- Email: [email protected]
Role of central immune organs in vaccinated rainbow trout in protection against Yersinia ruckeri infection
Abstracts / Fish & Shellfish Immunology 34 (2013) 1692–1752
colony-forming units ml1). Additionally, phorbol myristate acetate was added as a soluble stimulant of the superoxide anion production and served as a positive control, while lipopolysaccharides (LPS) from E. coli (serotype 0111:B4; 10 mg ml1) were added to cell monolayers for nitric oxide production. Head-kidney leucocytes were also incubated in 24-well plates with: medium alone (controls), LPS, UV killed T. maritimum, and a mixed leucocyte reaction (MLR) was carried out by co-incubation of leucocytes from 3 specimens in triplicate. After 4, 24 and 48 h of incubation, leucocytes were washed, pooled and the cDNA obtained. A significant increase in both the superoxide anion and NO production was observed after stimulation with the strain ACC13.1 compared to ACC6.1. Although headkidney cells incubated with LPS did not show significant differences in the NO production, hepcidin antimicrobial peptide (HAMP), IL-8 and g-type lysozyme transcripts increased after stimulation with LPS at 4 and 24 h. HAMP was also up-regulated in cells stimulated with T. maritimum after 4 and 24 h. The level of expression of HAMP and g-type lysozyme increased to peak levels at 24 and 48 h during the MLR, respectively. Moreover, IL-8 transcripts showed the opposite pattern with higher expression levels (20fold increase) already at 4 h. Results show different degrees of stimulation by several pathogen-associated molecular patterns. Not surprisingly, stimulation with LPS or bacteria led to a significant up-regulation of HAMP, in agreement with the major role described for hepcidin in the immune response to bacteria. Since both ACC6.1 and ACC13.1 belong to the same serotype, it is suggested that different leucocyte responses against these strains are probably related to the existence of genetic heterogeneity. * Corresponding author. E-mail address: [email protected] (B. Costas)
P-430. Identification of microRNAs related to the immune response of Atlantic salmon Salmo salar L. fry against infectious pancreatic necrosis virus C. Da Silva-Duarte 1, *, M. Bekaert 1, K.D. Thompson 1, J.E. Bron 1, R.D. Houston 2, A. Adams 1, R.H. Richards 1, J.B. Taggart 1. 1 Institute of Aquaculture, School of Natural Sciences, University of Stirling, Stirling FK9 4LA, UK; 2 The Roslin Institute, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, UK
Abstract Infectious pancreatic necrosis (IPN) is a viral disease with a significant negative impact on the global aquaculture of Atlantic salmon. Outbreaks of IPN can occur during specific windows of both the freshwater and seawater stages of the salmon life cycle. Previous research has shown that the variation in resistance to IPN in Atlantic salmon is partly related to host genetics. Identification of the immune (and other) genes involved in the defence of Atlantic salmon against IPNV is crucial for understanding this resistance. Currently we are carrying out molecular analyses on archived tissue samples obtained from successful IPN challenges on Atlantic salmon fry, involving families and individuals known to be resistant or susceptible to IPNV. One aspect of this work is focused on exploring the potential role that microRNAs may play in modulating genetic resistance to this disease. MicroRNAs (miRNAs) are endogenous non-protein coding short RNA molecules (18-26 nucleotides) that play an important role in the posttranscriptional regulation of gene expression via binding to the 30 UTR region of the target mRNAs. We have constructed whole fish miRNA libraries for 36 individual juveniles (resistant and susceptible genotypes, prior to and one day post IPNV challenge) and extensively characterised miRNA expression profiles under these conditions by Illumina highthroughput sequencing. Details of the extensive and, in part, novel miRNA repertoire identified for Atlantic salmon will be reported. Initial results of qPCR-based validation and further characterisation of miRNAs found to be differentially expressed between resistant and susceptible genotype will be detailed and discussed. This work may lead to the identification of key genes and pathways involved in controlling immune gene expression against IPNV infection and lead to a better understanding of the
