This work reveals distinct roles of the two-cysteine-containing type I IFNs, IFNa and IFNd, and the four-cysteine-containing IFNb and IFNc in antiviral immunity of Atlantic salmon. IFNa and IFNc ...showed similar antiviral activities and ability to induce antiviral genes, IFNb was less active, and IFNd showed no activity. Expression of IFNs was compared by treatment of cells or fish with the dsRNA polyinosinic-polycytidylic acid poly(I:C), which induces IFNs via the viral RNA receptors MDA5 and TLR3/TLR22 and with the imidazoquinoline R848, which induces IFNs via TLR7. Poly(I:C) strongly induced IFNa in cell lines, whereas the other IFNs showed little response, indicating that IFNa is the main IFN subtype induced through the RIG-I/MDA5 pathway. In contrast, IFNb and IFNc are the main IFNs induced through the TLR7 pathway because R848 induced high transcript levels of IFNb and IFNc and low transcript levels of IFNa in the head kidney and spleen. IFNd was constitutively expressed in cells and organs but showed no response to poly(I:C) or R848. Fluorescence in situ hybridization studies showed that poly(I:C) induced IFNa and IFNc in a variety of cells in the head kidney, spleen, gills, liver, and heart, whereas R848 induced coexpression of IFNb and IFNc in distinct cells in head kidney and spleen. These cells are likely to be specialized high IFN producers because they were few in numbers despite high IFNb/IFNc transcript levels in the same organs. High IFN expression in response to TLR7 ligation is a feature shared by mammalian plasmacytoid dendritic cells.
Background
Interferon (IFN) responses are critical in the resolution of viral infections and are actively targeted by many viruses. They also play a role in inducing protective responses after ...vaccination and have been successfully tested as vaccine adjuvants. IFN responses are well conserved and function very similar in teleosts and mammals. Like in mammals, IFN responses in piscine cells are initiated by intracellular detection of the viral infection by different pattern recognition receptors. Upon the recognition of viral components, IFN responses are rapidly induced to combat the infection. However, many viruses may still replicate and be able to inhibit or circumvent the IFN response by different means.
Methods
By employing CRISPR Cas9 technology, we have disrupted proteins that are central for IFN signaling in the salmonid cell line CHSE-214. We successfully generated KO clones for the mitochondrial antiviral signaling protein MAVS, the transcription factors IRF3 and IRF7-1, as well as a double KO for IRF7-1/3 using an optimized protocol for delivery of CRISPR-Cas ribonucleoproteins through nucleofection.
Results
We found that MAVS and IRF3 KOs inhibited IFN and IFN-stimulated gene induction after intracellular poly I:C stimulation as determined through gene expression and promoter activation assays. In contrast, the IRF7-1 KO had no clear effect. This shows that MAVS and IRF3 are essential for initiation of intracellular RNA-induced IFN responses in CHSE-214 cells. To elucidate viral interference with IFN induction pathways, the KOs were infected with Salmon alphavirus 3 (SAV3) and infectious pancreatic necrosis virus (IPNV). SAV3 infection in control and IRF7-1 KO cells yielded similar titers and no cytopathic effect, while IRF3 and MAVS KOs presented with severe cytopathic effect and increased titers 6 days after SAV 3 infection. In contrast, IPNV yields were reduced in IRF3 and MAVS KOs, suggesting a dependency on interactions between viral proteins and pattern recognition receptor signaling components during viral replication.
Conclusion
Aside from more insight in this signaling in salmonids, our results indicate a possible method to increase viral titers in salmonid cells.
Type I interferons (IFN–I) play a pivotal role in vertebrate innate immunity against viruses. This study is an analysis of IFN-I genes in an updated version of the Atlantic salmon genome published in ...2021 (version Ssal_v3.1), revealing 47 IFN-I genes in the Atlantic salmon genome. The GH1 locus of chromosome (Chr) 3 harbors 9 IFNa genes, 5 IFNb genes, 6 IFNc genes, 11 IFNe genes and 1 IFNf gene. The GH2 locus on Chr6 contains 1 IFNa gene, 12 IFNc genes and 1 IFNf gene while Chr19 carries a single IFNd gene. Intraperitoneal injection of Atlantic salmon presmolts with poly I:C, a mimic of virus double-stranded RNA, significantly up-regulated IFNc genes from both Chr3 and Chr6 in heart, with lower expression in head kidney. IFNe expression increased in the heart, but not in the head kidney while IFNf was strongly up-regulated in both tissues. Antiviral activity of selected IFNs was assessed by transfection of salmon cells with IFN-expressing plasmids followed by infectious pancreatic necrosis virus infection, and by injection of fish with IFN-plasmids followed by measuring expression of the antiviral Mx1 gene. The results demonstrated that IFNc from both Chr3 and Chr6 provided full protection of cells against virus infection, whereas IFNe and IFNf showed lesser protection. IFNc from Chr3 and Chr6 along with IFNe and IFNf, up-regulated the Mx1 gene in the muscle, while only the IFNcs caused induction of Mx1 in liver. Overall, this study reveals that Atlantic salmon possesses an even more potent innate immune defense against viruses than previously understood.
