Macrophages in the Adult Murine Heart Figure 6. Differential gene expression by GFP+ populations from adult Cx3cr1GFP/+ mouse tissues determined by qRT-PCR. Means for respective genes from various tissues are normalised to the means obtain for heart (IL1b, IL6, CXCL1, CXCL2, IL10, IGF1, MMP13, Timp2, and Hmox1), lung (Angpt1), or peritoneum (PT; Arg1). Histograms show mean 6 SEM (n = 3 independent cell and RNA isolation experiments). RNA for 3 biological replicate samples of GFP+ cells of various tissues were prepared in 3 independent isolation procedures for each tissue. For all tissues except spleen, GFP+ cells were isolated from 4–7 mice in each independent cell isolation. Splenic GFP+ cells were isolated from groups of 2–4 mice per replicate. doi:10.1371/journal.pone.0036814.g006 Complement secretion and modulation by cTMs points to another mechanism whereby these cells might contribute to myocardial homeostasis. cTM expression of Vsig4, a complement binding protein that negatively regulates T-cell activation [28], indicates that cTMs may suppress T-cell activation within the resting myocardium. Moreover, C1q deposition is important for clearance of apoptotic debris by macrophages in vitro and in vivo and can dampen inflammation while facilitating macrophage phagocytic activity [29], a function also attributed to other cTM- expressed genes, such as the M2-gene TREM2 [30]. Thus, cTMs may play a significant role in dampening local tissue inflammation. Indeed, loss of complement receptors C1 and C2 results in more severe virally induced myocarditis [31]. Consistently, myocarditis is a common manifestation of systemic lupus erythematosus arising from C1q deficiency. Gene expression analysis of cTMs indicates that they may share a number of characteristics with adipose tissue macrophages, another M2-like macrophage population [22,32]. In mouse adipose tissue, deficiency of the cytokines IL4 and IL13, important for M2 macrophage polarisation, results in tissue macrophages presenting a classically activated M1 phenotype [33], coinciding with tissue inflammation [34,35]. Similarly, IL13 deficiency leads to more severe cardiac inflammation compared to controls, resulting in dilated cardiomyopathy after experimentally induced myocarditis [36]. Thus a loss of M2-phenotype in cTMs is likely to have a similarly detrimental inflammatory effect on the heart. Another similarity identified between adipose tissue macrophages and cTMs is the expression of Lyve-1, which in adipose tissue is implicated in hemangiogenesis [37]. Likewise, cTMs express a number of proangiogenic genes including the proangiogenic M2- genes Retnla/Fizz1 [38] and IGF1 [39,40]. These data are consistent with the established role of macrophages in angiogenesis [41] and their direct role in capillary anastomosis [42]. However, additional microarray analyses show that VEGF isoforms (a, b and c) are not highly expressed in cTMs (data not shown), indicating that cTMs may participate in angiogenesis by non-canonical VEGF-independent pathways [43]. IGF-1 expression in cTMs and GFP+ cells from adipose tissue indicates that these cells are also likely to have a tissue salutary role. IGF-1 exerts protective effects in a number of tissues including heart muscle [44], skeletal muscle [45,46] and skin [47], and has immunomodulatory potential [44,46]. However, unlike GFP+ cells from adipose tissue, cTMs did not express Arg1, indicating a difference in tissue remodelling function. Consistently we found cTMs expressed the collagenolytic enzyme MMP13, implicated in scar-free liver regeneration [48]. These findings indicate that cTMs may have an antifibrotic role in the heart, distinct from adipose tissue macrophages. While we have characterised cTMs, with an emphasis on their unique gene expression profile and potential role in cardiac homeostasis, their importance in cardiac homeostasis remains to be confirmed by functional data. Elucidation of their functional role would require cTM-specific ablation strategies, which are currently unavailable. Existing mouse macrophage ablation strategies using clodronate-loaded liposomes [49] and myeloid- specific diphtheria toxin receptor expressing mice (such as CD11b- DTR/GFP mice) [50,51] are limited since they non-specifically ablate macrophages in many tissues [49,50,51]. Therefore, changes determined in cardiac development and homeostasis after ablating cTMs using these strategies are confounded due to systemic depletion of myeloid cell populations. A cTM-specific ablation strategy will help determine the role of cTMs in the induction and progression of cardiac inflammation following injury such as myocardial infarction, which recently has been found to involve a carefully orchestrated influx and efflux of monocytes, macrophages and other inflammatory cells [52]. PLoS ONE | www.plosone.org 9 May 2012 | Volume 7 | Issue 5 | e36814