Figure 8

ROS interact with Lyn but not Yrk to mediate macrophage M1-like activation. (A) Schedule of the experiment. Morpholinos targeting specifically lyn (MO Lyn) or yrk (MO Yrk) or morpholino control (MO CTRL) were injected in Tg(mfap4:mCherry/tnfa:GFP-F) at one-cell stage. At 3 dpf (71 hpf), larvae were treated with Apocynin or DMSO, 1 hour before fin fold amputation. At 6 hpA, tails were imaged using confocal microscopy. (B) Quantification of the percentage of tnfa⁺ macrophages in the recruited macrophage population at 6 hpA in CTRL morphants or lyn morphants which were either treated with DMSO or Apocynin (mean ± SEM, n_larvae is indicated in brackets). A significant interaction between morpholino and drug effect was determined by two-way ANOVA (2WA, **p<0. 01, F(1, 42) = 8.305). Then Mann Whitney test, two-tailed was performed to determine the significant difference between DMSO and Apocynin groups (ns-not significant, ***p<0.001). (C) Quantification of the percentage of tnfa⁺ macrophages in the recruited macrophage population at 6 hpA in CTRL morphants or yrk morphants which were treated with DMSO or Apocynin. (Representative experiment of two independent experiments, mean ± SEM, n_larvae is indicated in brackets). No significant (ns) interaction between morpholino and drug effect was determined by two-way ANOVA (2WA, F(1,29)=0.0002613). Then Mann Whitney test, two-tailed was performed to determine the significant difference between DMSO and Apocynin groups (**p<0.01, ***p<0.001). (D) A proposed model showing the role of early wound signals in macrophage activation. Fin fold amputation triggers different independent stimuli: 1/intracellular Ca²⁺ oscillations in epithelial cells at the wound margin, which further mediate both macrophage recruitment and pro-inflammatory activation. 2/ROS production (mainly H₂O₂) at the wound, which promotes macrophage pro-inflammatory activation and tnfa expression. 3/Activation of SFK Yrk, which promotes both macrophage recruitment and activation in an independent manner. Redox-sensitive transcription factor NFκB and SFK Lyn are activated by ROS and required for M1-like activation at the wound. Thapsigargin and PP2 treatments impair both macrophage recruitment and M1-like activation. Apocynin, VAS2870 and Bay11-7082 affect only macrophage M1-like activation.