ABSTRACT
Rab GTPases control trafficking of intracellular vesicles and are key regulators of endocytic and secretory pathways. Due to their specific distribution, they may serve as markers for different endolysosomal compartments. Since Rab GTPases are involved in uptake and trafficking of endocytosed ligands and cell receptors, as well as secretion of immune mediators, they have been implicated in diverse immunological processes and their functions are often exploited by intracellular pathogens such as viruses. While Rab proteins have been studied extensively in mammals, their functions in vesicle trafficking in teleosts are not well known. In the present work, Atlantic salmon Rab5c, Rab7a and Rab27a homologs were studied in terms of intracellular distribution and gene expression. Structured illumination microscopy demonstrated that transgenic, GFP-tagged salmon Rab5c and Rab7a are, predominantly, located within early endosomes and late endosomes/lysosomes, respectively. In contrast, Rab27a showed a broader distribution, which indicates that it associates with diverse intracellular vesicles and organelles. Infection of salmon with Salmonid alphavirus subtype 3 (SAV3) enhanced the mRNA levels of all of the studied Rab isoforms in heart and head kidney and most of them were upregulated in spleen. This may reflect the capacity of the virus to exploit the functions of these rab proteins. It is also possible that the transcriptional regulation of Rab proteins in SAV3-infected organs may play a role in the antiviral immune response. The latter was further supported by in vitro experiments with adherent head kidney leukocytes. The expression of Rab5c and Rab27a was upregulated in these cells following stimulation with TLR ligands including CpG oligonucleotides and polyI:C. The expression of most of the analyzed Rab isoforms in the primary leukocytes was also enhanced by stimulation with type I IFN. Interestingly, IFN-gamma had a negative effect on Rab7a expression which may be linked to the priming activity of this cytokine on monocytes and macrophages. Overall, these data demonstrate that the intracellular distribution of Rab5c, Rab7a and Rab27a is phylogenetically conserved within vertebrates and that these molecules might be implicated in viral infections and the regulation of the antiviral immune response in Atlantic salmon.
Subject(s)
Alphavirus Infections/veterinary , Fish Proteins/genetics , Salmo salar/genetics , rab GTP-Binding Proteins/genetics , rab27 GTP-Binding Proteins/genetics , rab5 GTP-Binding Proteins/genetics , Alphavirus , Alphavirus Infections/immunology , Animals , Cells, Cultured , Endosomes/genetics , Fish Proteins/immunology , Gene Expression , Gene Expression Regulation , Head Kidney/cytology , Head Kidney/immunology , Leukocytes/immunology , Lysosomes/genetics , Salmo salar/immunology , Sequence Homology , rab GTP-Binding Proteins/immunology , rab27 GTP-Binding Proteins/immunology , rab5 GTP-Binding Proteins/immunologyABSTRACT
BACKGROUND: Bacterial DNA is well-known for its potent immunostimulatory properties which have been attributed to the abundance of CpG dinucleotides within the genomes of prokaryotes. Research has found that mammalian TLR9 is a receptor which mediates the immune response to CpG DNA; however, its functional properties in non-mammalian vertebrates are still poorly characterized. Leukocytes isolated from lower vertebrates, including teleosts, respond to CpG DNA and TLR9 has been identified in many fish species; however, the ligand-binding properties of fish TLR9 have, so far, not been studied. The fact that some vertebrates, like chicken, lack TLR9 and use an alternative molecule (TLR21) as a receptor for CpGs has questioned the functional conservation of TLR9 within vertebrates. RESULTS: In the current study, TLR9 from Atlantic salmon (SsTLR9) has been found to interact with synthetic oligonucleotides via a CpG-independent but a pH-dependent mechanism. The endogenous receptor, expressed by primary mononuclear phagocytes colocalizes with CpG oligonucleotides (ODNs) in vesicles that appear to be endosomes. When overexpressed in salmonid cell lines, SsTLR9 spontaneously activates ISRE-containing promoters of genes involved in the IFN response; however, the transgenic receptor fails to translocate to CpG-containing endosomes. This indicates that only specific immune cell types have the ability to relocate the receptor to the appropriate cellular compartments where it may become activated by its ligand. In addition, through co-precipitation and mass spectrometry, two salmon proteins - hnRNPA0 and NCOA5, which both contain RNA-binding domains (RRM), were found to bind CpG ODNs, suggesting they may be involved in the CpG response in salmon leukocytes. CONCLUSION: The presented data are the first to demonstrate that the DNA-binding properties of TLR9 are conserved between teleosts and mammals. The current study also identifies additional molecules which may function as mediators of the immunostimulatory properties of foreign DNA.
