Publications by authors named "Nerea Roher"

38 Publications

Nanostructured recombinant protein particles raise specific antibodies against the nodavirus NNV coat protein in sole.

Fish Shellfish Immunol 2020 Apr 24;99:578-586. Epub 2020 Feb 24.

Institute of Biotechnology and Biomedicine (IBB), Universitat Autònoma de Barcelona, 08193, Barcelona, Spain; Department of Cell Biology, Animal Physiology and Immunology, Universitat Autònoma de Barcelona, 08193, Barcelona, Spain. Electronic address:

Nervous necrosis virus (NNV) reassortant strains RGNNV/SJNNV have emerged as a potent threat to the Mediterranean marine aquaculture industry, causing viral encephalopathy and retinopathy (VER) in Senegalese sole (Solea senegalensis). In this study, a cheap and practical vaccine strategy using bacterial inclusion bodies made of the coat protein of a virulent reassortant strain of this betanodavirus was devised. The nanostructured recombinant protein nanoparticles, VNNV-C, were administered without adjuvant to two groups of juvenile sole, one by intraperitoneal injection and the other by oral intubation. Specific antibodies were raised in vivo against the NNV coat protein via both routes, with a substantial specific antibody expansion in the injected group 30 days post homologous prime boost. Expression levels of five adaptive immune-related genes, cd8a, cd4, igm, igt and arg2, were also quantified in intestine, spleen and head kidney. Results showed cd4 and igm were upregulated in the head kidney of injected fish, indicating activation of an adaptive systemic response, while intubated fish exhibited a mucosal response in the intestine. Neither route showed significant differential expression of cd8a. The specific antibody response elicited in vivo and the lack of any signs of toxicity over the 6-week study period in young fish (n = 100), evidences the potential of the nanoparticle as a vaccine candidate.
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http://dx.doi.org/10.1016/j.fsi.2020.02.029DOI Listing
April 2020

Nanoliposomes encapsulating immunostimulants modulate the innate immune system and elicit protection in zebrafish larvae.

Fish Shellfish Immunol 2019 Sep 11;92:421-429. Epub 2019 Jun 11.

Institute of Biotechnology and Biomedicine (IBB), Universitat Autònoma de Barcelona, 08193, Barcelona, Spain; Department of Cell Biology, Animal Physiology and Immunology, Universitat Autònoma de Barcelona, 08193, Barcelona, Spain. Electronic address:

Here we present immunostimulant-loaded nanoliposomes (NL) as a strategy to protect zebrafish larvae against bacterial infection. The NL encapsulate crude lipopolysaccharide (LPS) from E. coli and polyinosinic:polycytidylic acid (Poly I:C), a synthetic analogue of viral dsRNA. Fluorescently-labeled NL were ingested by zebrafish larvae 4 days post fertilization, when administrated by bath immersion, and accumulated in the intestine. RT-qPCR analysis showed the expression of innate immune related genes (tnfα, il1β, nos2a, irf1a and ptgs2a) was significantly upregulated at 48 h post NL treatment. A zebrafish larvae infection model for Aeromonas hydrophila was set up by bath immersion, achieving bacterial-dose-dependent significant differences in survival at day 5 post infection in both injured and non-injured larvae. Using this model, NL protected non-injured zebrafish larvae against an A. hydrophila lethal infection. In contrast, neither the empty nanoliposomes nor the mixture of immunostimulants could protect larvae against lethal challenges. Our results demonstrate that nanoliposomes could be further developed as an efficient carrier, widening the scope for delivery of other immunostimulants in aquaculture.
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http://dx.doi.org/10.1016/j.fsi.2019.06.016DOI Listing
September 2019

Fish Red Blood Cells Modulate Immune Genes in Response to Bacterial Inclusion Bodies Made of TNFα and a G-VHSV Fragment.

Front Immunol 2019 22;10:1055. Epub 2019 May 22.

Departamento de Bioquímica y Biología Molecular, Instituto de Biología Molecular y Celular (IBMC), Universidad Miguel Hernández (UMH), Elche, Spain.

Fish Red-Blood Cells (RBCs) are nucleated cells that can modulate the expression of different sets of genes in response to stimuli, playing an active role in the homeostasis of the fish immune system. Nowadays, vaccination is one of the main ways to control and prevent viral diseases in aquaculture and the development of novel vaccination approaches is a focal point in fish vaccinology. One of the strategies that has recently emerged is the use of nanostructured recombinant proteins. Nanostructured cytokines have already been shown to immunostimulate and protect fish against bacterial infections. To explore the role of RBCs in the immune response to two nanostructured recombinant proteins, TNF and a G-VHSV protein fragment, we performed different and studies. We show for the first time that rainbow trout RBCs are able to endocytose nanostructured TNF and G-VHSV protein fragment , despite not being phagocytic cells, and in response to nanostructured TNF and G-VHSV fragment, the expression of different immune genes could be modulated.
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http://dx.doi.org/10.3389/fimmu.2019.01055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6538768PMC
August 2020

Metazoan evolution of glutamate receptors reveals unreported phylogenetic groups and divergent lineage-specific events.

Elife 2018 11 22;7. Epub 2018 Nov 22.

Molecular Physiology of the Synapse Laboratory, Biomedical Research Institute Sant Pau, Barcelona, Spain.

Glutamate receptors are divided in two unrelated families: ionotropic (iGluR), driving synaptic transmission, and metabotropic (mGluR), which modulate synaptic strength. The present classification of GluRs is based on vertebrate proteins and has remained unchanged for over two decades. Here we report an exhaustive phylogenetic study of GluRs in metazoans. Importantly, we demonstrate that GluRs have followed different evolutionary histories in separated animal lineages. Our analysis reveals that the present organization of iGluRs into six classes does not capture the full complexity of their evolution. Instead, we propose an organization into four subfamilies and ten classes, four of which have never been previously described. Furthermore, we report a sister class to mGluR classes I-III, class IV. We show that many unreported proteins are expressed in the nervous system, and that new Epsilon receptors form functional ligand-gated ion channels. We propose an updated classification of glutamate receptors that includes our findings.
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http://dx.doi.org/10.7554/eLife.35774DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6307864PMC
November 2018

Characterization of the TLR Family in and Discovery of a Novel TLR22-Like Involved in dsRNA Recognition in Amphioxus.

Front Immunol 2018 2;9:2525. Epub 2018 Nov 2.

