- Email: [email protected]
A closer look at Toll-like receptor 4 (TLR4) and toll-like receptor 20 (TLR20) of common carp (Cyprinus carpio)
Abstracts / Fish & Shellfish Immunology 34 (2013) 1635–1691
Abstract Interleukin (IL)-10 is an important regulatory cytokine that regulates both, innate and adaptive immunity. Mammalian IL-10 is produced by many cell types but primarily by antigen-specific T-lymphocytes and macrophages, and regulates the proliferation and differentiation of B- and T-lymphocytes after antigen recognition. IL-10 inhibits the production of reactive oxygen and nitrogen radicals in activated macrophages, and can redirect the immune response from a type-I (pro-inflammatory) to a type II (anti-inflammatory) immune response by suppressing the production of proinflammatory cytokines. A homologue of mammalian IL-10 has been identified in several fish species but very few studies have reported on its functional characterization. In the present study, we report on the production of recombinant carp IL-10 and its biological activities on carp leukocytes in vitro. Similar to mammalian IL-10, recombinant carp IL-10 shows to be a potent inhibitor of the respiratory burst induced by PMA and of nitric oxide production induced by LPS. IL-10 also downregulates the expression of pro-inflammatory cytokines such as interleukin-1beta. Upon binding to its receptor, IL-10 signals via the JAK/STAT pathway. The activation of JAK1 results in the phosphorylation of the cytosolic tails of the IL-10 receptor-1 (IL-10R1) which in turns facilitates the recruitment and phosphorylation of STAT3. Phospho-STAT3 induces the expression of SOCS3 leading to the downregulation of pro-inflammatory cytokines. A carp genome-based analysis shows the presence of duplicated genes for the IL-10R1, both possessing one conserved JAK1 docking site and two and maybe three STAT3 phosphorylation sites. Both genes are equally expressed in several carp tissues and cell types. Western blot analysis of head kidney leukocytes stimulated with recombinant carp IL-10, revealed increased levels of phospho-STAT3 within minutes after stimulation. We also observed an upregulation of SOCS3 gene expression in carp kidney leukocytes upon stimulation with recombinant IL-10. Altogether, our results suggest a conservation of the signaling pathway as well as anti-inflammatory activities of carp IL-10. * Corresponding author. E-mail address: [email protected] (C. Piazzon)
O-313. A closer look at Toll-like receptor 4 (TLR4) and toll-like receptor 20 (TLR20) of common carp (Cyprinus carpio) D. Pietretti*, M. Forlenza, I.R. Fink, G. Wiegertjes. Cell Biology and Immunology, Department of Animal Sciences, Wageningen University, Wageningen, The Netherlands Abstract Toll-like receptors (TLRs) constitute an important class of pattern-recognition receptors, which recognize a multitude of pathogen-associated molecular patterns (PAMPs). We focused on TLR4 and TLR20 of common carp (Cyprinus carpio), their signaling pathway and their possible functions as activating receptors involved in innate immune responses to various pathogens or immunostimulants. So far the expression of TLR4 and TLR20 genes has been reported only for few fish species of the Cypriniformes (zebrafish, grass carp, common carp) and close relatives Siluriformes (channel catfish). Multiple fish TLR4 genes, created by a (recent) duplication rather than speciation event, seem to exist. It is possible that these paralogs have evolved to express different ligand specificities. In mammals TLR4 recognizes lipopolysaccharides from Gram-negative bacteria. The ligand(s) for fish TLR4 have not been confirmed. Multiple TLR20 genes also seem to exist in zebrafish, but not in carp. Our phylogenetic analysis indicates an ancestral relationship could exist between TLR20 and TLR11/ TLR12, two TLRs found in mice but not in humans with ligand specificity for profilin from parasitic Toxoplasma gondii. At present, the ligand of TLR20 is unknown. We have cloned full-length coding sequences of carp TLR4a/b and carp TLR20 to study the function of these molecules. Expression analysis using real-time PCR show both TLR4 and TLR20 are mainly expressed in immune organs such as head kidney, gut and spleen with neutrophilic granulocytes
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as the primary source. We have also investigated the expression of TLR4 and of TLR20 after infection with protozoan parasites or after infection with spring vireamia of carp virus (SVCV). For TLR4, neither challenge with parasites nor with virus induced major changes in gene expression. However, we find high gene expression of TLR20 after infection with the protozoan parasites Trypanoplasma borreli or Trypanosoma carassii. We used the full-length sequences to create fluorescent protein-tagged TLRs for visualization of localization of these receptors by fluorescence microscopy. Our study suggests both receptors are located mainly in the cytoplasmic region. To define the unknown ligands of TLR4 and TLR20 we overexpressed these receptors in both human cell lines (HEK 293) and fish cell lines (EPC, CLC) stably transfected with a promoter of the transcription factor NF-kB and a luciferase reporter gene. These studies may help to identify ligands for carp TLR4 a/b and TLR20. * Corresponding author. E-mail address: [email protected] (D. Pietretti)