Subject(s)
Oligodeoxyribonucleotides/metabolism , RNA-Binding Proteins/metabolism , Salmo salar/metabolism , Toll-Like Receptor 9/metabolism , Animals , Animals, Genetically Modified , Cell Compartmentation/drug effects , Chromatography, Liquid , Cytoplasm/metabolism , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/drug effects , Embryo, Nonmammalian/metabolism , Endosomes/drug effects , Endosomes/metabolism , Head Kidney/cytology , Hydrogen-Ion Concentration/drug effects , Interferon-gamma/pharmacology , Ligands , Lysosomes/metabolism , Mass Spectrometry , Phagocytes/drug effects , Phagocytes/metabolism , Promoter Regions, Genetic/genetics , Protein Binding/drug effects , Protein Transport/drug effects , Recombinant Fusion Proteins/metabolism , Response Elements/genetics , Salmon/embryology , Transcription, Genetic/drug effects , Up-Regulation/drug effectsABSTRACT
The Mitogen-activated protein kinases (MAPK) are involved in transmitting intracellular signals downstream of diverse cell surface receptors and mediate the response to ligands such as growth factors, hormones and cytokines. In addition, MAPK are critically involved in the innate immune response to pathogen-derived substances, commonly referred to as pathogen-associated molecular patterns (PAMPs), such as bacterial lipopolysaccharide (LPS) and bacterial DNA rich in CpG dinucleotides. Currently, a great deal of knowledge is available about the involvement of MAPK in the innate immune response to PAMPs in mammals; however, little is known about the role of the different MAPK classes in the immune response to PAMPs in lower vertebrates. In the current study, p38 phosphorylation was induced by CpG oligonucleotides (ODNs) and LPS in primary salmon mononuclear phagocytes. Pre-treatment of the cells with a p38 inhibitor (SB203580) blocked the PAMP-induced p38 activity and suppressed the upregulation of most of the CpG- and LPS-induced transcripts highlighting the role of this kinase in the salmon innate immune response to PAMPs. In contrast to p38, the phosphorylation of extracellular signal-regulated kinase (ERK), a MAPK involved primarily in response to mitogens, was high in resting cells and, surprisingly, incubation with both CpG and control ODNs downregulated the phospho-ERK levels independently of p38 activation. The basal phospho-ERK level and the CpG-inducible p38 phosphorylation were greatly influenced by the length of in vitro incubation. The basal phospho-ERK level increased gradually throughout a 5-day culture period and was PI3K-dependent as demonstrated by its sensitivity to Wortmannin suggesting it is influenced by growth factors. Overall these data indicate that both basal and PAMP-induced activity of MAPKs might be greatly influenced by the differentiation status of salmon mononuclear phagocytes.
Subject(s)
Leukocytes/enzymology , Salmo salar/genetics , Salmo salar/immunology , Signal Transduction , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Blotting, Western/veterinary , Cell Differentiation , DNA, Bacterial/chemistry , Escherichia coli , Imidazoles/pharmacology , Immunity, Innate , Leukocytes/drug effects , Lipopolysaccharides/pharmacology , Microarray Analysis/veterinary , Oligodeoxyribonucleotides/pharmacology , Phagocytosis/drug effects , Phosphatidylinositol 3-Kinase/metabolism , Phosphorylation , Pyridines/pharmacology , Real-Time Polymerase Chain Reaction/veterinary , Salmo salar/metabolism , Up-RegulationABSTRACT
MyD88 is an intracellular adaptor protein that transmits signals downstream of immune receptors such as the IL-1 receptor and the majority of the known mammalian toll-like receptors. Homologs of MyD88 have been identified in many vertebrate species; however, the adaptor has been studied mostly in mammals, and little is known about its function in lower vertebrates. The results presented in the current paper demonstrate, for the first time, that the teleost MyD88, through its Toll/Interleukin-1 receptor domain, interacts with SsIRF3 and two SsIRF7 paralogs: transcription factors that are critically involved in the virus-induced IFN responses. The data further highlight the potential of transgenic SsMyD88 to modulate the IRF-induced type I IFN response as the adaptor synergized with SsIRF3 to activate IRF-E/IFN-stimulated response element-containing reporter gene constructs and endogenous myxovirus resistance homolog expression. Microscopy analyses demonstrated that, similar to mammalian MyD88, both endogenous and transgenic SsMyD88 accumulated in intracellular aggregates. However, unlike the endogenous SsMyD88 clusters, which co-localized with endocytosed CpGs and probably represented myddosomes, overexpressed SsMyD88 accumulated in aggresomes. Although these structures accumulated ubiquitinated proteins, they did not associate with the autophagosome markers p62 and light chain 3-like protein, indicating that they are most likely classical aggresomes rather than aggresome-like induced structures, aggregates of ubiquitinated proteins induced by toll-like receptor/MyD88 signaling in antigen-presenting cells. The significance of the accumulation of transgenic MyD88 in aggresomes is currently unknown; nevertheless it is tempting to speculate that it might represent a defense mechanism against the potentially harmful effects of excessive MyD88 signaling.
