The Repair of Skeletal Muscle Requires Iron Recycling through Macrophage Ferroportin Gianfranca Corna, Imma Caserta, Antonella Monno, Pietro Apostoli, Angelo A. Manfredi, Clara Camaschella and This information is current as Patrizia Rovere-Querini of September 25, 2021. J Immunol 2016; 197:1914-1925; Prepublished online 27 July 2016; doi: 10.4049/jimmunol.1501417 http://www.jimmunol.org/content/197/5/1914 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2016/07/26/jimmunol.150141 Material 7.DCSupplemental http://www.jimmunol.org/ References This article cites 53 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/197/5/1914.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 25, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *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 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology The Repair of Skeletal Muscle Requires Iron Recycling through Macrophage Ferroportin Gianfranca Corna,* Imma Caserta,*,† Antonella Monno,* Pietro Apostoli,‡ Angelo A. Manfredi,*,† Clara Camaschella,†,x and Patrizia Rovere-Querini*,† Macrophages recruited at the site of sterile muscle damage play an essential role in the regeneration of the tissue. In this article, we report that the selective disruption of macrophage ferroportin (Fpn) results in iron accumulation within muscle-infiltrating mac- rophages and jeopardizes muscle healing, prompting fat accumulation. Macrophages isolated from the tissue at early time points after injury express ferritin H, CD163, and hemeoxygenase-1, indicating that they can uptake heme and store iron. At later time points they upregulate Fpn expression, thus acquiring the ability to release the metal. Transferrin-mediated iron uptake by regen- erating myofibers occurs independently of systemic iron homeostasis. The inhibition of macrophage iron export via the silencing of Fpn results in regenerating muscles with smaller myofibers and fat accumulation. These results highlight the existence of a local Downloaded from pathway of iron recycling that plays a nonredundant role in the myogenic differentiation of muscle precursors, limiting the adipose degeneration of the tissue. The Journal of Immunology, 2016, 197: 1914–1925. esident macrophages are rare in healthy skeletal muscles. formation and assembly (1, 2). Accordingly, in mice carrying a Monocyte-derived macrophages are consistently detect- mutated Cebpb promoter, which selectively fail to express genes R able in the injured muscle (1) and, as demonstrated by associated with alternative activation, the clearance of remnants http://www.jimmunol.org/ depletion experiments using pharmacological and genetic tools is conserved, whereas myofiber regeneration is severely compro- (1–5), are required for muscle regeneration. mised (4). In agreement, IL-10, which is involved in macrophage- After damage, macrophages produce high levels of inflammatory alternative activation, has been recognized to have a role in muscle cytokines, such as TNF-a and IL-1b, and remove myofiber rem- regeneration (2, 6). Of importance, the pattern of macrophage ac- nants and apoptotic cells while sustaining the activation and the tivation controls the expression of iron-related genes and macro- proliferation of myogenic precursors (1). At later times during re- phage ability to store or release iron (7–9). Alternatively activated generation, alternatively activated macrophages predominate. They macrophages have a large intracellular labile iron pool but have secrete IL-10 and TGF-b, and favor myoblast fusion and neofiber limited storage ability, express the iron exporter ferroportin (Fpn), by guest on September 25, 2021 and spontaneously release the metal (7–9). Iron plays essential roles in many biological processes that *Division of Immunology, Transplantation and Infectious Diseases, San Raffaele reutilize the available molecules within a highly conservative Scientific Institute, 20132 Milan, Italy; †Vita-Salute San Raffaele University, 20132 Milan, Italy; ‡Department of Experimental and Applied Medicine, Section of Occu- metabolism (10). Splenic red pulp, bone marrow, and liver pational Health and Industrial Hygiene, University of Brescia, 25123 Brescia, Italy; macrophages phagocytose damaged and senescent RBCs, process and xDivision of Genetics and Cell Biology, San Raffaele Scientific Institute, 20132 Milan, Italy them to degrade hemoglobin-associated heme, and recycle the heme-associated iron (11–14) supplying the iron required for most ORCID: 0000-0003-1427-0143 (C.C.). of the daily needs (10, 13, 15, 16). Spleen and bone marrow Received for publication July 6, 2015. Accepted for publication June 26, 2016. macrophages accumulate intracellular heme and iron in hemolytic This work was supported by the Association Franc¸aise contre les Myopathies (Grant 15440 to P.R.