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mechanisms underlying resistance/susceptibility of Atlantic salmon to these pathogens. * Corresponding author. E-mail address: [email protected] (C. Da Silva-Duarte)
P-143. Proteomic approach to identify markers of resistance to bonamiosis in the proteins of the oyster Ostrea edulis haemolymph N.R. De la Ballina*, A. Ramilo, A. Villalba, A. Cao. Centro de Investigacións Mariñas (CIMA), Consellería do Medio Rural e do Mar, Xunta de Galicia, Apartado 13, 36620 Vilanova de Arousa, Spain Abstract European flat oyster Ostrea edulis production is dramatically constrained by bonamiosis. Two protozoan parasites, Bonamia ostreae and Bonamia exitiosa are responsible for this disease in Europe. Both are intracellular parasites able to survive and proliferate within haemocytes, the main cellular effectors of the immune system of molluscs. Both parasites are included in the list of notifiable diseases of the World Organisation for Animal Health. A proteomic approach was used to compare the protein expression in haemolymph between bonamiosis-affected and non-affected oysters O. edulis from 3 stocks with different susceptibility to this disease. The aim was to find molecular markers for resistance to bonamiosis, which could be used in selective breeding programmes to produce oyster resistant strains. Oysters produced in CIMA hatchery facilities and on-grown in a raft in the ría de Arousa (Galicia, NW Spain) were used in the study. Those oysters derived from two different brood-stocks, one stock derived from a selective breeding programme for resistance to bonamiosis and the other stock consisted of non-selected oysters taken from a natural bed in the ría de Pontevedra. The oysters deriving from each stock shared a common environment since the larval spawning and the selected stock showed higher resistance to bonamiosis. Additionally, oysters produced within an Irish selective breeding programme for resistance to bonamiosis, Rossmore oysters, were involved in the study. Pools of haemolymph from 7 oysters from each stock and each health condition (B. ostreae-infected, B. exitiosa-infected, and non-infected oysters) were obtained. Haemolymph proteins were separated by bi-dimensional electrophoresis (2D-PAGE); four gels were produced from each pool and analysis of spot patterns in gels and comparison between treatments (stock and health condition) were performed with PD Quest software. The comparison of the gels from Galician oyster stocks, considering all the spots that were common in various treatments and the spots exclusive of each treatment, allowed detecting 52 spots exclusive of non-infected selected oysters and 10 spots exclusive of bonamiosis-affected selected oysters. In the case of Rossmore stock the comparison between non-infected, very lightly-infected and more heavily-infected oysters allowed detecting 1 spot exclusive of noninfected oysters and 29 spots exclusive of very-lightly infected oysters. Next step is to identify the proteins in the selected spots. That should improve our knowledge in the host-parasite interaction, which hopefully will provide key information to recover the European flat oyster natural beds and production. * Corresponding author. E-mail address: [email protected] (N.R. De la Ballina)
P-388. Role of central immune organs in vaccinated rainbow trout in protection against Yersinia ruckeri infection S. Deshmukh 1,*, J.K. Chetrri 1, A.M. Bojesen 2, K. Buchmann 1. 1
Laboratory of Aquatic Pathobiology, Section of Biomedicine, Department of Veterinary Disease Biology, Faculty of Health and Medical Science, University of Copenhagen, Denmark;
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Abstracts / Fish & Shellfish Immunology 34 (2013) 1692–1752
2 Section of Microbiology, Department of Veterinary Disease Biology, Faculty of Health and Medical Science, University of Copenhagen, Denmark