Subject(s)
Interferon Type I/metabolism , Myeloid Differentiation Factor 88/chemistry , Animals , Cell Nucleus/metabolism , CpG Islands , Endocytosis , Evolution, Molecular , Gene Expression Regulation , HEK293 Cells , Humans , Immunity, Innate , Interferon Regulatory Factor-3/metabolism , Interferon Regulatory Factor-7/metabolism , Salmo salar , TransgenesABSTRACT
ß-Glucans (BG) are glucose polymers which are produced in bacteria and fungi but not in vertebrate organisms. Being recognized by phagocytic leukocytes including macrophages and neutrophils through receptors such as dectin-1 and Complement receptor 3 (CR3), the BG are perceived by the innate immune system of vertebrates as foreign substances known as Pathogen Associated Molecular Patterns (PAMPs). The yeast-derived BG has been recognized for its potent biological activity and it is used as an immunomodulator in human and veterinary medicine. The goal of the current study was to characterize the immunostimulatory activity of soluble yeast BG in primary cultures of Atlantic salmon (Salmo salar) head kidney leukocytes (HKLs) in which phagocytic cell types including neutrophils and mononuclear phagocytes predominate. The effect of BG on the secretome of HKL cultures, including secretion of extracellular vesicles (EVs) and soluble protein55s was characterized through western blotting and mass spectrometry. The results demonstrate that, along with upregulation of proinflammatory genes, BG induces secretion of ubiquitinated proteins (UbP), MHCII-containing EVs from professional antigen presenting cells as well as proteins derived from granules of polymorphonuclear granulocytes (PMN). Among the most abundant proteins identified in BG-induced EVs were beta-2 integrin subunits, including CD18 and CD11 homologs, which highlights the role of salmon granulocytes and mononuclear phagocytes in the response to soluble BG. Overall, the current work advances the knowledge about the immunostimulatory activity of yeast BG on the salmon immune system by shedding light on the effect of this PAMP on the secretome of salmon leukocytes.
Subject(s)
Immunity, Innate/immunology , Leukocytes/immunology , Phagocytes/immunology , Salmo salar/immunology , beta-Glucans/immunology , Animals , Extracellular Vesicles/immunology , Gene Expression Profiling , Head Kidney/immunology , Secretome/immunologyABSTRACT
A cDNA clone, designated sbIL-6 (seabream interleukin-6), was obtained from a cDNA library of enriched immune-stimulated sequences from gilthead seabream. The deduced sbIL-6 protein corresponds to a 225-amino acid protein with a putative 24-amino acid signal peptide, four conserved alpha helices and one N-linked glycosylation site. At the amino acid level sbIL-6 shares 23-26% identity with mammalian IL-6 sequences and 30-51% identity with other fish IL-6 sequences. The structure of the sbIL-6 gene consisted of 5 exons and 4 introns, spanning 2.4 kb. Healthy fish expressed sbIL-6 in white muscle, skin, spleen, anterior intestine and stomach, while no expression was detected in brain, gill, head kidney, posterior intestine and adipose tissue. A significant up-regulation of sbIL-6 expression was observed after lipopolysaccharide (LPS), Vibrio anguillarum DNA (VaDNA) and peptidoglycan treatment in cultured seabream head kidney leukocytes. Using purified immune cells, sbIL-6 expression was induced similarly in macrophages and acidophilic granulocytes by VaDNA but LPS was more effective in inducing sbIL-6 expression in acidophilic granulocytes than in macrophages. Furthermore, in vivo infection of seabream with live V. anguillarum caused significant increases in sbIL-6 mRNA expression in the thymus, peritoneal exudate, head kidney and gills. In summary, our study provides further evidence for the existence of distinct IL-6 genes in lower vertebrates and for the strong induction of their expression by immune stimuli, supporting the notion of a potentially important role for this cytokine in fish.
