Pathogen-dependent role of turbot (Scophthalmus maximus) interferon-gamma

11 páginas, 7 figurasInterferon-gamma has been typically described as a pro-inflammatory cytokine playing an important role in the resolution of both viral and bacterial diseases. Nevertheless, some anti-inflammatory functions are also attributed to this molecule. In this work we have characterized for the first time the turbot (Scophthalmus maximus) interferon-gamma gene (ifng) and its expression pattern under basal conditions, after type I IFNs administration, and viral and bacterial infection. The intramuscular injection of an expression plasmid encoding turbot Ifng (pMCV1.4-ifng) was not able to affect the transcription of numerous immune genes directly related to the activity of IFN-gamma, with the exception of macrophage-colony stimulating factor (csf1). It was also unable to reduce the mortality caused by a Viral Hemorrhagic Septicemia Virus (VHSV) or Aeromonas salmonicida challenge. Interestingly, at 24 h post-infection, turbot previously inoculated with pMCV1.4-ifng and infected with VHSV showed an increase in the expression of pro-inflammatory cytokines and type I IFNs compared to those fish not receiving expression plasmid, indicating a synergic effect of Ifng and VHSV. On the other hand, some macrophage markers, such as the macrophage receptor with collagenous structure (marco), were down-regulated by Ifng during the viral infection. Ifng had the opposite effect in those turbot infected with the bacteria, showing a reduction in the transcription of pro-inflammatory and type I IFNs genes, and an increase in the expression of genes related to the activity of macrophagesThis work has been funded by the projects CSD2007-00002 “Aquagenomics”, 201230E057 (CSIC) and AGL2014-51773-C3 from the Spanish Ministerio de Economía y Competitividad. P. Pereiro received a predoctoral grant from the gs3:Ministerio de Educación [F.P.U. fellowship AP2010-2408]Peer reviewe

Conserved gene regulation during acute inflammation between zebrafish and mammals

9 páginas, 4 figuras, 1 tabla.-- This work is licensed under a Creative Commons Attribution 4.0 International LicenseZebrafish (Danio rerio), largely used as a model for studying developmental processes, has also emerged as a valuable system for modelling human inflammatory diseases. However, in a context where even mice have been questioned as a valid model for these analysis, a systematic study evaluating the reproducibility of human and mammalian inflammatory diseases in zebrafish is still lacking. In this report, we characterize the transcriptomic regulation to lipopolysaccharide in adult zebrafish kidney, liver, and muscle tissues using microarrays and demonstrate how the zebrafish genomic responses can effectively reproduce the mammalian inflammatory process induced by acute endotoxin stress. We provide evidence that immune signaling pathways and single gene expression is well conserved throughout evolution and that the zebrafish and mammal acute genomic responses after lipopolysaccharide stimulation are highly correlated despite the differential susceptibility between species to that compound. Therefore, we formally confirm that zebrafish inflammatory models are suited to study the basic mechanisms of inflammation in human inflammatory diseases, with great translational impact potentialThis work was funded by the projects CSD2007–00002 “Aquagenomics” and AGL2014-51773-C3 from the Spanish Ministerio de Economía y Competitividad, and 201230E057 “Proyecto Intramural Especial, PIE”, Agencia Estatal Consejo Superior de Investigaciones Científicas (CSIC). P. Pereiro and M. Varela received predoctoral grants from the Ministerio de Educación (F.P.U. fellowship AP2010-2408) and the JAE Program (funded though the CSIC and European Social Funds), respectivelyPeer reviewe

High-Throughput Sequence Analysis of Turbot (Scophthalmus maximus) Transcriptome Using 454-Pyrosequencing for the Discovery of Antiviral Immune Genes

Turbot (Scophthalmus maximus L.) is an important aquacultural resource both in Europe and Asia. However, there is little information on gene sequences available in public databases. Currently, one of the main problems affecting the culture of this flatfish is mortality due to several pathogens, especially viral diseases which are not treatable. In order to identify new genes involved in immune defense, we conducted 454-pyrosequencing of the turbot transcriptome after different immune stimulations

Detection of cannabinoid receptor type 2 in native cells and zebrafish with a highly potent, cell-permeable fluorescent probe.

