Search

Biallelic mutations in the glucocerebrosidase (GBA1) gene cause Gaucher disease, characterized by lysosomal accumulation of glucosylceramide and glucosylsphingosine in macrophages. This and other lysosomal diseases occur with high frequency in Ashkenazi Jews. It has been proposed that the underlying mutations confer a selective advantage, in particular conferring protection against tuberculosis. Here, using a zebrafish Gaucher disease model, we find that the mutationGBA1N370S, predominant among Ashkenazi Jews, increases resistance to tuberculosis through the microbicidal activity of glucosylsphingosine in macrophage lysosomes. Consistent with lysosomal accumulation occurring only in homozygotes, heterozygotes remain susceptible to tuberculosis. Thus, our findings reveal a mechanistic basis for protection against tuberculosis byGBA1N370S and provide biological plausibility for its selection if the relatively mild deleterious effects in homozygotes were offset by significant protection against tuberculosis, a rampant killer of the young in Europe through the Middle Ages into the 19thcentury.Significance StatementGaucher disease is a recessively inherited disorder in which the lipids glucosylceramide and glucosylsphingosine accumulate in lysosomes of macrophages. Macrophages are the first immune cells to engulf infecting bacteria and we find that glucosylsphingosine increases their ability to killMycobacterium tuberculosisthat causes tuberculosis. Gaucher disease due to a particular mutation is frequent in Ashkenazi Jews. Since from the middle ages they were often confined to areas of high tuberculosis prevalence, it has been proposed that the mutation prevailed because heterozygotes, who do not accumulate lipids nor manifest Gaucher disease, were protected. Our findings raise the possibility that selection operated on homozygotes manifesting mild forms of Gaucher disease who were protected against tuberculosis which would often have been fatal.

Tumor necrosis factor (TNF) is a critical host resistance factor against tuberculosis. However, excess TNF produces susceptibility by increasing mitochondrial reactive oxygen species (mROS), which initiate a signaling cascade to cause pathogenic necrosis of mycobacterium-infected macrophages. In zebrafish, we identified the mechanism of TNF-induced mROS in tuberculosis. Excess TNF in mycobacterium-infected macrophages elevates mROS production by reverse electron transport (RET) through complex I. TNF-activated cellular glutamine uptake leads to an increased concentration of succinate, a Krebs cycle intermediate. Oxidation of this elevated succinate by complex II drives RET, thereby generating the mROS superoxide at complex I. The complex I inhibitor metformin, a widely used antidiabetic drug, prevents TNF-induced mROS and necrosis of Mycobacterium tuberculosis –infected zebrafish and human macrophages; metformin may therefore be useful in tuberculosis therapy.

Adjunctive treatment with antiinflammatory corticosteroids like dexamethasone increases survival in tuberculosis meningitis. Dexamethasone responsiveness associates with a C/T variant in Leukotriene A4 Hydrolase (LTA4H), which regulates expression of the proinflammatory mediator leukotriene B4 (LTB4). TT homozygotes, with increased expression of LTA4H, have the highest survival when treated with dexamethasone and the lowest survival without. While the T allele is present in only a minority of the world's population, corticosteroids confer modest survival benefit worldwide. Using Bayesian methods, we examined how pretreatment levels of cerebrospinal fluid proinflammatory cytokines affect survival in dexamethasone-treated tuberculous meningitis. LTA4H TT homozygosity was associated with global cytokine increases, including tumor necrosis factor. Association between higher cytokine levels and survival extended to non-TT patients, suggesting that other genetic variants may also induce dexamethasone-responsive pathological inflammation. These findings warrant studies that tailor dexamethasone therapy to pretreatment cerebrospinal fluid cytokine concentrations, while searching for additional genetic loci shaping the inflammatory milieu.

