Jorunn B. Jørgensen

Yeast β-glucan stimulates respiratory burst activity of Atlantic salmon (Salmo salar L.) macrophages

Previous studies have shown that head kidney macrophages isolated from glucan injected rainbow trout (Oncorhychus mykiss) and Atlantic salmon (Salmo salar L.) have increased ability to kill Aeromonas salmonicida. The present work was aimed at investigating the in vitro effects of glucan on the respiratory burst and bactericidal potential of Atlantic salmon head kidney macrophages. Salmon macrophages were incubated for 1-7 days with various concentrations of yeast beta-glucan (MacroGard) and tested for respiratory burst activity by the reduction of nitroblue-tetrazolium (NBT) after exposure to phorbol myristate acetate (PMA) or opsonized zymosan. The macrophages showed a marked increase in respiratory burst activity 4 to 7 days after addition of glucan. Macrophages treated with 0.1-1 microgram mL-1 gave a maximum respiratory burst response, whereas 10 micrograms mL-1 had no effect and 50 micrograms mL-1 was inhibitory. The glucan also triggered respiratory burst activity directly, but this occurred only at relative high concentrations with a maximal effect at > or = 200 micrograms mL-1. The validity of using the NBT-assay as a measure of respiratory burst activity was confirmed by using inhibitors of O2- production (superoxide dismutase, trifluoperazine and diphenylene iodonium). Despite the stimulatory effect of glucan on the respiratory burst activity of salmon macrophages, these cells did not show increased bactericidal activity against the avirulent and virulent strain of A. salmonicida. Upregulation of burst activity alone is thus apparently not sufficient to enhance bactericidal activity against this pathogen by Atlantic salmon macrophages.

CpG oligodeoxynucleotides and plasmid DNA stimulate Atlantic salmon (Salmo salar L.) leucocytes to produce supernatants with antiviral activity

Unmethylated CpG dinucleotides are more frequent in the genomes of bacteria and viruses than of vertebrates. We report herein that plasmid DNA and synthetic oliogodeoxynucleotides (ODNs) containing unmethylated CpG induce production of antiviral cytokine activity in Atlantic salmon leucocytes, whereas ODNs with an inverted motif (GpC) or with methylated cytosines have nearly no stimulatory effect. The adherent cell population, representing mainly macrophages, is directly activated by CpG-ODN, while the effect on the non-adherent population is weak. Since the peak antiviral activity in ODN-stimulated leucocytes is seen after 48h, this might indicate that the unmethylated DNA stimulates the adherent cells to produce co-stimulatory molecules, which in turn stimulates production of antiviral cytokines in the non-adherent cell population. The potent immune activation by CpG ODNs points to possible new applications as adjuvant in fish vaccines.

CpG DNA Induces Protective Antiviral Immune Responses in Atlantic Salmon (Salmo salar L.)

ABSTRACT Oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides within specific sequence contexts (CpG motifs) are detected, like bacterial or viral DNA, as a danger signal by the vertebrate immune system. CpG ODN show promise as vaccine adjuvants and immunoprotective agents in animal models. Here we report that pretreatment with CpG ODN in animals induces nonspecific protection against viral infection. A panel of different synthetic CpG ODN was tested for the in vitro effects in Atlantic salmon (Salmo salar L.) leukocytes. The ODN were tested for their capacity to stimulate proliferation of peripheral blood leukocytes and to induce production of interferon-like factors in head kidney leukocytes. These studies revealed that the sequence and number of the CpG motifs as well as the lengths of the ODN contribute to their stimulatory activity. ODN with the 6-mer CpG motif (5′-GTCGTT-3′) showed the highest stimulatory activity and were shown to induce protection against infectious pancreatic necrosis virus when injected in Atlantic salmon. Expression of the Mx transcript, as an indicator of alpha/beta interferon induction, was induced in the CpG-injected fish. These results suggest that CpG DNA in fish induces early, nonspecific antiviral protection.

Stimulation of respiratory burst and phagocytic activity in Atlantic salmon (Salmo salar L.) macrophages by lipopolysaccharide

