María P. Sepulcre

Evolution of Lipopolysaccharide (LPS) Recognition and Signaling: Fish TLR4 Does Not Recognize LPS and Negatively Regulates NF-κB Activation

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.

Evolution of the Inflammatory Response in Vertebrates: Fish TNF-α Is a Powerful Activator of Endothelial Cells but Hardly Activates Phagocytes

TNF-α is conserved in all vertebrate classes and has been identified in all taxonomic groups of teleost fish. However, its biological activities and its role in infection are largely unknown. Using two complementary fish models, gilthead seabream and zebrafish, we report here that the main proinflammatory effects of fish TNF-α are mediated through the activation of endothelial cells. Thus, TNF-α promotes the expression of E-selectin and different CC and CXC chemokines in endothelial cells, thus explaining the recruitment and activation of phagocytes observed in vivo in both species. We also found that TLR ligands, and to some extent TNF-α, were able to increase the expression of MHC class II and CD83 in endothelial cells, which might suggest a role for fish endothelial cells and TNF-α in Ag presentation. Lastly, we found that TNF-α increases the susceptibility of the zebrafish to viral (spring viremia of carp virus) and bacterial (Streptococcus iniae) infections. Although the powerful actions of fish TNF-α on endothelial cells suggest that it might facilitate pathogen dissemination, it was found that TNF-α increased antiviral genes and, more importantly, had little effect on the viral load in early infection. In addition, the stimulation of ZF4 cells with TNF-α resulted in increased viral replication. Together, these results indicate that fish TNF-α displays different sorts of bioactivity to their mammalian counterparts and point to the complexity of the evolution that has taken place in the regulation of innate immunity by cytokines.

Characterisation of gilthead seabream acidophilic granulocytes by a monoclonal antibody unequivocally points to their involvement in fish phagocytic response

Abstract. The various cell types involved in fish phagocytic defence have not been properly established because of the morphological heterogeneity of leucocytes and the lack of appropriate cell-surface markers. In this study, we report the production and characterisation of a monoclonal antibody, G7, which specifically recognises gilthead seabream acidophilic granulocytes, as assayed by immunofluorescence and immunoelectron microscopy. The antibody reacted with 40%–50% of head-kidney and peritoneal exudate leucocytes and 10%–20% of spleen and peripheral blood leucocytes. More importantly, G7+ acidophils constituted 85% of the head-kidney leucocytes showing phagocytic activity towards the fish pathogenic bacterium Vibrio anguillarum. The results are discussed in relation to the role played by this cell type in fish immune responses.

Zebrafish larvae are unable to mount a protective antiviral response against waterborne infection by spring viremia of carp virus.

Interferons (IFNs) and their receptors exist in all classes of vertebrates, where they represent early elements in innate and adaptive immunity. Both types I and II IFNs have been discovered in fish and type I IFN has recently been classified into two groups based on their primary protein sequences and biological activities. Thus, although groups I and II zebrafish IFN show powerful antiviral activities, only group I (IFNphi1) is able to protect the fish against bacterial infection. In addition, group II IFNs (IFNphi2 and IFNphi3) induce a rapid and transient expression of antiviral genes, while group I IFN exerts a slow but more powerful induction of several antiviral and pro-inflammatory genes. To gain further insight into the IFN system of fish, we have developed a waterborne infection model of zebrafish larvae with the spring viremia of carp virus (SVCV). Larvae were challenged 3 days post-fertilization by immersion, which considerably reduces the manipulation of fish and represents a more natural route of infection. Using this infection model, we unexpectedly found an inability on the part of zebrafish larvae to mount a protecting antiviral response to waterborne SVCV. Nevertheless, zebrafish larvae showed a functional antiviral system since ectopic expression of the cDNA of both groups I and II IFN was able to protect them against SVCV via the induction of IFN-stimulated genes (ISGs). Interestingly, group II IFNs also induced group I IFN, suggesting crosstalk between these two kinds of antiviral IFN. These results further confirm the antiviral activities of type I IFN in the zebrafish and provide the first viral infection model for zebrafish larvae using a natural route of infection. This model, in combination with the powerful gene overexpression and morpholino-mediated knockdown techniques, will help to illuminate the IFN system of teleost fish.