Infections by schistosomes result in granulomatous lesions around parasite eggs entrapped within the host tissues. The host and parasite determinants of the Schistosoma mansoni egg-induced granulomatous response are areas of active investigation. Some studies in mice implicate Tumor Necrosis Factor (TNF) produced in response to the infection whereas others fail to find a role for it. In addition, in the mouse model, the S. mansoni secreted egg antigen omega-1 is found to induce granulomas but the underlying mechanism remains unknown. We have recently developed the zebrafish larva as a model to study macrophage recruitment and granuloma formation in response to Schistosoma mansoni eggs. Here we use this model to investigate the mechanisms by which TNF and omega-1 shape the early granulomatous response. We find that TNF, specifically signaling through TNF receptor 1, is not required for macrophage recruitment to the egg and granuloma initiation but does mediate granuloma enlargement. In contrast, omega-1 mediates initial macrophage recruitment, with this chemotactic activity being dependent on its RNase activity. Our findings further the understanding of the role of these host- and parasite-derived factors and show that they impact distinct facets of the granulomatous response to the schistosome egg., Author summary Schistosomiasis is a disease caused by parasitic flatworms which lay eggs within the veins of their human host. Upon sensing the parasite egg, macrophages, the first line defense cells, aggregate tightly around the egg to encapsulate it within an immune structure known as a granuloma. These granulomas are the key pathological structures which determine both host disease outcome and parasite transmission. Studies in mice have implicated omega-1, a secreted parasite protein. Omega-1 is an RNase, an enzyme that degrades host RNA. Mouse studies have also suggested that a host defense protein, Tumor Necrosis Factor (TNF), is required to form granulomas around the egg. We used the small and transparent zebrafish larva to examine the requirement of omega-1 and TNF for granuloma formation. We find that omega-1 induces rapid macrophage migration and that its RNase activity is required for this. In contrast, TNF is not involved in the initial recruitment of macrophages. Rather, it enlarges granulomas after they are initiated. These findings improve our understanding of the role of omega-1 and TNF, and show that they impact distinct facets of granuloma formation around Schistosoma eggs.

Adjunctive treatment with anti-inflammatory corticosteroids like dexamethasone increases survival in tuberculosis meningitis. Dexamethasone responsiveness associates with a C/T variant inLeukotriene A4 Hydrolase (LTA4H), which regulates expression of the pro-inflammatory mediator leukotriene B4 (LTB4). TT homozygotes, with increased LTB4, have the highest survival when treated with dexamethasone and the lowest survival without. While the T allele is present in only a minority of the world’s population, corticosteroids confer modest survival benefit worldwide. Using Bayesian methods, we examined how pre-treatment levels of cerebrospinal fluid (CSF) pro-inflammatory cytokines affect survival in dexamethasone-treated tuberculous meningitis.LTA4HTT homozygosity was associated with global cytokine increases, including TNF. Association between higher cytokine levels and survival extended to non-TT patients, suggesting that other genetic variants may also induce dexamethasone-responsive pathological inflammation. These findings warrant studies that tailor dexamethasone therapy to pre-treatment CSF cytokine concentrations, while searching for additional genetic loci shaping the inflammatory milieu.

Infections by schistosomes result in granulomatous lesions around parasite eggs entrapped within the host tissues. The host and parasite determinants of theSchistosoma mansoniegg-induced granulomatous response are areas of active investigation. Some studies in mice implicate Tumor Necrosis Factor (TNF) produced in response to the infection whereas others fail to find a role for it. In addition, in the mouse model, theS. mansonisecreted egg antigen omega-1 is found to induce granulomas but the underlying mechanism remains unknown. We have recently developed the zebrafish larva as a model to study macrophage recruitment and granuloma formation in response toSchistosoma mansonieggs. Here we use this model to investigate the mechanisms by which TNF and omega-1 shape the early granulomatous response. We find that TNF, specifically signaling through TNF receptor 1, is not required for macrophage recruitment to the egg and granuloma initiation but does mediate granuloma enlargement. In contrast, omega-1 mediates initial macrophage recruitment, with this chemotactic activity being dependent on its RNase activity. Our findings further the understanding of the role of these host- and parasite-derived factors and show that they impact distinct facets of the granulomatous response to the schistosome egg.