Abstract We investigated, by the use of histology, the changes of tissue architecture and transformation of various resident cell types in immune organs of vaccinated and unvaccinated fish responding to Yersinia ruckeri serotype 01, biotype 2 infection. The protection of vaccinated trout was shown to be based primarily on spleen involvement and to a lesser degree head kidney reactions. Additional immunohistochemical studies revealed differential occurrence of CD8a and IgM positive cells in protected and nonprotected fish. The study identifies protective cell reactions in vaccinated rainbow trout. The systemic infection was confirmed by fluorescent in situ hybridization technique (FISH) using an Yersinia ruckeri specific probe. * Corresponding author. E-mail address: [email protected] (S. Deshmukh)
P-536. The warm temperature acclimation protein (Wap65) has an important role in the inflammatory response of turbot (Scophthalmus maximus) P. Diaz-Rosales x, P. Pereiro x, S. Dios, A. Figueras, B. Novoa. Institute of Marine Research (CSIC), Eduardo Cabello 6, 36208, Vigo, Spain Abstract The warm temperature acclimation protein (Wap65) is a plasma glycoprotein similar to the mammalian hemopexin. Hemopexin is a serum glycoprotein produced by the liver and its primary role is to bind free heme and prevent oxidative damage whilst sequestering iron away from bacteria. It has been reported an important role of hemopexin in the inflammation as an acute-phase protein and its production is up-regulated by proinflammatory cytokines. Most teleost species presents two Wap65 genes but their physiological functions have not been completely elucidated; in fact, the transcriptional patterns of Wap65 genes to stimulatory treatments are variable and contradictory. In the present study Wap65 has been characterized for the first time in turbot (Scophthalmus maximus) to understand the role of Wap65 in thermal physiology and innate immunity for this species. Two types of Wap65 were identified and the differential modulation by thermal treatments, immune challenges (bacterial and viral), as well as iron supplementation, has been investigated. On the other hand, the protein was purified from the turbot serum using hemin-agarose affinity chromatography and the effect of purified Wap65 protein was studied to clarify its role on immune response.
E-mail address: [email protected] (P. Diaz-Rosales) xThese authors have contributed equally to this work.
P-122. Regulation by resveratrol of turbot inflammatory response induced by vaccines B. Domínguez 1, M. Noia 1, J. Leiro 2, J. Lamas 1, *. 1 Departamento de Biología Celular y Ecología, Universidad de Santiago de Compostela, Santiago de Compostela, Spain; 2 Departamento de Microbiología y Parasitología, Instituto de Análisis Alimentarios, Universidad de Santiago de Compostela, Santiago de Compostela, Spain
Abstract Resveratrol is a polyphenol that exerts anti-inflammatory activity in mammals. The effect is apparently mediated by inhibition of the production of reactive oxygen and nitrogen species, inhibition of the synthesis and release of pro-inflammatory mediators and modulation of
the expression of many genes, including transcription factors such as nuclear factor kappaB, a pro-inflammatory transcription factor involved in many cell activities. Adjuvants are substances that are added to vaccines with the aim of enhancing the immune response to the antigen. Oil-based adjuvants are commonly used in fish and usually induce good immune responses, as indicated by the production of polyclonal antibodies. However, adjuvants may also induce an extensive inflammatory reaction, granulomas and several pathological changes in vaccinated fish. In the present study, we evaluated the effects of resveratrol on the inflammatory response and on antibody production induced in turbot by administration of a vaccine containing an oilbased adjuvant. Fish were injected i.p. with the antigen (Philasterides dicentrarchi), adjuvant, or antigen plus adjuvant, in the presence or absence of resveratrol. Cell migration, changes in gene expression in peritoneal exudate cells and in head kidney leukocytes and the serum antibody response were determined in i.p. vaccinated fish. In fish injected with the vaccine plus resveratrol, fewer peritoneal cells were observed on days 1, 3 and 5 and more cells were observed on day 7 than in fish injected with the vaccine alone. This suggests that resveratrol inhibited cell migration but that the inhibition lasted for only a few days. We also observed inhibition of expression of genes involved in inflammatory responses, mainly on the first day of treatment. However, the administration of resveratrol with the vaccine did not affect serum antibody levels, suggesting that the resveratrol did not decrease the immune response elicited by the vaccine. * Corresponding author. E-mail address: [email protected] (J. Lamas)
