中国科学技术大学Li-kun Ma等研究人员合作发现，巨噬细胞通过GMCSF/CCL2/CCR2和表型转换促进小鼠心肌缺血再灌注损伤向心肌纤维化的转变。这一研究成果于2024年1月15日在线发表在国际学术期刊《中国药理学报》上。

研究人员揭示了巨噬细胞在缺血后心肌纤维化中的作用，并探索了心肌纤维化的治疗靶点。雄性小鼠左冠状动脉被结扎30分钟。研究人员首先检测了心脏组织中招募免疫细胞浸润心脏的趋化因子水平，发现小鼠心脏微血管内皮细胞（MCMEC）释放的粒细胞-巨噬细胞集落刺激因子（GMCSF）在再灌注后6小时达到峰值，GMCSF诱导的巨噬细胞释放的c-c模体趋化因子配体2（CCL2）在再灌注后24小时达到峰值。在BMDM与MCMEC的共培养中，研究人员证实MCMEC产生的GMCSF可刺激BMDM释放CCL2，并有效促进BMDM的迁移。

研究人员还证实，GMCSF在体外促进了巨噬细胞的M1极化，而GMCSF中和抗体（NTAB）阻断了CCL2/CCR2信号传导。在心肌缺血再灌注（MIR）小鼠心脏中，研究人员发现GMCSF激活了CCL2/CCR2信号传导，促进了NLRP3/caspase-1/IL-1β介导的炎症损伤并扩大了炎症损伤。敲低CC趋化因子受体2基因（CCR2^-/-）或给予特异性CCR2抑制剂RS102895（5mg/kg每12h，腹腔注射，MIR前一天，并持续到实验结束）可有效减少心肌梗死面积，并下调炎症介质和NLRP3/Caspase-1/IL-1β信号传导。质谱法证实，M2巨噬细胞在纤维化过程中发挥了重要作用，而巨噬细胞缺失的小鼠在MIR后心脏组织中的转化生长因子-β（Tgf-β）水平显著降低。

在巨噬细胞与成纤维细胞的共培养中，用重组小鼠CCL2处理可刺激巨噬细胞释放大量Tgf-β，并促进成纤维细胞释放Col1α1。这种作用在CCR2^-/-小鼠的BMDM中减弱。在敲除或抑制CCR2基因后，Tgf-β 的水平显著降低，心肌纤维化的水平也显著降低，心脏功能得到保护。这项研究证实，小鼠MIR后急性损伤向慢性纤维化转变是由巨噬细胞中的GMCSF/CCL2/CCR2信号通过NLRP3炎症级联和表型转换介导的。

据介绍，急性MIR后，巨噬细胞会浸润受损的心脏组织，并改变其极化表型，以应对急性炎症和慢性纤维化重塑。

附：英文原文

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

Author: Shen, Shi-chun, Xu, Jie, Cheng, Cheng, Xiang, Xin-jian, Hong, Bao-yu, Zhang, Meng, Gong, Chen, Ma, Li-kun

Issue&Volume: 2024-01-15

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 30min. 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 6h after reperfusion, and c-c motif chemokine ligand 2 (CCL2) released by GMCSF-induced macrophages peaked at 24h 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 (5mg/kg per 12h, 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.

DOI: 10.1038/s41401-023-01222-3

Source: https://www.nature.com/articles/s41401-023-01222-3