Department of Cell Biology, Animal Physiology and Immunology, Institute of Biotechnology and Biomedicine (IBB), Universitat Autònoma de Barcelona, Bellaterra, Spain.

Toll-like receptors (TLRs) are important for raising innate immune responses in both invertebrates and vertebrates. Amphioxus belongs to an ancient chordate lineage which shares key features with vertebrates. The genomic research on TLR genes in and reveals the expansion of TLRs in amphioxus. However, the repertoire of TLRs in has not been studied and the functionality of amphioxus TLRs has not been reported. We have identified from transcriptomic data 30 new putative TLRs in and all of them are transcribed in adult amphioxus. Phylogenetic analysis showed that the repertoire of TLRs consists of both non-vertebrate and vertebrate-like TLRs. It also indicated a lineage-specific expansion in orthologous clusters of the vertebrate TLR11 family. We did not detect any representatives of the vertebrate TLR1, TLR3, TLR4, TLR5 and TLR7 families. To gain insight into these TLRs, we studied in depth a particular TLR highly similar to a gene annotated as bbtTLR1. The phylogenetic analysis of this novel BlTLR showed that it clusters with the vertebrate TLR11 family and it might be more related to TLR13 subfamily according to similar domain architecture. Transient and stable expression in HEK293 cells showed that the BlTLR localizes on the plasma membrane, but it did not respond to the most common mammalian TLR ligands. However, when the ectodomain of BlTLR is fused to the TIR domain of human TLR2, the chimeric protein could indeed induce NF-κB transactivation in response to the viral ligand Poly I:C, also indicating that in amphioxus, specific accessory proteins are needed for downstream activation. Based on the phylogenetic, subcellular localization and functional analysis, we propose that the novel BlTLR might be classified as an antiviral receptor sharing at least partly the functions performed by vertebrate TLR22. TLR22 is thought to be viral teleost-specific TLR but here we demonstrate that teleosts and amphioxus TLR22-like probably shared a common ancestor. Additional functional studies with other lancelet TLR genes will enrich our understanding of the immune response in amphioxus and will provide a unique perspective on the evolution of the immune system.
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http://dx.doi.org/10.3389/fimmu.2018.02525DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6224433PMC
October 2019

Oral Intubation of Adult Zebrafish: A Model for Evaluating Intestinal Uptake of Bioactive Compounds.

J Vis Exp 2018 09 27(139). Epub 2018 Sep 27.

Institute of Biotechnology and Biomedicine (IBB-UAB), Universitat Autònoma de Barcelona; Department of Cell Biology, Animal Physiology and Immunology, Universitat Autònoma de Barcelona;

Most pathogens invade organisms through their mucosa. This is particularly true in fish as they are continuously exposed to a microbial-rich water environment. Developing effective methods for oral delivery of immunostimulants or vaccines, which activate the immune system against infectious diseases, is highly desirable. In devising prophylactic tools, good experimental models are needed to test their performance. Here, we show a method for oral intubation of adult zebrafish and a set of procedures to dissect and prepare the intestine for cytometry, confocal microscopy and quantitative polymerase chain reaction (qPCR) analysis. With this protocol, we can precisely administer volumes up to 50 µL to fish weighing approximately 1 g simply and quickly, without harming the animals. This method allows us to explore the direct in vivo uptake of fluorescently labelled compounds by the intestinal mucosa and the immunomodulatory capacity of such biologics at the local site after intubation. By combining downstream methods such as flow cytometry, histology, qPCR and confocal microscopy of the intestinal tissue, we can understand how immunostimulants or vaccines are able to cross the intestinal mucosal barriers, pass through the lamina propria, and reach the muscle, exerting an effect on the intestinal mucosal immune system. The model could be used to test candidate oral prophylactics and delivery systems or the local effect of any orally administered bioactive compound.
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http://dx.doi.org/10.3791/58366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6235345PMC
September 2018

Protein Nanoparticles Made of Recombinant Viral Antigens: A Promising Biomaterial for Oral Delivery of Fish Prophylactics.

Front Immunol 2018 18;9:1652. Epub 2018 Jul 18.

Department of Cellular Biology, Physiology and Immunology, Institute of Biotechnology and Biomedicine (IBB), Universitat Autònoma de Barcelona, Barcelona, Spain.

In the search for an eminently practical strategy to develop immunostimulants and vaccines for farmed fish, we have devised recombinant viral antigens presented as "nanopellets" (NPs). These are inclusion bodies of fish viral antigenic proteins produced in . Soluble recombinant proteins are too labile to endure the environment and maintain full functionality, and therefore require encapsulation strategies. Yet when they are produced as nanostructures, they can withstand the wide range of gastrointestinal pH found in fish, high temperatures, and lyophilization. Moreover, these nanomaterials are biologically active, non-toxic to fish, cost-effective regarding production and suitable for oral administration. Here, we present three versions of NPs formed by antigenic proteins from relevant viruses affecting farmed fish: the viral nervous necrosis virus coat protein, infectious pancreatic necrosis virus viral protein 2, and a viral haemorrhagic septicemia virus G glycoprotein fragment. We demonstrate that the nanoparticles are taken up by zebrafish ZFL cells and by intubating zebrafish as a proof of concept for oral delivery. Encouragingly, analysis of gene expression suggests these NPs evoke an antiviral innate immune response in ZFL cells and in rainbow trout head kidney macrophages. They are therefore a promising platform for immunostimulants and may be candidates for vaccines should protection be demonstrated.
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http://dx.doi.org/10.3389/fimmu.2018.01652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6060434PMC
July 2018

Functional evidence for the inflammatory reflex in teleosts: A novel α7 nicotinic acetylcholine receptor modulates the macrophage response to dsRNA.

Dev Comp Immunol 2018 07 1;84:279-291. Epub 2018 Mar 1.

Institut de Biotecnologia i de Biomedicina (IBB), Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain; Departament de Biologia Cel·lular, Fisiologia Animal i Immunologia, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain. Electronic address:

The inflammatory reflex modulates the innate immune system, keeping in check the detrimental consequences of overstimulation. A key player controlling the inflammatory reflex is the alpha 7 acetylcholine receptor (α7nAChR). This receptor is one of the signalling molecules regulating cytokine expression in macrophages. In this study, we characterize a novel teleost α7nAChR. Protein sequence analysis shows a high degree of conservation with mammalian orthologs and trout α7nAChR has all the features and essential amino acids to form a fully functional receptor. We demonstrate that trout macrophages can bind α-bungarotoxin (α-BTX), a competitive antagonist for α7nAChRs. Moreover, nicotine stimulation produces a decrease in pro-inflammatory cytokine expression after stimulation with poly(I:C). These results suggest the presence of a functional α7nAChR in the macrophage plasma membrane. Further, in vivo injection of poly(I:C) induced an increase in serum ACh levels in rainbow trout. Our results manifest for the first time the functional conservation of the inflammatory reflex in teleosts.
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http://dx.doi.org/10.1016/j.dci.2018.02.020DOI Listing
July 2018

Complex Particulate Biomaterials as Immunostimulant-Delivery Platforms.