P-121. Modulation by resveratrol of inflammatory and immune gene expression in turbot leucocytes B. Domínguez 1, x, B.G. Pardo 2,x, M. Noia 1, A. Millán 2, A. Gómez-Tato 3, P. Martínez 2, J. Leiro 4, J. Lamas 1, *. 1 Departamento de Biología Celular y Ecología, Universidad de Santiago de Compostela, Santiago de Compostela, Spain; 2 Departamento de Genética, Universidad de Santiago de Compostela, Lugo, Spain; 3 Departamento de Geometría y Topología, Universidad de Santiago de Compostela, Santiago de Compostela, Spain; 4 Departamento de Microbiología y Parasitología, Instituto de Investigación y Análisis Alimentarios, Universidad de Santiago de Compostela, Santiago de Compostela, Spain
Abstract In previous studies, we showed that the polyphenol resveratrol exerted a dose-dependent inhibitory effect on the migratory response and on the production of reactive oxygen species in turbot leukocytes. RESV also inhibited enzymatic activity and myeloperoxidase gene expression, decreased TNF-a gene expression and reduced the generation of the proinflammatory mediator prostaglandin E2. In the present study, we used a DNA microarray enriched in genes involved in inflammatory and immune responses to investigate the anti-inflammatory activity of resveratrol and to examine its effects on gene expression in turbot head kidney leucocytes. Leucocytes were cultured in L-15 medium, for 0, 3, 6 and 24 h, in the presence or absence of resveratrol, or were stimulated with the membrane fraction of the parasite Philasterides dicentrarchi or with the membrane fraction plus resveratrol. Important changes in the expression of genes associated with the cytoskeleton and with inflammatory and immune responses were observed in control cells: 29, 38 and 119 genes were downregulated and 15, 32 and 48 genes were upregulated after incubation times of 3, 6 and 24 h, respectively. Gene expression was very similar in control cells and in cells stimulated with the P. dicentrarchi membrane fraction. Treatment with resveratrol induced changes in the expression (mostly downregulation) of 42, 135 and 63 genes after respectively 3, 6 and 24 h. Several genes involved in inflammation, the transcription factor PU.1, pentraxin-multidomain protein, heme oxygenase 1, and the signal transducer and activator of transcription 4, were downregulated after the three incubation times. Downregulation of several genes
colony-forming units ml1). Additionally, phorbol myristate acetate was added as a soluble stimulant of the superoxide anion production and served as a positive control, while lipopolysaccharides (LPS) from E. coli (serotype 0111:B4; 10 mg ml1) were added to cell monolayers for nitric oxide production. Head-kidney leucocytes were also incubated in 24-well plates with: medium alone (controls), LPS, UV killed T. maritimum, and a mixed leucocyte reaction (MLR) was carried out by co-incubation of leucocytes from 3 specimens in triplicate. After 4, 24 and 48 h of incubation, leucocytes were washed, pooled and the cDNA obtained. A significant increase in both the superoxide anion and NO production was observed after stimulation with the strain ACC13.1 compared to ACC6.1. Although headkidney cells incubated with LPS did not show significant differences in the NO production, hepcidin antimicrobial peptide (HAMP), IL-8 and g-type lysozyme transcripts increased after stimulation with LPS at 4 and 24 h. HAMP was also up-regulated in cells stimulated with T. maritimum after 4 and 24 h. The level of expression of HAMP and g-type lysozyme increased to peak levels at 24 and 48 h during the MLR, respectively. Moreover, IL-8 transcripts showed the opposite pattern with higher expression levels (20fold increase) already at 4 h. Results show different degrees of stimulation by several pathogen-associated molecular patterns. Not surprisingly, stimulation with LPS or bacteria led to a significant up-regulation of HAMP, in agreement with the major role described for hepcidin in the immune response to bacteria. Since both ACC6.1 and ACC13.1 belong to the same serotype, it is suggested that different leucocyte responses against these strains are probably related to the existence of genetic heterogeneity. * Corresponding author. E-mail address: [email protected] (B. Costas)
