- Email: [email protected]
Fluoride influences haemocytes functionality in the venus clam Venerupis philippinarum
Abstracts / Fish & Shellfish Immunology 34 (2013) 1692–1752
often poorly immunogenic and may require adjuvants. Adjuvants stimulate components of the innate immune system, e.g. Toll-like receptors (TLRs), and lead to increased antigen immunogenicity. In humans, TLRs are studied in great detail and their ligands are used as adjuvants. Fish genome projects demonstrate a large number of TLRs. However, the functional characterization is lagging behind, and functional agonists are only verified for four of the 18 identified fish TLRs. With a more detailed understanding of fish TLRs, their agonists may find their way into fish vaccinology. To increase this understanding we are establishing a nuclear factor kappa B (NF-kB) luciferase reporter system for TLR signaling. The assay is based on transfection of HEK293 cells with a reporter plasmid containing luciferase under control of a NF-kB promoter. We have established a positive control which includes human TLR2 and two of its known ligands. The system is now ready for expression of TLRs from different fish species (salmon, trout and zebrafish). Cells can then be stimulated with potential agonists (from TLR agonist libraries and pathogenic salmon viruses) and assayed for NF-kB activation and TLR activation. We have also assayed salmon cells transfected with the reporter construct which showed NF-kB activation for several ligands known to activate human TLRs. This system reveals information that is important for a more detailed understanding of fish TLRs. * Corresponding author. E-mail address: [email protected] (M. Arnemo)
P-368. Arginine or glutamine supplementation to culture media modulates innate immune mechanisms of Senegalese sole (Solea senegalensis) head-kidney leukocytes R. Azeredo 1, 2, A. Afonso 1, 3, A. Oliva-Teles 1, 2, B. Costas 1, *. 1 CIMAR/CIIMAR – Centro Interdisciplinar de Investigação Marinha e Ambiental, 4050-123 Porto, Portugal; 2 Departamento de Biologia, Faculdade de Ciências da Universidade do Porto, 4169-007 Porto, Portugal; 3 ICBAS – Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal
Abstract Emerging evidence shows that many amino acids regulate key metabolic pathways that are crucial to immune responses. For instance, a large body of evidence from animal studies indicates that arginine or glutamine supplementation enhances immune function in various models of immunological challenges. The present study aimed to evaluate to what extent media supplemented with these amino acids can influence some aspects of the innate immune system in Senegalese sole (Solea senegalensis). Therefore, in vitro assays were performed using head-kidney leukocytes treated either with two levels of glutamine or arginine or with medium alone. Primary cell cultures were used to analyze the respiratory burst by quantifying extracellular superoxide anion and nitric oxide levels following infection with Tenacibaculum maritimum (2 106 colony-forming units ml 1). Additionally, phorbol myristate acetate was added as a soluble stimulant of the superoxide anion production and served as a positive control, while lipopolysaccharides from Escherichia coli (serotype 0111:B4) at two different concentrations were added to cell monolayers for nitric oxide production. A significant increase in the superoxide anion production was observed after stimulation with bacteria in cells treated with glutamine (1 mM) as well as in those treated with arginine (0.5 and 1 mM). However, head-kidney cells incubated with either lipopolysaccharides (1 and 10 mg ml 1) or T. maritimum for 72 h did not show significant differences in the nitric oxide production at any treatment. Preliminary results suggest that glutamine or arginine levels beyond those required for normal cell growth and differentiation may lead to a better leucocyte performance against antigens. Glutamine is a major energy substrate for leukocytes and is responsible for the activation of macrophage functions, while arginine is a precursor substrate for polyamine biosynthesis. Regarding nitric oxide production, the absence of different responses among treatments could be due to the incubation period since distinct responses have been observed in cells incubated for 48 h. Further studies
1695
are now being carried out in our laboratory to study the gene expression profile in head-kidney cells following different stimuli and media supplementation. * Corresponding author. E-mail address: [email protected] (B. Costas)
