CCR2+ Monocyte-Derived Infiltrating Macrophages Are Required for Adverse Cardiac Remodeling During Pressure Overload

Figures

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  Figure 1. CCR2+ Macrophage Infiltration After TAC (A) Mouse LV gene expression of CCR2 chemokine ligands (left) and proinflammatory and anti-inflammatory cytokines (right) 1 week after transverse aortic constriction (TAC) or sham operation. (B) Identification of CD45+F4/80+MerTK+CD64+ cardiac macrophages using flow cytometry, and representative gate plots of CCR2− resident and CCR2+ infiltrating macrophages in TAC and sham mice. (C) Quantitation of CD45+ leukocytes, F4/80+MerTK+CD64+ macrophages, and CCR2+ and CCR2− macrophage subsets isolated from mouse hearts 1 week after TAC or sham operation. (D) Left, representative flow cytometry gate plots of Ly6Chi and Ly6Clow blood monocytes pre-gated on CD45+CD11b+Ly6G− cells from TAC and sham-operated mice (left) and quantitation of monocyte subsets (right). Ly6Chi monocytes uniformly express CCR2. *p < 0.05, **p < 0.01 versus sham; n = 6 per group.

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  Figure 2. Pharmacological CCR2 Antagonism Prevents Cardiac Macrophage Infiltration in Early Pressure-Overload (A) Experimental design for treatment with the CCR2 antagonist RS-504393 after transverse aortic constriction (TAC). (B) Quantitative group data for CCR2+ and CCR2− cardiac macrophages in mice treated with CCR2 antagonist or vehicle 1 week after TAC or sham operation. Statistical comparisons for CCR2+ macrophages were performed after logarithmic data transformation as described in the text. Group data for LV ICAM-1 and VCAM-1 gene expression (C) and Ly6Chi blood monocytes (D) in the same experimental groups. (E) Quantitative group data for total CD3+, CD3+CD4+, and CD3+CD8+ T-lymphocytes in mediastinal lymph nodes (MLNs) from the same animal groups. *p < 0.05, **p < 0.01, ***p < 0.001; n = 6 to 9 per group.

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  Figure 3. Early Pharmacological CCR2 Antagonism After TAC Blunts Late Cardiac Remodeling (A) Experimental protocol for treatment with the CCR2 antagonist RS-504393 after TAC. HW to TL ratios (B) and cardiomyocyte area measured using wheat germ agglutinin staining (C) in mice treated with CCR2 antagonist or vehicle 4 weeks after TAC or sham operation. (D) LV gene expression of hypertrophic markers atrial and B-type natriuretic peptide (ANP and BNP, respectively) from the same experimental groups. Statistical comparisons for ANP expression were performed after logarithmic data transformation as described in text. (E) Representative Masson’s trichrome stains of the LV and fibrosis quantitation for the same groups. Scale bar = 50 μm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; n = 5 to 7 per group.

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  Figure 4. Ly6Chi Monocyte Depletion Early During Pressure-Overload Ameliorates Late Adverse Pressure-Overload LV Remodeling (A) Experimental design for treatment with the anti-CCR2 monoclonal antibody (MC21) or control IgG after TAC. (B) Representative flow cytometry gate plots and quantitation of circulating Ly6ChiCCR2+ monocytes in IgG or MC21 treated mice 24 h after the final dose (n = 8 to 9 per group). (C) Quantitation of circulating CD11b+Ly6G+ neutrophils from the same groups at the same time point. (D) Gross heart images and quantitative heart weight (HW) to tibia length (TL) ratios in mice treated with MC21 or IgG 4 weeks after TAC or sham operation (n = 13 to 16 per group). Representative wheat germ agglutinin staining of the LV and quantitation of cardiomyocyte area (scale bar = 25 μm) (E) and Masson’s trichrome stains and quantitation of LV fibrosis (scale bar = 50 μm) (F) for the same experimental groups (n = 5 to 8 per group). *p < 0.05, **p < 0.01, ***p < 0.001. For E only, statistical comparisons were performed using individual unpaired Student t test or Mann-Whitney test (as appropriate) between the 4 specific groups, with Bonferroni post hoc correction as detailed in the…

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  Figure 5. Ly6ChiCCR2+ Monocyte Depletion Suppresses Cardiac Profibrotic Gene Expression During Chronic Pressure-Overload HF LV gene expression of collagen I, collagen III, collagen 4α3, vimentin, and fibronectin (A) and TGF-β and its 2 receptors (B) in mice treated with MC21 or IgG 4 weeks after TAC or sham operation. *p < 0.05, **p < 0.01; n = 6 per group. HF = heart failure.

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  Figure 6. Ly6ChiCCR2+ Monocyte Depletion Suppresses Cardiac and Lymphoid T-Cell Expansion During Chronic Pressure-Overload HF (A) Flow cytometry gating strategy for identification of cardiac T cells and CD4+ Th1 (IFN-γ+), Th2 (IL-4+), and T-regulatory (Foxp3+) subsets. (B) Representative gate plots of cardiac CD4+ T cells and corresponding tissue quantitation in mice treated with MC21 or IgG 4 weeks after TAC or sham operation. Statistical comparisons for CD4+ T cells performed after logarithmic data transformation as described in text. Quantitation of cardiac CD4+ T-cell subsets (C) and CD3+CD4−CD8+ cardiac T cells (D) in the same groups. (E) Top, representative gate plots of CD4+ and CD8+ T cells in mediastinal lymph nodes (MLN) from IgG- and MC21-treated TAC mice. Bottom, quantitation of CD3+, CD3+CD4+CD8−, and CD3+CD4−CD8+ T cells in the MLN of mice treated with MC21 or IgG 4 weeks after TAC or sham operation. *p < 0.05; n = 6 to 8 per group.

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  Figure 7. CCR2+ Macrophages and Pressure-Overload Cardiac Hypertrophy and Failure The model integrates the findings from the current study with prior work 5, 7, 9, 10. During the early compensated phase of pressure overload, there is myocardial up-regulation of chemokine ligands for CCR2 (CCL2, CCL7, CCL12) and innate immune cell expansion including: CCR2+ macrophages (accompanying Ly6Chi monocytosis), dendritic cells, and resident CCR2− macrophages. The CCR2+ macrophage population contributes importantly to early compensatory hypertrophy, antigen presentation, and CD4+ and CD8+ T-cell activation and expansion in the heart and mediastinal lymph nodes (MLNs). CD4+ T cells, alone or together with CCR2+ macrophages, subsequently engender tissue-injurious responses including pathological hypertrophy, interstitial fibrosis, and left ventricular systolic dysfunction. Schematic representations of the heart, LNs, blood vessel, and monocytes/macrophages were adapted from Servier Medical Art (Creative Commons license).