Subject(s)
Interleukin-6/genetics , Sea Bream/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Gene Expression Profiling , Gene Expression Regulation , Interleukin-6/chemistry , Interleukin-6/metabolism , Molecular Sequence Data , Phylogeny , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sea Bream/microbiology , Sequence Alignment , VibrioABSTRACT
Due to their ability to present foreign antigens and prime naïve T cells, macrophages, and dendritic cells (DCs) are referred to as professional antigen-presenting cells (APCs). Although activated macrophages may function as APCs, these cells are particularly effective at directly engaging pathogens through phagocytosis, and production of antimicrobial compounds. On the other hand, DCs possess superb antigen-presenting and costimulatory capacity and they are essential for commencement and regulation of adaptive immune responses. In in vitro models, development of mature mammalian DCs from monocytes requires sequential exposure to growth factors (including GM-CSF and IL-4) and proinflammatory stimuli such as toll-like receptor (TLR) ligands. Currently, except for IL-4/13, neither orthologs nor functional analogs of the growth factors which are essential for the differentiation of mammalian DCs (including GM-CSF and FLT3) have been identified in teleosts and data about differentiation of piscine APCs is scant. In the present study, primary salmon mononuclear phagocytes (MPs) stimulated in vitro for 5-7 days with a B-class CpG oligodeoxynucleotides (ODN 2006PS) underwent morphological differentiation and developed "dendritic" morphology, characterized by long, branching pseudopodia. Transcriptional profiling showed that these cells expressed high levels of proinflammatory mediators characteristic for M1 polarized MPs. However, the cells treated with CpGs for 7 days downregulated their surface MHCII molecules as well as their capacity to endocytose ovalbumin and exhibited attenuated allostimulatory activity. This concurred with transcriptional downregulation of costimulatory CD80/86 and upregulation of inhibitory CD274 (B7-H1) genes. Despite their exhausted allostimulatory activity, these cells were still responsive to re-stimulation with gardiquimod (a TLR7/8 ligand) and further upregulated a wide array of immune genes including proinflammatory mediators such as intereukin-1 beta and tumor necrosis factor. Overall, the presented data highlight the disparate effects TLR ligands may have on the proinflammatory status of APCs, on one side, and their antigen-presenting/costimulatory functions, on the other. These findings also indicate that despite the poor phylogenetic conservation of the growth factors involved in the differentiation of DCs, some of the processes that orchestrate the development and the differentiation of professional APCs are conserved between teleosts in mammals.
Subject(s)
Cell Differentiation/immunology , Dendrites , Mononuclear Phagocyte System/cytology , Mononuclear Phagocyte System/metabolism , Oligodeoxyribonucleotides/immunology , Salmo salar/genetics , Salmo salar/immunology , Transcriptome , Animals , Biomarkers , Cells, Cultured , Gene Expression Profiling , Inflammation Mediators , Mononuclear Phagocyte System/immunology , Phagocytosis/genetics , Phagocytosis/immunology , Salmo salar/metabolismABSTRACT
Members of the Toll-like receptor family 9 (TLR9) subfamily sense viral and bacterial DNA present in the endosomal compartment. Here we describe the cloning and regulation of a TLR9 gene from Atlantic salmon (Salmo salar). The salmon TLR9 cDNA encodes 1075 amino acids and analysis of the inferred protein sequence shows that several amino acid residues known to be important for the functions of TLR9 in mammals are conserved in salmon. Furthermore, TLR9 expression was elevated in head kidney leukocytes after in vitro treatment with CpG ODNs and recombinant trout interferon (IFN)-gamma. IFN-gamma was the strongest inducer of TLR9 expression. Together, the results indicate that the structure, the expression and possibly the function of TLR9 are conserved across the teleost and mammalian lineages.
Subject(s)
Interferon-gamma/metabolism , Salmo salar/genetics , Toll-Like Receptor 9/genetics , Toll-Like Receptor 9/metabolism , Amino Acid Sequence , Animals , Cloning, Molecular , Leukocytes/cytology , Leukocytes/metabolism , Molecular Sequence Data , Oligodeoxyribonucleotides/immunology , Oligodeoxyribonucleotides/metabolism , Phylogeny , Salmo salar/immunology , Sequence Alignment , Sequence Homology, Nucleic Acid , Toll-Like Receptor 9/immunology , Up-RegulationABSTRACT
BACKGROUND: The aim of this study was to identify differentially expressed ovarian genes during primary and early secondary oocyte growth in coho salmon, a semelparous teleost that exhibits synchronous follicle development. METHODS: Reciprocal suppression subtractive hybridization (SSH) libraries were generated from ovaries with perinucleolus (P) or cortical alveolus (CA) stage follicles and selected genes were assessed with quantitative PCR (qPCR). An assessment of changes in RNA composition during oocyte growth and its relationship to transcript levels was also conducted. RESULTS: SSH revealed several differentially expressed genes during early oogenesis, some which will not likely be utilized until 1-3 years later in salmon. Zona pellucida glycoprotein (zp) genes, vitellogenin receptor (vldlr) isoforms, cathepsin B (ctsba), cyclin E (ccne), a DnaJ transcript (dnaja2), and a ferritin subunit (fth3) were significantly elevated at the P stage, while a C-type lectin, retinol dehydrogenase (rdh1), and a coatomer protein subunit (cope) were upregulated at the CA stage. Putative follicle cell transcripts such as anti-Müllerian hormone (amh), lipoprotein lipase (lpl), apolipoprotein E (apoe), gonadal soma-derived growth factor (gsdf) and follicle-stimulating hormone receptor (fshr) also increased significantly at the CA stage. The analysis of RNA composition during oocyte growth showed that the total RNA yield and proportion of messenger RNA relative to non-polyadenylated RNAs declined as oogenesis progressed. This influenced apparent transcript levels depending on the type of RNA template used and normalization method. CONCLUSION: In coho salmon, which exhibit a dramatic change in oocyte size and RNA composition during oogenesis, use of messenger RNA as template and normalization of qPCR data to a housekeeping gene, ef1a, yielded results that best reflected transcript abundance within the ovarian follicle. Synthesis of zp transcripts and proteins involved in yolk incorporation and processing occurred during primary growth, while increased expression of a CA component and genes related to lipid incorporation occurred concomitant with the appearance of CA, but prior to lipid accumulation. Significant increases in transcripts for fshr, gsdf, and amh at the CA stage suggest a role of FSH and TGFbeta peptides in previtellogenic oocyte growth and puberty onset in female salmon.