PLoS One 2016 7;11(10):e0164073. Epub 2016 Oct 7.

Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain.

The control of infectious diseases is a major current challenge in intensive aquaculture. Most commercial vaccines are based on live attenuated or inactivated pathogens that are usually combined with adjuvants, oil emulsions being as the most widely used for vaccination in aquaculture. Although effective, the use of these oil emulsions is plagued with important side effects. Thus, the development of alternative safer and cost-effective immunostimulants and adjuvants is highly desirable. Here we have explored the capacity of inclusion bodies produced in bacteria to immunostimulate and protect fish against bacterial infections. Bacterial inclusion bodies are highly stable, non-toxic protein-based biomaterials produced through fully scalable and low-cost bio-production processes. The present study shows that the composition and structured organization of inclusion body components (protein, lipopolysaccharide, peptidoglycan, DNA and RNA) make these protein biomaterials excellent immunomodulators able to generically protect fish against otherwise lethal bacterial challenges. The results obtained in this work provide evidence that their inherent nature makes bacterial inclusion bodies exceptionally attractive as immunostimulants and this opens the door to the future exploration of this biomaterial as an alternative adjuvant for vaccination purposes in veterinary.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0164073PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5055299PMC
June 2017

Nanostructured recombinant cytokines: A highly stable alternative to short-lived prophylactics.

Biomaterials 2016 11 27;107:102-14. Epub 2016 Aug 27.

Institut de Biotecnologia i de Biomedicina (IBB), Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain; Departament de Biologia Cel·lular, Fisiologia Animal i Immunologia, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain. Electronic address:

Cytokines have been widely used as adjuvants and therapeutic agents in treatments of human diseases. Despite their recognized potential as drugs, the medical use of cytokines has considerable drawbacks, mainly related to their low stability and short half-life. Such intrinsic limitations imply the administration of high doses, often prompting toxicity, undesirable side effects and greater production costs. Here, we describe a new category of mechanically stable nanostructured cytokines (TNFα and CCL4/MIP-1β) that resist harsh physicochemical conditions in vitro (pH and temperature), while maintaining functionality. These bio-functional materials are produced in recombinant cell factories through cost-effective and fully scalable processes. Notably, we demonstrate their prophylactic potential in vivo showing they protect zebrafish from a lethal infection by Pseudomonas aeruginosa.
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http://dx.doi.org/10.1016/j.biomaterials.2016.08.043DOI Listing
November 2016

Nanodelivery Systems as New Tools for Immunostimulant or Vaccine Administration: Targeting the Fish Immune System.

Biology (Basel) 2015 Oct 19;4(4):664-96. Epub 2015 Oct 19.

Institut de Biotecnologia i de Biomedicina-Parc de Recerca UAB, Universitat Autònoma de Barcelona, Bellaterra 08193, Spain.

Fish disease treatments have progressed significantly over the last few years and have moved from the massive use of antibiotics to the development of vaccines mainly based on inactivated bacteria. Today, the incorporation of immunostimulants and antigens into nanomaterials provide us with new tools to enhance the performance of immunostimulation. Nanoparticles are dispersions or solid particles designed with specific physical properties (size, surface charge, or loading capacity), which allow controlled delivery and therefore improved targeting and stimulation of the immune system. The use of these nanodelivery platforms in fish is in the initial steps of development. Here we review the advances in the application of nanoparticles to fish disease prevention including: the type of biomaterial, the type of immunostimulant or vaccine loaded into the nanoparticles, and how they target the fish immune system.
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http://dx.doi.org/10.3390/biology4040664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4690013PMC
October 2015

Decoding the genetic and functional diversity of the DSF quorum-sensing system in Stenotrophomonas maltophilia.

Front Microbiol 2015 28;6:761. Epub 2015 Jul 28.

Institut de Biotecnologia i de Biomedicina (IBB), Universitat Autònoma de Barcelona (UAB) Barcelona, Spain ; Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona Barcelona, Spain.

Stenotrophomonas maltophilia uses the Diffusible Signal Factor (DSF) quorum sensing (QS) system to mediate intra- and inter-specific signaling and regulate virulence-related processes. The components of this system are encoded by the rpf cluster, with genes rpfF and rpfC encoding for the DSF synthase RpfF and sensor RpfC, respectively. Recently, we have shown that there exist two variants of the rpf cluster (rpf-1 and rpf-2), distinguishing two groups of S. maltophilia strains. Surprisingly, only rpf-1 strains produce detectable DSF, correlating with their ability to control biofilm formation, swarming motility and virulence. The evolutive advantage of acquiring two different rpf clusters, the phylogenetic time point and mechanism of this acquisition and the conditions that activate DSF production in rpf-2 strains, are however not known. Examination of this cluster in various species suggests that its variability originated most probably by genetic exchange between rhizosphere bacteria. We propose that rpf-2 variant strains make use of a strategy recently termed as "social cheating." Analysis of cellular and extracellular fatty acids (FAs) of strains E77 (rpf-1) and M30 (rpf-2) suggests that their RpfFs have also a thioesterase activity that facilitates the release of unspecific FAs to the medium in addition to DSF. Production of DSF in rpf-1 strains appears in fact to be modulated by some of these extracellular FAs in addition to other factors such as temperature and nutrients, while in rpf-2 strains DSF biosynthesis is derepressed only upon detection of DSF itself, suggesting that they require cohabitation with DSF-producer bacteria to activate their DSF regulatory machinery. Finally, we show that the mixed rpf-1/rpf-2 population presents synergism in DSF production and virulence capacity in an in vivo infection model. Recovery and quantification of DSF from co-infected animals correlates with the observed mortality rate.
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http://dx.doi.org/10.3389/fmicb.2015.00761DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4517397PMC
August 2015

AIM/CD5L: a key protein in the control of immune homeostasis and inflammatory disease.