Despite its essential role in the (patho)physiology of several diseases, CB2R tissue expression profiles and signaling mechanisms are not yet fully understood. We report the development of a highly potent, fluorescent CB2R agonist probe employing structure-based reverse design. It commences with a highly potent, preclinically validated ligand, which is conjugated to a silicon-rhodamine fluorophore, enabling cell permeability. The probe is the first to preserve interspecies affinity and selectivity for both mouse and human CB2R. Extensive cross-validation (FACS, TR-FRET and confocal microscopy) set the stage for CB2R detection in endogenously expressing living cells along with zebrafish larvae. Together, these findings will benefit clinical translatability of CB2R based drugs

El pez cebra como modelo de inflamación

209 páginasEn organismos vertebrados, la correcta activación de la respuesta inflamatoria es de gran importancia para mantener la homeostasis tisular y la eficiente eliminación de patógenos. La inflamación es un proceso complejo perpetrado por la inmunidad innata en respuesta a perturbaciones del organismo en el que intervienen moléculas efectoras (péptidos antimicrobianos, sistema del complemento…) y células especializadas (fundamentalmente fagocitos) con el objetivo de crear un ambiente protector en el tejido afectado. Pese a ser un proceso generalmente benigno y crucial para la defensa del organismo, el exceso de inflamación tiene un gran potencial destructivo en el tejido del propio hospedador. La incapacidad de resolver eficientemente el proceso inflamatorio conlleva la aparición de patologías relacionadas con la inflamación, tanto aguda, como la sepsis, como crónica, como la inflamación producida por factores metabólicos. La modelización eficiente de la respuesta inflamatoria y su regulación es necesaria para aumentar nuestro conocimiento sobre las enfermedades relacionadas con la inflamación.El pez cebra, Danio rerio (Hamilton 1822), es un pez teleósteo de la familia de los ciprínidos ampliamente utilizado como modelo animal en investigación biomédica. Su extendido uso se debe a que presenta ventajas propias de los modelos invertebrados (pequeño tamaño, rápido ciclo vital, maduración externa y transparencia de los embriones, facilidad de manutención y manipulación genética…) siendo un organismo vertebrado, por lo que dispone de un sistema inmune altamente desarrollado. Estas características convierten al pez cebra, especialmente en sus etapas de desarrollo larvario, en un organismo idóneo para realizar estudios in vivo usando el organismo entero, lo que permite estudiar en detalle los procesos relacionados con el sistema inmune o la toxicidad considerando el ambiente formado por la interacción entre las diferentes células y tejidos del organismo. No obstante, debido a la presión evolutiva durante más de 400 millones de años, la modelización de la respuesta inmune y enfermedades humanas en el pez cebra también presenta problemas que solventar. Por ejemplo, el genoma de los peces teleósteos presenta una duplicación total del genoma adicional a la de otros vertebrados. La evolución asimétrica de estos genes duplicados permite que una copia del gen mute libremente y adquiera nuevas funciones. Este proceso, clave en la gran adaptabilidad a diferentes medios de los peces teleósteos, dificulta la predicción de ortología entre humanos y pez cebra. Pese a que el conocimiento de la práctica totalidad del genoma del pez cebra nos permite estudiar y tener en cuenta estas duplicaciones en los estudios con este organismo, la caracterización funcional de estas duplicaciones aún es necesaria para asegurar relaciones de ortología entre los genes de pez cebra y humanos.En esta Tesis hemos intentado consolidar y profundizar en el conocimiento sobre el uso del pez cebra para el estudio del proceso inflamatorio. Por ello, hemos analizado la respuesta transcriptómica y su regulación del pez cebra en modelos de inflamación aguda y crónica en el contexto de dos patologías humanas: el choque séptico y la Enfermedad del Hígado Graso No Alcohólico. Además, por su importancia central en la inducción y resolución del proceso inflamatorio, hemos caracterizado las copias del gen C3 presentes en el genoma de pez cebra.The work presented in this thesis has been funded by the projects: ‘AQUAGENOMICS’ (CSD2007-00002), 201230E057 ‘Proyecto Intramural Especial, PIE’, Agencia Estatal Consejo Superior de Investigaciones Científicas (CSIC), Spanish Ministerio de Ciencia e Innovación (MICINN) and the European project 289209 ‘FISHFORPHARMA’Peer reviewe

