An Abundant Tissue Macrophage Population in the Adult Murine Heart with a Distinct Alternatively-Activated Macrophage Profile Alexander R. Pinto1,2*, Rosa Paolicelli1, Ekaterina Salimova1, Janko Gospocic1, Esfir Slonimsky1, Daniel Bilbao-Cortes1, James W. Godwin2, Nadia A. Rosenthal1,2,3* 1 European Molecular Biology Laboratory (EMBL) Mouse Biology Unit, Monterotondo (Rome), Italy, 2 Australian Regenerative Medicine Institute (ARMI), Monash University, Clayton, Victoria, Australia, 3 Harefield Heart Science Centre, National Heart and Lung Institute, Imperial College London, London, United Kingdom Abstract Cardiac tissue macrophages (cTMs) are a previously uncharacterised cell type that we have identified and characterise here as an abundant GFP+ population within the adult Cx3cr1GFP/+ knock-in mouse heart. They comprise the predominant myeloid cell population in the myocardium, and are found throughout myocardial interstitial spaces interacting directly with capillary endothelial cells and cardiomyocytes. Flow cytometry-based immunophenotyping shows that cTMs exhibit canonical macrophage markers. Gene expression analysis shows that cTMs (CD45+CD11b+GFP+) are distinct from mononuclear CD45+CD11b+GFP+ cells sorted from the spleen and brain of adult Cx3cr1GFP/+ mice. Gene expression profiling reveals that cTMs closely resemble alternatively-activated anti-inflammatory M2 macrophages, expressing a number of M2 markers, including Mrc1, CD163, and Lyve-1. While cTMs perform normal tissue macrophage homeostatic functions, they also exhibit a distinct phenotype, involving secretion of salutary factors (including IGF-1) and immune modulation. In summary, the characterisation of cTMs at the cellular and molecular level defines a potentially important role for these cells in cardiac homeostasis. Citation: Pinto AR, Paolicelli R, Salimova E, Gospocic J, Slonimsky E, et al. (2012) An Abundant Tissue Macrophage Population in the Adult Murine Heart with a Distinct Alternatively-Activated Macrophage Profile. PLoS ONE 7(5): e36814. doi:10.1371/journal.pone.0036814 Editor: David L. Boone, University of Chicago, United States of America Received January 31, 2012; Accepted April 6, 2012; Published May 10, 2012 Copyright: 2012 Pinto et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by grants to NR from the European Union (EUCOMM: LSHM-CT-2005-018931; EUMODIC: LSHG-CT-2006-037188; EUCOMMTOOLS: HEALTH-F4-2010-261492; and HeartRepair: Heart Failure and Repair LSHM-CT-2005-018630). The Australian Regenerative Medicine Institute is supported by grants from the Australian Government and State Government of Victoria. NR is an NHMRC Australia Fellow. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: nadia.rosenthal@monash.edu (NAR); alex.pinto@monash.edu (ARP) Introduction Macrophages and dendritic cells (DCs) are mononuclear phagocytes (MPs), playing an important role in tissue homeostasis and serve as sentinels for tissue damage and foreign antigens. Tissue MPs, comprised of tissue macrophages (TMs) and DCs, exhibit significant heterogeneity in their phenotype depending on the local environment [1,2]. As components of the mononuclear phagocytic system, TMs play an important role in inflammation, tissue remodelling and clearing tissue debris by acting as sentinels for foreign antigens and tissue damage. However to date, a systematic analysis of MPs in the mammalian heart has not been undertaken. The identification of MPs has been significantly aided by the Cx3cr1GFP/+ transgenic mouse, where one allele of the Cx3cr1 gene, the receptor for the membrane tethered chemokine fractalkine/ Cx3cl1 expressed specifically in MPs, has been replaced by the gene encoding enhanced green fluorescent protein (GFP) [3]. Expression of GFP within these mice has been used to identify tissue MPs from a wide array of tissues including the central nervous system (microglia) [3,4,5], kidney [6], liver [7], skin [8], intestine [9] and blood vessels [10]. Analysis of resident GFP- expressing cells from these tissues has led to key insights regarding MP characteristics in tissue homeostatic conditions and MP responses to tissue damage and invasion by pathogens. In addition, these studies have highlighted the heterogeneity of MPs from various tissues. Although a number of studies have characterised different tissue MPs, this cell population has not been systematically investigated in myocardial homeostasis and the specific features of these cells in the heart have remained, until now, unexplored. Activated macrophages can generally be categorised based on their functional phenotypes [11], designated M1 for classically-activated and M2 for alternatively-activated. Both in vitro and in vivo studies have established that M1 macrophages have an inflammatory phenotype coinciding with early-phases of tissue injury, whereas M2 macrophages have an anti-inflammatory, pro-angiogenic and tissue remodelling phenotype coinciding with late-phases of tissue injury [1,12]. Although this categorisation is over-simplistic, it is useful in characterising MP phenotypes when considering their tissue functions. Using the Cx3cr1GFP/+ transgenic mouse model, we describe an abundant cardiac tissue macrophage (cTM) population within the adult mouse myocardium. Gene expression analysis reveals several defining characteristics of these cells, which closely resemble M2 macrophages in their gene expression signature. The analysis presented here provides new evidence that cTMs participate in PLoS ONE | www.plosone.org 1 May 2012 | Volume 7 | Issue 5 | e36814