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. 2012;7(5):e36814.
doi: 10.1371/journal.pone.0036814. Epub 2012 May 10.

An abundant tissue macrophage population in the adult murine heart with a distinct alternatively-activated macrophage profile

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An abundant tissue macrophage population in the adult murine heart with a distinct alternatively-activated macrophage profile

Alexander R Pinto et al. PLoS One. 2012.

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 Cx(3)cr1(GFP/+) 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 Cx(3)cr1(GFP/+) 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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Detection of GFP+ cells within adult Cx3cr1GFP/+ mouse hearts.
(A) Scatter profiles showing CD45+ cells in the adult mouse heart (left panel) and the expression of CD11b and GFP within this population (right panel). (B) Scatter profile of GFP+ cells (green), with CD45+GFP (black) and CD45GFP cells and debris (grey) shown (top left panel). (Top right panel) scatter profile of CD45+ cells (as shown in figure 1A left panel) with GFP+ cells, GFP cells and debris indicated (green, black and grey respectively; based on gating from figure 1A). (Bottom left panel) scatter profile of CD11b and CD45 expression within GFP+ cells. (C) 2 MDa dextran uptake by CD45+CD11b+GFP+ cells. (D) 45 µm projection view of a cross sectional image of the adult Cx3cr1GFP/+ mouse heart left ventricle. Scale bar = 150 µm. (E) 3D projections of typical GFP+ (green) cells from the left ventricular myocardium with isosurface rendering of nuclei (blue). Figures representative of at least 3 independent experiments. (F) Magnified projection view of GFP+ cells with perivascular position shown (capillaries stained with IB4; nuclei stained with DAPI). Scale bar = 5 µm. (G) Optical section of GFP+ cells with perivascular (capillaries stained with IB4) and pericardiomyocyte position (cardiomyocyte cell surface stained with WGA; nuclei stained with DAPI). Scale bar = 10 µm. All images representative of at least 3 similar independent experiments.
Figure 2
Figure 2. Immunophenotype of cTMs within adult Cx3cr1GFP/+ mouse hearts.
Representative histograms for myeloid (F4/80, CD11c, MHC-II (IAb), CD14, CD86) and lymphoid (CD3ε, B220) related markers detected in CD45+CD11b+GFP+ population and CD45+CD11bGFP population. Red line shows isotype control staining. Histograms representative of at least 4 experiments, with mouse group sizes of at least 4 mice.
Figure 3
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 ± 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.
Figure 4
Figure 4. Expression of M2 macrophage markers by cTMs.
(A) Heat map of M2 related gene expression by cTMs and GFP+ cells from the spleen and brain by microarray analysis. (B) Scatter/correlation profile of M2 related genes expressed by cTMs (heart) and genes expressed in GFP+ cells from the spleen and brain. (C) Micrographs of adult Cx3cr1GFP/+ mouse heart sections stained for GFP, Mrc1, CD163 and Lyve-1. Scale bar = 30 µm. (D) Flow cytometry histogram showing expression of M2 markers Mrc1 and Ly6C/G by cTMs. Histograms are representative of at least 4 independent experiments. (E) qRT-PCR analysis of Lyve-1 expression by GFP+ cells from different tissues. Mean values determined for respective tissues are normalised to means obtained for heart. Histograms show mean ± SEM (n = 3).
Figure 5
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.
Figure 6
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 (IL1β, IL6, CXCL1, CXCL2, IL10, IGF1, MMP13, Timp2, and Hmox1), lung (Angpt1), or peritoneum (PT; Arg1). Histograms show mean ± 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.

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