An immune-enriched oligo-microarray analysis of gene expression in Manila clam (Venerupis philippinarum) haemocytes after a Perkinsus olseni challenge

12 páginas, 7 figuras, 3 tablas, 1 apéndiceParasites of the genus Perkinsus cause high mortality and economic losses in bivalves commonly produced in global aquaculture. Although the immune responses of oysters and clams naturally infected with Perkinsus marinus or Perkinsus olseni have been extensively studied, there is not much information on host response at the early stages of infection. In this study, we analysed how P. olseni influences the gene expression profiles of haemocytes from the Manila clam (Venerupis philippinarum) using temporal experimental infections and an immune-enriched microarray. We identified an early phase of infection that was characterised by no mortality and by the increased expression of genes associated with pathogen recognition, production of nitrogen radicals and antimicrobial activity. Cellular processes such as inhibition of serine proteases and proliferation were also involved in this early response. This phase was followed by an intermediate stage, when the pathogen was most likely multiplying and infecting new areas of the body, and animals began to die. In this stage, many genes related to cell movement were over-expressed. Thirty days after infection metabolic pathway genes were the most affected. Apoptosis appears to be important during pathogenesis. Our results provide novel observations of the broader innate immune response triggered by P. olseni at different infection stagesThis work was partially funded by the EU Project REPROSEED (245119) and European structural funds (FEDER)/Ministerio de Ciencia e Innovación (CSIC08-1E-102). RM thanks the Spanish MICINN for an FPI Spanish research grant (BES-2009-029765).Peer reviewe

Advantajes and disadvantajes of zebrafish as a model of inflammation

3rd International Symposium on Fish & Shellfish Immunology, June 16th-20th 2019, Las Palmas de Gran Canaria (Spain)Zebrafish (Danio rerio), largely used as a model for studying developmental processes, has also emerged as a valuable system for modelling human disease. Zebrafish possesses a complex immune system comparable to those of mammalian models. Nevertheless, whole-genome duplication event and subfunction specialization of gene duplicates results in a more intricate relationship among the components implicated in the immune response and as a consequence in the inflammatory response. This is the case of the of the complement component c3 with 8 different genes in the zebrafish genome and different functions. This aspect, that could be considered an inconvenient, contrasts with the clear advantages that this model offers. We show how the real-time imaging and the use of the whole animal are excellent tools to visualize the in vivo interaction of a pathogen with the immune system. Also, we demonstrate how the genomic responses of adult zebrafish tissues can effectively reproduce the mammalian inflammatory process induced by acute endotoxin stress. Immune signalling has been well conserved throughout evolution and zebrafish and mammal genomic responses after lipopolysaccharide stimulation are highly correlated. Therefore, we confirm that zebrafish is an ideal model to study the basic mechanisms of inflammation and to model human inflammatory diseasesPeer reviewe

Proinflammatory Caspase A Activation and an Antiviral State Are Induced by a Zebrafish Perforin after Possible Cellular and Functional Diversification from a Myeloid Ancestor

