing. Additionally, Arg1 action limits Antibodies arginine availability to bystander proliferating T cells, leading to The anti-FRβ monoclonal antibody (mAb) (clone EM-35) (17); their suppression (10). While these key metabolic differences was provided by EXBIO (Vestec, Czech Republic), either as between M1 and M2 Mϕs are widely accepted, the metabolic