Selective expansion of cardiac macrophage subtypes distinguishes their functional roles in disease and homeostasis
Rajesh K. Kasam, Ronald J. Vagnozzi, Yasuhide Kuwabara, Anne Katrine Z. Johansen, N. Scott Blair, Vikram Prasad, Suh-Chin J. Lin, Akanksha Rajput, Michelle Nieman, Jeffery D. Molkentin
Rajesh K. Kasam, Ronald J. Vagnozzi, Yasuhide Kuwabara, Anne Katrine Z. Johansen, N. Scott Blair, Vikram Prasad, Suh-Chin J. Lin, Akanksha Rajput, Michelle Nieman, Jeffery D. Molkentin
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Research Article Cardiology Immunology

Selective expansion of cardiac macrophage subtypes distinguishes their functional roles in disease and homeostasis

Abstract

Cardiac macrophages are broadly studied as 2 subtypes, tissue-resident CX3C chemokine motif receptor 1 positive (CX3CR1+) that are also CC motif chemokine receptor 2 negative (CCR2–) and monocyte-derived CCR2+. Previous systemic loss-of-function approaches suggested unique roles for each subtype in the heart, with CCR2+ being inflammatory and CX3CR1+ being prohealing. Here, we employed a cardiac-specific gain-of-function approach to selectively enhance either macrophage subtype. A robust increase in basal CCR2+ macrophages in the heart by targeted CC chemokine ligand 2 (Ccl2) expression did not induce inflammation, cause fibroblast activation, or impair cardiac function. However, increased CCR2+ macrophages reciprocally diminished self-renewing tissue-resident macrophages and worsened cardiac fibrosis due to pressure overload stimulation. Conversely, augmented expression of colony-stimulating factor-1 (Csf1) in the heart promoted selective expansion of resident CX3CR1+ macrophages, which exerted no pathophysiological consequences at steady state. However, pressure overload in these mice with expanded CX3CR1+ macrophages showed a CCR2+ macrophage–dependent inflammation leading to exacerbated cardiac dysfunction, simultaneously protecting from adverse remodeling and cardiac fibrosis. In conclusion, cardiac-specific selective enrichment of macrophage subtypes shows their intricate interplay and unique functional roles in regulating myocardial inflammation and fibrosis during hypertrophy and at homeostasis.

Authors

Rajesh K. Kasam, Ronald J. Vagnozzi, Yasuhide Kuwabara, Anne Katrine Z. Johansen, N. Scott Blair, Vikram Prasad, Suh-Chin J. Lin, Akanksha Rajput, Michelle Nieman, Jeffery D. Molkentin

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Figure 2

Cardiac-specific CCR2+ macrophage enrichment does not induce pathology in mice.

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Cardiac-specific CCR2+ macrophage enrichment does not induce pathology i...
(A) Experimental scheme showing AAV9-Ccl2 vector injected into Cx3cr1+/GFP Ccr2+/RFP (reporter) pups at postnatal day 3 (P3), with tissue harvested for analysis at 2 and 6 months of age. (B) Quantification of CCL2 cytokine levels by ELISA in cardiac tissue from AAV9-Empty– or AAV9-Ccl2–injected mice at 2 months of age. n = 3–5 mice per group, and error bars denote ± SEM. *P < 0.05, 2-tailed unpaired Student’s t test. (C) Quantification of serum CCL2 levels by ELISA in the indicated groups at 2 months of age. n = 3–5 mice per group, and error bars denote ± SEM. *P < 0.05, 2-tailed unpaired Student’s t test. (D) Representative immunofluorescence images of wheat germ agglutinin (WGA; membrane stain in purple) and RFP (red) to show CCR2+ cells from heart histological sections of the indicated groups of mice at 2 months of age. Scale bar = 200 μm. n = 4–5 mice per group. (E–H) Flow cytometry quantification of cardiac macrophage subtypes shown in each graph from the 2 groups of mice at 2 and 6 months of age by flow cytometry. n = 5–6 mice per group, and error bars denote ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by 2-tailed unpaired Student’s t test. (I) Flow cytometry quantification of indicated immune cell populations in the heart from the 2 groups of mice at 2 months of age by flow cytometry (Neutro = neutrophils; NK = natural killer cells). n = 5 mice per group. Error bars denote ± SEM. Two-tailed unpaired Student’s t test. (J) Flow cytometry quantification of cardiac fibroblasts as CD45–CD31–MEFSK4+ from the hearts of the 2 groups of mice at 2 months of age. n = 6 mice per group, and error bars denote ± SEM. Two-tailed unpaired Student’s t test. (K) Representative immunofluorescence images of heart histological sections from the 2 groups of mice showing the fibroblast marker PDGFRα (purple) and nuclear stain DAPI (blue) at 2 months of age. Scale bar = 500 μm. n = 4 mice per group. (L and M) Echocardiography assessment of cardiac (L) ejection fraction percentage (EF%) and (M) left ventricular inner diameter at diastole (LVIDd) in the 2 groups of mice at 2 and 6 months of age. n = 6–16 mice per group. Error bars denote ± SEM. Two-tailed unpaired Student’s t test. (N) Ventricular weight to body weight (VW/BW) ratio from hearts of the 2 groups of mice at 12 months of age, injected at P3 with indicated viral vector. n = 4 mice per group, and error bars denote ± SEM. Two-tailed unpaired Student’s t test.