•47 IFN-I genes were identified in the 2021 version of the Atlantic salmon genome.•The GH1 locus of Chr3 contains 9 IFNa genes, 5 IFNb genes, 6 IFNc genes, 11 IFNe genes and 1 IFNf gene.•The GH2 locus of Chr6 contains 1 IFNa gene, 12 IFNc genes and 1 IFNf gene.•IFNc and IFNf genes showed strong expression and IFNe weak expression in organs of Atlantic salmon in response to poly I:C.•Antiviral activity was demonstrated for IFNc, IFNe and IFNf genes.
•The full-length cDNA sequences of Atlantic salmon SOCS1, two SOCS2-isoforms and CISH were cloned.•SOCS1 demonstrated strong negative regulatory activity on type I and II IFN signalling.•A direct ...interaction between SOCS1 and STAT1 and SOCS1 and Tyk2 were detected.•SOCS1 also inhibited STAT1 nuclear localization upon IFNγ-treatment.
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.
Glycerophosphocholine choline phosphodiesterase (GPC-Cpde) is a glycosylphosphatidylinositol (GPI)-anchored alkaline hydrolase that is expressed in the brain and kidney. In brain the hydrolase is ...synthesized by the oligodendrocytes and expressed on the myelin membrane. There are two forms of brain GPC-Cpde, a membrane-linked (mGPC-Cpde) and a soluble (sGPC-Cpde). Here we report the characterisation sGPC-Cpde from bovine brain. The amino acid sequence was identical to ectonucleotide pyrophosphatase/phosphodiesterase 6 (eNPP6) precursor, lacking the N-terminal signal peptide region and a C-terminal stretch, suggesting that the hydrolase was solubilised by C-terminal proteolysis, releasing the GPI-anchor. sGPC-Cpde existed as two isoforms, a homodimer joined by a disulfide bridge linking C414 from each monomer, and a monomer resulting from proteolysis N-terminally to this disulfide bond. The only internal disulfide bridge, linking C142 and C154, stabilises the choline-binding pocket. sGPC-Cpde was specific for lysosphingomyelin, displaying 1 to 2 orders of magnitude higher catalytic activity than towards GPC and lysophosphatidylcholine, suggesting that GPC-Cpde may function in the sphingomyelin signaling, rather than in the homeostasis of acylglycerophosphocholine metabolites. The truncated high mannose and bisected hybrid type glycans linked to N118 and N341 of sGPC-Cpde is a hallmark of glycans in lysosomal glycoproteins, subjected to GlcNAc-1-phosphorylation
en route
through Golgi. Thus, sGPC-Cpde may originate from the lysosomes, suggesting that lysosomal sorting contributes to the level of mGPC-Cpde on the myelin membrane.
Mx proteins are antiviral GTPases, which are induced by type I IFN and virus infection. Analysis of the Atlantic salmon genome revealed the presence of 9 Mx genes localized to three chromosomes. A ...cluster of three Mx genes (SsaMx1 – SsaMx3), which includes previously cloned Mx genes, is present on chromosome (Chr) 12. A cluster of five Mx genes (SsaMx4-SsaMx8) is present on Chr25 while one Mx gene (SsaMx9) is present on Chr9. Phylogenetic and gene synteny analyses showed that SsaMx1-SsaMx3 are most closely related to the main group of teleost Mx proteins. In contrast, SsaMx 4-SsaMx9 formed a separate group together with zebrafish MxD and MxG and eel MxB. The Mx cluster in Chr25 showed gene synteny similar to a Mx gene cluster in the gar genome. Expression of Mx genes in cell lines stimulated with recombinant IFNs showed that Mx genes in Chr12 responded more strongly to type I IFN than to type II IFN (IFN gamma) whilst Mx genes in Chr25 responded more strongly to IFN gamma than to type I IFNs. SsaMx9 showed no response to the IFNs.
