Macrophages in the Adult Murine Heart Figure 3. Microarray gene expression analysis of cTMs and GFP+ cells from the spleen and brain. (A) Venn-diagram of differentially expressed genes from cTMs (blue) and GFP+ cells from the spleen (green) and brain (red). (B) Heat map of genes enriched greater than 20-fold in cTMs compared to GFP+ cells from the spleen and brain. Heat map displays data from each individually isolated GFP+ populations. (C) qRT-PCR histograms conducted on selected genes to determine unique enrichment in cTMs (Lyve-1, IGF1, MMP13, IL10, IL6, IL1b, CXCL1, and CXCl2), enrichment in both cTMs and GFP+ cells from the brain (Stab1, Timp2), enrichment in GFP+ cells from the spleen (Angpt1) and expression in all three populations (Hmox1). Histograms show mean 6 SEM. For microarray analysis, 3 biological replicate samples of GFP+ cells from the heart, spleen and brain were prepared in 3 independent isolation procedures for each tissue. Cardiac and brain tissue from 4 mice were used to isolate sufficient number of cells in each independent cell isolation, whereas splenic cells were isolated from at least 2–4 mice. doi:10.1371/journal.pone.0036814.g003 Table S2). M2 probe sets not expressed or expressed at low levels in cTMs included arginase 1 (Arg1), triggering receptor expressed on myeloid-2 (TREM2), fibronectin 1 (Fn1) and Cadherin 1 (Cdh1). Of these however, one of the two probe sets corresponding to Fn1 was expressed in cTMs at high levels, and one of two probe sets corresponding to Trem2 was expressed at intermediate levels (indicating both genes are expressed in cTMs). Scatter profiles of the M2-related genes examined in the three GFP+ populations revealed that cTMs expressed significantly more M2 genes than did splenic and brain GFP+ cells (Fig. 4 b). Although splenic GFP+ cells were found to express several M2-genes, brain GFP+ cells were found to express very few, indicating a difference in phenotype in the three GFP+ populations analysed, with cTMs strongly resembling M2-polarised macrophages. Expression of the canonical M2-macrophage genes, Mrc1 and CD163, was confirmed by confocal fluorescence microscopy (Fig. 4 c and Fig. S2). Expression of Lyve-1, considered another M2-macro- phage related gene [21,22], and a gene enriched in tumour infiltrating TIE-2 expressing monocytes (TEMs) [23], was also confirmed by confocal fluorescence microscopy (Fig. 4 c and Fig. S2). The M2-phenotype of cTMs was further demonstrated by flow cytometry, by determining expression levels of LY6C/G, which is expressed in high levels in M1 and low levels in M2 macrophages [24]. Additionally, we confirmed expression of Mrc1 and low levels of LY6C/G in cTMs, with almost all cells comprising the low expression peak, and a small fraction of cells being present in the high expression peak (Fig. 4 d). Consistent with the LY6C/ Glo expression, almost all cTMs expressed Mrc1. To determine if cTMs have a distinct gene expression profile amongst GFP+ cells from various tissues in Cx3cr1GFP/+ mice, we determined expression levels of Lyve-1 from isolated GFP+ cells from other tissues (Fig. 4 PLoS ONE | www.plosone.org 6 May 2012 | Volume 7 | Issue 5 | e36814