The morpho-kinetic landscape of macrophage modes during wound healing in zebrafish.

Macrophages play an essential role in wound healing due to their dynamic nature and functional plasticity, exhibiting highly heterogeneous morpho-kinetic behaviors depending on their activation states. However, quantitative analysis of macrophage behavior in in vivo settings remains limited, largely due to the complexity of their diverse morphologies and motility patterns over time. In this study, we present an analytic workflow to investigate macrophage dynamics in zebrafish. By computing a comprehensive set of morpho-kinetic features, we observe that M1-like (pro-inflammatory) and M2-like (anti-inflammatory) macrophages exhibit distinct behaviors, such as reduced shape elongation, more directed movement, and less random-like motion in M1-like compared to M2-like macrophages. Based on these features, we classify macrophages in the transition period into cM1-like (classified M1-like) and cM2-like (classified M2-like) groups. We compare and analyze their behaviors, which allows us to estimate the timing of the phenotypic switch. In addition, macrophages not expressing Tumor Necrosis Factor (TNF) are located significantly farther from the wound compared to M1-like macrophages. While macrophages unstimulated by wound signaling exhibit some cM2-like features, they differ notably in shape elongation and migration speed. In summary, this study provides a quantitative analysis of macrophage behavior during wound healing and suggests distinct behavioral landscapes across different macrophage activation states.