J Leukoc Biol 2015 Aug 5;98(2):173-84. Epub 2015 Jun 5.

*Innate Immunity Group, Health Sciences Research Institute Germans Trias i Pujol, Badalona, Spain; Evolutive Immunology Group, Institute of Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, Barcelona, Spain; Nuclear Receptor Group, Department of Physiology and Immunology, School of Biology, University of Barcelona, Barcelona, Spain; and Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Barcelona, Spain

CD5L, a soluble protein belonging to the SRCR superfamily, is expressed mostly by macrophages in lymphoid and inflamed tissues. The expression of this protein is transcriptionally controlled by LXRs, members of the nuclear receptor family that play major roles in lipid homeostasis. Research undertaken over the last decade has uncovered critical roles of CD5L as a PRR of bacterial and fungal components and in the control of key mechanisms in inflammatory responses, with involvement in processes, such as infection, atherosclerosis, and cancer. In this review, we summarize the current knowledge of CD5L, its roles at the intersection between lipid homeostasis and immune response, and its potential use as a diagnostic biomarker in a variety of diseases, such as TB and liver cirrhosis.
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http://dx.doi.org/10.1189/jlb.3RU0215-074RDOI Listing
August 2015

In Vivo Molecular Responses of Fast and Slow Muscle Fibers to Lipopolysaccharide in a Teleost Fish, the Rainbow Trout (Oncorhynchus mykiss).

Biology (Basel) 2015 Feb 4;4(1):67-87. Epub 2015 Feb 4.

Departament de Fisiologia i Immunologia, Facultat de Biologia, Universitat de Barcelona and Institut de Biomedicina de la Universitat de Barcelona (IBUB), Barcelona 08028, Spain.

The physiological consequences of the activation of the immune system in skeletal muscle in fish are not completely understood. To study the consequences of the activation of the immune system by bacterial pathogens on skeletal muscle function, we administered lipopolysaccharide (LPS), an active component of Gram-negative bacteria, in rainbow trout and performed transcriptomic and proteomic analyses in skeletal muscle. We examined changes in gene expression in fast and slow skeletal muscle in rainbow trout at 24 and 72 h after LPS treatment (8 mg/kg) by microarray analysis. At the transcriptional level, we observed important changes in metabolic, mitochondrial and structural genes in fast and slow skeletal muscle. In slow skeletal muscle, LPS caused marked changes in the expression of genes related to oxidative phosphorylation, while in fast skeletal muscle LPS administration caused major changes in the expression of genes coding for glycolytic enzymes. We also evaluated the effects of LPS administration on the fast skeletal muscle proteome and identified 14 proteins that were differentially induced in LPS-treated trout, primarily corresponding to glycolytic enzymes. Our results evidence a robust and tissue-specific response of skeletal muscle to an acute inflammatory challenge, affecting energy utilization and possibly growth in rainbow trout.
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http://dx.doi.org/10.3390/biology4010067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4381218PMC
February 2015

Zebrafish liver (ZFL) cells are able to mount an anti-viral response after stimulation with Poly (I:C).

Comp Biochem Physiol B Biochem Mol Biol 2015 Apr 20;182:55-63. Epub 2014 Dec 20.

Institut de Biotecnologia i de Biomedicina - Parc de Recerca UAB, Universitat Autònoma de Barcelona, Bellaterra, Spain; Departament de Biologia Cel·lular, Fisiologia Animal i Immunologia, Universitat Autònoma de Barcelona, Bellaterra, Spain. Electronic address:

The zebrafish (Danio rerio) is a widely used model species for biomedical research and is also starting to be a model for aquaculture research. The ZFL cell line, established from zebrafish liver, has been mostly used in toxicological and ecotoxicological studies. However, no studies have previously characterised this cell line in regard to its immunological response. The aim of this work was to study the gene expression response of the ZFL cell line after incubation with different prototypical immune stimuli, such as lipopolysaccharide (LPS), peptidoglycan (PGN), zymosan, and with a special focus on the dsRNA Poly (I:C). Using PCR, microarrays, and confocal microscopy we have explored the response of the ZFL cells against Poly (I:C). This study shows that the ZFL is able to uptake very efficiently the Poly (I:C) and mount a strong anti-viral response. We can conclude that ZFL could be used not only in toxicological studies, but also in studying anti-viral responses in zebrafish.
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http://dx.doi.org/10.1016/j.cbpb.2014.12.002DOI Listing
April 2015

Synthesis, culture medium stability, and in vitro and in vivo zebrafish embryo toxicity of metal-organic framework nanoparticles.

Chemistry 2015 Feb 11;21(6):2508-18. Epub 2014 Dec 11.

ICN2 (ICN-CSIC), Institut Catala de Nanociencia i Nanotecnologia, Esfera UAB, 08193 Bellaterra, Barcelona (Spain); Institut de Biotecnologia i de Biomedicina, Parc de Recerca UAB, Campus Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona (Spain).

Metal-organic frameworks (MOFs) are among the most attractive porous materials available today. They have garnered much attention for their potential utility in many different areas such as gas storage, separation, catalysis, and biomedicine. However, very little is known about the possible health or environmental risks of these materials. Here, the results of toxicity studies on sixteen representative uncoated MOF nanoparticles (nanoMOFs), which were assessed for cytotoxicity to HepG2 and MCF7 cells in vitro, and for toxicity to zebrafish embryos in vivo, are reported. Interestingly, there is a strong correlation between their in vitro toxicity and their in vivo toxicity. NanoMOFs were ranked according to their respective in vivo toxicity (in terms of the amount and severity of phenotypic changes observed in the treated zebrafish embryos), which varied widely. Altogether these results show different levels of toxicity of these materials; however, leaching of solubilized metal ions plays a main role.
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http://dx.doi.org/10.1002/chem.201405380DOI Listing
February 2015

Amyloid formation by human carboxypeptidase D transthyretin-like domain under physiological conditions.

J Biol Chem 2014 Dec 7;289(49):33783-96. Epub 2014 Oct 7.

