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1.
Front Immunol ; 12: 736964, 2021.
Article in English | MEDLINE | ID: mdl-34917074

ABSTRACT

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/immunology
2.
Fish Shellfish Immunol ; 99: 119-129, 2020 Apr.
Article in English | MEDLINE | ID: mdl-32014587

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/immunology
3.
Front Immunol ; 10: 378, 2019.
Article in English | MEDLINE | ID: mdl-30918507

ABSTRACT

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/metabolism
4.
FEBS Open Bio ; 4: 858-71, 2014.
Article in English | MEDLINE | ID: mdl-25379383

ABSTRACT

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.

5.
Dev Comp Immunol ; 45(1): 177-89, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24582990

ABSTRACT

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 Activation
6.
Dev Comp Immunol ; 41(4): 553-63, 2013 Dec.
Article in English | MEDLINE | ID: mdl-23872231

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 Activation
7.
Fish Shellfish Immunol ; 35(4): 1079-85, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23872471

ABSTRACT

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-Regulation
8.
Front Immunol ; 4: 137, 2013.
Article in English | MEDLINE | ID: mdl-23761795

ABSTRACT

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.

9.
BMC Immunol ; 14: 12, 2013 Mar 01.
Article in English | MEDLINE | ID: mdl-23452377

ABSTRACT

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 effects
10.
Dev Comp Immunol ; 38(3): 416-30, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22889889

ABSTRACT

The CD40L/CD40 signalling pathway is critically involved in the final stage of the maturation of DCs. This paper reports the identification and functional characterization of CD40L and CD40 from Atlantic salmon (Salmo salar). Salmon CD40L is a type II membrane-bound protein with a TNF homology domain in its extracellular C-terminal region, while CD40 is a type I membrane-bound receptor with a sequence pattern of four cysteine-rich domains in its extracellular N-terminal region. The salmon CD40L and CD40 were widely expressed, particularly in immune tissues, and while CD40L expression was induced by in vitro stimulation of HKLs with PHA and ConA, CpG increased CD40 expression. A CD40L construct was overexpressed in the CHSE-214 cell line and co-cultivation of the CD40L-CHSE transfectants with HKL induced a rapid and long-lasting upregulation of important costimulatory molecules like CD40, CD83, B7-H1 and the cytokines IL-12p40, IL-10, IL-1ß and IFNs, which all are involved in T-helper cell responses. Furthermore, the CD40L transfected cells increased the percentage of HKLs expressing surface MHCIIß but unlike other APC maturation stimuli, like CpG, they did not reduce the capacity to internalise antigen. Our results provide the first evidence for the existence of a functional CD40L mediated costimulatory pathway in Atlantic salmon.


Subject(s)
Antigen-Presenting Cells/immunology , CD40 Antigens/immunology , CD40 Ligand/immunology , Leukocytes/immunology , Salmo salar/immunology , Amino Acid Sequence , Animals , Antigen-Presenting Cells/metabolism , CD40 Antigens/biosynthesis , CD40 Antigens/chemistry , CD40 Ligand/chemistry , CD40 Ligand/metabolism , Concanavalin A/immunology , Dinucleoside Phosphates/immunology , Head Kidney/cytology , Head Kidney/immunology , Lymphocyte Activation , Molecular Sequence Data , Phylogeny , Phytohemagglutinins/immunology , Protein Structure, Tertiary , Salmo salar/genetics , Sequence Alignment
11.
Vaccine ; 30(32): 4828-34, 2012 Jul 06.
Article in English | MEDLINE | ID: mdl-22634299

ABSTRACT

CpG oligonucleotides and polyinosinic:polycytidylic acid (poly I:C) are toll-like receptor (TLR) agonists that mimic the immunostimulatory properties of bacterial DNA and double-stranded viral RNA respectively, and which have exhibited potential to serve as vaccine adjuvants in previous experiments. Here, a combination of CpGs and poly I:C together with water- or oil-formulated Salmonid Alphavirus (SAV) antigen preparations has been used for a vaccine in Atlantic salmon and tested for protection in SAV challenge trial. The results demonstrate that vaccination with a high dose of the SAV antigen induced protection against challenge with SAV which correlated with production of neutralizing antibodies (NAbs). As the high antigen dose alone induced full protection, no beneficial effect from the addition of CpG and poly I:C could be observed. Nevertheless, these TLR ligands significantly enhanced the levels of NAbs in serum of vaccinated fish. Interestingly, gene expression analysis demonstrated that while addition of oil suppressed the CpG/poly I:C-induced expression of IFN-γ, the upregulation of IFNa1 was substantially enhanced. A low dose of the SAV antigen combined with oil did not induce any detectable levels of NAbs either with or without TLR ligands present, however the addition of CpG and poly I:C to the low SAV antigen dose formulation significantly enhanced the protection against SAV suggesting that CpG/poly I:C may have enhanced a cytotoxic response - a process which is dependent on the up-regulation of type I IFN. These results highlight the immunostimulatory properties of the tested TLR ligands and will serve as a ground for further, more detailed studies aimed to investigate their capacity to serve as adjuvants in vaccine formulations for Atlantic salmon.