SUMMARY Necrosis of infected macrophages constitutes a critical pathogenetic event in tuberculosis releasing mycobacteria into the extracellular environment where they can grow unrestricted. In zebrafish infected with Mycobacterium marinum, a close relative of Mycobacterium tuberculosis, excess Tumor Necrosis Factor triggers programmed necrosis of infected macrophages through the production of mitochondrial reactive oxygen species (ROS) and the participation of cyclophilin D, a component of the mitochondrial permeability transition pore. Here we show that this necrosis pathway is not mitochondrion-intrinsic but rather results from an interorganellar circuit initiating and culminating in the mitochondrion. Mitochondrial ROS induce production of lysosomal ceramide which ultimately activates the cytosolic protein BAX. BAX promotes calcium flow from the endoplasmic reticulum into the mitochondrion through ryanodine receptors. The resultant mitochondrial calcium overload triggers cyclophilin D-mediated necrosis. We identify ryanodine receptors and plasma membrane L-Type calcium channels as specific druggable targets to intercept mitochondrial calcium overload so as to inhibit macrophage necrosis.

Summary Necrosis of infected macrophages constitutes a critical pathogenetic event in tuberculosis by releasing mycobacteria into the growth-permissive extracellular environment. In zebrafish infected with Mycobacterium marinum or Mycobacterium tuberculosis, excess tumor necrosis factor triggers programmed necrosis of infected macrophages through the production of mitochondrial reactive oxygen species (ROS) and the participation of cyclophilin D, a component of the mitochondrial permeability transition pore. Here, we show that this necrosis pathway is not mitochondrion-intrinsic but results from an inter-organellar circuit initiating and culminating in the mitochondrion. Mitochondrial ROS induce production of lysosomal ceramide that ultimately activates the cytosolic protein BAX. BAX promotes calcium flow from the endoplasmic reticulum into the mitochondrion through ryanodine receptors, and the resultant mitochondrial calcium overload triggers cyclophilin-D-mediated necrosis. We identify ryanodine receptors and plasma membrane L-type calcium channels as druggable targets to intercept mitochondrial calcium overload and necrosis of mycobacterium-infected zebrafish and human macrophages., Graphical Abstract, Highlights • TNF induces mitochondrial ROS to cause necrosis of mycobacterium-infected macrophages • Mitochondrial ROS activate lysosomal enzymes that lead to BAX activation • BAX activates ER ryanodine receptors to cause Ca2+ flow into the mitochondrion • Drugs preventing mitochondrial Ca2+ overload prevent pathogenic macrophage necrosis in TB, During TB infection, the necrosis of infected macrophages involves an inter organelle signaling relay involving druggable targets such as ryanodine receptors and plasma membrane L-type Ca2+ channels.

Cell death is of pivotal importance in the regulation of the immune response and has a direct impact in disease resistance. Fish are becoming an interesting model organism to study the immune response since they hold a key phylogenetic position and many species are of high economic interest. The role of cell death in the immune response has recently been investigated in fish and the molecules and pathways orchestrating cell death in this group of animals have begun to be elucidated. In this study, we will summarize the different molecular strategies displayed by major fish bacterial and viral pathogens to interfere with programmed cell death of the host as well as the relevance of cell death in the resolution of the infectious diseases caused by these pathogens.

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.

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.

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.

Summary Two major professional phagocyte populations have been described in fish, namely granulocytes and monocytes/macrophages. Although the distribution and localization of macrophages have been documented in several teleost species using mainly light and/or electron microscopy, the lack of appropriate markers for these cells has hampered our in-depth knowledge of their biology. We report here the generation of a monospecific rabbit polyclonal antibody against the gilthead seabream macrophage colony-stimulating factor receptor (Mcsfr), which is an excellent marker of macrophages in mammals and the zebrafish. The anti-Mcsfr has been found to be very useful in immunohistochemistry (IHC) to specifically immunostain the purified macrophages (adherent cells) obtained from the head-kidney as well as different cell populations in paraffin-embedded organs, including the head-kidney, spleen, thymus, gills and liver. Unexpectedly, however, no Mcsfr immunoreactive (Mcsfr + ) cells were observed in the brain and intestine of the gilthead seabream. We also show that the distribution of Mcsfr + cells in the head-kidney and the spleen is unaltered following infection with the fish pathogenic bacterium Vibrio anguillarum and that the Il1b-producing cells in these two organs after infection are exclusively acidophilic granulocytes. Finally, as the epitope recognized by the anti-Mcsfr is well conserved, we illustrate the potential usefulness of this antibody in other teleost species, such as the European seabass.

