Macrophages in the Adult Murine Heart Figure 5. Expression of complement opsonisation components cTMs and GFP+ cells from the spleen and brain. Microarray analysis derived heat maps of complement pathway related gene expression by cTMs and GFP+ cells from the spleen and brain identified by microarray gene expression analysis. (A) Secreted complement factors with probes corresponding to C1q members highlighted. (B) Complement receptors. doi:10.1371/journal.pone.0036814.g005 cells, with low or no transcripts in GFP+ cells from other tissues. In addition, matrix metalloprotease 13 (MMP13) was detected in cTMs and GFP+ cells from the kidney, with low or no levels in other tissues, whereas Heme oxygenase 1 (Hmox1) and tissue inhibitor of matrix metalloproteases 2 (TIMP2) were detected in all tissues. Thus cTMs have a distinctive gene expression profile compared to TMs from other tissue sources. Discussion This report characterises a novel, abundant population of macrophages within cardiac tissue (cTMs) marked by GFP expression in the Cx3cr1GFP/+ knock-in mouse. Immunophenotyp- ing and gene expression analysis confirm that these cells are cardiac tissue macrophages expressing canonical macrophage genes such as F4/80, CD14 and Csf1-r. These findings show that cTMs are distinct from aortic and cardiac valve mononuclear phagocytes, which are reported to exhibit a dendritic cell (DC) cell surface profile [25]. Since migration to local lymph nodes after antigen exposure has not been investigated, we cannot formally exclude the possibility that cTMs represent a DC population [26]. The finding that cTMs occupy an interstitial space, and are closely associated with capillary endothelial cells suggests that cTMs might participate in endocrine signalling through the coronary vasculature. Moreover, the direct contact between cTMs and cardiomyocytes indicates that both cells would be receptive to reciprocal paracrine signalling, pointing to a potentially important role for cTMs in cardiomyocyte homeostasis. Indeed, the finding that cTMs have an M2-like gene expression signature suggests that cTMs perform an immunomodulatory and a tissue salutary function in the heart. The rigorous examination of the M2 phenotype by gene array analysis, flow cytometry and microscopy techniques excludes the possibility that M2 gene expression may be induced as an artefact of the cTM isolation procedure. In addition, of the 24 M2 macrophage markers assessed, the canonical M2-markers Mrc1, Fizz1, Ym1, Mgl1, and Mgl2 [12,20] were expressed. The absence of Arg1, a canonical marker of mouse M2-macrophages, is understandable considering its role in the production of polyamines that are essential for collagen deposition and fibrosis, which characterise M2 macrophage function following injury [27]. Thus these results indicate that, unlike mouse M2 macrophages involved at later phases of inflammation, cTMs in the undamaged heart have a unique phenotype and function. PLoS ONE | www.plosone.org 8 May 2012 | Volume 7 | Issue 5 | e36814