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 4

Controlled expansion of tissue-resident macrophages in the heart.

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Controlled expansion of tissue-resident macrophages in the heart. (A) Sc...
(A) Scheme of the MyoAAV-Csf1 recombinant MyoAAV expression system driven by the cardiac troponin T (cTnT) promoter injected retro-orbitally in adult reporter mice at 8 weeks of age and harvested 3 weeks later. (B) Cardiac tissue CSF1 levels measured by ELISA in the 2 groups of mice. n = 4 mice per group. CSF1 levels were not detectable in MyoAAV-Empty samples, but for visualization purposes it is shown as 0. (C) Representative immunofluorescence images from heart tissue sections of GFP (CX3CR1+) cells and WGA (membrane stain, purple) in the 2 groups of mice 3 weeks after control or MyoAAV-Csf1 injection. n = 4 mice per group, scale bar = 200 μm. (D–G) Flow cytometry quantification from hearts of the indicated cardiac macrophage subtypes in both groups of mice shown as (D) CX3CR1+ macrophages, (E) CX3CR1+MHC-IIlo macrophages, (F) CX3CR1+MHC-IIhi, and (G) CCR2+MHC-IIhi. n = 4 mice per group, and error bars denote ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, by 2-tailed unpaired Student’s t test. (H) Flow cytometry quantification of the indicated immune cell populations from hearts of both groups of mice 3 weeks after MyoAAV injection (Neutro = neutrophils, NK = natural killer). n = 4 mice per group, and error bars denote ± SEM by 2-tailed unpaired Student’s t test. (I) Experimental scheme with tamoxifen (TAM) was given in Cx3cr1 lineage tracing mice (Cx3cr1+/CreERT Rosa26+/tdTom) for 10 days followed by 4 weeks of washout before injecting MyoAAV-Empty or MyoAAV-Csf1, followed by analysis 3 weeks later. (J) Representative immunofluorescence cardiac histological images of Cx3cr1-labeled (tdTom, red) macrophages also stained for CD68 (white) in mice 3 weeks after control or MyoAAV-Csf1 injection. n = 4–5 mice per group. Scale bar = 100 μm. (K) Flow cytometry quantification of CX3CR1 labeled (tdTom+) CCR2-negative cardiac macrophages by flow cytometry, 3 weeks after MyoAAV injection. n = 4–5 mice per group. Error bars denote ± SEM. **P < 0.01 by 2-tailed unpaired Student’s t test. (L and M) Echocardiography assessment of cardiac fractional shortening percentage (FS%) and diastolic left ventricular dimension 3 weeks after control or Csf1 vector delivery. n = 12–13 mice per group. Error bars denote ± SEM. Two-tailed unpaired Student’s t test. (N) Representative histological images of hearts for fibrosis (blue) with Masson’s trichrome staining 3 weeks after MyoAAV-Empty or MyoAAV-Csf1 injection. Scale bar = 100 μm. (O) Cardiac fibroblast flow cytometry quantification as shown in Figure 2J from hearts of the 2 groups of mice. n = 4 mice per group and error bars denote ± SEM. Two-tailed unpaired Student’s t test.