Subject(s)
Adjuvants, Immunologic/pharmacology , Alphavirus Infections/veterinary , Fish Diseases/prevention & control , Salmon/immunology , Viral Vaccines/immunology , Alphavirus/immunology , Alphavirus Infections/prevention & control , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Fish Diseases/virology , Interferon Type I/immunology , Oligodeoxyribonucleotides/pharmacology , Poly I-C/pharmacology , Salmon/virology , Toll-Like Receptors/agonists , Virus Inactivation
12.
J Biol Chem ; 286(49): 42715-42724, 2011 Dec 09.
Article in English | MEDLINE | ID: mdl-21990356

ABSTRACT

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 , Transgenes
13.
Dev Comp Immunol ; 35(11): 1116-27, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21527278

ABSTRACT

Both CpG oligodeoxynucleotides and double-stranded RNA (poly I:C) have documented effects as treatments against several viral diseases in fish. However, as stand-alone treatments their effects have been modest. We have tested here whether CpG and poly I:C, alone or in combination induce protection against Salmonid Alphavirus (SAV), the causative agent of pancreas disease in Atlantic salmon. Our results revealed a significant reduction of viraemia 2 weeks after ip injection of the combined treatment and 1 week after challenge with SAV subtype 3, followed by reduced SAV induced heart pathology 3 weeks later. The SAV titers in blood samples from the combination group were lower as compared to single treatments with either CpG or poly I:C. Surprisingly, reduced SAV levels could also be found in fish as long as 7 weeks after receiving the combination treatment. The expression of IFNγ and to a lesser extent IFNa and Mx was up-regulated in head kidney and spleen 5 days after the fish had been treated with CpG and poly I:C. Furthermore, the complement factor C4 was depleted in serum 8 weeks post treatment, suggesting complement activation leading to C4 consumption. We hypothesize that the CpG/poly I:C-induced protection against SAV3 is mediated by mechanisms involving type I and type II IFN induced antiviral activity and complement mediated protective responses.


Subject(s)
Alphavirus Infections/veterinary , Antiviral Agents/administration & dosage , Fish Diseases/immunology , Oligodeoxyribonucleotides/administration & dosage , Pancreatic Diseases/veterinary , Poly I-C/administration & dosage , Salmo salar/immunology , Alphavirus/immunology , Alphavirus/pathogenicity , Alphavirus Infections/immunology , Alphavirus Infections/virology , Animals , Antiviral Agents/immunology , Complement C4/analysis , Dinucleoside Phosphates , Fish Diseases/virology , Head Kidney/drug effects , Head Kidney/immunology , Immunity, Innate , Interferon Type I/biosynthesis , Interferon-gamma/biosynthesis , Oligodeoxyribonucleotides/immunology , Pancreatic Diseases/immunology , Pancreatic Diseases/virology , Poly I-C/immunology , Salmo salar/virology , Spleen/drug effects , Spleen/immunology
14.
Dev Comp Immunol ; 34(1): 29-41, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19665478