Abstract The present paper describes the effect of lipopolysaccharides (LPS) from Aeromonas salmonicida and other Gram-negative bacteria on the respiratory burst, phagocytosis and bactericidal activity of head kidney macrophages from Atlantic salmon ( Salmo salar L.) in vitro . Macrophages were first cultured in the presence of various concentrations of LPS from A. salmonicida for 1, 2 and 5 days and then tested for respiratory burst activity (reduction of nitroblue tetrazolium) after exposure to phorbol myristate acetate (PMA). The most marked increase in respiratory burst activity of LPS-treated macrophages was observed after 5 days of incubation with 1, 10 and 100 μ g LPS ml −1 . The increase appeared to be dose-dependent with a maximal response at 10 μ g ml −1 . At this LPS-concentration and incubation time the respiratory burst activity was 3·9 times larger in the treated macrophages than in the control macrophages. LPS from three other Gram-negative bacterial salmon pathogens and two non-fish pathogens also enhanced the respiratory burst activity of salmon macrophages. Macrophages incubated with 10 and 50 μ g LPS ml −1 also showed a significant increase in PMA-stimulated H 2 O 2 -production after 5 days of incubation. LPS also stimulated the phagocytic activity of Atlantic salmon macrophages against opsonized and nonopsonized glucan particles, and glutaraldehyde-fixed sheep red blood cells. LPS-treated macrophages showed an increased ability to kill an avirulent A-layer lacking strain of A. salmonicida , but not a virulent A-layer positive strain.

Identification and characterization of TLR8 and MyD88 homologs in Atlantic salmon (Salmo salar).

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.

Antiviral Activity of Salmonid Gamma Interferon against Infectious Pancreatic Necrosis Virus and Salmonid Alphavirus and Its Dependency on Type I Interferon

ABSTRACT We investigated the antiviral activity and gene induction properties of interferon gamma (IFN-γ) compared to type I IFN (IFNa1) in Atlantic salmon. IFN-γ protected salmon cells against infectious pancreatic necrosis virus (IPNV)-induced cytopathic effect (CPE), reduced virus titers, and inhibited the synthesis of the viral structural protein VP3. Moreover, IFN-γ showed potent antiviral activity against salmonid alphavirus 3 (SAV3) measured as a reduction in virus nsP1 transcripts. IFN-γ (a type II IFN) had less specific antiviral activity against IPNV than IFNa1, showing a half-maximal effective concentration of 1.6 ng/ml versus 31 pg/ml determined in the CPE reduction assay. Compared to IFNa1, IFN-γ was a more effective inducer of the antiviral protein GBP, several interferon regulatory transcription factors (IRFs), and the chemokine IP-10. The antiviral activity of IFN-γ may also in part be ascribed to upregulation of Mx, ISG15, and viperin. These are typical type I IFN-induced genes in mammals and were also more strongly induced by IFNa1 than by IFN-γ in salmon cells. Fish and mammalian IFN-γ thus show strikingly similar gene induction properties. Interestingly, the antiviral activity of IFN-γ against IPNV and SAV3 and its ability to induce Mx and ISG15 markedly decreased in the presence of neutralizing antiserum against IFNa1. In contrast, antiIFNa1 had no effect on the induction of IRF-1 and IP-10 by IFN-γ. This suggests that the antiviral activity of IFN-γ is partially dependent on IFNa induction. However, because antiIFNa1 could not abolish the IFN-γ-mediated induction of Mx and ISG15 completely, IFN-γ may possibly also induce such genes directly.

Double-stranded RNA- and CpG DNA-induced immune responses in Atlantic salmon: comparison and synergies.

Several TLR agonists are shown to activate piscine immunity and are interesting adjuvant candidates in vaccine development. To test the outcome of stimulating Atlantic salmon with CpG DNA and poly I:C, ligands for TLR9 and 3, respectively, we have measured the in vivo expression of different immune molecules in spleen and head kidney. The expression profiles for individual treatments with CpGs or poly I:C not only showed similarities but they also displayed unique profiles. When combining them a synergistic up-regulation of the genes interferon (IFN)-alpha1/alpha2, Mx, CXCL10, IL-1beta, IFN-gamma and CD83 was detected. Interestingly, synergies between two different CpG ODNs classes also resulted in pronounced IFN-alpha1/alpha2 and IFN-gamma transcripts levels. To our knowledge this is the first study showing synergy by combining two different TLR9 ligands. In conclusion, detection of dsRNA and CpG DNA in fish may mimic viral recognition, resulting in an enhanced innate immune response that could be used for vaccination.

IPNV with high and low virulence: host immune responses and viral mutations during infection