Subject(s)
Gene Expression , Oncorhynchus kisutch/physiology , Oocytes/growth & development , Ovary/chemistry , Animals , Anti-Mullerian Hormone/genetics , Female , Gene Expression Profiling , Gene Expression Regulation, Developmental , Gene Library , Nucleic Acid Hybridization , Oogenesis/genetics , Polymerase Chain Reaction , RNA/analysis , RNA, Messenger/analysisABSTRACT
A partial cDNA with significant similarity to IL-6 was identified in rainbow trout. Rapid amplification of cDNA ends was used to obtain the full sequence of the trout IL-6 homolog which contains 1180 nucleotides. The transcript encodes a predicted protein of 219 amino acids and eight instability motifs in the 3'UTR. While the complete sequence of the trout IL-6 is poorly conserved, the protein contains a distinct IL-6/G-CSF/MGF family consensus pattern and predicted characteristic alpha-helical tertiary structure. However, like in fugu, trout IL-6 lacks a pair of cysteine residues, which in mammals are involved in formation of a disulphide bond. The expression of the IL-6 homolog in trout mononuclear phagocytes was highly up-regulated by LPS but not poly(I:C) as demonstrated by Northern analysis. Using RT-PCR the IL-6 expression was detected in trout spleen, gill, gastrointestinal tract, ovary and brain. The highest transcript levels were detected in the ovary suggesting that IL-6 may perform specific functions within this organ.
Subject(s)
Interleukin-6/metabolism , Oncorhynchus mykiss/immunology , Amino Acid Sequence , Animals , Cloning, Molecular , Conserved Sequence , Gene Expression , Interleukin-6/classification , Interleukin-6/genetics , Molecular Sequence Data , Oncorhynchus mykiss/genetics , Phylogeny , RNA, Messenger/analysis , RNA, Messenger/metabolism , Sequence Homology, Amino Acid , Tissue DistributionABSTRACT
Toll-like receptors (TLRs) are a small family of type-I glycoproteins that bind to and are activated by conserved non-self molecular signatures carried by microorganisms. Toll-like receptor 4 is triggered by most lipopolysaccharides (LPS). LPS is a complex amphipathic saccharolipidic glycan derived from Gram-negative bacteria. Unique among TLRs, TLR4 activity and interaction with its natural ligand(s) strictly depends on the presence of the extracellular adaptor MD-2. MD-2 is a small secreted glycoprotein that binds with cytokine-like affinities to both the hydrophobic portion of LPS and to the extracellular domain of TLR4. The interaction between MD-2 and LPS induces a triggering event on TLR4, which involves the molecular rearrangement of the receptor complex and its homotypic aggregation. In silico analysis suggests that MD-2 and MD-1 are paralogs derived from a common predecessor at the level of early vertebrates. In this review, we summarize the current state of knowledge concerning MD-2.
Subject(s)
Lymphocyte Antigen 96/physiology , Amino Acid Sequence , Animals , Bacteria/immunology , Humans , Immunity, Innate/physiology , Inflammation/immunology , Lipopolysaccharides/immunology , Lymphocyte Antigen 96/genetics , Molecular Sequence Data , Toll-Like Receptor 4/physiologyABSTRACT
Rainbow trout (Oncorhynchus mykiss) cells of a monocyte-macrophage lineage (rtMOCs) were used to characterize the ability of the trout innate immune system to recognize and respond to different pathogen associated molecular pattern (PAMP) bearing substances. Compared to what has been reported for mammalian macrophages, rtMOCs responded with lower sensitivity to lipopolysaccharide (LPS) from Escherichia coli (EC-LPS) and Pseudomonas aeruginosa (PA-LPS). The sensitivity of rtMOCs to LPS was not influenced by the presence of serum which suggests that the resistance to endotoxic shock in fish may be due to the lack of serum-borne factors that confer sensitivity to LPS in mammals. The time course of the response to PAMPs could be separated into two patterns. EC-LPS induced stable cytokine expression whereas PA-LPS, zymosan and muramyl dipeptide induced transient TNF2 expression. By analogy to the type of stimulation observed in mammals it can be hypothesized that different signaling pathways, possibly initiated by different receptors, may be involved in the recognition of these PAMPs by rtMOCs.