Interleukin-1beta (IL-1beta) is the prototypic pro-inflammatory cytokine. All the biological effects of IL-1beta are mediated through interaction with type 1 IL-1 receptor (IL-1RI), whereas another receptor, called type 2 IL-1R (IL-1RII), lacks an intracellular signalling domain and acts as a decoy receptor that down-regulates responses to IL-1beta. Although both receptors are present in bony fish, their expression and biological role in the regulation of IL-1beta activity in non-mammalian vertebrates remain to be established. In this study, a homologue of mammalian IL-1RII was isolated and characterized in the gilthead seabream (Sparus aurata). The seabream IL-1RII harboured two Ig-like domains in its extracellular region and a short cytoplasmic tail lacking a signalling domain. The seabream IL-1RII cDNA showed an unexpectedly long 3'UTR compared with that from other species and contained three ATTTA instability motifs, which seem to be responsible for its relatively short half-life (less than 2h). The expression of seabream IL-1RII was dramatically up-regulated after infection with Vibrio anguillarum in all the immune tissues examined and was even more strongly induced than the IL-1beta gene in the head kidney, spleen and liver. Strikingly, the mRNA levels of IL-1RII were 15-fold higher than those of IL-1beta in the liver, suggesting a role for this organ in the neutralization of IL-1beta leaking into the systemic circulation from the sites of inflammation. In vitro, bacterial DNA and flagellin increased the mRNA levels of IL-1RII in macrophages, while only flagellin was able to weakly induce its expression in acidophilic granulocytes. Finally, the seabream IL-1RII was localized in the plasma membrane when expressed in HEK293 cells and was able to bind IL-1beta.

The overproduction of cytokines, such us interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha), contributes to the pathological complications observed in many inflammatory diseases caused by bacterial endotoxins. The synthesis of these cytokines is tightly regulated at both transcriptional and post-transcriptional levels. Post-transcriptional regulation of gene expression depends on specific cis-acting sequences and trans-acting factors. Thus, the presence of adenylate- and uridylate-rich (AU-rich) elements (AREs) has been described in the 3'-untranslated regions (UTRs) of many unstable mammalian mRNAs. Although, it represents the most widespread, phylogenetically conserved and efficient determinant of mRNA stability among those so far characterized in mammalian cells, no studies are available on the functional relevance of this sequence in non-mammalian vertebrates. In this contribution, we study the enzymatic activity of various luciferase reporter constructs, containing or lacking the 3'UTR of IL-1beta and TNFalpha from different fish species, and report the finding that bony fish AREs are able to decrease luciferase activity but are less potent than their mammalian counterparts. Surprisingly, the 3'UTR of the IL-1beta from the cartilaginous fish small spotted catshark had the greatest ability to decrease luciferase activity. Lastly, the functional significance of the above was confirmed by measuring the half-life of IL-1beta and TNFalpha mRNAs in gilthead seabream leukocytes by blocking transcription with actinomycin D. Both cytokine mRNAs were unstable with an estimated half-life of about 45 min in control and activated cells.