14 pages, 9 figuresIn mammals, perforins play a central role in the granule-dependent cell death induced by natural killer T cells and cytotoxic T lymphocytes, and participate both in the defense against virus-infected and neoplastic cells and in the recognition of nonself molecules by the immune system. Little is known about fish perforin genes. We examined the zebrafish with the aim of increasing our knowledge about the role of perforins. We characterized 6 perforin genes in the zebrafish genome, and we studied them at the evolutionary level in combination with expression patterns in several tissues and cell populations, during both larval development and in the course of a viral infection. Our results suggest the specialization of different cell types in the production of perforins. Moreover, functional diversification during the evolution of these molecules could be inferred from this study. In particular, one of the genes, prf19b , which is mainly produced by myeloid cells, seemed to be involved in antiviral defense, conferring protection after an in vivo infectionThis work was funded by the projects CSD2007–00002 ‘Aquagenomics’ and AGL2014–51773-C3 from the Spanish Ministerio de Economía y Competitividad. M. Varela received a predoctoral grant from the JAE Program (funded by the CSIC and European Social Funds).Peer reviewe

Obesity and the liver immune response

12 pages, 4 figures, 2 tablesObesity- and metabolic syndrome-related diseases are becoming important medical challenges for the western world. Non-alcoholic fatty liver disease (NAFLD) is a manifestation of these altered conditions in the liver, and inflammation appears to be a factor that is tightly connected to its evolution. In this study, we used a diet-induced obesity approach in zebrafish (Danio rerio) based on overfeeding to analyze liver transcriptomic modulation in the disease and to determine how obesity affects the immune response against an acute inflammatory stimulus such as lipopolysaccharide (LPS). Overfed zebrafish developed an obese phenotype, showed signs of liver steatosis, and its modulation profile resembled that observed in humans, with overexpression of tac4, col4a3, col4a5, lysyl oxidases, and genes involved in retinoid metabolism. In response to LPS, healthy fish exhibited a typical host defense reaction comparable to that which occurs in mammals, whereas there was no significant gene modulation when comparing expression in the liver of LPS-stimulated and non-stimulated obese zebrafish at the same statistical level. The stimulation of obese fish represents a double-hit to the already damaged liver and can help understand the evolution of the disease. Finally, a comparison of the differential gene activation between stimulated healthy and obese zebrafish revealed the expected difference in the metabolic state between healthy and diseased liver. The differentially modulated genes are currently being studied as putative new pathological markers in NAFLD-stimulated liver in humansThis work was supported by the projects CSD2007-00002 ‘AQUAGENOMICS’ of the program Consolider-Ingenio 2010, Spanish Ministerio de Ciencia e Innovación (MICINN); 289209 ‘FISHFORPHARMA’ (EU); and 201230E057 ‘Proyecto Intramural Especial, PIE’, Agencia Estatal Consejo Superior de Investigaciones Científicas (CSIC). M V received a predoctoral grant from the JAE Program (CSIC and European structural Funds)Peer reviewe

The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection.

Mycobacterial pathogens are the causative agents of chronic infectious diseases like tuberculosis and leprosy. Autophagy has recently emerged as an innate mechanism for defense against these intracellular pathogens. In vitro studies have shown that mycobacteria escaping from phagosomes into the cytosol are ubiquitinated and targeted by selective autophagy receptors. However, there is currently no in vivo evidence for the role of selective autophagy receptors in defense against mycobacteria, and the importance of autophagy in control of mycobacterial diseases remains controversial. Here we have used Mycobacterium marinum (Mm), which causes a tuberculosis-like disease in zebrafish, to investigate the function of two selective autophagy receptors, Optineurin (Optn) and SQSTM1 (p62), in host defense against a mycobacterial pathogen. To visualize the autophagy response to Mm in vivo, optn and p62 zebrafish mutant lines were generated in the background of a GFP-Lc3 autophagy reporter line. We found that loss-of-function mutation of optn or p62 reduces autophagic targeting of Mm, and increases susceptibility of the zebrafish host to Mm infection. Transient knockdown studies confirmed the requirement of both selective autophagy receptors for host resistance against Mm infection. For gain-of-function analysis, we overexpressed optn or p62 by mRNA injection and found this to increase the levels of GFP-Lc3 puncta in association with Mm and to reduce the Mm infection burden. Taken together, our results demonstrate that both Optn and p62 are required for autophagic host defense against mycobacterial infection and support that protection against tuberculosis disease may be achieved by therapeutic strategies that enhance selective autophagy