It has long been established that lower vertebrates, most notably fish and amphibians, are resistant to the toxic effect of LPS. Furthermore, the lack of a TLR4 ortholog in some fish species and the lack of the essential costimulatory molecules for LPS activation via TLR4 (i.e., myeloid differentiation protein 2 (MD-2) and CD14) in all the fish genomes and expressed sequence tag databases available led us to hypothesize that the mechanism of LPS recognition in fish may be different from that of mammals. To shed light on the role of fish TLRs in LPS recognition, a dual-luciferase reporter assay to study NF-κB activation in whole zebrafish embryos was developed and three different bony fish models were studied: 1) the gilthead seabream (Sparus aurata, Perciformes), an immunological-tractable teleost model in which the presence of a TLR4 ortholog is unknown; 2) the spotted green pufferfish (Tetraodon nigroviridis, Tetraodontiformes), which lacks a TLR4 ortholog; and 3) the zebrafish (Danio rerio, Cypriniformes), which possesses two TLR4 orthologs. Our results show that LPS signaled via a TLR4- and MyD88-independent manner in fish, and, surprisingly, that the zebrafish TLR4 orthologs negatively regulated the MyD88-dependent signaling pathway. We think that the identification of TLR4 as a negative regulator of TLR signaling in the zebrafish, together with the absence of this receptor in most fish species, explains the resistance of fish to endotoxic shock and supports the idea that the TLR4 receptor complex for LPS recognition arose after the divergence of fish and tetrapods.

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.

All animals develop in association with complex microbial communities. It is now well established that commensal microbiota is essential for the correct functionality of each organ in the host. Particularly, the commensal gastro-intestinal microbiota (CGIM) is a key factor for development, immunity and nutrient conversion, rendering them bio-available for various uses. Thus, nutritional inputs generate a positive loop in maintaining host health and are essential in shaping the composition of the CGIM communities. Probiotics, which are live exogenous microorganisms, selectively provided to the host, are a promising concept for manipulating the microbiota and thus for increasing the host health status. Nevertheless, most mechanisms induced by probiotics to fortify the immune system are still a matter of debate. Alternatively, prebiotics, which are non-digestible food ingredients, can favor the growth of specific target groups of CGIM. Several metabolites are produced by the CGIM, one of the most important are the short-chain fatty acids (SCFAs), which emerge from the fermentation of complex carbohydrates. SCFAs have been recognized as key players in triggering beneficial effects elicited by simple diffusion and by specific receptors present thus far only in epithelial cells of higher vertebrates at different GI locations. However, both strategies have shown to provide resistance against pathogens during periods of high stress. In fish, knowledge about the action of pro- and prebiotics and SCFAs is still limited. Thus, in this review, we briefly summarize the mechanisms described on this topic for higher vertebrates and discuss why many of them may operate in the fish gut representing a model for different mucosal tissues.

The IFNs and their receptors have existed in early chordates for ∼500 million years and represent the early elements in innate and adaptive immunity. Both types I and II IFNs have been discovered in fish, and type I has recently been classified into two groups based on their primary protein sequences. However, the biological activities of fish IFNs and their roles in infection are largely unknown. Using the zebrafish and manageable bacterial (Streptococcus iniae) and viral (spring viremia of carp virus) infection models, we are reporting in this study that zebrafish IFN (zfIFN) γ failed to induce antiviral and proinflammatory genes when administered in vivo, which correlates with its inability to protect the fish against bacterial and viral infections. We also found that, although both group I (i.e., zfIFN1) and group II zfIFNs (i.e., zfIFN2 and zfIFN3) displayed strong in vivo antiviral activities, only group I zfIFN was able to protect the fish against bacterial infection, which may reflect the different patterns and kinetics of immune-related genes elicited by these two groups of IFNs. Thus, group II zfIFNs induced a rapid and transient expression of antiviral genes, whereas group I zfIFN exerted a slow but more powerful induction of several antiviral and proinflammatory genes. Collectively, our results suggest nonredundant, complementary roles of type I zfIFNs in viral infections and provide evidence for a pivotal role of the recently identified group II IFN of fish in the early stages of viral infections.