From the Institut de Biotecnologia i Biomedicina, Departaments de Bioquimica i Biologia Molecular and

Protein aggregation is linked to a growing list of diseases, but it is also an intrinsic property of polypeptides, because the formation of functional globular proteins comes at the expense of an inherent aggregation propensity. Certain proteins can access aggregation-prone states from native-like conformations without the need to cross the energy barrier for unfolding. This is the case of transthyretin (TTR), a homotetrameric protein whose dissociation into its monomers initiates the aggregation cascade. Domains with structural homology to TTR exist in a number of proteins, including the M14B subfamily carboxypeptidases. We show here that the monomeric transthyretin-like domain of human carboxypeptidase D aggregates under close to physiological conditions into amyloid structures, with the population of folded but aggregation-prone states being controlled by the conformational stability of the domain. We thus confirm that the TTR fold keeps a generic residual aggregation propensity upon folding, resulting from the presence of preformed amyloidogenic β-strands in the native state. These structural elements should serve for functional/structural purposes, because they have not been purged out by evolution, but at the same time they put proteins like carboxypeptidase D at risk of aggregation in biological environments and thus can potentially lead to deposition diseases.
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http://dx.doi.org/10.1074/jbc.M114.594804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4256313PMC
December 2014

The involvement of cholesterol in sepsis and tolerance to lipopolysaccharide highlighted by the transcriptome analysis of zebrafish (Danio rerio).

Zebrafish 2014 Oct 2;11(5):421-33. Epub 2014 Sep 2.

1 Instituto de Investigaciones Marinas (IIM)-Consejo Superior de Investigaciones Científicas (CSIC) , Vigo, Spain .

Septic shock is the most common cause of death in intensive care units due to an aggressive inflammatory response that leads to multiple organ failure. However, a lipopolysaccharide (LPS) tolerance phenomenon (a nonreaction to LPS), is also often described. Neither the inflammatory response nor the tolerance is completely understood. In this work, both of these responses were analyzed using microarrays in zebrafish. Fish that were 4 or 6 days postfertilization (dpf) and received a lethal dose (LD) of LPS exhibited 100% mortality in a few days. Their transcriptome profile, even at 4 dpf, resembled the profile in humans with severe sepsis. Moreover, we selected 4-dpf fish to set up a tolerance protocol: fish treated with a nonlethal concentration of Escherichia coli LPS exhibited complete protection against the LD of LPS. Most of the main inflammatory molecules described in mammals were represented in the zebrafish microarray experiments. Additionally and focusing on this tolerance response, the use of cyclodextrins may mobilize cholesterol reservoirs to decrease mortality after a LD dose of LPS. Therefore, it is possible that the use of the whole animal could provide some clues to enhance the understanding of the inflammatory/tolerance response and to guide drug discovery.
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http://dx.doi.org/10.1089/zeb.2014.0995DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4172467PMC
October 2014

Targeting and stimulation of the zebrafish (Danio rerio) innate immune system with LPS/dsRNA-loaded nanoliposomes.

Vaccine 2014 Jun 14;32(31):3955-62. Epub 2014 May 14.

Institut de Biotecnologia i de Biomedicina - Parc de Recerca UAB and Dep. de Biologia Cellular, Immunologia i Fisiologia Animal, Universitat Autònoma de Barcelona, Bellaterra, Spain. Electronic address:

Herein we report the use of immunostimulant-loaded nanoliposomes (called NLcliposomes) as a strategy to protect fish against bacterial and/or viral infections. This work entailed developing a method for in vivo tracking of the liposomes administered to adult zebrafish that enables evaluation of their in vivo dynamics and characterisation of their tissue distribution. The NLc liposomes, which co-encapsulate poly(I:C) and LPS, accumulate in immune tissues and in immunologically relevant cells such as macrophages, as has been assessed in trout primary cell cultures. They protect zebrafish against otherwise lethal bacterial (Pseudomonas aeruginosa PAO1) and viral (Spring Viraemia of Carp Virus) infections regardless of whether they are administered by injection or by immersion, as demonstrated in a series of in vivo infection experiments with adult zebrafish. Importantly, protection was not achieved in fish that had been treated with empty liposomes or with a mixture of the free immunostimulants. Our findings indicate that stimulation of the innate immune system with co-encapsulated immunostimulants in nano-liposomes is a promising strategy to simultaneously improve the levels of protection against bacterial and viral infections in fish.
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http://dx.doi.org/10.1016/j.vaccine.2014.05.010DOI Listing
June 2014

Two different rpf clusters distributed among a population of Stenotrophomonas maltophilia clinical strains display differential diffusible signal factor production and virulence regulation.

J Bacteriol 2014 Jul 25;196(13):2431-42. Epub 2014 Apr 25.

Institut de Biotecnologia i de Biomedicina (IBB), Universitat Autònoma de Barcelona (UAB), Cerdanyola del Vallès (Barcelona), Spain Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona (UAB), Cerdanyola del Vallès (Barcelona), Spain

The quorum-sensing (QS) system present in the emerging nosocomial pathogen Stenotrophomonas maltophilia is based on the signaling molecule diffusible signal factor (DSF). Production and detection of DSF are governed by the rpf cluster, which encodes the synthase RpfF and the sensor RpfC, among other components. Despite a well-studied system, little is known about its implication in virulence regulation in S. maltophilia. Here, we have analyzed the rpfF gene from 82 S. maltophilia clinical isolates. Although rpfF was found to be present in all of the strains, it showed substantial variation, with two populations (rpfF-1 and rpfF-2) clearly distinguishable by the N-terminal region of the protein. Analysis of rpfC in seven complete genome sequences revealed a corresponding variability in the N-terminal transmembrane domain of its product, suggesting that each RpfF variant has an associated RpfC variant. We show that only RpfC-RpfF-1 variant strains display detectable DSF production. Heterologous rpfF complementation of ΔrpfF mutants of a representative strain of each variant suggests that RpfF-2 is, however, functional and that the observed DSF-deficient phenotype of RpfC-RpfF-2 variant strains is due to permanent repression of RpfF-2 by RpfC-2. This is corroborated by the ΔrpfC mutant of the RpfC-RpfF-2 representative strain. In line with this observations, deletion of rpfF from the RpfC-RpfF-1 strain leads to an increase in biofilm formation, a decrease in swarming motility, and relative attenuation in the Caenorhabditis elegans and zebrafish infection models, whereas deletion of the same gene from the representative RpfC-RpfF-2 strain has no significant effect on these virulence-related phenotypes.
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http://dx.doi.org/10.1128/JB.01540-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4054175PMC
July 2014

A novel liposome-based nanocarrier loaded with an LPS-dsRNA cocktail for fish innate immune system stimulation.

PLoS One 2013 18;8(10):e76338. Epub 2013 Oct 18.

Institut de Biotecnologia i de Biomedicina, Parc de Recerca UAB, Universitat Autònoma de Barcelona, Barcelona, Spain.