BackgroundInfectious pancreatic necrosis virus (IPNV) is an aquatic member of the Birnaviridae family that causes widespread disease in salmonids. IPNV is represented by multiple strains with markedly different virulence. Comparison of isolates reveals hyper variable regions (HVR), which are presumably associated with pathogenicity. However little is known about the rates and modes of sequence divergence and molecular mechanisms that determine virulence. Also how the host response may influence IPNV virulence is poorly described.MethodsIn this study we compared two field isolates of IPNV (NFH-Ar and NFH-El). The sequence changes, replication and mortality were assessed following experimental challenge of Atlantic salmon. Gene expression analyses with qPCR and microarray were applied to examine the immune responses in head kidney.ResultsSignificant differences in mortality were observed between the two isolates, and viral load in the pancreas at 13 days post infection (d p.i.) was more than 4 orders of magnitude greater for NFH-Ar in comparison with NFH-El. Sequence comparison of five viral genes from the IPNV isolates revealed different mutation rates and Ka/Ks ratios. A strong tendency towards non-synonymous mutations was found in the HRV of VP2 and in VP3. All mutations in VP5 produced precocious stop codons. Prior to the challenge, NFH-Ar and NFH-El possessed high and low virulence motifs in VP2, respectively. Nucleotide substitutions were noticed already during passage of viruses in CHSE-214 cells and their accumulation continued in the challenged fish. The sequence changes were notably directed towards low virulence. Co-ordinated activation of anti-viral genes with diverse functions (IFN-a1 and c, sensors - Rig-I, MDA-5, TLR8 and 9, signal transducers - Srk2, MyD88, effectors - Mx, galectin 9, galectin binding protein, antigen presentation - b2-microglobulin) was observed at 13 d p.i. (NFH-Ar) and 29 d p.i. (both isolates).ConclusionsMortality and expression levels of the immune genes were directly related to the rate of viral replication, which was in turn associated with sequences of viral genes. Rapid changes in the viral genome that dramatically reduced virus proliferation might indicate a higher susceptibility to protective mechanism employed by the host. Disease outbreak and mortality depend on a delicate balance between host defence, regulation of signalling cascades and virus genomic properties.

Serum amyloid A transcription in Atlantic salmon (Salmo salar L.) hepatocytes is enhanced by stimulation with macrophage factors, recombinant human IL-1β, IL-6 and TNFα or bacterial lipopolysaccharide

Serum amyloid A (A-SAA) has previously been reported to be an acute-phase protein in salmonids. Hepatocytes represent a major source of A-SAA in salmonids, but nothing is known about hepatocyte SAA synthesis in fish. In the present work, the expression of A-SAA transcripts in primary cultures of Atlantic salmon hepatocytes in response to macrophage derived cytokines, human recombinant cytokines and bacterial lipopolysaccharide (LPS) was studied by Northern blot analysis. The macrophage supernatants were prepared by stimulating Atlantic salmon head kidney macrophages with LPS, yeast glucan or a leukocyte derived macrophage activating factor (MAF). The supernatants from glucan- or MAF-stimulated macrophages had no effect on A-SAA expression of the hepatocytes, while supernatants from LPS-stimulated macrophages gave about a 2-fold increase in expression. The combination of either glucan and MAF, or LPS and MAF were more effective and these supernatants gave a 3.4- and 5.2-fold increase in A-SAA expression, respectively. The hepatocytes were also treated with the human recombinant cytokines TNFalpha, IL-1beta and IL-6, alone or in combination. The A-SAA response to each of them alone was modest, but TNFalpha and IL-6 or IL-1beta and IL-6 in combination gave a higher response than each cytokine alone. These data suggest that the expression of A-SAA by hepatocytes from Atlantic salmon is induced by cytokine-like molecules. Interestingly, hepatocytes treated directly with LPS gave a more than 10-fold increase in SAA mRNA expression, but it is not known if this is a direct effect of LPS on the hepatocytes or if it is mediated by other contaminating cell types.

Structural and functional studies of STAT1 from Atlantic salmon (Salmo salar)

BackgroundType I and type II interferons (IFNs) exert their effects mainly through the JAK/STAT pathway, which is presently best described in mammals. STAT1 is involved in signaling pathways induced by both types of IFNs. It has a domain-like structure including an amino-terminus that stabilizes interaction between STAT dimers in a promoter-binding situation, a coiled coil domain facilitating interactions to other proteins, a central DNA-binding domain, a SH2 domain responsible for dimerization of phosphorylated STATs and conserved phosphorylation sites within the carboxy terminus. The latter is also the transcriptional activation domain.ResultsA salmon (Salmo salar) STAT1 homologue, named ssSTAT1a, has been identified and was shown to be ubiquitously expressed in various cells and tissues. The ssSTAT1a had a domain-like structure with functional motifs that are similar to higher vertebrates. Endogenous STAT1 was shown to be phosphorylated at tyrosine residues both in salmon leukocytes and in TO cells treated with recombinant type I and type II IFNs. Also ectopically expressed ssSTAT1 was phosphorylated in salmon cells upon in vitro stimulation by the IFNs, confirming that the cloned gene was recognized by upstream tyrosine kinases. Treatment with IFNs led to nuclear translocation of STAT1 within one hour. The ability of salmon STAT1 to dimerize was also shown.ConclusionsThe structural and functional properties of salmon STAT1 resemble the properties of mammalian STAT1.