The Src family kinase Lyn is identified as a physiological redox sensor that mediates the initial attraction of leukocytes to wounds in zebrafish larvae. It is known that hydrogen peroxide (H2O2) — often an ingredient in over-the-counter medicines for cleaning cuts — is generated at injured tissue and that it attracts immune cells (neutrophils) that promote wound healing. How immune cells sense the H2O2 secreted from wounds was not known. This study identifies an Src family kinase, Lyn, as a redox sensor that mediates initial neutrophil recruitment. Exposure to H2O2 directly oxidizes a single cysteine residue is indispensable in Lyn, and inhibition of Lyn attenuates neutrophil wound recruitment. Tissue wounding induces the rapid recruitment of leukocytes1. Wounds and tumours—a type of ‘unhealed wound’2—generate hydrogen peroxide (H2O2) through an NADPH oxidase (NOX). This extracellular H2O2 mediates recruitment of leukocytes, particularly the first responders of innate immunity, neutrophils, to injured tissue3,4,5,6. However, the sensor that neutrophils use to detect the redox state at wounds is unknown. Here we identify the Src family kinase (SFK) Lyn as a redox sensor that mediates initial neutrophil recruitment to wounds in zebrafish larvae. Lyn activation in neutrophils is dependent on wound-derived H2O2 after tissue injury, and inhibition of Lyn attenuates neutrophil wound recruitment. Inhibition of SFKs also disrupted H2O2-mediated chemotaxis of primary human neutrophils. In vitro analysis identified a single cysteine residue, C466, as being responsible for direct oxidation-mediated activation of Lyn. Furthermore, transgenic-tissue-specific reconstitution with wild-type Lyn and a cysteine mutant revealed that Lyn C466 is important for the neutrophil wound response and downstream signalling in vivo. This is the first identification, to our knowledge, of a physiological redox sensor that mediates leukocyte wound attraction in multicellular organisms.

Bacterial pathogens utilize toxins to modify or kill host cells. The bacterial ADP-ribosyltransferases are a family of protein toxins that covalently transfer the ADP-ribose portion of NAD to host proteins. Each bacterial ADP-ribosyltransferase toxin modifies a specific host protein(s) that yields a unique pathology. These toxins possess the capacity to enter a host cell or to use a bacterial Type III apparatus for delivery into the host cell. Advances in our understanding of bacterial toxin action parallel the development of biophysical and structural biology as well as our understanding of the mammalian cell. Bacterial toxins have been utilized as vaccines, as tools to dissect host cell physiology, and more recently for the development of novel therapies to treat human disease.

Motile cells navigate through complex tissue environments that include both attractive and repulsive cues. In response to tissue wounding, neutrophils, primary cells of the innate immune response, exhibit bidirectional migration that is orchestrated by chemokines and their receptors. Although progress has been made in identifying signals that mediate the recruitment phase, the mechanisms that regulate neutrophil reverse migration remain largely unknown. Here, we visualize bidirectional neutrophil migration to sterile wounds in zebrafish larvae and identify specific roles for the chemokine receptors Cxcr1 and Cxcr2 in neutrophil recruitment to sterile injury and infection. Notably, we also identify Cxcl8a/Cxcr2 as a specific ligand-receptor pair that orchestrates neutrophil chemokinesis in interstitial tissues during neutrophil reverse migration and resolution of inflammation. Taken together, our findings identify distinct receptors that mediate bidirectional leukocyte motility during interstitial migration depending on the context and type of tissue damage in vivo.

MicroRNA-223 is known as a myeloid-enriched anti-inflammatory microRNA that is dysregulated in numerous inflammatory conditions. Here, we report that neutrophilic inflammation (wound response) is augmented in miR-223-deficient zebrafish, due primarily to elevated activation of the canonical nuclear factor κB (NF-κB) pathway. NF-κB over-activation is restricted to the basal layer of the surface epithelium, although miR-223 is detected throughout the epithelium and in phagocytes. Not only phagocytes but also epithelial cells are involved in miR-223-mediated regulation of neutrophils' wound response and NF-κB activation. Cul1a/b, Traf6, and Tab1 are identified as direct targets of miR-223, and their levels rise in injured epithelium lacking miR-223. In addition, miR-223 is expressed in cultured human bronchial epithelial cells, where it also downregulates NF-κB signaling. Together, this direct connection between miR-223 and the canonical NF-κB pathway provides a mechanistic understanding of the multifaceted role of miR-223 and highlights the relevance of epithelial cells in dampening neutrophil activation.