Development of novel systems of vaccine delivery is a growing demand of the aquaculture industry. Nano- and micro- encapsulation systems are promising tools to achieve efficient vaccines against orphan vaccine fish diseases. In this context, the use of liposomal based-nanocarriers has been poorly explored in fish; although liposomal nanocarriers have successfully been used in other species. Here, we report a new ∼125 nm-in-diameter unilamellar liposome-encapsulated immunostimulant cocktail containing crude lipopolysaccharide (LPS) from E. coli and polyinosinic:polycytidylic acid [poly (I:C)], a synthetic analog of dsRNA virus, aiming to be used as a non-specific vaccine nanocarrier in different fish species. This liposomal carrier showed high encapsulation efficiencies and low toxicity not only in vitro using three different cellular models but also in vivo using zebrafish embryos and larvae. We showed that such liposomal LPS-dsRNA cocktail is able to enter into contact with zebrafish hepatocytes (ZFL cell line) and trout macrophage plasma membranes, being preferentially internalized through caveolae-dependent endocytosis, although clathrin-mediated endocytosis in ZFL cells and macropinocytocis in macrophages also contribute to liposome uptake. Importantly, we also demonstrated that this liposomal LPS-dsRNA cocktail elicits a specific pro-inflammatory and anti-viral response in both zebrafish hepatocytes and trout macrophages. The design of a unique delivery system with the ability to stimulate two potent innate immunity pathways virtually present in all fish species represents a completely new approach in fish health.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0076338PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3799751PMC
August 2014

Behavioural fever is a synergic signal amplifying the innate immune response.

Proc Biol Sci 2013 Sep 7;280(1766):20131381. Epub 2013 Sep 7.

Institut de Biotecnologia i de Biomedicina, Universitat Autonoma de Barcelona, , Bellaterra (Barcelona) 08193, Spain.

Behavioural fever, defined as an acute change in thermal preference driven by pathogen recognition, has been reported in a variety of invertebrates and ectothermic vertebrates. It has been suggested, but so far not confirmed, that such changes in thermal regime favour the immune response and thus promote survival. Here, we show that zebrafish display behavioural fever that acts to promote extensive and highly specific temperature-dependent changes in the brain transcriptome. The observed coupling of the immune response to fever acts at the gene-environment level to promote a robust, highly specific time-dependent anti-viral response that, under viral infection, increases survival. Fish that are not offered a choice of temperatures and that therefore cannot express behavioural fever show decreased survival under viral challenge. This phenomenon provides an underlying explanation for the varied functional responses observed during systemic fever. Given the effects of behavioural fever on survival and the fact that it exists across considerable phylogenetic space, such immunity-environment interactions are likely to be under strong positive selection.
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http://dx.doi.org/10.1098/rspb.2013.1381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3730603PMC
September 2013

Abundance of the Quorum-Sensing Factor Ax21 in Four Strains of Stenotrophomonas maltophilia Correlates with Mortality Rate in a New Zebrafish Model of Infection.

PLoS One 2013 26;8(6):e67207. Epub 2013 Jun 26.

Institut de Biotecnologia i de Biomedicina (IBB), Universitat Autònoma de Barcelona (UAB), Cerdanyola del Vallès, Barcelona, Spain.

Stenotrophomonas maltophilia is a Gram-negative pathogen with emerging nosocomial incidence. Little is known about its pathogenesis and the genomic diversity exhibited by clinical isolates complicates the study of pathogenicity and virulence factors. Here, we present a strategy to identify such factors in new clinical isolates of S. maltophilia, incorporating an adult-zebrafish model of S. maltophilia infection to evaluate relative virulence coupled to 2D difference gel electrophoresis to explore underlying differences in protein expression. In this study we report upon three recent clinical isolates and use the collection strain ATCC13637 as a reference. The adult-zebrafish model shows discrimination capacity, i.e. from very low to very high mortality rates, with clinical symptoms very similar to those observed in natural S. maltophilia infections in fish. Strain virulence correlates with resistance to human serum, in agreement with previous studies in mouse and rat and therefore supporting zebrafish as a replacement model. Despite its clinical origin, the collection strain ATCC13637 showed obvious signs of attenuation in zebrafish, with null mortality. Multilocus-sequence-typing analysis revealed that the most virulent strains, UV74 and M30, exhibit the strongest genetic similitude. Differential proteomic analysis led to the identification of 38 proteins with significantly different abundance in the three clinical strains relative to the reference strain. Orthologs of several of these proteins have been already reported to have a role in pathogenesis, virulence or resistance mechanisms thus supporting our strategy. Proof of concept is further provided by protein Ax21, whose abundance is shown here to be directly proportional to mortality in the zebrafish infection model. Indeed, recent studies have demonstrated that this protein is a quorum-sensing-related virulence factor.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0067207PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3693955PMC
October 2017

RNA-Seq reveals an integrated immune response in nucleated erythrocytes.

PLoS One 2011 27;6(10):e26998. Epub 2011 Oct 27.

Institute of Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, Barcelona, Spain.

Background: Throughout the primary literature and within textbooks, the erythrocyte has been tacitly accepted to have maintained a unique physiological role; namely gas transport and exchange. In non-mammalian vertebrates, nucleated erythrocytes are present in circulation throughout the life cycle and a fragmented series of observations in mammals support a potential role in non-respiratory biological processes. We hypothesised that nucleated erythrocytes could actively participate via ligand-induced transcriptional re-programming in the immune response.

Methodology/principal Findings: Nucleated erythrocytes from both fish and birds express and regulate specific pattern recognition receptor (PRR) mRNAs and, thus, are capable of specific pathogen associated molecular pattern (PAMP) detection that is central to the innate immune response. In vitro challenge with diverse PAMPs led to de novo specific mRNA synthesis of both receptors and response factors including interferon-alpha (IFNα) that exhibit a stimulus-specific polysomal shift supporting active translation. RNA-Seq analysis of the PAMP (Poly (I:C), polyinosinic:polycytidylic acid)-erythrocyte response uncovered diverse cohorts of differentially expressed mRNA transcripts related to multiple physiological systems including the endocrine, reproductive and immune. Moreover, erythrocyte-derived conditioned mediums induced a type-1 interferon response in macrophages thus supporting an integrative role for the erythrocytes in the immune response.