O-175. Temperature-induced transcription of inflammatory mediators and the influence of HSP70 following LPS stimulation of southern bluefin tuna leukocytes M. Polinski*, A. Bridle, B. Nowak. National Centre for Marine Conservation and Resource Sustainability, University of Tasmania, Launceston, Tasmania, Australia
Abstract Temperature is known to influence inflammatory signaling in mammals, but far less understood in fish. The aim of the present study was to explore the potential effects of temperature on innate immune signaling in head kidney and leukocyte populations of the economically important southern bluefin tuna through the identification and utilization of gene expression targets in vitro. Here, we identified the mRNA sequences of five potential inflammatory mediators – TNFa (1 and 2), IL-1b, IL-8, and Cox2 – and demonstrate induction of four – TNFa (2), IL-1b, IL-8, and Cox2 – following LPS stimulation of both peripheral blood leukocytes and head kidney homogenates in vitro by real-time quantitative PCR. Comparison of transcriptional expression in cultures held at 18 and 25 C (both within the presumed natural temperature range of this heterothermic species) showed accelerated transcription of cytokines TNFa, IL-1b and IL-8 following LPS stimulation at 25 C in both tissue types. Peak induction reached comparable levels for each transcript at both temperatures during the 24 h test period with only limited (if any) protraction in expression resulting from cold temperature (18 C) incubation. Partial mRNA sequences were also identified for both the constitutively expressed and heat inducible chaperone proteins Hsc70 and Hsp70, and 24h incubation at 25 C was sufficient to induce Hsp70 transcription in leukocyte but not in head kidney cell populations. Taken together these findings suggest that temperature exerts influence in the timing but not the degree of an innate inflammatory response in bluefin tuna and that different cell populations have differential responsiveness to heat shock in this heterothermic species. Further, LPS stimulation failed to induce Hsp70 at either incubation temperature in leukocytes; whereas 25 C incubation caused Hsp70 upregulation in leukocytes with or without the presence of LPS. This suggests that Hsp70 does not play a direct role in immune responsiveness for this species and that an environmental temperature of 25 C in excess of 24 h initiates a cellular stress response in blood cells of this organism. Lastly, a strong correlation between Hsp70 and IL-8 transcriptional expression was observed following LPS/heat shock stimulation of leukocytes and five potential heat shock response elements were subsequently identified on the gene promoter region of IL-8 indicating that heat shock co-activation of this chemokine previously identified in mammals is also likely present in fish. * Corresponding author. E-mail address: [email protected] (M. Polinski)
Abstract Interleukin (IL)-10 is an important regulatory cytokine that regulates both, innate and adaptive immunity. Mammalian IL-10 is produced by many cell types but primarily by antigen-specific T-lymphocytes and macrophages, and regulates the proliferation and differentiation of B- and T-lymphocytes after antigen recognition. IL-10 inhibits the production of reactive oxygen and nitrogen radicals in activated macrophages, and can redirect the immune response from a type-I (pro-inflammatory) to a type II (anti-inflammatory) immune response by suppressing the production of proinflammatory cytokines. A homologue of mammalian IL-10 has been identified in several fish species but very few studies have reported on its functional characterization. In the present study, we report on the production of recombinant carp IL-10 and its biological activities on carp leukocytes in vitro. Similar to mammalian IL-10, recombinant carp IL-10 shows to be a potent inhibitor of the respiratory burst induced by PMA and of nitric oxide production induced by LPS. IL-10 also downregulates the expression of pro-inflammatory cytokines such as interleukin-1beta. Upon binding to its receptor, IL-10 signals via the JAK/STAT pathway. The activation of JAK1 results in the phosphorylation of the cytosolic tails of the IL-10 receptor-1 (IL-10R1) which in turns facilitates the recruitment and phosphorylation of STAT3. Phospho-STAT3 induces the expression of SOCS3 leading to the downregulation of pro-inflammatory cytokines. A carp genome-based analysis shows the presence of duplicated genes for the IL-10R1, both possessing one conserved JAK1 docking site and two and maybe three STAT3 phosphorylation sites. Both genes are equally expressed in several carp tissues and cell types. Western blot analysis of head kidney leukocytes stimulated with recombinant carp IL-10, revealed increased levels of phospho-STAT3 within minutes after stimulation. We also observed an upregulation of SOCS3 gene expression in carp kidney leukocytes upon stimulation with recombinant IL-10. Altogether, our results suggest a conservation of the signaling pathway as well as anti-inflammatory activities of carp IL-10. * Corresponding author. E-mail address: [email protected] (C. Piazzon)
