Damage-Induced Calcium Signaling and Reactive Oxygen Species Mediate Macrophage Activation in Zebrafish

Immediately after a wound, macrophages are activated and change their phenotypes in reaction to danger signals released from the damaged tissues. The cues that contribute to macrophage activation after wounding in vivo are still poorly understood. Calcium signaling and Reactive Oxygen Species (ROS), mainly hydrogen peroxide, are conserved early wound signals that emanate from the wound and guide neutrophils within tissues up to the wound. However, the role of these signals in the recruitment and the activation of macrophages is elusive. Here we used the transparent zebrafish larva as a tractable vertebrate system to decipher the signaling cascade necessary for macrophage recruitment and activation after the injury of the caudal fin fold. By using transgenic reporter lines to track pro-inflammatory activated macrophages combined with high-resolutive microscopy, we tested the role of Ca²⁺ and ROS signaling in macrophage activation. By inhibiting intracellular Ca²⁺ released from the ER stores, we showed that macrophage recruitment and activation towards pro-inflammatory phenotypes are impaired. By contrast, ROS are only necessary for macrophage activation independently on calcium. Using genetic depletion of neutrophils, we showed that neutrophils are not essential for macrophage recruitment and activation. Finally, we identified Src family kinases, Lyn and Yrk and NF-κB as key regulators of macrophage activation in vivo, with Lyn and ROS presumably acting in the same signaling pathway. This study describes a molecular mechanism by which early wound signals drive macrophage polarization and suggests unique therapeutic targets to control macrophage activity during diseases.

(E) Quantification of total macrophages in uninjured Tg(mfap4:mCherry/tnfa:GFP-F) larvae at 3 dpf after DMSO or Thapsigargin treatments. Thapsigargin does not influence the total number of macrophages. Mean ± SEM, nlarvae is indicated in brackets, two-tailed t-test, ns -not significant. Figure S2. ROS release at the wound mediates macrophage activation but not recruitment (A) Schedule of the experiment. Larvae were injected in the yolk with VAS2870 or DMSO at 3 dpf, and 20 min after incubated in CellROX solution. After 1 h, fin folds were amputated. CellROX staining was removed at 20 min pA, and larvae were immediately imaged using epi-fluorescence microscopy.
(B) Representative images of the CellROX fluorescence in uncut or cut fin folds, after the treatment with DMSO or VAS2870 at 20 min pA, showing ROS production at the wound. Rainbow color scale was applied to images, emphasizing the differences in signal intensity. The white lines outline the fin fold and the notochord. Scale bar: 100 μm.
(C) Quantification of signal intensity the CellROX fluorescence by mean gray value. Representative experiment of three independent experiments, mean ± SEM, nlarvae is indicated in brackets, upper graph: Mann Whitney test, two-tailed, ns -not significant, bottom graph: two-tailed t-test, ***p<0.001.
(D) Schedule of the experiment. Tg(mfap4:mCherry-F/tnfa:GFP-F) larvae were injected in the yolk with VAS2870 or DMSO at 3 dpf. Fin folds were amputated 20 min later and imaged at 6 hpA using confocal microscopy.
(F) Quantification of recruited macrophages (up) and tnfa + recruited macrophages (middle) in in DMSO or VAS2870 treated larvae at 6 hpA. Representative experiment of two independent experiments, mean ± SEM, nlarvae is indicated in brackets, upper graph: two-tailed t-test, ns -not significant, bottom graph: Mann Whitney test, two-tailed, **p<0.01. were removed at 20 min pA, and larvae were immediately imaged using epi-fluorescence microscopy.
(B) Representative images of the CellROX fluorescence in uncut or cut fin folds, after the treatment without or with H2O2 at 20 min pA, showing ROS production at the wound. Rainbow color scale was applied to images, emphasizing the differences in signal intensity. The white lines outline the fin fold and the notochord. The yellow arrowheads show the intensity increase at the fin fold or wound area.
(C) Quantification of signal intensity of the CellROX fluorescence by mean gray value. Representative experiment of three independent experiments, mean ± SEM, nlarvae is indicated in brackets, Mann Whitney test, two-tailed, **p<0.01, ***p<0.001.
(D) Schedule of the experiment. Immediately after the fin fold amputation at 3 dpf, Tg(mfap4:mCherry-F/tnfa:GFP-F) larvae were incubated in H2O2 (2mM) or fish water for 6 h. Larvae were imaged at 6 hours post amputation (hpA) using confocal microscopy.
(F) Quantification of total macrophages in whole larvae at 3 dpf in indicated conditions. H2O2 treatment does not influence total number of macrophages. Mean ± SEM, nlarvae is indicated in brackets, twotailed t-test, ns -not significant.
(G) Quantification of recruited macrophages (up) and percentage of tnfa + macrophages in the recruited population (down) after incubation in H2O2 or fish water (Control) at 6 hpA. Representative experiment of three independent experiments, mean ± SEM, nlarvae is indicated in brackets, Mann Whitney test, two-tailed, ***p<0.001. (F) Quantification of recruited macrophages (up) and tnfa + recruited macrophages (down) in controls and in p47 phox morphants at 6 hpA. Two independent experiments merged, mean ± SEM, nlarvae is indicated in brackets, upper graph: two-tailed t-test, bottom graph: two-tailed t-test with Welch's correction, ns -not significant. (B) Quantification of total macrophages in whole larvae at 3 dpf in indicated conditions. PP2 treatment does not influence total number of macrophages. Mean ± SEM, nlarvae is indicated in brackets, twotailed t-test, ns -not significant.    (A) Schedule of the experiment. 1h before the fin fold amputation at 3 dpf, larvae were incubated in Thapsigargin or DMSO, containing CellROX solution for the detection of the ROS production. Drug and staining were both removed at 20 min pA, and larvae were immediately imaged using epi-fluorescent microscopy.
(B) Representative images of the CellROX fluorescence in uncut or cut fin folds, after the treatment with DMSO or Thapsigargin at 20 min pA. Rainbow color scale was applied to images, emphasizing the differences in signal intensity. The white lines outline the fin fold and the notochord. Scale bar: 100 μm.
(C) Quantification of signal intensity of the CellROX fluorescence by mean grey value. Representative experiment of two independent experiments, mean ± SEM, nlarvae is indicated in brackets, two-tailed ttest, ns -not significant.
(D) Schedule of the experiment. Tg(NFκB-RE:GFP) at 3 dpf was treated either with Apocynin 1h before the fin fold amputation until 6 hpA, or with Thapsigargin immediately after the amputation during 1 h.
Larvae were imaged at 6 hpA using epi-fluorescent microscopy.
(E) Representative images of the GFP fluorescence in cut fin folds of Tg(NFκB-RE:GFP) at 6 hpA, after the treatment with DMSO, Apocynin or Thapsigargin, detecting the NF-κB activation. Rainbow color scale was applied to images, emphasizing the differences in signal intensity. Scale bar: 100 μm.

(F) Quantification of signal intensity of GFP fluorescence of Tg(NFκB-RE:GFP) line by mean grey value.
Representative experiment of two independent experiments, mean ± SEM, nlarvae is indicated in brackets, two tailed t-test, ns -not significant, ***p<0.001.