P-430. Identification of microRNAs related to the immune response of Atlantic salmon Salmo salar L. fry against infectious pancreatic necrosis virus C. Da Silva-Duarte 1, *, M. Bekaert 1, K.D. Thompson 1, J.E. Bron 1, R.D. Houston 2, A. Adams 1, R.H. Richards 1, J.B. Taggart 1. 1 Institute of Aquaculture, School of Natural Sciences, University of Stirling, Stirling FK9 4LA, UK; 2 The Roslin Institute, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, UK
Abstract Infectious pancreatic necrosis (IPN) is a viral disease with a significant negative impact on the global aquaculture of Atlantic salmon. Outbreaks of IPN can occur during specific windows of both the freshwater and seawater stages of the salmon life cycle. Previous research has shown that the variation in resistance to IPN in Atlantic salmon is partly related to host genetics. Identification of the immune (and other) genes involved in the defence of Atlantic salmon against IPNV is crucial for understanding this resistance. Currently we are carrying out molecular analyses on archived tissue samples obtained from successful IPN challenges on Atlantic salmon fry, involving families and individuals known to be resistant or susceptible to IPNV. One aspect of this work is focused on exploring the potential role that microRNAs may play in modulating genetic resistance to this disease. MicroRNAs (miRNAs) are endogenous non-protein coding short RNA molecules (18-26 nucleotides) that play an important role in the posttranscriptional regulation of gene expression via binding to the 30 UTR region of the target mRNAs. We have constructed whole fish miRNA libraries for 36 individual juveniles (resistant and susceptible genotypes, prior to and one day post IPNV challenge) and extensively characterised miRNA expression profiles under these conditions by Illumina highthroughput sequencing. Details of the extensive and, in part, novel miRNA repertoire identified for Atlantic salmon will be reported. Initial results of qPCR-based validation and further characterisation of miRNAs found to be differentially expressed between resistant and susceptible genotype will be detailed and discussed. This work may lead to the identification of key genes and pathways involved in controlling immune gene expression against IPNV infection and lead to a better understanding of the
1703
mechanisms underlying resistance/susceptibility of Atlantic salmon to these pathogens. * Corresponding author. E-mail address: [email protected] (C. Da Silva-Duarte)
P-143. Proteomic approach to identify markers of resistance to bonamiosis in the proteins of the oyster Ostrea edulis haemolymph N.R. De la Ballina*, A. Ramilo, A. Villalba, A. Cao. Centro de Investigacións Mariñas (CIMA), Consellería do Medio Rural e do Mar, Xunta de Galicia, Apartado 13, 36620 Vilanova de Arousa, Spain Abstract European flat oyster Ostrea edulis production is dramatically constrained by bonamiosis. Two protozoan parasites, Bonamia ostreae and Bonamia exitiosa are responsible for this disease in Europe. Both are intracellular parasites able to survive and proliferate within haemocytes, the main cellular effectors of the immune system of molluscs. Both parasites are included in the list of notifiable diseases of the World Organisation for Animal Health. A proteomic approach was used to compare the protein expression in haemolymph between bonamiosis-affected and non-affected oysters O. edulis from 3 stocks with different susceptibility to this disease. The aim was to find molecular markers for resistance to bonamiosis, which could be used in selective breeding programmes to produce oyster resistant strains. Oysters produced in CIMA hatchery facilities and on-grown in a raft in the ría de Arousa (Galicia, NW Spain) were used in the study. Those oysters derived from two different brood-stocks, one stock derived from a selective breeding programme for resistance to bonamiosis and the other stock consisted of non-selected oysters taken from a natural bed in the ría de Pontevedra. The oysters deriving from each stock shared a common environment since the larval spawning and the selected stock showed higher resistance to bonamiosis. Additionally, oysters produced within an Irish selective breeding programme for resistance to bonamiosis, Rossmore oysters, were