While this study characterises cTMs in the adult murine heart, further studies are required to chart the appearance of cTMs within the myocardium during cardiac development, although previous reports have used Cx3cr1GFP/+ knock-in mice to detect macrophage subsets as early as embryonic days 7.5 to 8.5 of development [53,54]. The cTM gene expression analysis and comparisons with other adult tissue macrophage populations presented here provides a valuable starting point for the generation of new reporters and cTM-specific drivers that will permit further study of cTMs in cardiac development, homeostasis and injury. In summary, we have shown that the adult mouse heart hosts an abundant population of tissue macrophages. We have shown that these cells exhibit an M2-macrophage-like phenotype, and have the capacity to secrete salutary factors, clear tissue debris and dampen inflammation, suggesting that they may be important for tissue homeostasis. Future studies will characterise these cells during development, cardiac injury and other pathological states. Notably, the adult mammalian heart retains a number of resident stem cell populations with varying pleuripotency and regenerative capacity, which may be sensitive to a number of cTM secreted factors such as IGF-1, influencing stem cell mobility and differentiation in a wide array of tissue contexts including muscle [55,56,57]. Moreover, the quality and behaviour of cTMs in clinically relevant states in the obese, diabetic and elderly context need to be examined. Manipulation of these cells during cardiac repair and regeneration, and in chronic inflammatory states, may represent a novel therapeutic avenue for restoring normal cardiac homeostasis after tissue damage or stress. Supporting Information Figure S1 Scatter/correlation profile of microarray data. House-keeping and canonical macrophage genes expressed by cTMs (heart) vs GFP+ cells from the brain and spleen (top and bottom panels respectively) are shown in the respective magnified views (right panels). (TIF) Figure S2 Heterogeneity of Mrc1, CD163 and Lyve-1 expression in cTMs. Histogram of proportion of GFP+ cells References 1. Auffray C, Sieweke MH, Geissmann F (2009) Blood monocytes: development, heterogeneity, and relationship with dendritic cells. Annu Rev Immunol 27: 669–692. 2. Gordon S, Taylor PR (2005) Monocyte and macrophage heterogeneity. Nat Rev Immunol 5: 953–964. 3. Jung S, Aliberti J, Graemmel P, Sunshine MJ, Kreutzberg GW, et al. (2000) Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion. Mol Cell Biol 20: 4106–4114. 4. Davalos D, Grutzendler J, Yang G, Kim JV, Zuo Y, et al. (2005) ATP mediates rapid microglial response to local brain injury in vivo. Nat Neurosci 8: 752–758. 5. 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Macrophages in the Adult Murine Heart from adult Cx3cr1GFP/+ mouse heart sections that stain for Mrc1, CD163 and Lyve-1. Data obtained from confocal micrographs of stained tissue sections as shown in figure 4C. Histograms show mean 6 SEM. For each marker, data was obtained from three independent confocal micrograph fields from the mouse myocar- dium. (TIF) Table S1 Genes 20-fold or greater enriched in cTMs vs GFP+ cells from the spleen and brain. Acronyms H, B and S denote expression values derived from CD45+CD11b+GFP+ cells from the heart, brain and spleen respectively. HvS denotes a ratio of expression values for CD45+CD11b+GFP+ cells from the heart versus spleen. HvB denotes a similar ratio except CD45+CD11b + GFP+ cells from the heart versus brain. (TIF) Table S2 Expression of M2-related genes in cTMs and GFP+ cells from the spleen and brain. Acronyms are the same as in Table S1. 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