•Salmon possesses 9 Mx genes located on three chromosomes: Chr12 (SsaMx1-SsaMx3), Chr25 (SsaMx4-SsaMx8) and Chr9 (SsaMx9).•Phylogenetic and gene synteny analyses showed that SsaMx1-SsaMx3 belong to the main group of teleost Mx genes.•SsaMx4-SsaMx9 formed a separate group together with zebrafish MxD and MxG and eel MxB.•Mx genes in Chr12 responded more strongly to type I IFN than to IFNγ.•Mx genes in Chr25 responded more strongly to IFN gamma than to type I IFNs while SsaMx9 showed no response to IFNs.
•The transcription factors STAT2 and IRF9 from Atlantic salmon have been identified.•STAT2 co-expressed with STAT1 translocates to the nucleus upon IFN stimulation.•STAT2 are tyrosine phosphorylated ...upon type I IFN and IFNγ stimulation.•STAT2 and STAT1, as well as IRF9, activates GAS elements upon IFNγ stimulation.•Our results suggest novel roles of salmon STAT2 and IRF9 in IFNγ-mediated signaling.
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.
•Type I IFN receptors CRFB1a, CRFB1b, CRFB2, CRFB5a, CRFB5b, CRFB5c and CRFB5x were identified in Atlantic salmon.•Clusters of CRFBs were found on chromosomes 21 and 25.•Salmon has a larger ...repertoire of type I IFN receptors than mammals and zebrafish.•Mx reporter assays suggested that IFNa, IFNb and IFNc bind to different CRFB5 members.•The relative expression of CRFB1, CRFB2 and CRFB5 is tissue dependent.
Mammalian type I interferons (IFNs) signal through a receptor composed of the IFNAR1 and IFNAR2 chains. In zebrafish two-cysteine IFNs utilize a receptor composed of CRFB1 and CRFB5, while four-cysteine IFNs signal through a receptor formed by CRFB2 and CRFB5. In the present work two CRFB clusters were identified in different chromosomes of Atlantic salmon. Genes of three CRFB5s, one CRFB1, one CRFB2 and the novel CRFB5x were identified, cloned and studied functionally. All CRFBs were expressed in 10 different organs, but the relative expression of CRFBs varied. Mx-reporter assay was used to study which CRFBs might be involved in receptors for salmon IFNa, IFNb and IFNc. The results of Mx-reporter assays suggest that IFNa signals through a receptor composed of CRFB1a as the long chain and either CRFB5a, CRFB5b or CRFB5c as the short chain; IFNc signals through a receptor with CRFB5a or CRFB5c as the short chain while IFNb may signal through a receptor with CRFB5x as a short chain. Taken together, the present work demonstrates that Atlantic salmon has a more diverse repertoire of type I IFN receptors compared to zebrafish or mammals.
The development and persistence of antibody secreting cells (ASC) after antigenic challenge remain inadequately understood in teleosts. In this study, intraperitoneal (ip) injection of Atlantic ...salmon (Salmo salar) with salmonid alphavirus (WtSAV3) increased the total ASC response, peaking 3–6 weeks post injection (wpi) locally in the peritoneal cavity (PerC) and in systemic lymphoid tissues, while at 13 wpi the response was only elevated in PerC. At the same time point a specific ASC response was induced by WtSAV3 in PerC and systemic tissues, with the highest frequency in PerC, suggesting a local role. Inactivated SAV (InSAV1) induced comparatively lower ASC responses in all sites, and specific serum antibodies were only induced by WtSAV3 and not by InSAV1. An InSAV1 boost did not increase these responses. Expression of immune marker genes implies a role for PerC adipose tissue in the PerC immune response. Overall, the study suggests the Atlantic salmon PerC as a secondary immune site and an ASC survival niche.
•Infection with salmonid alphavirus induced a specific IgM secreting cell response.•The local B cell response in the peritoneal cavity was pronounced and long lasting.•Gene expression suggests peritoneal adipose tissue is involved in the B cell response.•The B cell response to inactivated virus was modest or absent, also after a boost.
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