TNFα is a pleiotropic pro-inflammatory cytokine with a key role in the activation of the immune system to fight viral infections. Despite its antiviral role, a few viruses might utilize the host produced TNFα to their benefit. Some recent reports have shown that anti-TNFα therapies could be utilized to treat certain viral infections. However, the underlying mechanisms by which TNFα can favor virus replication have not been identified. Here, a rhabdoviral infection model in zebrafish allowed us to identify the mechanism of action by which Tnfa has a deleterious role for the host to combat certain viral infections. Our results demonstrate that Tnfa signals through its receptor Tnfr2 to enhance viral replication. Mechanistically, Tnfa does not affect viral adhesion and delivery from endosomes to the cytosol. In addition, the host interferon response was also unaffected by Tnfa levels. However, Tnfa blocks the host autophagic response, which is required for viral clearance. This mechanism of action provides new therapeutic targets for the treatment of SVCV-infected fish, and advances our understanding of the previously enigmatic deleterious role of TNFα in certain viral infections., Author Summary Tumor necrosis factor alpha (TNFα) is one of the main pro-inflammatory cytokines produced in response to a broad type of infections [1]. Although TNFα has a crucial role in protecting the host organism from pathogens, its deregulation can promote susceptibility to pathogens by impairing pathogen clearance and, ultimately, promoting maintenance of infection and death. In addition, some viruses might utilize the host produced TNFα to their benefit. Thus, anti-TNFα therapies could be utilized to treat certain viral infections. However, the underlying mechanisms by which TNFα can favor certain virus replication have not been identified. Here, we have used a viral infection model in zebrafish to identify the mechanism of action by which TNFα has a deleterious role for the host to combat certain viral infections. Our results demonstrate that Tnfa does not affect viral ability to infect host cells or to antagonize the main host antiviral pathway, namely the interferon pathway. However, Tnfa impairs viral clearance by blocking the host autophagy response, which is usually used by host cells to degrade unnecessary or dysfunctional cellular components, and that we found to be critical to eliminate intracellular viral particles. This mechanism of action provides new therapeutic targets for the treatment of SVCV-infected fish in aquaculture and probably to other viral infection affecting cattle industry and human.

Recent work in a variety of animal models, including mice, zebrafish, and macaques, as well as in humans, has led to a reassessment of several tenets of mycobacterial infection. In this review, we highlight new findings about the composition and dynamics of the tuberculous granuloma, the central host structure in mycobacterial infection, as well as inflammatory mediators that drive a successful anti-microbial response on one hand and pathological inflammation on the other. We highlight granuloma heterogeneity that emerges in the context of infection, the functional consequences of angiogenesis in tuberculous granulomas, and data that balanced inflammation in humans, with a central role for tumor necrosis factor, appears to play a key role in optimal defense against mycobacterial infection. These findings have suggested new and specific host-directed therapies that await further clinical exploration.

While tuberculosis susceptibility has historically been ascribed to failed inflammation, it is now known that an excess of leukotriene A4 hydrolase (LTA4H), which catalyzes the final step in leukotriene B4 (LTB4) synthesis, produces a hyperinflammatory state and tuberculosis susceptibility. Here we show that the LTB4-inactivating enzyme leukotriene B4 dehydrogenase/prostaglandin reductase 1 (LTB4DH/PTGR1) restricts inflammation and independently confers resistance to tuberculous infection. LTB4DH overexpression counters the susceptibility resulting from LTA4H excess while ltb4dh-deficient animals can be rescued pharmacologically by LTB4 receptor antagonists. These data place LTB4DH as a key modulator of TB susceptibility and suggest new tuberculosis therapeutic strategies.

SummarySusceptibility to tuberculosis is historically ascribed to an inadequate immune response that fails to control infecting mycobacteria. In zebrafish, we find that susceptibility to Mycobacterium marinum can result from either inadequate or excessive acute inflammation. Modulation of the leukotriene A4 hydrolase (LTA4H) locus, which controls the balance of pro- and anti-inflammatory eicosanoids, reveals two distinct molecular routes to mycobacterial susceptibility converging on dysregulated TNF levels: inadequate inflammation caused by excess lipoxins and hyperinflammation driven by excess leukotriene B4. We identify therapies that specifically target each of these extremes. In humans, we identify a single nucleotide polymorphism in the LTA4H promoter that regulates its transcriptional activity. In tuberculous meningitis, the polymorphism is associated with inflammatory cell recruitment, patient survival and response to adjunctive anti-inflammatory therapy. Together, our findings suggest that host-directed therapies tailored to patient LTA4H genotypes may counter detrimental effects of either extreme of inflammation.

