CpG oligonucleotides bind TLR9 and RRM-containing proteins in Atlantic salmon (Salmo salar).

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.

Antiviral activity of salmonid gamma interferon against infectious pancreatic necrosis virus and salmonid alphavirus and its dependency on type I interferon.

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.

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

BACKGROUND: Infectious 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. METHODS: In 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. RESULTS: Significant 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). CONCLUSIONS: Mortality 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.

Development of a novel beta-glucan supplemented hydrogel spray formulation and wound healing efficacy in a db/db diabetic mouse model.

To relieve the severe economic and social burdens and patient suffering caused by the increasing incidence of chronic wounds, more effective treatments are urgently needed. In this study, we focused on developing a novel sprayable wound dressing with the active ingredient ß-1,3/1,6-glucan (ßG). Since ßG is already available as the active ingredient in a commercial wound healing product provided as a hydrogel in a tube (ßG-Gel), the sprayable format should bring clinical benefit by being easily sprayed onto wounds; whilst retaining ßG-Gel's physical stability, biological safety and wound healing efficacy. Potentially sprayable ßG hydrogels were therefore formulated, based on an experimental design setup. One spray formulation, named ßG-Spray, was selected for further investigation, as it showed favorable rheological and spraying properties. The ßG-Spray was furthermore found to be stable at room temperature for more than a year, retaining its rheological properties and sprayability. The cytotoxicity of ßG-Spray in keratinocytes in vitro, was shown to be promising even at the highest tested concentration of 100 µg/ml. The ßG-Spray also displayed favorable fluid affinity characteristics, with a capacity to both donate and absorb close to 10% fluid relative to its own weight. Finally, the ßG-Spray was proven comparably effective to the commercial product, ßG-Gel, and superior to both the water and the carrier controls (NoßG-Spray), in terms of its ability to promote wound healing in healing-impaired animals. Contraction was found to be the main wound closure mechanism responsible for the improvement seen in the ßG-treatment groups (ßG-Spray and ßG-Gel). In conclusion, the novel sprayable ßG formulation, confirmed its potential to expand the clinical use of ßG as wound dressing.

A TLR9 homolog that is up-regulated by IFN-gamma in Atlantic salmon (Salmo salar).

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.

Beta-glucan-loaded nanofiber dressing improves wound healing in diabetic mice.

The increased prevalence of chronic wounds requires novel treatment options. The aim of this study was to develop a beta-glucan (ßG)-loaded nanofiber wound dressing. Nanofibers were prepared using the needle-free Nanospider™ technology, an electrospinning method which enables the production of nanofibers at an industrial scale. The ßG was selected as active ingredient based on its confirmed wound healing potential in both animals and humans. Hydroxypropyl methylcellulose (HPMC) and polyethylene oxide (PEO) were included as copolymers. Rheological profiles of spinning solutions containing HPMC, PEO, ßG, ethanol and water, were optimized. The nanofiber formation was confirmed by Field Emission Scanning Electron Microscopy (FE-SEM), and both nanofibers with (ßG-nanofibers) or without ßG (NoßG-nanofibers) were evaluated by their swelling index and FT-IR spectroscopy. The formulations, active ingredient and excipients were tested for their possible in vitro toxicity in keratinocytes. Finally, the wound healing potential of the nanofibers was tested in externally induced excisional wounds in male diabetic db/db mice. Three different doses of ßG-nanofibers and the ßG-free, NoßG-nanofibers, were evaluated for their in vivo wound healing efficacy. All nanofiber-treatments provided improved wound healing as compared to the negative control (water). All ßG-nanofiber treated groups exhibited significantly improved wound healing as compared to the NoßG-nanofiber treated group, indicating the potential of ßG-nanofibers as wound dressing.

Sprayable Carbopol hydrogel with soluble beta-1,3/1,6-glucan as an active ingredient for wound healing - Development and in-vivo evaluation.

Chronic wounds represent a significant health problem worldwide. There is a need for advanced- and cost-efficient wound healing products able to increase patient comfort and reduce the healing time. The aim of this study was to develop a sprayable hydrogel dressing with beta-glucan (ßG) as the active ingredient, targeting future application in the treatment of both chronic and burn wounds. The ßG was chosen as an active ingredient because of its promising wound healing capabilities, whereas Carbopol 971P NF (Carbopol) was chosen as the thickening agent in the formulation due to several attractive characteristics such as its low viscosity, low toxicity, high transparency and good ion tolerance. Four different hydrogel formulations were prepared with varying Carbopol concentrations. The higher Carbopol concentration, 0.5% (w/w), was used to prepare three formulations comprising the HighCP:NoßG, HighCP:LowßG and the HighCP:MediumßG formulation, respectively. Lower Carbopol concentration, 0.25% (w/w), was used to prepare the LowCP:HighßG formulation. The content of ßG varied from 0.25% in the HighCP:LowßG, 0.5% in the HighCP:MediumßG and 1.0% (w/w) in the LowCP:HighßG formulation, respectively. The first part of the study focused on the rheological characterization of the hydrogels and the fluid affinity testing. All formulations were confirmed to be stable gels; the ßG was shown to augment the gel strength by increasing the yield strength of the gel in a dose dependent manner. The stability of the formulations containing either Carbopol alone or in a combination with ßG did not deteriorate over 26weeks, and the fluid donation and absorption study indicated a fluid donation profile, which favors healing of dry wounds. The in vivo efficacy of the formulations, evaluated in the modified diabetic male mice (db/db mice), showed that Carbopol alone was unable to induce improved healing and caused adverse reactions in some wounds. The inclusion of ßG increased the epithelialization and wound contraction in the db/db mice when given at high ßG:Carbopol ratio. The positive effect of ßG was, however, not sufficient to counteract the adverse effect of Carbopol, thus a more suitable thickening agent should be investigated for further development of a sprayable wound care product.

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.

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.