Conclusions/significance: We demonstrate that nucleated erythrocytes in non-mammalian vertebrates spanning significant phylogenetic distance participate in the immune response. RNA-Seq studies highlight a mRNA repertoire that suggests a previously unrecognized integrative role for the erythrocytes in other physiological systems.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0026998PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3203173PMC
March 2012

Tumor necrosis factor alpha may act as an intraovarian mediator of luteinizing hormone-induced oocyte maturation in trout.

Biol Reprod 2012 Jan 10;86(1):1-12. Epub 2012 Jan 10.

Departament de Fisiologia i Immunologia, Facultat de Biologia, Universitat de Barcelona and Institut de Biomedicina de la Universitat de Barcelona, Spain.

In fish, like in other vertebrates, luteinizing hormone (Lh) is an essential hormone for the completion of oocyte maturation. In salmonid fish (i.e., salmon and trout), oocyte maturation is induced by Lh through its stimulation of the production of the maturation-inducing steroid, 17alpha,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P). In mammals, several factors, including ovarian cytokines and growth factors, have been reported to contribute to the regulation of oocyte maturation. In fish, growing evidence suggests that tumor necrosis factor alpha (hereafter referred to as Tnf) could play multiple physiological roles in the control of ovarian function. In the present study, we have investigated the possible involvement of Tnf in the regulation of oocyte maturation in brown trout (Salmo trutta). Our results show that in vitro treatment of brown trout preovulatory follicles with coho salmon (Oncorhynchus kisutch) Lh (sLh) significantly increased oocyte maturation, as assessed by germinal vesicle breakdown (GVBD), and that this effect was blocked by TAPI-1 (an inhibitor of Tnf-converting enzyme or Tace/Adam17). Furthermore, treatment of preovulatory follicles with sLh increased the expression of tnf and tace/adam17 as well as the secretion of the Tnf protein. Importantly, recombinant trout Tnf (rtTnf) significantly increased GVBD in vitro. Our results also show that the stimulatory effects of rtTnf on oocyte maturation may be the result of the direct involvement of rtTnf in stimulating the production of the maturation-inducing steroid as evidenced, first, by the stimulatory effects of rtTnf on 17,20beta-P production in vitro and on the expression of cholesterol side-chain cleavage P450 cytochrome (p450scc) and 20beta-hydroxysteroid dehydrogenase/carbonyl reductase 1 (cbr1), the enzyme responsible for the production of 17,20beta-P, and, second, by the ability of TAPI-1 to block the stimulatory effects of sLh on 17,20beta-P production and cbr1 expression. Furthermore, sLh and rtTnf increased the expression of the Lh receptor (lhr) and decreased the expression of aromatase (cyp19a1), and TAPI-1 completely blocked the effects of sLh. These results strongly suggest that Tnf may contribute to the regulation of oocyte maturation by Lh in trout.
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http://dx.doi.org/10.1095/biolreprod.111.094433DOI Listing
January 2012

PAMPs, PRRs and the genomics of gram negative bacterial recognition in fish.

Dev Comp Immunol 2011 Dec 29;35(12):1195-203. Epub 2011 Mar 29.

Institute of Biotechnology and Biomedicine, Dep. Biologia Cel·lular, Immunologia i Fisiologia Animal, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain.

Understanding the mechanisms that underpin pathogen recognition and subsequent orchestration of the immune response in fish is an area of significant importance for both basic research and management of health in aquaculture. In recent years much attention has been given to the identification of pattern recognition receptors (PRRs) in fish, however, characterisation of interactions with specific pathogen-associated molecular patterns (PAMPs) is still incomplete. Microarray studies have significantly contributed to functional studies and early descriptions of PAMP-PRR driven activation of specific response cassettes in the genome have been obtained although much is left to be done. In this review we will address gram negative (G-negative) bacterial recognition in fish addressing contributing factors such as structure-function relationships between G-negative PAMPs, current knowledge of fish PRRs and the input achieved by microarray-based studies ranging from in vivo infection studies to directed in vitro PAMP-cell studies. Finally we revisit the endotoxic recognition paradigm in fish and suggest a series of future perspectives that could contribute toward the further elucidation of G-negative bacterial recognition across the highly diverse group of vertebrates that encompass the fishes.
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http://dx.doi.org/10.1016/j.dci.2011.02.010DOI Listing
December 2011

Direct involvement of tumor necrosis factor-α in the regulation of glucose uptake in rainbow trout muscle cells.

Am J Physiol Regul Integr Comp Physiol 2011 Mar 29;300(3):R716-23. Epub 2010 Dec 29.

Departament de Fisiologia, Facultat de Biologia, Universitat de Barcelona, Institut de Biomedicina de la Universitat de Barcelona, Spain.

The proinflammatory cytokine TNF-α is known to have a direct action on skeletal muscle in mammals. However, little is known regarding the potential effects of cytokines on nonimmune tissues, particularly in skeletal muscle, in fish. The aim of this study was to investigate the effects of recombinant trout TNF-α (rtTNF-α) on skeletal muscle carbohydrate metabolism in rainbow trout (Oncorhynchus mykiss). We used a primary cell culture of muscle cells from rainbow trout to show that rtTNF-α stimulates glucose uptake in myoblasts and myotubes at concentrations that do not affect the viability of the cells, requiring de novo protein synthesis as shown by the impairment of rtTNF-α-stimulated glucose uptake by cycloheximide. With the use of specific inhibitors, we show that rtTNF-α-stimulated glucose uptake is mediated by the p38MAPK, NF-κB, and JNK pathways. Additionally, we provide evidence that the stimulatory effects of rtTNF-α on glucose uptake in trout skeletal muscle cells may be caused, at least in part, by an increase in the amount of GLUT4 at the plasma membrane. Incubation of trout muscle cells with conditioned medium from LPS-stimulated trout macrophages, enriched in TNF-α, increased glucose uptake. Our results indicate that recombinant, as well as native trout TNF-α, directly stimulates glucose uptake in trout muscle cells and provide evidence, for the first time in nonmammalian vertebrates, for a potential regulatory role of TNF-α in skeletal muscle metabolism.
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http://dx.doi.org/10.1152/ajpregu.00514.2010DOI Listing
March 2011

Cellular and molecular evidence for a role of tumor necrosis factor alpha in the ovulatory mechanism of trout.

Reprod Biol Endocrinol 2010 Apr 12;8:34. Epub 2010 Apr 12.

Departament de Fisiologia, Facultat de Biologia, Universitat de Barcelona and Institut de Biomedicina de la Universitat de Barcelona (IBUB), 08028 Barcelona, Spain.