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. This work was supported by the Spanish Ministry of Science and Innovation [grant numbers BIO2008-01379, BIO2011-23400, CSD2007-00002 to V.M.; RYC-2006001694 to N.M.; 05/0087, PIO6/00369 to M.L.C.; fellowship to S.C.), the Fundación Séneca-Murcia [grant numbers 04538/GERM/06 to V.M.; 08760/PI/08 to M.L.C.; fellowship to R.E.], the University of Murcia [fellowship to F.A.-P.] and the Fundación Marcelino Botín [grant to V.M.]. Sí

In mammals, IFNγ is the only type II IFN member, whereas most bony fish species have two IFNγ genes, namely IFNγ1 and IFNγ2. We report that both zebrafish IFNγ genes were unable to protect zebrafish larvae against viral infection, despite the fact that they moderately induced the expression of antiviral genes, strongly induced pro-inflammatory and antigen processing and presentation genes, and increased neutrophil numbers. Although both zebrafish IFNγs induced a similar set of immune genes, IFNγ1 was more powerful at inducing pro-inflammatory genes than IFNγ2, which correlated with its ability to promote larval death. Strikingly, IFNγ1-induced larval death was prevented by genetic ablation of the myeloid transcription factor SPI1 but not IL-1β or TNFα, suggesting that professional phagocytes are also one of the main targets of IFNγ in fish. In addition, the usefulness of the zebrafish for the identification of IFNγ-target genes is illustrated by the identification of several genes whose expression is also regulated in murine macrophages by IFNγ, namely two membrane-spanning 4-domain family members and the opioid growth factor receptor. Finally, we found for the first time that the thymic specific proteasome subunit PSMB11/β5t is regulated by IFNγ. Collectively, our data throw light on partially redundant functions of fish IFNγ genes, demonstrate that the pro-inflammatory and antigen presentation functions of IFNγ have been conserved during vertebrate evolution, and highlight the fact that zebrafish is an excellent model for studying IFNγ biology.

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.

12 páginas, 6 figuras, 2 tablas, The tumor necrosis factor (TNF) superfamily is composed by several proteins with similar structure and functions. One of the main representatives of this family is TNF-alpha (TNF ), a proinflammatory cytokine which is produced by different immune cells and presents a wide variety of activities. Using the RACE technique, we have cloned and sequenced the turbot TNF cDNA. The analysis of its sequence showed several conserved motifs characteristic of members of the TNF family. A phylogenetic tree constructed with different TNFs of fish and mammals grouped our sequence within the fish TNF cluster. Therefore, the turbot TNF here studied was identified as TNF . The complete TNF gene was obtained by gene walking, and, similarly to the other known fish TNF genes, presented three introns and four exons. A PCR was designed to study the turbot TNF expression in vivo using as stimulus the bacteria Vibrio pelagius strain Hq222 and virus VHSV. The expression of the cytokine happened early after injection, and it was dependent on the pathogen injected and organ analyzed. Virus induced a higher TNF expression, but this response was shorter in time than that induced by bacteria. In addition, TNF expression was in general higher in kidney than in liver, as expected since the former is the haematopoietic organ of fish. The turbot recombinant TNF (rTNF ) was obtained by IPTG induction of bacteria transformed with the pET15b-TNF construct, and it was purified in native conditions. The recombinant protein was approximately 20 kDa in size, and its biological activity was assessed in vitro. No effect of the rTNF neither alone nor in combination with LPS was observed on the chemiluminescence activity of turbot macrophages at any time tested. However, NO production was enhanced by the recombinant protein alone or with LPS 72 h after the addition of the treatments. Finally, turbot rTNF was able to recruit and activate inflammatory cells when injected in gilthead seabream, although to a lesser extent than gilthead seabream rTNF ., This research has been supported by the Ministerio de Ciencia y Tecnología (BIO2001 2324-C02), Xunta de Galicia (PGIDIT02PXIC40204PN) and the Canadian-Spanish program NRC-MEC. Dr. Ordás thanks the Consejo Superior de Investigaciones Científicas for her I3P post-doctoral contract, and Ms. Costa thanks the Ministerio de Educación y Ciencia for her predoctoral fellowship.