Subject(s)
Macrophage Activation/drug effects , Macrophage-Activating Factors/pharmacology , Macrophages/drug effects , Monocytes/physiology , Oncorhynchus mykiss/metabolism , Animals , Cell Lineage , Cells, Cultured , Cytokines/metabolism , Escherichia coli/immunology , Kinetics , Lipopolysaccharides/pharmacology , Oncorhynchus mykiss/immunology , Pseudomonas aeruginosa/immunology , Sensitivity and Specificity , Stimulation, Chemical , Tumor Necrosis Factor-alpha/metabolismABSTRACT
The interaction between pathogens and their multicellular hosts is initiated by activation of pathogen recognition receptors (PRRs). These receptors, that include most notably members of the toll-like receptor (TLR) family, recognize specific pathogen-associated molecular patterns (PAMPs). TLR4 is a central part of the receptor complex that is involved in the activation of the immune system by lipopolysaccharide (LPS) through the specific recognition of its endotoxic moiety (Lipid A). This is a critical event that is essential for the immune response to Gram-negative bacteria as well as the etiology of endotoxic shock. Interestingly, compared to mammals, fish are resistant to endotoxic shock. This in vivo resistance concurs with in vitro studies demonstrating significantly lowered sensitivity of fish leukocytes to LPS activation. Further, our in vitro analyses demonstrate that in trout mononuclear phagocytes, LPS fails to induce antiviral genes, an event that occurs downstream of TLR4 and is required for the development of endotoxic shock. Finally, an in silico approach that includes mining of different piscine genomic and EST databases, reveals the presence in fish of all of the major TLR signaling elements except for the molecules specifically involved in TLR4-mediated endotoxin recognition and signaling in mammals. Collectively, our analysis questions the existence of TLR4-mediated cellular responses to LPS in fish. We further speculate that other receptors, in particular beta-2 integrins, may play a primary role in the activation of piscine leukocytes by LPS.
Subject(s)
Endotoxins/immunology , Fishes/immunology , Receptors, Pattern Recognition/immunology , Animals , Biological Evolution , Endotoxins/pharmacology , Phagocytes/drug effects , Toll-Like Receptors/genetics , Toll-Like Receptors/immunologyABSTRACT
In the current study, the first non-mammalian growth/differentiation factor (GDF) 11-like homolog was cloned from zebrafish. At the nucleotide level, zebrafish GDF11 is most similar to human GDF11 (79%), while the peptide is most similar to mouse GDF11 (78%). Phylogenetic analysis showed that the zebrafish GDF11 clusters with mammalian GDF11s. This study also cloned a second MSTN form in zebrafish most similar to Salmonid MSTN2 forms. Based on real time PCR, GDF11 is expressed in multiple adult tissues, with levels highest in whole heads and gonads, and expression is less ubiquitous when compared to MSTN expression. During embryonic development, real time PCR demonstrated increasing GDF11 mRNA levels 10 h post-fertilization (hpf), while MSTN mRNA levels remain low until 48 hpf. This is the first report of a transforming growth factor (TGF)-beta superfamily member in a non-mammalian species that is more closely related to GDF11 than MSTN, and also a second form of MSTN in zebrafish; suggesting that a more complex TGF-beta superfamily array exists in primitive vertebrates than previously thought.
Subject(s)
Bone Morphogenetic Proteins/genetics , Transforming Growth Factor beta/genetics , Zebrafish Proteins/genetics , Animals , Base Sequence , Bone Morphogenetic Proteins/isolation & purification , Gene Components , Growth Differentiation Factors , Molecular Sequence Data , Myostatin , Phylogeny , RNA, Messenger/analysis , Sequence Alignment , Tissue Distribution , Transforming Growth Factor beta/isolation & purification , Zebrafish Proteins/isolation & purificationABSTRACT
A primary cell culture system was used to obtain differentiated rainbow trout (Oncorhynchus mykiss) macrophages that were stimulated with Escherichia coli lipopolysaccharide (LPS-10 microg/ml) for 12 h in vitro. Messenger RNA from the LPS-stimulated cells was used to create two cDNA libraries from which a total of 1048 sequences were analyzed. A large number of cDNAs were obtained that could be related to immune function including structural proteins, proteases and antiproteases, regulators of transcription and translation, cell death regulators, receptors, lectins and immunoglobulins, cytokines and chemokines, cell surface antigens, signal transduction proteins, antimicrobial peptides, and enzymes involved in eicosanoid synthesis. Selected genes that were analyzed by RT-PCR and real time PCR and found to be upregulated by LPS, included vascular cell adhesion molecule, the CCAAT/enhancer binding protein beta, the inhibitor of NF-kB alpha, CD209, a major histocompatibility class II-invariant chain protein, cyclin L1, acute phase serum amyloid A, and prostaglandin endoperoxide synthase 2.
