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-kB 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.