Background: The relevance of immune-endocrine interactions to the regulation of ovarian function in teleosts is virtually unexplored. As part of the innate immune response during infection, a number of cytokines such as tumor necrosis factor alpha (TNF alpha) and other immune factors, are produced and act on the reproductive system. However, TNF alpha is also an important physiological player in the ovulatory process in mammals. In the present study, we have examined for the first time the effects of TNF alpha in vitro in preovulatory ovarian follicles of a teleost fish, the brown trout (Salmo trutta).

Methods: To determine the in vivo regulation of TNF alpha expression in the ovary, preovulatory brook trout (Salvelinus fontinalis) were injected intraperitoneally with either saline or bacterial lipopolysaccharide (LPS). In control and recombinant trout TNF alpha (rtTNF alpha)-treated brown trout granulosa cells, we examined the percentage of apoptosis by flow cytometry analysis and cell viability by propidium iodide (PI) staining. Furthermore, we determined the in vitro effects of rtTNF alpha on follicle contraction and testosterone production in preovulatory brown trout ovarian follicles. In addition, we analyzed the gene expression profiles of control and rtTNF alpha-treated ovarian tissue by microarray and real-time PCR (qPCR) analyses.

Results: LPS administration in vivo causes a significant induction of the ovarian expression of TNF alpha. Treatment with rtTNF alpha induces granulosa cell apoptosis, decreases granulosa cell viability and stimulates the expression of genes known to be involved in the normal ovulatory process in trout. In addition, rtTNF alpha causes a significant increase in follicle contraction and testosterone production. Also, using a salmonid-specific microarray platform (SFA2.0 immunochip) we observed that rtTNF alpha induces the expression of genes known to be involved in inflammation, proteolysis and tissue remodeling. Furthermore, the expression of kallikrein, TOP-2, serine protease 23 and ADAM 22, genes that have been postulated to be involved in proteolytic and tissue remodeling processes during ovulation in trout, increases in follicles incubated in the presence of rtTNF alpha.

Conclusions: In view of these results, we propose that TNF alpha could have an important role in the biomechanics of follicle weakening, ovarian rupture and oocyte expulsion during ovulation in trout, primarily through its stimulation of follicular cell apoptosis and the expression of genes involved in follicle wall proteolysis and contraction.
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http://dx.doi.org/10.1186/1477-7827-8-34DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2873445PMC
April 2010

Peptidoglycan, not endotoxin, is the key mediator of cytokine gene expression induced in rainbow trout macrophages by crude LPS.

Mol Immunol 2010 Apr 20;47(7-8):1450-7. Epub 2010 Mar 20.

Institute of Biotechnology and Biomedicine, Dep. Biologia Cellular, Immunologia i Fisiologia Animal, Universitat Autónoma de Barcelona, 08193 Barcelona, Spain.

In rainbow trout macrophages, phenol-extracted lipopolysaccharide (LPS) preparations stimulate proinflammatory cytokine gene expression but ultrapure preparations of LPS are inactive. Crude LPS preparations could potentially have a number of contaminants including peptidoglycans (PGNs), nucleic acids and lipoproteins. Thus, in the current study we individually tested potentially contaminating pathogen associated molecular patterns (PAMPs) on rainbow trout (Oncorhynchus mykiss) macrophages to determine which ones could induce proinflammatory cytokine expression. We found that PGNs derived from Gram-negative bacteria (Escherichia coli 0111:B4 and K12), are potent inducers of IL-1beta and IL-6 gene expression and were equal to, or more potent than, crude LPS. On the other hand, PGNs of Gram-positive bacteria, DNA, RNA and lipoteichoic acid were weak stimulators, and lipid A, lipoprotein (Pam3CSK4) and ultrapure LPS were nonstimulatory. More importantly, crude LPS treated with lysozyme to degrade PGNs, exhibited greatly reduced activity in stimulating IL-1beta and IL-6 gene expression, indicating that PGNs in the crude LPS are responsible for a significant amount of the proinflammatory activity. Finally, we showed that PGN treatment induces expression of COX-2 and the subsequent synthesis and release of prostaglandin E(2) (PGE(2)), an important mediator of inflammatory processes. The strong stimulatory effect of E. coli PGNs by themselves on trout macrophages suggests that the recognition of Gram-negative bacteria in trout is through PGNs in the bacterial wall, and indicates that the systems responsible for bacterial recognition in invertebrates (e.g., Drosophila) may also be conserved in some vertebrates.
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http://dx.doi.org/10.1016/j.molimm.2010.02.009DOI Listing
April 2010

Endotoxin recognition in fish results in inflammatory cytokine secretion not gene expression.

Innate Immun 2011 Feb 18;17(1):16-28. Epub 2010 Jan 18.

Departament Biologia Cellular, Immunologia i Fisiologia Animal Universitat Autónoma de Barcelona, Barcelona, Spain.

Macrophages are phagocytes that have a central role in the organization of the immune system after an infection. These cells can recognize specific molecular components of micro-organisms (pathogen-associated molecular patterns, PAMPs) via specific receptors (PRRs) and elicit specific cellular responses. In the past, the expression of immune genes in response to different PAMPs has been characterized in different fish species. However, little is known about actual cytokine release. We characterized the secretion of tumour necrosis factor (TNF)-α in primary macrophage cultures of rainbow trout (Oncorhynchus mykiss) in response to several PAMPs by Western blot and compared this to the induction of TNF-α gene expression as well as other pro-inflammatory cytokines such as interleukin (IL)-1β and IL-6 and anti-viral molecules such as INF-α and Mx protein (Mx). We show that lipopolysaccharide (LPS) and zymosan are major inducers of TNF-α secretion, which is not initially linked to the induction of TNF-α mRNA expression. The secretion of TNF-α, but intriguingly not the expression, is also stimulated by ultrapure LPS meaning that, in fish, contaminants of commercial LPS preparations are better inducers of the inflammatory response. Moreover, we have characterized the signaling pathways that are activated by different PAMPs and the link between those pathways and the final step of TNF-α secretion: TNF-α shedding by TNF-α converting enzyme (TACE/ ADAM17). For the first time, we show that, in fish macrophages, TNF-α is processed by TACE-like activity and this cleavage is dependent upon the activation of ERK, p38MAPK and JNK signaling pathways by LPS.
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http://dx.doi.org/10.1177/1753425909348232DOI Listing
February 2011