Subject(s)
Gene Expression Regulation/immunology , Lipopolysaccharides/pharmacology , Macrophages/metabolism , Animals , Cells, Cultured , Gene Expression Regulation/drug effects , Gene Library , Genes , Immunity/genetics , Macrophages/immunology , Oncorhynchus mykiss , RNA, Messenger/analysis , Up-Regulation/drug effects , Up-Regulation/immunologyABSTRACT
The full-length cDNA for the cod (Gadus morhua) StAR was cloned by RT-PCR and library screening using ovarian RNA. From the library screening, 2 size classes of cDNA were obtained; a 1577 bp cDNA (cStAR1) and a 2851 bp cDNA (cStAR2). The cStAR1 cDNA presumably encodes a protein of 286 amino acids. The cStAR2 cDNA was composed of 6 separated sequences that contained all of the coding regions of cStAR1 when added together, but also contained 5 noncoding regions not observed in cStAR1. Polymerase chain reactions of cod genomic DNA produced products slightly larger than cStAR2. The sequence of these products were the same as cStAR2 but revealed one additional noncoding region (intron). Thus, the fish StAR gene contains the same number of exons (7) and introns (6) as observed in mammals, but is approximately half the size of the mammalian gene. Using Northern analysis and RT-PCR, cStAR1 expression was observed only in testes, ovaries and head kidneys. Polymerase chain reaction products were also observed using cDNA from steroidogenic tissues and primers designed to regions specific for cStAR2, indicating that cStAR2 is expressed in tissues and may account for the presence of larger transcripts observed on Northern blots.
Subject(s)
Fishes/genetics , Gene Components , Phosphoproteins/genetics , Animals , Base Sequence , Cloning, Molecular , DNA, Complementary/genetics , Exons , Introns , Molecular Sequence Data , Organ Specificity , Phosphoproteins/metabolism , RNA, Messenger/analysis , Tissue DistributionABSTRACT
Suppressor of cytokine signaling (SOCS) proteins are crucially involved in the control of inflammatory responses through their impact on various signaling pathways including the JAK/STAT pathway. Although all SOCS protein family members are identified in teleost fish, their functional properties in non-mammalian vertebrates have not been extensively studied. To gain further insight into SOCS functions in bony fish, we have identified and characterized the Atlantic salmon (Salmo salar) SOCS1, SOCS2 and CISH genes. These genes exhibited sequence conservation with their mammalian counterparts and they were ubiquitously expressed. SOCS1 in mammalian species has been recognized as a key negative regulator of interferon (IFN) signaling and recent data for the two model fish Tetraodon (Tetraodon nigroviridis) and zebrafish (Danio rerio) suggest that these functions are conserved from teleost to mammals. In agreement with this we here demonstrate a strong negative regulatory activity of salmon SOCS1 on type I and type II IFN signaling, while SOCS2a and b and CISH only moderately affected IFN responses. SOCS1 also inhibited IFNγ-induced nuclear localization of STAT1 and a direct interaction between SOCS1 and STAT1 and between SOCS1 and the Tyk2 kinase was found. Using SOCS1 mutants lacking either the KIR domain or the ESS, SH2 and SOCS box domains showed that all domains affected the ability of SOCS1 to inhibit IFN-mediated signaling. These results are the first to demonstrate that SOCS1 is a potent inhibitor of IFN-mediated JAK-STAT signaling in teleost fish.