ABSTRACT

Major histocompatibility complex class II (MHCII) is encoded by polymorphic genes present in vertebrates and expressed predominately in leukocytes. Upon leukocyte differentiation, intracellular MHCII is dynamically redistributed within the cells and it is expressed at maximal levels on mature antigen presenting cells (APCs). In addition, APCs secrete MHCII within endosome-derived vesicles known as exosomes which possess diverse immunomodulatory properties. Genetic and biochemical data have confirmed that piscine leukocytes express the MHCII components as well as costimulatory molecules that are necessary for the function of APCs. However data concerning the biosynthesis and the distribution of the MHCII complex within leukocytes of lower vertebrates is scarce. The presented data demonstrates for the first time that salmon leukocytes secrete vesicles that contain exosomal markers and the abundance of MHCII indicates that these exosomes are released by APCs. The secretion was specifically induced by CpG stimulation in vitro and it was observed only in head kidney leukocytes but not in splenocyte cultures. Flow cytometry revealed that, unlike splenocytes, the majority of the MHCII-positive head kidney leukocytes were Ig-negative and a population of cells expressing high levels of surface MHCII underwent degranulation upon CpG stimulation suggesting that the MHCII-containing exosomes were derived from maturing salmon APCs. Gene expression analyses have further demonstrated that CpG-B, despite its relatively weak proinflammatory activity compared to LPS, induced expression of a larger group of genes involved in regulation of the adaptive immune response.


Subject(s)
Adjuvants, Immunologic/pharmacology , Antigen-Presenting Cells/drug effects , Antigen-Presenting Cells/immunology , Dinucleoside Phosphates/pharmacology , Exosomes/metabolism , Histocompatibility Antigens Class II/metabolism , Leukocytes/metabolism , Animals , Cells, Cultured , Flow Cytometry , Gene Expression Regulation/drug effects , Immunohistochemistry , Lipopolysaccharides/pharmacology , Microscopy, Electron, Transmission , Reverse Transcriptase Polymerase Chain Reaction , Salmo salar , Spleen/cytology
15.
Dev Comp Immunol ; 33(9): 1011-7, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19422846

ABSTRACT

Toll-like receptor 8 (TLR 8) belongs to a subgroup of the TLR family that recognizes nucleic acids and that is involved in the protection against viruses. In mammals, TLR7 and 8 have been characterized as receptors for viral and synthetic single-stranded RNA. Here we describe the cloning of a TLR8 homolog in Atlantic salmon (Salmo salar) and its proximal adaptor protein MyD88. The mRNA expression of SsTLR8 was tissue-restricted and its highest level was detected in the spleen while SsMyD88 was expressed in all of the tested organs. SsTLR8 and SsMyD88 mRNAs were up-regulated in TO cells treated with recombinant IFN alpha1 and IFN gamma. In vivo, the expression of SsTLR8 was not significantly affected following challenge with salmon alphavirus subtype 3 (SAV3). By contrast, infection with SAV3 up-regulated SsMyD88 transcripts on day 7 post-challenge and the expression remained elevated at day 28. The SsMyD88 expression in vivo paralleled type I IFN expression. In vitro stimulation of salmon head kidney leukocytes with CpG ODNs and IFN gamma also up-regulated SsMyD88 mRNA. Furthermore, ectopic expression of SsMyD88 in HEK cells was able to activate a NF-kappaB reporter construct indicating that the cloned salmon molecule was a functional MyD88 homologue.


Subject(s)
Leukocytes/metabolism , Myeloid Differentiation Factor 88/biosynthesis , Salmo salar/immunology , Toll-Like Receptor 8/biosynthesis , Alphavirus , Alphavirus Infections/immunology , Alphavirus Infections/veterinary , Animals , Cell Line , Fish Diseases/immunology , Fish Diseases/virology , Gene Expression , Humans , Immunologic Factors/pharmacology , Interferon-alpha/pharmacology , Interferon-gamma/pharmacology , Leukocytes/drug effects , Leukocytes/immunology , Leukocytes/virology , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/immunology , NF-kappa B/metabolism , Oligodeoxyribonucleotides/pharmacology , Salmo salar/virology , Toll-Like Receptor 8/genetics , Transfection
16.
Mol Immunol ; 45(12): 3363-70, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18513800

ABSTRACT

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 , Vibrio
17.
Reprod Biol Endocrinol ; 6: 2, 2008 Jan 18.
Article in English | MEDLINE | ID: mdl-18205936

ABSTRACT

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/analysis
18.
Dev Comp Immunol ; 32(6): 603-7, 2008.
Article in English | MEDLINE | ID: mdl-18068763

ABSTRACT

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-Regulation
19.
Mol Immunol ; 44(7): 1803-7, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17045654

ABSTRACT

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 Distribution
20.
Immunobiology ; 211(6-8): 437-47, 2006.
Article in English | MEDLINE | ID: mdl-16920483

ABSTRACT

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/physiology
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