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Macrophages promote the transition from myocardial ischemia reperfusion injury to cardiac fibrosis in mice through GMCSF/CCL2/CCR2 and phenotype switching

Acta Pharmacologica Sinica volume 45, pages 959–974 (2024)Cite this article

Abstract

Following acute myocardial ischemia reperfusion (MIR), macrophages infiltrate damaged cardiac tissue and alter their polarization phenotype to respond to acute inflammation and chronic fibrotic remodeling. In this study we investigated the role of macrophages in post-ischemic myocardial fibrosis and explored therapeutic targets for myocardial fibrosis. Male mice were subjected to ligation of the left coronary artery for 30 min. We first detected the levels of chemokines in heart tissue that recruited immune cells infiltrating into the heart, and found that granulocyte-macrophage colony-stimulating factor (GMCSF) released by mouse cardiac microvascular endothelial cells (MCMECs) peaked at 6 h after reperfusion, and c-c motif chemokine ligand 2 (CCL2) released by GMCSF-induced macrophages peaked at 24 h after reperfusion. In co-culture of BMDMs with MCMECs, we demonstrated that GMCSF derived from MCMECs stimulated the release of CCL2 by BMDMs and effectively promoted the migration of BMDMs. We also confirmed that GMCSF promoted M1 polarization of macrophages in vitro, while GMCSF neutralizing antibodies (NTABs) blocked CCL2/CCR2 signaling. In MIR mouse heart, we showed that GMCSF activated CCL2/CCR2 signaling to promote NLRP3/caspase-1/IL-1β-mediated and amplified inflammatory damage. Knockdown of CC chemokine receptor 2 gene (CCR2−/−), or administration of specific CCR2 inhibitor RS102895 (5 mg/kg per 12 h, i.p., one day before MIR and continuously until the end of the experiment) effectively reduced the area of myocardial infarction, and down-regulated inflammatory mediators and NLRP3/Caspase-1/IL-1β signaling. Mass cytometry confirmed that M2 macrophages played an important role during fibrosis, while macrophage-depleted mice exhibited significantly reduced transforming growth factor-β (Tgf-β) levels in heart tissue after MIR. In co-culture of macrophages with fibroblasts, treatment with recombinant mouse CCL2 stimulated macrophages to release a large amount of Tgf-β, and promoted the release of Col1α1 by fibroblasts. This effect was diminished in BMDMs from CCR2−/− mice. After knocking out or inhibiting CCR2-gene, the levels of Tgf-β were significantly reduced, as was the level of myocardial fibrosis, and cardiac function was protected. This study confirms that the acute injury to chronic fibrosis transition after MIR in mice is mediated by GMCSF/CCL2/CCR2 signaling in macrophages through NLRP3 inflammatory cascade and the phenotype switching.

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Fig. 1: Macrophages infiltration during the transition from acute MIR injury to fibrosis.
Fig. 2: Cytokine levels in mouse heart tissue at different time points after MIR.
Fig. 3: MCMECs releases GMCSF under starvation treatment, inducing macrophage migration and releasing CCL2.
Fig. 4: GMCSF/CCL2/CCR2 promotes the transition from MIR injury to cardiac fibrosis through the NLRP3 signaling pathway.
Fig. 5: CCL2 reverses M1 macrophage-induced by GMCSF to the M2 macrophage, which releases Tgf-β to promote the transformation of fibroblasts into myofibroblasts.
Fig. 6: The effect of inhibiting CCR2 on fibrosis and cardiac function after MIR injury.
Fig. 7: Mass cytometry detection of the cardiac immune microenvironment status of mouse on the seventh day after MIR.
Fig. 8: Mass cytometry detection of the cardiac immune microenvironment status of mouse on the 7th day after MIR.
Fig. 9: After myocardial ischemic injury, cardiac microvascular endothelial cells release a large amount of GMCSF to attract monocytes to migrate to the heart, which differentiate into macrophages and transform into pro-inflammatory M1 phenotype under GMCSF induction, releasing a large amount of inflammatory factors and CCL2.

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Acknowledgements

The work was supported by the National Natural Science Foundation of China (82170263, 82200282), the National Key Research and Development Program of China (2021YFA0804904), the Natural Science Foundation of Hefei City (2022043).

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Authors and Affiliations

  1. Department of Cardiology, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, 230001, China

    Shi-chun Shen, Jie Xu & Li-kun Ma

  2. Department of Cardiology, The Second Affiliated Hospital of Anhui Medical University, Hefei, 230601, China

    Cheng Cheng & Meng Zhang

  3. Department of Plastic and Reconstructive Surgery, the Second Affiliated Hospital of Anhui Medical University, Hefei, 230601, China

    Xin-jian Xiang

  4. Department of Pediatrics, The Second Affiliated Hospital of Anhui Medical University, Hefei, 230601, China

    Bao-yu Hong

  5. Department of Pediatrics, The First Affiliated Hospital of Anhui Medical University, Hefei, 230022, China

    Chen Gong

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Contributions

SCS and LKM designed the experiments. SCS and CG performed most of the experiments. JX, CC, XJX, BYH and MZ performed some experiments. SCS and MZ analyzed the results. SCS, CG and LKM wrote the paper with help from other authors.

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Correspondence to Chen Gong or Li-kun Ma.

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Shen, Sc., Xu, J., Cheng, C. et al. Macrophages promote the transition from myocardial ischemia reperfusion injury to cardiac fibrosis in mice through GMCSF/CCL2/CCR2 and phenotype switching. Acta Pharmacol Sin 45, 959–974 (2024). https://doi.org/10.1038/s41401-023-01222-3

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