P-259. Fluoride influences haemocytes functionality in the venus clam Venerupis philippinarum L. Ballarin*, L. Masiero, V. Covre, S. Casellato. Department of Biology, University of Padova, Padova, Italy Abstract Invertebrates rely only on innate immunity to cope with foreign, potentially pathogenic microorganisms having entered the organism and haemocytes play a pivotal role in immune responses, as they are involved in phagocytosis, encapsulation, secretion of lectins, cytokines and cytotoxic factors. Therefore, adverse effects on haemocytes can compromise the survival of the organism. In the present study, we analysed the effects of the exposure of haemocytes of the venus clam Venerupis philippinarum to fluoride on cell morphology and functionality. Indeed, the increasing concentration of fluoride in many aquatic ecosystems, as a consequence of human activities, is a matter of great concern and the main negative effect of fluoride are related to its inhibition of metabolic processes, such as glycolysis and protein synthesis. Haemocytes, collected from the adductor muscles, were exposed to NaF (10, 50 and 250 mg/L) in filtered seawater (FSW), for 60 min. We observed a significant (p < 0.01) increase in cell mortality, using the Trypan Blue assay, for all the concentrations used in our experiments. The Neutral Red assay indicated a decrease in the stability of the internal membranes after exposure to 250 mg/L as the dye, usually accumulating inside acid compartments, such as lysosomes, leaks from these compartments into the cytoplasm so that the cells assume a pinkish colour. We also reported a significant (p < 0.01) reduction in the fraction of phagocytosing cells which suggests an effect of fluoride ions on the cytoskeleton or the hampering of the interaction with foreign particles. The observed increase in the cell shape factor in haemocytes exposed to 250 mg/L, indicating a decrease in the capability of haemocytes to assume a spreading morphology, fits the former above hypothesis. Future experiments will be directed to assess further the effects of fluoride on haemocyte functionality. * Corresponding author. E-mail address: [email protected] (L. Ballarin)
P-113. The pyloric caeca area is a major site for IgM+ and IgT+ B cell recruitment in response to oral vaccination in rainbow trout N.A. Ballesteros 1, S.S.R. Saint-Jean 1, S.I. Perez-Prieto 1, R. Castro 2, B. Abos 2, C. Tafalla 2, *. 1
Centro de Investigaciones Biológicas, (CSIC), Dpto. Microbiología Molecular y Biología de la Infección, 28040 Madrid, Spain; 2 Centro de Investigación en Sanidad Animal (CISA-INIA). 28130 Valdeolmos (Madrid), Spain Abstract Although previous studies have characterized some aspects of the immune response of the teleost gut in response to diverse pathogens or stimuli, most studies have focused on the posterior segments exclusively, and only a few have addressed the study comparatively throughout different segments of the digestive tract. Moreover, although the presence of IgM+ B cells had been described in the gut of several fish species, a recent publication identified IgT+ B cells as the main B cell specialized in mucosal immunity. However, there are still many details of how teleost intestinal immunity is regulated along the tract that remain unsolved, including the
often poorly immunogenic and may require adjuvants. Adjuvants stimulate components of the innate immune system, e.g. Toll-like receptors (TLRs), and lead to increased antigen immunogenicity. In humans, TLRs are studied in great detail and their ligands are used as adjuvants. Fish genome projects demonstrate a large number of TLRs. However, the functional characterization is lagging behind, and functional agonists are only verified for four of the 18 identified fish TLRs. With a more detailed understanding of fish TLRs, their agonists may find their way into fish vaccinology. To increase this understanding we are establishing a nuclear factor kappa B (NF-kB) luciferase reporter system for TLR signaling. The assay is based on transfection of HEK293 cells with a reporter plasmid containing luciferase under control of a NF-kB promoter. We have established a positive control which includes human TLR2 and two of its known ligands. The system is now ready for expression of TLRs from different fish species (salmon, trout and zebrafish). Cells can then be stimulated with potential agonists (from TLR agonist libraries and pathogenic salmon viruses) and assayed for NF-kB activation and TLR activation. We have also assayed salmon cells transfected with the reporter construct which showed NF-kB activation for several ligands known to activate human TLRs. This system reveals information that is important for a more detailed understanding of fish TLRs. * Corresponding author. E-mail address: [email protected] (M. Arnemo)
P-368. Arginine or glutamine supplementation to culture media modulates innate immune mechanisms of Senegalese sole (Solea senegalensis) head-kidney leukocytes R. Azeredo 1, 2, A. Afonso 1, 3, A. Oliva-Teles 1, 2, B. Costas 1, *. 1 CIMAR/CIIMAR – Centro Interdisciplinar de Investigação Marinha e Ambiental, 4050-123 Porto, Portugal; 2 Departamento de Biologia, Faculdade de Ciências da Universidade do Porto, 4169-007 Porto, Portugal; 3 ICBAS – Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal
Abstract Emerging evidence shows that many amino acids regulate key metabolic pathways that are crucial to immune responses. For instance, a large body of evidence from animal studies indicates that arginine or glutamine supplementation enhances immune function in various models of immunological challenges. The present study aimed to evaluate to what extent media supplemented with these amino acids can influence some aspects of the innate immune system in Senegalese sole (Solea senegalensis). Therefore, in vitro assays were performed using head-kidney leukocytes treated either with two levels of glutamine or arginine or with medium alone. Primary cell cultures were used to analyze the respiratory burst by quantifying extracellular superoxide anion and nitric oxide levels following infection with Tenacibaculum maritimum (2 106 colony-forming units ml 1). Additionally, phorbol myristate acetate was added as a soluble stimulant of the superoxide anion production and served as a positive control, while lipopolysaccharides from Escherichia coli (serotype 0111:B4) at two different concentrations were added to cell monolayers for nitric oxide production. A significant increase in the superoxide anion production was observed after stimulation with bacteria in cells treated with glutamine (1 mM) as well as in those treated with arginine (0.5 and 1 mM). However, head-kidney cells incubated with either lipopolysaccharides (1 and 10 mg ml 1) or T. maritimum for 72 h did not show significant differences in the nitric oxide production at any treatment. Preliminary results suggest that glutamine or arginine levels beyond those required for normal cell growth and differentiation may lead to a better leucocyte performance against antigens. Glutamine is a major energy substrate for leukocytes and is responsible for the activation of macrophage functions, while arginine is a precursor substrate for polyamine biosynthesis. Regarding nitric oxide production, the absence of different responses among treatments could be due to the incubation period since distinct responses have been observed in cells incubated for 48 h. Further studies
1695
are now being carried out in our laboratory to study the gene expression profile in head-kidney cells following different stimuli and media supplementation. * Corresponding author. E-mail address: [email protected] (B. Costas)
P-259. Fluoride influences haemocytes functionality in the venus clam Venerupis philippinarum L. Ballarin*, L. Masiero, V. Covre, S. Casellato. Department of Biology, University of Padova, Padova, Italy Abstract Invertebrates rely only on innate immunity to cope with foreign, potentially pathogenic microorganisms having entered the organism and haemocytes play a pivotal role in immune responses, as they are involved in phagocytosis, encapsulation, secretion of lectins, cytokines and cytotoxic factors. Therefore, adverse effects on haemocytes can compromise the survival of the organism. In the present study, we analysed the effects of the exposure of haemocytes of the venus clam Venerupis philippinarum to fluoride on cell morphology and functionality. Indeed, the increasing concentration of fluoride in many aquatic ecosystems, as a consequence of human activities, is a matter of great concern and the main negative effect of fluoride are related to its inhibition of metabolic processes, such as glycolysis and protein synthesis. Haemocytes, collected from the adductor muscles, were exposed to NaF (10, 50 and 250 mg/L) in filtered seawater (FSW), for 60 min. We observed a significant (p < 0.01) increase in cell mortality, using the Trypan Blue assay, for all the concentrations used in our experiments. The Neutral Red assay indicated a decrease in the stability of the internal membranes after exposure to 250 mg/L as the dye, usually accumulating inside acid compartments, such as lysosomes, leaks from these compartments into the cytoplasm so that the cells assume a pinkish colour. We also reported a significant (p < 0.01) reduction in the fraction of phagocytosing cells which suggests an effect of fluoride ions on the cytoskeleton or the hampering of the interaction with foreign particles. The observed increase in the cell shape factor in haemocytes exposed to 250 mg/L, indicating a decrease in the capability of haemocytes to assume a spreading morphology, fits the former above hypothesis. Future experiments will be directed to assess further the effects of fluoride on haemocyte functionality. * Corresponding author. E-mail address: [email protected] (L. Ballarin)
P-113. The pyloric caeca area is a major site for IgM+ and IgT+ B cell recruitment in response to oral vaccination in rainbow trout N.A. Ballesteros 1, S.S.R. Saint-Jean 1, S.I. Perez-Prieto 1, R. Castro 2, B. Abos 2, C. Tafalla 2, *. 1
Centro de Investigaciones Biológicas, (CSIC), Dpto. Microbiología Molecular y Biología de la Infección, 28040 Madrid, Spain; 2 Centro de Investigación en Sanidad Animal (CISA-INIA). 28130 Valdeolmos (Madrid), Spain Abstract Although previous studies have characterized some aspects of the immune response of the teleost gut in response to diverse pathogens or stimuli, most studies have focused on the posterior segments exclusively, and only a few have addressed the study comparatively throughout different segments of the digestive tract. Moreover, although the presence of IgM+ B cells had been described in the gut of several fish species, a recent publication identified IgT+ B cells as the main B cell specialized in mucosal immunity. However, there are still many details of how teleost intestinal immunity is regulated along the tract that remain unsolved, including the