Neutrophils play a pivotal role in the innate immune response. The small cytokine CXCL8 (also known as IL-8) is known to be one of the most potent chemoattractant molecules that, among several other functions, is responsible for guiding neutrophils through the tissue matrix until they reach sites of injury. Unlike mice and rats that lack a CXCL8 homolog, zebrafish has two distinct CXCL8 homologs: Cxcl8-l1 and Cxcl8-l2. Cxcl8-l1 is known to be upregulated under inflammatory conditions caused by bacterial or chemical insult but until now the role of Cxcl8s in neutrophil recruitment has not been studied. In this study we show that both Cxcl8 genes are upregulated in response to an acute inflammatory stimulus, and that both are crucial for normal neutrophil recruitment to the wound and normal resolution of inflammation. Additionally, we have analyzed neutrophil migratory behavior through tissues to the site of injury in vivo, using open-access phagocyte tracking software PhagoSight. Surprisingly, we observed that in the absence of these chemokines, the speed of the neutrophils migrating to the wound was significantly increased in comparison with control neutrophils, although the directionality was not affected. Our analysis suggests that zebrafish may possess a subpopulation of neutrophils whose recruitment to inflamed areas occurs independently of Cxcl8 chemokines. Moreover, we report that Cxcl8-l2 signaled through Cxcr2 for inducing neutrophil recruitment. Our study, therefore, confirms the zebrafish as an excellent in vivo model to shed light on the roles of CXCL8 in neutrophil biology.

How fish larvae are protected from infection before the maturation of adaptive immunity, a process which may take up to several weeks in most species, has long been a matter of speculation. Using a germ-free model, we show that colonization by commensals in newly hatched zebrafish primes neutrophils and induces several genes encoding proinflammatory and antiviral mediators, increasing the resistance of larvae to viral infection. Commensal microbe recognition was found to be mediated mainly through a TLR/MyD88 signaling pathway, and professional phagocytes were identified as the source of these immune mediators. However, the induction of proinflammatory and antiviral genes, but not of antimicrobial effector genes, also required the covalent modification of histone H3 at gene promoters. Interestingly, chromatin modifications were not altered by commensal microbes or hatching. Taken together, our results demonstrate that gene-specific chromatin modifications are associated with the protection of zebrafish larvae against infectious agents before adaptive immunity has developed and prevent pathologies associated with excessive inflammation during development.

Non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH) is an increasing clinical problem associated with progression to hepatocellular carcinoma (HCC). The effect of a high-fat diet on the early immune response in HCC is poorly understood, while the role of metformin in treating NAFLD and HCC remains controversial. Herein, we visualized the early immune responses in the liver and the effect of metformin on progression of HCC using optically transparent zebrafish. We used live imaging to visualize liver inflammation and disease progression in a NAFLD/NASH-HCC zebrafish model. We combined a high-fat diet with a transgenic zebrafish HCC model induced by hepatocyte-specific activated beta-catenin and assessed liver size, angiogenesis, micronuclei formation and inflammation in the liver. In addition, we probed the effects of metformin on immune cell composition and early HCC progression. We found that a high-fat diet induced an increase in liver size, enhanced angiogenesis, micronuclei formation and neutrophil infiltration in the liver. Although macrophage number was not affected by diet, a high-fat diet induced changes in macrophage morphology and polarization with an increase in liver associated TNFα-positive macrophages. Treatment with metformin altered macrophage polarization, reduced liver size and reduced micronuclei formation in NAFLD/NASH-associated HCC larvae. Moreover, a high-fat diet reduced T cell density in the liver, which was reversed by treatment with metformin. These findings suggest that diet alters macrophage polarization and exacerbates the liver inflammatory microenvironment and cancer progression in a zebrafish model of NAFLD/NASH-associated HCC. Metformin specifically affects the progression induced by diet and modulates the immune response by affecting macrophage polarization and T cell infiltration, suggesting possible effects of metformin on tumor surveillance. This paper reports a new zebrafish model that can be used to study the effects of diet on liver cancer. We found that a high-fat diet promotes non-resolving inflammation in the liver and enhances cancer progression. In addition, we found that metformin, a drug used to treat diabetes, inhibits high-fat diet-induced cancer progression in this model, by reducing diet-induced non-resolving inflammation and potentially restoring tumor surveillance.

Dual oxidase 1 (Duox1) is the NADPH oxidase responsible for the H₂O₂ gradient formed in tissues after injury to trigger the early recruitment of leukocytes. Little is known about the signals that modulate H₂O₂ release from DUOX1 and whether the H₂O₂ gradient can orchestrate the inflammatory response in vivo. In this study, we report on a dominant-negative form of zebrafish Duox1 that is able to inhibit endogenous Duox1 activity, H₂O₂ release and leukocyte recruitment after tissue injury, with none of the side effects associated with morpholino-mediated Duox1 knockdown. Using this specific tool, we found that ATP release following tissue injury activates purinergic P2Y receptors, and modulates Duox1 activity through phospholipase C (PLC) and intracellular calcium signaling in vivo. Furthermore, Duox1-derived H₂O₂ is able to trigger the NF-κB inflammatory signaling pathway. These data reveal that extracellular ATP acting as an early danger signal is responsible for the activation of Duox1 via a P2YR/PLC/Ca²⁺ signaling pathway and the production of H₂O₂, which, in turn, is able to modulate in vivo not only the early recruitment of leukocytes to the wound but also the inflammatory response through activation of the NF-κB signaling pathway.

In the sixties and seventies, erythrocytes or red blood cells (RBCs) were extensively studied. Much has been learnt particularly concerning their metabolism and gas transporter function. In the past decade, the use of new approaches and methodologies