Understanding the roles of neutrophils and macrophages in fighting bacterial infections is a critical issue in human pathologies. Although phagocytic killing has been extensively studied, little is known about how bacteria are eliminated extracellularly in live vertebrates. We have recently developed an infection model in the zebrafish embryo in which leukocytes cannot reach the injected bacteria. When Escherichia coli bacteria are injected within the notochord, both neutrophils and macrophages are massively recruited during several days, but do not infiltrate the infected tissue presumably because of its tough collagen sheath. Nevertheless, the bacteria are killed during the first 24 hours, and we report here that neutrophils, but not macrophages are involved in the control of the infection. Using genetic and chemical approaches, we show that even in absence of phagocytosis, the bactericidal action relies on NADPH oxidase-dependent production of superoxide in neutrophils. We thus reveal a host effector mechanism mediated by neutrophils that eliminates bacteria that cannot be reached by phagocytes and that is independent of macrophages, NO synthase or myeloperoxidase.

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While considered an extracellular pathogen, Pseudomonas aeruginosa has been reported to be engulfed by macrophages in cellular and animal models. However, the role of macrophages in P. aeruginosa clearance in vivo remains poorly studied. The major outer membrane porin OprF has been recently shown to be involved in P. aeruginosa fate within cultured macrophages and analysis of an oprF mutant may thus provide insights to better understand the relevance of this intramacrophage stage during infection. In the present study, we investigated for the first time the virulence of a P. aeruginosa oprF mutant in a vertebrate model that harbors functional macrophages, the zebrafish (Danio rerio) embryo, which offers powerful tools to address macrophage–pathogen interactions. We established that P. aeruginosa oprF mutant is attenuated in zebrafish embryos in a macrophage-dependent manner. Visualization and quantification of P. aeruginosa bacteria phagocytosed by macrophages after injection into closed cavities suggested that the attenuated phenotype of oprF mutant is not linked to higher macrophage recruitment nor better phagocytosis than wild-type strain. Using cultured macrophages, we showed an intramacrophage survival defect of P. aeruginosa oprF mutant, which is correlated with elevated association of bacteria with acidic compartments. Notably, treatment of embryos with bafilomycin, an inhibitor of acidification, increased the sensibility of embryos towards both wild-type and oprF mutant, and partially suppressed the attenuation of oprF mutant. Taken together, this work supports zebrafish embryo as state-of-the-art model to address in vivo the relevance of P. aeruginosa intramacrophage stage. Our results highlight the contribution of macrophages in the clearance of P. aeruginosa during acute infection and suggest that OprF protects P. aeruginosa against macrophage clearance by avoiding bacterial elimination in acidified phagosomes.

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.

Background and Purpose Specialized pro‐resolving mediators (SPMs) are a family of lipids controlling the resolution of inflammation and playing a role in many processes including organ protection and tissue repair. While SPMs are potent bioactive molecules in vivo, their role in epimorphic regeneration of organs in vertebrates has not been tested. Using the zebrafish larva as a robust regenerative vertebrate system, we studied the role of the SPM neuroprotectin/protectin D1 (PD1) during the caudal fin fold regeneration. Experimental Approach Regeneration of the fin fold was analysed when exposed to a synthetic PD1. The effect of PD1 on immune cell recruitment and activation was further investigated using live imaging combined with fluorescent reporter lines. Using genetic and pharmacological approaches, we dissected the role of neutrophils and macrophages on driving the pro‐regenerative effect of PD1. Key Results We showed that PD1 improves fin fold regeneration. Acting in a narrow time window during regeneration, PD1 accelerates the resolution of inflammation without affecting the initial kinetic of neutrophil recruitment but instead, promotes their reverse migration potential. In addition, PD1 induces macrophage polarization switch towards non‐inflammatory states in both zebrafish and mammalian system. Finally, macrophages but not neutrophils are essential for PD1‐mediated regeneration. Conclusion and Implications These results reveal the pro‐regenerative action of PD1 and its role in regulating neutrophil and macrophage response in vertebrates. These findings strongly support the development of pro‐resolving mediators as natural therapeutic candidates for degenerative disorders and the use of the zebrafish as a tool to investigate pro‐regenerative drugs.

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The innate immune response to inflammation and infections are complex and represent major challenges for developing much needed new treatments for chronic inflammatory diseases and drug resistant infections. To be ultimately successful, the immune response must be balanced to allow pathogen clearance without excess tissue damage, processes controlled by pro- and anti-inflammatory signals. The roles of anti-inflammatory signalling in raising an appropriate immune response are underappreciated, representing overlooked potential drugs targets. This is especially true in neutrophils, a difficult cell type to study ex vivo due to a short lifespan, dogmatically seen as being highly pro-inflammatory. Here, we have generated and describe the first zebrafish transgenic line that labels expression of an anti-inflammatory gene (TgBAC(arg2:eGFP)sh571) and show that a subpopulation of neutrophils upregulate arginase early after immune challenge with injury and infection. At wound healing stages, arg2:GFP is expressed in subsets of neutrophils and macrophages, potentially representing anti-inflammatory, polarised immune cell populations. Our findings identify nuanced responses to immune challenge in vivo, responses that represent new opportunities for therapeutic interventions during inflammation and infection.