Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan.
Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.
Elife. 2023 Jul 27;12:e84679. doi: 10.7554/eLife.84679.
Zebrafish exhibit a robust ability to regenerate their hearts following injury, and the immune system plays a key role in this process. We previously showed that delaying macrophage recruitment by clodronate liposome (-1d_CL, macrophage-delayed model) impairs neutrophil resolution and heart regeneration, even when the infiltrating macrophage number was restored within the first week post injury (Lai et al., 2017). It is thus intriguing to learn the regenerative macrophage property by comparing these late macrophages vs. control macrophages during cardiac repair. Here, we further investigate the mechanistic insights of heart regeneration by comparing the non-regenerative macrophage-delayed model with regenerative controls. Temporal RNAseq analyses revealed that -1d_CL treatment led to disrupted inflammatory resolution, reactive oxygen species homeostasis, and energy metabolism during cardiac repair. Comparative single-cell RNAseq profiling of inflammatory cells from regenerative vs. non-regenerative hearts further identified heterogeneous macrophages and neutrophils, showing alternative activation and cellular crosstalk leading to neutrophil retention and chronic inflammation. Among macrophages, two residential subpopulations ( Mac and Mac 3) were enriched only in regenerative hearts and barely recovered after +1d_CL treatment. To deplete the resident macrophage without delaying the circulating macrophage recruitment, we established the resident macrophage-deficient model by administrating CL earlier at 8 d (-8d_CL) before cryoinjury. Strikingly, resident macrophage-deficient zebrafish still exhibited defects in revascularization, cardiomyocyte survival, debris clearance, and extracellular matrix remodeling/scar resolution without functional compensation from the circulating/monocyte-derived macrophages. Our results characterized the diverse function and interaction between inflammatory cells and identified unique resident macrophages prerequisite for zebrafish heart regeneration.
斑马鱼在受伤后表现出强大的心脏再生能力，而免疫系统在这个过程中起着关键作用。我们之前曾表明，通过使用氯膦酸盐脂质体（-1d_CL，巨噬细胞延迟模型）延迟巨噬细胞募集会损害中性粒细胞的清除和心脏再生，即使在损伤后第一周内恢复了浸润巨噬细胞数量（Lai 等人，2017）。因此，通过比较心脏修复过程中的这些晚期巨噬细胞与对照巨噬细胞，了解再生巨噬细胞的特性是很有趣的。在这里，我们通过比较非再生性巨噬细胞延迟模型与再生性对照模型，进一步研究心脏再生的机制见解。时间 RNAseq 分析表明，-1d_CL 处理导致心脏修复过程中炎症反应的清除、活性氧稳态和能量代谢受到破坏。对来自再生性和非再生性心脏的炎症细胞进行比较单细胞 RNAseq 分析进一步鉴定了异质性巨噬细胞和中性粒细胞，显示出替代性激活和细胞串扰，导致中性粒细胞滞留和慢性炎症。在巨噬细胞中，仅在再生性心脏中富集了两个常驻亚群（Mac 和 Mac3），并且在 +1d_CL 处理后几乎没有恢复。为了在不延迟循环巨噬细胞募集的情况下耗尽常驻巨噬细胞，我们通过在冷冻损伤前 8 天（-8d_CL）更早地给予 CL 建立了常驻巨噬细胞缺陷模型。令人惊讶的是，常驻巨噬细胞缺陷型斑马鱼仍然表现出血管生成、心肌细胞存活、碎片清除和细胞外基质重塑/疤痕消退缺陷，而循环/单核细胞衍生的巨噬细胞没有功能补偿。我们的研究结果描述了炎症细胞之间的多种功能和相互作用，并确定了独特的常驻巨噬细胞是斑马鱼心脏再生的必要条件。
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