Table 7_Temporal dynamics of macrophage transcriptional profiles during zebrafish wound healing.xlsx

IntroductionMacrophages participate in wound healing by contributing to host defense and by orchestrating inflammation and repair. They adapt dynamically to the wound microenvironment by adopting diverse polarization states, which are determinant of wound outcome, influencing whether healing is successful or becomes chronic. The zebrafish embryo, widely used for live imaging of immune responses, is a powerful model to study macrophage behavior after injury. However, the transcriptional landscape of polarized macrophages in this model during wound healing remains insufficiently characterized.
MethodsHere, we employed bulk RNA sequencing on FACS-sorted zebrafish macrophages at different times following tail fin wounding in larvae, a robust model for studying sterile inflammation. We analyzed gene expression changes over the time by comparing wounded versus unwounded conditions to characterize macrophage transcriptional programs during wound healing.
ResultsOur findings reveal that zebrafish macrophages undergo large transcriptomic changes along different wound healing phases, particularly between 2 and 5 hours post-amputation, suggesting a fast reprogramming leading to different functional states. We further show that, at 2h, macrophages acquire a pro-inflammatory profile with a gene signature closed to M1-like program. At 5h, macrophages express genes involved in immunoregulation and healing, associated with shutoff of pro-inflammatory pathways and the activation of glucose and glycogen metabolism. Finally, we show that macrophage reprogramming becomes deeply attenuated by 29h post injury.
DiscussionOur findings provide a foundation for understanding macrophage polarization in zebrafish, revealing underpinning molecular mechanisms that include both specific and evolutionarily conserved pathways with a potential impact on translational medicine.

引言
巨噬细胞通过参与宿主防御、协调炎症与修复过程参与伤口愈合。它们可动态适配伤口微环境，呈现多样化的极化状态，而这些状态是决定伤口转归的关键因素，直接影响愈合能否成功或是否进展为慢性伤口。
斑马鱼胚胎因被广泛应用于免疫应答的活体成像研究，是探究损伤后巨噬细胞行为的理想模型。然而，目前针对该模型中伤口愈合过程内极化巨噬细胞的转录调控图谱，仍缺乏充分的表征。

方法
本研究针对无菌性炎症研究的经典模型——斑马鱼幼体的尾鳍损伤后不同时间点的荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）纯化巨噬细胞，开展批量RNA测序。我们通过对比损伤组与未损伤组的基因表达变化，分析随时间推移的基因表达动态，以表征伤口愈合过程中巨噬细胞的转录调控程序。

结果
本研究结果显示，斑马鱼巨噬细胞随伤口愈合的不同阶段发生显著的转录组变化，尤其在截尾后2至5小时之间，提示快速重编程过程可产生不同的功能状态。我们进一步发现，在截尾后2小时，巨噬细胞呈现促炎表型，其基因特征与M1样极化程序高度相似；在截尾后5小时，巨噬细胞表达参与免疫调节与愈合过程的基因，同时伴随促炎通路的关闭以及葡萄糖与糖原代谢通路的激活。最后，我们证实，在损伤后29小时，巨噬细胞的重编程过程已显著减弱。

讨论
本研究结果为理解斑马鱼巨噬细胞极化过程提供了重要的研究基础，揭示了其背后的分子机制——这些机制既包含特异性通路，也包含进化保守的通路，对转化医学研究具有潜在的应用价值。