Subject(s)
Fish Proteins/genetics , Interferon Type I/physiology , Interferon-gamma/physiology , Signal Transduction , Suppressor of Cytokine Signaling Proteins/genetics , Active Transport, Cell Nucleus , Animals , Cell Nucleus/metabolism , Fish Proteins/metabolism , Gene Expression , HEK293 Cells , Humans , Janus Kinases/metabolism , Organ Specificity , Phylogeny , Promoter Regions, Genetic , Protein Binding , RNA, Messenger/genetics , RNA, Messenger/metabolism , STAT Transcription Factors/metabolism , Salmo salar , Suppressor of Cytokine Signaling Proteins/metabolism , Transcriptional ActivationABSTRACT
Mammalian IRF9 and STAT2, together with STAT1, form the ISGF3 transcription factor complex, which is critical for type I interferon (IFN)-induced signaling, while IFNγ stimulation is mediated by homodimeric STAT1 protein. Teleost fish are known to possess most JAK and STAT family members, however, description of their functional activity in lower vertebrates is still scarce. In the present study we have identified two different STAT2 homologs and one IRF9 homolog from Atlantic salmon (Salmo salar). Both proteins have domain-like structures with functional motifs that are similar to higher vertebrates, suggesting that they are orthologs to mammalian STAT2 and IRF9. The two identified salmon STAT2s, named STAT2a and STAT2b, showed high sequence identity but were divergent in their transactivation domain (TAD). Like STAT1, ectopically expressed STAT2a and b were shown to be tyrosine phosphorylated by type I IFNs and, interestingly, also by IFNγ. Microscopy analyses demonstrated that STAT2 co-localized with STAT1a in the cytoplasm of unstimulated cells, while IFNa1 and IFNγ stimulation seemed to favor their nuclear localization. Overexpression of STAT2a or STAT2b together with STAT1a activated a GAS-containing reporter gene construct in IFNγ-stimulated cells. The highest induction of GAS promoter activation was found in IFNγ-stimulated cells transfected with IRF9 alone. Taken together, these data suggest that salmon STAT2 and IRF9 may have a role in IFNγ-induced signaling and promote the expression of GAS-driven genes in bony fish. Since mammalian STAT2 is primarily an ISGF3 component and not involved in IFNγ signaling, our finding features a novel role for STAT2 in fish.
ABSTRACT
Tyk2, a member of the Janus Kinase (JAK) family of protein tyrosine kinases, is required for interferon-α/ß binding and signaling in higher vertebrates. Currently, little is known about the role of the different JAKs in signaling responses to interferon (IFN) in lower vertebrates including fish. In this paper we report the identification and characterization of Atlantic salmon (Salmo salar) Tyk2. Four cDNA sequences, two containing an open reading frame encoding full-length Tyk protein and two with an up-stream in frame stop codon, were identified. The deduced amino acid sequences of the salmon full-length Tyk2 proteins showed highest identity with Tyk2 from other species and their transcripts were ubiquitously expressed. Like in mammals the presented data suggests that salmon Tyk2 is auto-phosporylated when ectopically expressed in cells. In our experiments, full-length salmon Tyk2 overexpressed in CHSE-cells phosphorylated itself, while both a kinase-deficient mutant and the truncated Tyk2 (Tyk-short) were inactive. Interestingly, the overexpression of full length Tyk2 was shown to up-regulate the transcript levels of the IFN induced gene Mx, thus indicating the involvement of salmon Tyk2 in the salmon IFN I pathway.
Subject(s)
Fish Proteins/metabolism , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Salmo salar/metabolism , TYK2 Kinase/metabolism , Amino Acid Sequence , Animals , Cells, Cultured , Cloning, Molecular/methods , DNA, Complementary/genetics , Fish Proteins/biosynthesis , Fish Proteins/genetics , Gene Expression , Interferons/metabolism , Molecular Sequence Data , Phosphorylation , Phylogeny , Salmo salar/genetics , Sequence Homology, Amino Acid , Signal Transduction , TYK2 Kinase/biosynthesis , TYK2 Kinase/genetics , Transcriptional ActivationABSTRACT
Lymph nodes and spleen are major organs where mammalian antigen-presenting cells (APCs) initiate and orchestrate Ag-specific immune responses. Unlike mammals, teleosts lack lymph nodes and an interesting question is whether alternative organs may serve as sites for antigen presentation in teleosts. In the current study, fluorescent ovalbumin (Ova) and CpG oligonucleotides (ODNs) injected intra-abdominally were detected in significant numbers of salmon head kidney (HK) MHCII+ cells over a period of 2 weeks while in spleen the percentage of these was transient and declined from day 1 post injection. In vitro studies further shed light on the properties of the diverse MHCII+ cell types found in HK. The ultrastructure of a subpopulation of MHCII+ cells with a high capacity to endocytose and process Ova indicated that these were able to perform constitutive macropinocytosis. Upon stimulation with CpG ODNs these cells upregulated CD86 and gave very high levels of TNF mRNA indicating that these are professional APCs, related to macrophages and dendritic cells (DCs). A subpopulation of HK granulocytes expressed high levels of surface MHCII and upon CpG stimulation upregulated most of the tested APC marker genes. Although these granulocytes expressed TNF weakly, they had relatively high basal levels of IL-1ß mRNA and the CpG stimulation upregulated IL-1ß, along with its signaling and decoy receptors, to the highest levels as compared to other HK cell types. Interestingly, the high expression of IL-1ß mRNA in the granulocytes correlated with a high autophagy flux as demonstrated by LC3-II conversion. Autophagy has recently been found to be implicated in IL-1ß processing and secretion and the presented data suggests that granulocytes of salmon, and perhaps other teleost species, may serve as a valuable model to study the involvement of autophagy in regulation of the vertebrate immune response.