Mulero Méndez, Victoriano Francisco

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Mulero Méndez, Victoriano Francisco

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Mulero Méndez, Victoriano Francisco

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Universidad de Murcia. Departamento de Biología Celular e Histología

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The colony-stimulating factor-1 receptor is a specific marker of macrophages from the bony fish gilthead seabream
(Elsevier, 2005-08-30) Lopez-Castejón, Gloria; Meseguer Peñalver, J.; Mulero Méndez, Victoriano Francisco; Sepulcre Cortés, María Pilar; Roca Soler, Francisco José; Bioquímica y Biología Molecular B e Inmunología
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.
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Evolution of Lipopolysaccharide (LPS) Recognition and Signaling: Fish TLR4 Does Not Recognize LPS and Negatively Regulates NF-κB Activation
(American Association of Immunologists Oxford University Press, 2009-02-15) Alcaraz-Perez, Francisca; López-Muñoz, Azucena; Meseguer Peñalver, J.; Cayuela Fuentes, Maria Luisa; Mulero Méndez, Victoriano Francisco; Sepulcre Cortés, María Pilar; Roca Soler, Francisco José; Bioquímica y Biología Molecular B e Inmunología
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.
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New insights into the evolution of IFNs: zebrafish group II IFNs induce a rapid and transient expression of IFN-dependent genes and display powerful antiviral activities
(American Association of Immunologists, Oxford University Press, 2009-03-15) López-Muñoz, Azucena; Meseguer Peñalver, J.; Mulero Méndez, Victoriano Francisco; Roca Soler, Francisco José; Bioquímica y Biología Molecular B e Inmunología
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 proin-flammatory 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.
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Evolution of the inflammatory response in vertebrates: fish TNF-α is a powerful activator of endothelial cells but hardly activates phagocytes
(2008-10-01) Mulero Méndez, Iván; López-Muñoz, Azucena; Renshaw, Stephen A.; Meseguer Peñalver, J.; Mulero Méndez, Victoriano Francisco; Sepulcre Cortés, María Pilar; Roca Soler, Francisco José; Bioquímica y Biología Molecular B e Inmunología
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.
Open Access
Acidophilic granulocytes of the marine fish gilthead seabream (Sparus aurata L.) produce interleukin-1b following infection with Vibrio anguillarum
(Springer, 2004) Chaves-Pozo, Elena; García-Castillo, Jesús; Meseguer, José; García Ayala, Alfonsa; Mulero Méndez, Victoriano Francisco; Pelegrín Vivancos, Pablo; Bioquímica y Biología Molecular B e Inmunología
The fish immune response to Gram-negative bacteria is poorly understood. In this study, we use a monoclonal antibody (mAb) specific to acidophilic granulocytes from the marine fish gilthead seabream (Sparus aurata L.), together with an antiserum specific to interleukin-1b (IL-1b) from this species, in order to investigate whether these cells are involved in the immune response against the pathogenic bacterium Vibrio anguillarum and, in particular, in the production of the pro-inflammatory cytokine IL-1b. We found that gilthead seabream head- kidney, peritoneal exudate and peripheral blood leukocytes accumulated proIL-1b intracellularly when challenged in vitro with V. anguillarum, whereas only peritoneal exudate and blood leukocytes were able to accumulate proIL-1b following infection. Importantly, the blood leukocytes from infected animals that accumulated proIL-1b were shown to be the acidophilic granulocytes. A rapid mobilization of such cells from the head-kidney to the site of inflammation following infection with V. anguillarum was also observed.
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Post-transcriptional regulation of cytokine genes in fish: A role for conserved AU-rich elements located in the 3′-untranslated region of their mRNAs
(Elsevier, 2006-04-03) Cayuela Fuentes, Maria Luisa; Secombes, Chris. J.; Meseguer Peñalver, J.; Mulero Méndez, Victoriano Francisco; Roca Soler, Francisco José; Bioquímica y Biología Molecular B e Inmunología
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.
Open Access
The tumor necrosis factor a of the bony fish seabream exhibits the in vivo proinflammatory and proliferative activities of its mammalian counterparts, yet it functions in a species-specific manner
(Springer, 2004) García-Castillo, Jesús; Chaves-Pozo, Elena; Olivares, Pedro; Meseguer, José; Mulero Méndez, Victoriano Francisco; Pelegrín Vivancos, Pablo; Bioquímica y Biología Molecular B e Inmunología
Information on the bioactivities of non-mammalian cytokines is scant due to the lack of the recombinant molecules and specific antibodies. We produced the mature predicted peptide of tumor necrosis factor a (TNFa) from the bony fish gilthead seabream (Sparus aurata L.) (sbTNFa), and its biological role was determined in vitro and in vivo. We first demonstrated by analytical size-exclusion chromatography that sbTNFa is an oligomeric protein but the dimer appears to predominate over the trimeric form, in contrast to mammalian TNFa. Intraperitoneal injection of native sbTNFa resulted in (i) priming of the respiratory burst of the peritoneal exudate and head-kidney (HK) leukocytes, the latter being the bone marrow equivalent in fish; (ii) rapid recruitment of phagocytic granulocytes to the injection site, and (iii) induction of granulopoiesis in the HK. Interestingly, sbTNFa was able to induce a strong proliferation of HK cells in vitro, whereas human TNFa did not. Conversely, sbTNFa was not cytotoxic for murine L929 fibroblasts.