Evolution of inflammasome functions in vertebrates: Inflammasome and caspase-1 trigger fish macrophage cell death but are dispensable for the processing of IL-1β.

Members of the nucleotide binding and oligomerization domain-like receptors (NLRs) and the PYD and CARD domain containing adaptor protein (PYCARD) assemble into multi-protein platforms, termed inflammasomes, to mediate in the activation of caspase-1 and the subsequent secretion of IL-1β and IL-18, and the induction of pyroptotic cell death. While the recognition site for caspase-1 is well conserved in mammals, most of the non-mammalian IL-1β genes cloned so far lack this conserved site. We report here that stimulation or infection of seabream macrophages (MØ) led to the caspase-1-independent processing and release of IL-1β. In addition, several classical activators of the NLRP3 inflammasome failed to activate caspase-1 and to induce the processing and release of IL-1β. Furthermore, the processing of IL-1β in seabream MØ is not prevented by caspase-1 or pan-caspase inhibitors, and recombinant seabream caspase-1 failed to process IL-1β. However, the pharmacological inhibition of caspase-1 impaired Salmonella enterica sv. Typhimurium-induced cell death. These results suggest a role for the inflammasome and caspase-1 in the regulation of pyroptotic cell death in fish and support the idea that its use as a molecular platform for the processing of pro-inflammatory cytokines arose after the divergence of fish and tetrapods.

Evolution of a multifunctional gene: The warm temperature acclimation protein Wap65 in the European seabass Dicentrarchus labrax

The warm temperature acclimation protein Wap65 has been shown to be involved in temperature acclimation, in immune response as well as in development. In teleosts, two types of Wap65 proteins, Wap65-1 and Wap65-2 are found, both acting as a multifunctional agent in several biological processes. In the present study we identified both transcripts Wap65-1 and Wap65-2 for the European seabass (Dicentrarchus labrax), examined their evolutionary rate and performed selection tests. The two paralogues were shown to be under moderate positive selection indicating their evolutionary adaptation. This functional diversification was further explored through expression studies. Both transcripts were differentially expressed during development as well as in various tissues and pathogen challenges, showing that Wap65-1 and Wap65-2 have evolved diverse functions. These results direct to the hypothesis that Wap65 proteins may, similarly to heat-shock proteins, have a general role in cell physiology.

TLR agonists extend the functional lifespan of professional phagocytic granulocytes in the bony fish gilthead seabream and direct precursor differentiation towards the production of granulocytes

Neutrophils are major cells participants in innate host responses. They are short-lived leukocytes, although microbial products activate intracellular signaling cascades that prolong their survival by inhibiting constitutive apoptosis. To gain insight into the phylogeny of this important cell type, we examined the ability of toll-like receptor agonists to extend the lifespan of gilthead seabream (Sparus aurata L.) acidophilic granulocytes, which are the functional equivalent of mammalian neutrophils. The results obtained demonstrated that apoptosis was also the default state of seabream acidophilic granulocytes and that toll-like receptor agonists were able to dramatically extend their functional lifespan (up to 10 days) by inhibiting apoptosis and inducing a long lasting activation of phagocytic and respiratory burst activities, together with the expression of genes coding for several proinflammatory molecules. This process was independent on contaminating cells and interleukin-1β production. In addition, the results showed that p38 mitogen-activated protein kinase, but not nuclear factor κB, c-Jun terminal kinase or phosphatidylinositol 3-kinase, was involved in the inhibition of acidophilic granulocyte apoptosis following toll-like receptor engagement. Finally, stimulation of head kidney hematopoietic precursor cells with toll-like receptor agonists promoted their terminal differentiation to acidophilic granulocytes. These results demonstrated that the extension of neutrophil lifespan by microbial products is conserved in lower vertebrates although the magnitude of the response is much higher in fish.