We report the molecular cloning of the colony-stimulating factor-1 receptor gene from the bony fish gilthead seabream (sbCSF-1R). The deduced sbCSF-1R shows a predicted signal sequence, a transmembrane domain and a tyrosine kinase domain, all in conserved positions. A transcript showing a premature stop codon that predicted the removal of 84 C-terminal amino acids was also found. RT-PCR expression studies demonstrate that, although the sbCSF-1R transcripts are found in different immune tissues, including gill, liver, spleen, blood, peritoneal exudate, thymus and head-kidney (HK), their expression is confined to the monocyte/macrophage lineage. Furthermore, the expression of sbCSF-1R might be modulated by the activation stage of the macrophages, since both the infection of fish and the in vitro activation of leukocytes resulted in the down-regulation of gene expression. These data indicate that the CSF-1R may be used as a specific probe for cells of the monocyte/macrophage lineage in the gilthead seabream, an immunological tractable fish model. In addition, the functional characterisation of the CSF-1R and its ligand may shed light into the mechanisms of proliferation and the pathways of differentiation of macrophages in bony fish.

IL-1β is a potent pro-inflammatory cytokine which is translated as a leaderless 33 kDa precursor protein (proIL-1β) that is processed into its 17 kDa active form by the IL-1β-converting enzyme (ICE, caspase-1). Several stimuli have been reported to activate caspase-1, including the engagement of P2X7 receptors by extracellular ATP and infection with invasive bacteria. 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. In addition, we have previously shown that extracellular ATP fails to induce the processing and release of this cytokine in the bony fish gilthead seabream. In this study, we report that the stimulation of seabream leukocytes with flagellin or bacterial DNA leads to the processing of proIL-1β into a 20 kDa form, although it largely remains intracellular. However, the infection of leukocytes with salmonella results in the efficient and specific release of the mature form. Interestingly, the processing of IL-1β in this species is not prevented by a specific inhibitor of caspase-1 or a pan-caspase inhibitor. Whatever the case, our results suggest that the processing and release of IL-1β in early vertebrates is mediated by cell death upon bacterial infection by a caspase-1 independent mechanism, while a more sophisticated mechanism operated by activation of ATP-gated P2X7 receptors appeared later in evolution. This work was supported by the Spanish Ministry of Education and Science (grant BIO2005-05078 to V.M. and fellowship to G.L.-C.).

SummaryTumor necrosis factor (TNF) constitutes a critical host defense against tuberculosis, but its excess is also implicated in tuberculosis pathogenesis in zebrafish and humans. Using the zebrafish, we elucidate the pathways by which TNF mediates tuberculosis pathogenesis. TNF excess induces mitochondrial reactive oxygen species (ROS) in infected macrophages through RIP1-RIP3-dependent pathways. While initially increasing macrophage microbicidal activity, ROS rapidly induce programmed necrosis (necroptosis) and release mycobacteria into the growth-permissive extracellular milieu. TNF-induced necroptosis occurs through two pathways: modulation of mitochondrial cyclophilin D, implicated in mitochondrial permeability transition pore formation, and acid sphingomyelinase-mediated ceramide production. Combined genetic blockade of cyclophilin D and acid sphingomyelinase renders the high TNF state hyperresistant by preventing macrophage necrosis while preserving increased microbicidal activity. Similarly, the cyclophilin D-inhibiting drug alisporivir and the acid sphingomyelinase-inactivating drug, desipramine, synergize to reverse susceptibility, suggesting the therapeutic potential of these orally active drugs against tuberculosis and possibly other TNF-mediated diseases.