involved in the study. Pools of haemolymph from 7 oysters from each stock and each health condition (B. ostreae-infected, B. exitiosa-infected, and non-infected oysters) were obtained. Haemolymph proteins were separated by bi-dimensional electrophoresis (2D-PAGE); four gels were produced from each pool and analysis of spot patterns in gels and comparison between treatments (stock and health condition) were performed with PD Quest software. The comparison of the gels from Galician oyster stocks, considering all the spots that were common in various treatments and the spots exclusive of each treatment, allowed detecting 52 spots exclusive of non-infected selected oysters and 10 spots exclusive of bonamiosis-affected selected oysters. In the case of Rossmore stock the comparison between non-infected, very lightly-infected and more heavily-infected oysters allowed detecting 1 spot exclusive of noninfected oysters and 29 spots exclusive of very-lightly infected oysters. Next step is to identify the proteins in the selected spots. That should improve our knowledge in the host-parasite interaction, which hopefully will provide key information to recover the European flat oyster natural beds and production. * Corresponding author. E-mail address: [email protected] (N.R. De la Ballina)
P-388. Role of central immune organs in vaccinated rainbow trout in protection against Yersinia ruckeri infection S. Deshmukh 1,*, J.K. Chetrri 1, A.M. Bojesen 2, K. Buchmann 1. 1
Laboratory of Aquatic Pathobiology, Section of Biomedicine, Department of Veterinary Disease Biology, Faculty of Health and Medical Science, University of Copenhagen, Denmark;
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Abstracts / Fish & Shellfish Immunology 34 (2013) 1692–1752
2 Section of Microbiology, Department of Veterinary Disease Biology, Faculty of Health and Medical Science, University of Copenhagen, Denmark
Abstract We investigated, by the use of histology, the changes of tissue architecture and transformation of various resident cell types in immune organs of vaccinated and unvaccinated fish responding to Yersinia ruckeri serotype 01, biotype 2 infection. The protection of vaccinated trout was shown to be based primarily on spleen involvement and to a lesser degree head kidney reactions. Additional immunohistochemical studies revealed differential occurrence of CD8a and IgM positive cells in protected and nonprotected fish. The study identifies protective cell reactions in vaccinated rainbow trout. The systemic infection was confirmed by fluorescent in situ hybridization technique (FISH) using an Yersinia ruckeri specific probe. * Corresponding author. E-mail address: [email protected] (S. Deshmukh)
P-536. The warm temperature acclimation protein (Wap65) has an important role in the inflammatory response of turbot (Scophthalmus maximus) P. Diaz-Rosales x, P. Pereiro x, S. Dios, A. Figueras, B. Novoa. Institute of Marine Research (CSIC), Eduardo Cabello 6, 36208, Vigo, Spain Abstract The warm temperature acclimation protein (Wap65) is a plasma glycoprotein similar to the mammalian hemopexin. Hemopexin is a serum glycoprotein produced by the liver and its primary role is to bind free heme and prevent oxidative damage whilst sequestering iron away from bacteria. It has been reported an important role of hemopexin in the inflammation as an acute-phase protein and its production is up-regulated by proinflammatory cytokines. Most teleost species presents two Wap65 genes but their physiological functions have not been completely elucidated; in fact, the transcriptional patterns of Wap65 genes to stimulatory treatments are variable and contradictory. In the present study Wap65 has been characterized for the first time in turbot (Scophthalmus maximus) to understand the role of Wap65 in thermal physiology and innate immunity for this species. Two types of Wap65 were identified and the differential modulation by thermal treatments, immune challenges (bacterial and viral), as well as iron supplementation, has been investigated. On the other hand, the protein was purified from the turbot serum using hemin-agarose affinity chromatography and the effect of purified Wap65 protein was studied to clarify its role on immune response.
E-mail address: [email protected] (P. Diaz-Rosales) xThese authors have contributed equally to this work.