Prolactin-Induced Production of Reactive Oxygen Species and IL-1β in Leukocytes from the Bony Fish Gilthead Seabream Involves Jak/Stat and NF-κB Signaling Pathways

Prolactin (PRL), a peptide hormone produced by the pituitary gland, was shown to play an important role in the modulation of the immune system of lower and higher vertebrates. To further investigate the effects of PRL on the activation of professional phagocytes of bony fish, we stimulated head kidney leukocytes and purified macrophages from the gilthead seabream (Sparus aurata L.) with various physiological concentrations of native salmon PRL for 2 and 16 h and analyzed the respiratory burst activity and proinflammatory cytokine expression profile. The results showed that PRL was able to induce the production of reactive oxygen species and the expression of IL-1β and TNF-α in a similar way to two major pathogen-associated molecular patterns: polyinosinic–polycytidylic acid and genomic DNA from the bacterium Vibrio anguillarum. Interestingly, when the leukocytes were stimulated with suboptimal concentrations of PRL in the presence of bacterial DNA, the expression of IL-1β was synergistically induced. More importantly, all PRL activities were blocked by neutralizing Abs to PRL, as well as by pharmacological inhibitors of the Jak/Stat and NF-κB signaling pathways. In addition, EMSA and HPLC/mass spectrometry further confirmed that Stat and NF-κB were involved in the activation of seabream leukocytes by PRL. Collectively, our data identified PRL as a key regulator of the activation of fish professional phagocytes and demonstrated a cross-talk between TLR/NF-κB and PRLR/Jak/Stat signaling pathways. In addition, to the best of our knowledge, this is the first report showing that PRL modulates the activation of phagocyte NADPH oxidase through the Jak/Stat pathway in vertebrates.

A fish cell surface receptor defined by a mAb mediates leukocyte aggregation and deactivation

Cell adhesion molecules play a key role in the inflammatory response. Selectins, integrins and immunoglobulin gene superfamily adhesion receptors mediate the different steps of leukocyte migration from the blood-stream towards inflammatory foci. In addition to their adhesive function, these receptors modulate major cellular processes such as cell activation, growth, differentiation and death. To characterise the fish molecules involved in cell adhesion, a panel of mAbs was raised by immunising mice with macrophages from the marine fish gilthead seabream (Sparus aurata L.). One of these mAbs, which we named anti-Aggregatin, was found to induce a rapid heterotypic aggregation of seabream leukocytes. Anti-Aggregatin defined a 140-kDa cell surface receptor which was highly expressed by macrophages and was up-regulated after co-stimulation with LPS and MAF. Functionally, the cell adhesion which occurred upon exposure to anti-Aggregatin required Ca(2+), an intact cytoskeleton and an active cell metabolism. More importantly, Aggregatin engagement resulted in strong inhibition of the phagocyte respiratory burst, although the cells showed neither loss of viability nor DNA fragmentation. The results are discussed in relation to the potential role of cell adhesion molecules in fish immune responses.

TNF receptors regulate vascular homeostasis in zebrafish through a caspase-8, caspase-2 and P53 apoptotic program that bypasses caspase-3

SUMMARY Although it is known that tumor necrosis factor receptor (TNFR) signaling plays a crucial role in vascular integrity and homeostasis, the contribution of each receptor to these processes and the signaling pathway involved are still largely unknown. Here, we show that targeted gene knockdown of TNFRSF1B in zebrafish embryos results in the induction of a caspase-8, caspase-2 and P53-dependent apoptotic program in endothelial cells that bypasses caspase-3. Furthermore, the simultaneous depletion of TNFRSF1A or the activation of NF-κB rescue endothelial cell apoptosis, indicating that a signaling balance between both TNFRs is required for endothelial cell integrity. In endothelial cells, TNFRSF1A signals apoptosis through caspase-8, whereas TNFRSF1B signals survival via NF-κB. Similarly, TNFα promotes the apoptosis of human endothelial cells through TNFRSF1A and triggers caspase-2 and P53 activation. We have identified an evolutionarily conserved apoptotic pathway involved in vascular homeostasis that provides new therapeutic targets for the control of inflammation- and tumor-driven angiogenesis.