O-313. A closer look at Toll-like receptor 4 (TLR4) and toll-like receptor 20 (TLR20) of common carp (Cyprinus carpio) D. Pietretti*, M. Forlenza, I.R. Fink, G. Wiegertjes. Cell Biology and Immunology, Department of Animal Sciences, Wageningen University, Wageningen, The Netherlands Abstract Toll-like receptors (TLRs) constitute an important class of pattern-recognition receptors, which recognize a multitude of pathogen-associated molecular patterns (PAMPs). We focused on TLR4 and TLR20 of common carp (Cyprinus carpio), their signaling pathway and their possible functions as activating receptors involved in innate immune responses to various pathogens or immunostimulants. So far the expression of TLR4 and TLR20 genes has been reported only for few fish species of the Cypriniformes (zebrafish, grass carp, common carp) and close relatives Siluriformes (channel catfish). Multiple fish TLR4 genes, created by a (recent) duplication rather than speciation event, seem to exist. It is possible that these paralogs have evolved to express different ligand specificities. In mammals TLR4 recognizes lipopolysaccharides from Gram-negative bacteria. The ligand(s) for fish TLR4 have not been confirmed. Multiple TLR20 genes also seem to exist in zebrafish, but not in carp. Our phylogenetic analysis indicates an ancestral relationship could exist between TLR20 and TLR11/ TLR12, two TLRs found in mice but not in humans with ligand specificity for profilin from parasitic Toxoplasma gondii. At present, the ligand of TLR20 is unknown. We have cloned full-length coding sequences of carp TLR4a/b and carp TLR20 to study the function of these molecules. Expression analysis using real-time PCR show both TLR4 and TLR20 are mainly expressed in immune organs such as head kidney, gut and spleen with neutrophilic granulocytes
1673
as the primary source. We have also investigated the expression of TLR4 and of TLR20 after infection with protozoan parasites or after infection with spring vireamia of carp virus (SVCV). For TLR4, neither challenge with parasites nor with virus induced major changes in gene expression. However, we find high gene expression of TLR20 after infection with the protozoan parasites Trypanoplasma borreli or Trypanosoma carassii. We used the full-length sequences to create fluorescent protein-tagged TLRs for visualization of localization of these receptors by fluorescence microscopy. Our study suggests both receptors are located mainly in the cytoplasmic region. To define the unknown ligands of TLR4 and TLR20 we overexpressed these receptors in both human cell lines (HEK 293) and fish cell lines (EPC, CLC) stably transfected with a promoter of the transcription factor NF-kB and a luciferase reporter gene. These studies may help to identify ligands for carp TLR4 a/b and TLR20. * Corresponding author. E-mail address: [email protected] (D. Pietretti)
O-175. Temperature-induced transcription of inflammatory mediators and the influence of HSP70 following LPS stimulation of southern bluefin tuna leukocytes M. Polinski*, A. Bridle, B. Nowak. National Centre for Marine Conservation and Resource Sustainability, University of Tasmania, Launceston, Tasmania, Australia
Abstract Temperature is known to influence inflammatory signaling in mammals, but far less understood in fish. The aim of the present study was to explore the potential effects of temperature on innate immune signaling in head kidney and leukocyte populations of the economically important southern bluefin tuna through the identification and utilization of gene expression targets in vitro. Here, we identified the mRNA sequences of five potential inflammatory mediators – TNFa (1 and 2), IL-1b, IL-8, and Cox2 – and demonstrate induction of four – TNFa (2), IL-1b, IL-8, and Cox2 – following LPS stimulation of both peripheral blood leukocytes and head kidney homogenates in vitro by real-time quantitative PCR. Comparison of transcriptional expression in cultures held at 18 and 25 C (both within the presumed natural temperature range of this heterothermic species) showed accelerated transcription of cytokines TNFa, IL-1b and IL-8 following LPS stimulation at 25 C in both tissue types. Peak induction reached comparable levels for each transcript at both temperatures during the 24 h test period with only limited (if any) protraction in expression resulting from cold temperature (18 C) incubation. Partial mRNA sequences were also identified for both the constitutively expressed and heat inducible chaperone proteins Hsc70 and Hsp70, and 24h incubation at 25 C was sufficient to induce Hsp70 transcription in leukocyte but not in head kidney cell populations. Taken together these findings suggest that temperature exerts influence in the timing but not the degree of an innate inflammatory response in bluefin tuna and that different cell populations have differential responsiveness to heat shock in this heterothermic species. Further, LPS stimulation failed to induce Hsp70 at either incubation temperature in leukocytes; whereas 25 C incubation caused Hsp70 upregulation in leukocytes with or without the presence of LPS. This suggests that Hsp70 does not play a direct role in immune responsiveness for this species and that an environmental temperature of 25 C in excess of 24 h initiates a cellular stress response in blood cells of this organism. Lastly, a strong correlation between Hsp70 and IL-8 transcriptional expression was observed following LPS/heat shock stimulation of leukocytes and five potential heat shock response elements were subsequently identified on the gene promoter region of IL-8 indicating that heat shock co-activation of this chemokine previously identified in mammals is also likely present in fish. * Corresponding author. E-mail address: [email protected] (M. Polinski)