P-122. Regulation by resveratrol of turbot inflammatory response induced by vaccines B. Domínguez 1, M. Noia 1, J. Leiro 2, J. Lamas 1, *. 1 Departamento de Biología Celular y Ecología, Universidad de Santiago de Compostela, Santiago de Compostela, Spain; 2 Departamento de Microbiología y Parasitología, Instituto de Análisis Alimentarios, Universidad de Santiago de Compostela, Santiago de Compostela, Spain
Abstract Resveratrol is a polyphenol that exerts anti-inflammatory activity in mammals. The effect is apparently mediated by inhibition of the production of reactive oxygen and nitrogen species, inhibition of the synthesis and release of pro-inflammatory mediators and modulation of
the expression of many genes, including transcription factors such as nuclear factor kappaB, a pro-inflammatory transcription factor involved in many cell activities. Adjuvants are substances that are added to vaccines with the aim of enhancing the immune response to the antigen. Oil-based adjuvants are commonly used in fish and usually induce good immune responses, as indicated by the production of polyclonal antibodies. However, adjuvants may also induce an extensive inflammatory reaction, granulomas and several pathological changes in vaccinated fish. In the present study, we evaluated the effects of resveratrol on the inflammatory response and on antibody production induced in turbot by administration of a vaccine containing an oilbased adjuvant. Fish were injected i.p. with the antigen (Philasterides dicentrarchi), adjuvant, or antigen plus adjuvant, in the presence or absence of resveratrol. Cell migration, changes in gene expression in peritoneal exudate cells and in head kidney leukocytes and the serum antibody response were determined in i.p. vaccinated fish. In fish injected with the vaccine plus resveratrol, fewer peritoneal cells were observed on days 1, 3 and 5 and more cells were observed on day 7 than in fish injected with the vaccine alone. This suggests that resveratrol inhibited cell migration but that the inhibition lasted for only a few days. We also observed inhibition of expression of genes involved in inflammatory responses, mainly on the first day of treatment. However, the administration of resveratrol with the vaccine did not affect serum antibody levels, suggesting that the resveratrol did not decrease the immune response elicited by the vaccine. * Corresponding author. E-mail address: [email protected] (J. Lamas)
P-121. Modulation by resveratrol of inflammatory and immune gene expression in turbot leucocytes B. Domínguez 1, x, B.G. Pardo 2,x, M. Noia 1, A. Millán 2, A. Gómez-Tato 3, P. Martínez 2, J. Leiro 4, J. Lamas 1, *. 1 Departamento de Biología Celular y Ecología, Universidad de Santiago de Compostela, Santiago de Compostela, Spain; 2 Departamento de Genética, Universidad de Santiago de Compostela, Lugo, Spain; 3 Departamento de Geometría y Topología, Universidad de Santiago de Compostela, Santiago de Compostela, Spain; 4 Departamento de Microbiología y Parasitología, Instituto de Investigación y Análisis Alimentarios, Universidad de Santiago de Compostela, Santiago de Compostela, Spain
Abstract In previous studies, we showed that the polyphenol resveratrol exerted a dose-dependent inhibitory effect on the migratory response and on the production of reactive oxygen species in turbot leukocytes. RESV also inhibited enzymatic activity and myeloperoxidase gene expression, decreased TNF-a gene expression and reduced the generation of the proinflammatory mediator prostaglandin E2. In the present study, we used a DNA microarray enriched in genes involved in inflammatory and immune responses to investigate the anti-inflammatory activity of resveratrol and to examine its effects on gene expression in turbot head kidney leucocytes. Leucocytes were cultured in L-15 medium, for 0, 3, 6 and 24 h, in the presence or absence of resveratrol, or were stimulated with the membrane fraction of the parasite Philasterides dicentrarchi or with the membrane fraction plus resveratrol. Important changes in the expression of genes associated with the cytoskeleton and with inflammatory and immune responses were observed in control cells: 29, 38 and 119 genes were downregulated and 15, 32 and 48 genes were upregulated after incubation times of 3, 6 and 24 h, respectively. Gene expression was very similar in control cells and in cells stimulated with the P. dicentrarchi membrane fraction. Treatment with resveratrol induced changes in the expression (mostly downregulation) of 42, 135 and 63 genes after respectively 3, 6 and 24 h. Several genes involved in inflammation, the transcription factor PU.1, pentraxin-multidomain protein, heme oxygenase 1, and the signal transducer and activator of transcription 4, were downregulated after the three incubation times. Downregulation of several genes