-Q.), the Italian Ministry of Health (Fondo per gli Investimenti della conditions, including autoimmune anemia and sickle cell disease Ricerca di Base-IDEAS to P.R.-Q.; Grant Ricerca Finalizzata 2011 to P.R.-Q. and (17). Of interest, heme recognition induces the expression of Fpn (18, A.A.M.), and the Italian Ministry of University and Research (Grant Progetto di Ricerca di Rilevanza Nazionale 2010 to A.A.M.). 19), thus delivering the metal to plasma, where it becomes available for tissue uptake and ensuing synthesis of novel heme groups G.C. designed experiments, performed experiments, analyzed the data, and wrote the manuscript; I.C. performed experiments; A.M. performed immunohistochemistry and and biogenesis of iron sulfur clusters in mitochondria (20, 21). immunofluorescence; P.A. was responsible for macrophage iron content measure- Iron homeostasis during skeletal muscle regeneration has not ment; C.C. discussed the results and wrote the manuscript; A.A.M. discussed the results and wrote the manuscript; and P.R.-Q. designed experiments and wrote the been extensively characterized. The tissue contains from 10 to 15% manuscript. of body iron, prevalently in the form of heme-iron bound myo- Address correspondence and reprint requests to Dr. Gianfranca Corna and Dr. Patrizia globin. Iron released during muscle fiber damage must be promptly Rovere-Querini, Ospedale San Raffaele, DIBIT-3A1, via Olgettina 58, 20132 Milano, removed to limit its oxidative effect; however, iron must become Italy. E-mail addresses: [email protected] (G.C.) and [email protected] (P.R.-Q.) available during skeletal muscle repair to allow the synthesis of The online version of this article contains supplemental material. myoglobin and prosthetic groups of the catalytic site of many Abbreviations used in this article: BMP, bone morphogenetic protein; CSA, cross- enzymes instrumental for fiber survival and function (22). Because sectional area; CTX, cardiotoxin; DFO, Desferal; DT, diphtheria toxin; FAP, fibroa- of their natural ability to uptake and release iron, macrophages are dipogenic precursor; Fpn, ferroportin; FtH, ferritin H; HIF1a, hypoxia inducible factor 1a; HO-1, hemeoxygenase-1; IF, immunofluorescence; IHC, immunohisto- strong candidates to accomplish these homeostatic functions: re- chemistry; KD, knock down; PB, peripheral blood; Q, quadriceps femoris; rm, moval of potentially dangerous free iron and transfer to regener- recombinant murine; Scr, scramble; shRNA, short hairpin RNA; TA, tibialis anterior; ating myofibers. Tf, transferrin; TfR1, Tf receptor I. In this study, we show that muscle-infiltrating macrophages after Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 acute sterile muscle damage undergo a time-dependent functional www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501417 The Journal of Immunology 1915 modification of their ability to handle iron. By depletion of en- differentiation medium (IMDM supplemented with 2% horse serum, dogenous macrophages and adoptive transfer of cells characterized 100 U/ml penicillin, and 100 mg/ml streptomycin) to allow differentiation. by the selective inactivation of Fpn via short hairpin RNA (shRNA) In selected experiments differentiation was carried out in the presence of DFO. After 24 h of differentiation, cells were fixed with 4% PFA and interference, we show that Fpn expression by macrophages at the immunostained using an anti-sarcomeric myosin mAb (MF20; Develop- site of injury represents a requirement for appropriate activation of mental Studies Hybridoma Bank). Nuclei were stained with Hoechst 33342 myogenic precursors and eventual healing of the injured skeletal (Invitrogen). Samples were examined with a Nikon Eclipse 55i microscope muscle. (Nikon), and images were captured with a Digital Sight DS-5 M digital camera (Nikon). Fusion index was determined as the number of myosin- expressing myotubes with 2, 3–4, or $5 nuclei versus the total number of Materials