Virus-specific antibody secreting cells reside in the peritoneal cavity and systemic immune sites of Atlantic salmon (Salmo salar L.) challenged intraperitoneally with salmonid alphavirus.

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 L.) with salmonid alphavirus (WtSAV3) increased the total ASC response, peaking 3 to 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 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.

There and back again? A B cell's tale on responses and spatial distribution in teleosts.

Teleost B cells are of special interest due to their evolutionary position and involvement in vaccine-induced adaptive immune responses. While recent progress has revealed uneven distribution of B cell subsets across the various immune sites and that B cells are one of the early responders to infection, substantial knowledge gaps persist regarding their immunophenotypic profile, functional mechanisms, and what factors lead them to occupy different immune niches. This review aims to assess the current understanding of B cell diversity, their spatial distribution in various systemic and peripheral immune sites, how B cell responses initiate, the sites where these responses develop, their trafficking, and the locations where long-term B cell responses take place.

Transcriptomics of early responses to purified Piscine orthoreovirus-1 in Atlantic salmon (Salmo salar L.) red blood cells compared to non-susceptible cell lines.

Piscine red blood cells (RBC) are nucleated and have been characterized as mediators of immune responses in addition to their role in gas exchange. Salmonid RBC are major target cells of Piscine orthoreovirus-1 (PRV-1), the etiological agent of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). PRV-1 replicates in RBC ex vivo, but no viral amplification has been possible in available A. salmon cell lines. To compare RBC basal transcripts and transcriptional responses to PRV-1 in the early phase of infection with non-susceptible cells, we exposed A. salmon RBC, Atlantic salmon kidney cells (ASK) and Salmon head kidney cells (SHK-1) to PRV-1 for 24 h. The RNA-seq analysis of RBC supported their previous characterization as pluripotent cells, as they expressed a wide repertoire of genes encoding pattern recognition receptors (PRRs), cytokine receptors, and genes implicated in antiviral activities. The comparison of RBC to ASK and SHK-1 revealed immune cell features exclusively expressed in RBC, such as genes involved in chemotactic activity in response to inflammation. Differential expression analysis of RBC exposed to PRV-1 showed 46 significantly induced genes (≥ 2-fold upregulation) linked to the antiviral response pathway, including RNA-specific PRRs and interferon (IFN) response factors. In SHK-1, PRV induced a more potent or faster antiviral response (213 genes induced). ASK cells showed a differential response pattern (12 genes induced, 18 suppressed) less characterized by the dsRNA-induced antiviral pathway. Despite these differences, the RIG-I-like receptor 3 (RLR3) in the family of cytosolic dsRNA receptors was significantly induced in all PRV-1 exposed cells. IFN regulatory factor 1 (IRF1) was significantly induced in RBC only, in contrast to IRF3/IRF7 induced in SHK-1. Differences in IRF expression and activity may potentially affect viral propagation.

CRISPR-Cas- induced IRF3 and MAVS knockouts in a salmonid cell line disrupt PRR signaling and affect viral replication.

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.

Secretome Profiling of Atlantic Salmon Head Kidney Leukocytes Highlights the Role of Phagocytes in the Immune Response to Soluble ß-Glucan.

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.

The importance of the Atlantic salmon peritoneal cavity B cell response: Local IgM secreting cells are predominant upon Piscirickettsia salmonis infection.

The intraperitoneal route is favored for administration of inactivated and attenuated vaccines in Atlantic salmon. Nevertheless, the immune responses in the teleost peritoneal cavity (PerC) are still incompletely defined. In this study, we investigated the B cell responses after intraperitoneal Piscirickettsia salmonis (P. salmonis) challenge of Atlantic salmon, focusing on the local PerC response versus responses in the lymphatic organs: spleen and head kidney. We observed a major increase of leukocytes, total IgM antibody secreting cells (ASC), and P. salmonis-specific ASC in the PerC at 3- and 6-weeks post infection (wpi). The increase in ASC frequency was more prominent in the spleen and PerC compared to the head kidney during the observed 6 wpi. The serum antibody response included P. salmonis-specific antibodies and non-specific antibodies recognizing the non-related bacterial pathogen Yersinia ruckeri and the model antigen TNP-KLH. Finally, we present evidence that supports a putative role for the adipose tissue in the PerC immune response.

Piscine Orthoreovirus (PRV)-3, but Not PRV-2, Cross-Protects against PRV-1 and Heart and Skeletal Muscle Inflammation in Atlantic Salmon.

Heart and skeletal muscle inflammation (HSMI), caused by infection with Piscine orthoreovirus-1 (PRV-1), is a common disease in farmed Atlantic salmon (Salmo salar). Both an inactivated whole virus vaccine and a DNA vaccine have previously been tested experimentally against HSMI and demonstrated to give partial but not full protection. To understand the mechanisms involved in protection against HSMI and evaluate the potential of live attenuated vaccine strategies, we set up a cross-protection experiment using PRV genotypes not associated with disease development in Atlantic salmon. The three known genotypes of PRV differ in their preference of salmonid host species. The main target species for PRV-1 is Atlantic salmon. Coho salmon (Oncorhynchus kisutch) is the target species for PRV-2, where the infection may induce erythrocytic inclusion body syndrome (EIBS). PRV-3 is associated with heart pathology and anemia in rainbow trout, but brown trout (S. trutta) is the likely natural main host species. Here, we tested if primary infection with PRV-2 or PRV-3 in Atlantic salmon could induce protection against secondary PRV-1 infection, in comparison with an adjuvanted, inactivated PRV-1 vaccine. Viral kinetics, production of cross-reactive antibodies, and protection against HSMI were studied. PRV-3, and to a low extent PRV-2, induced antibodies cross-reacting with the PRV-1 σ1 protein, whereas no specific antibodies were detected after vaccination with inactivated PRV-1. Ten weeks after immunization, the fish were challenged through cohabitation with PRV-1-infected shedder fish. A primary PRV-3 infection completely blocked PRV-1 infection, while PRV-2 only reduced PRV-1 infection levels and the severity of HSMI pathology in a few individuals. This study indicates that infection with non-pathogenic, replicating PRV could be a future strategy to protect farmed salmon from HSMI.

Salmonid Alphavirus Subtype 3 Induces Prolonged Local B Cell Responses in Atlantic Salmon (Salmo salar) After Intraperitoneal Infection.

B cell responses are a crucial part of the adaptive immune response to viral infection. Infection by salmonid alphavirus subtype 3 (SAV3) causes pancreas disease (PD) in Atlantic salmon (Salmo salar) and is a serious concern to the aquaculture industry. In this study, we have used intraperitoneal (IP) infection with SAV3 as a model to characterize local B cell responses in the peritoneal cavity (PerC) and systemic immune tissues (head kidney/spleen). Intraperitoneal administration of vaccines is common in Atlantic salmon and understanding more about the local PerC B cell response is fundamental. Intraperitoneal SAV3 infection clearly induced PerC B cell responses as assessed by increased frequency of IgM+ B cells and total IgM secreting cells (ASC). These PerC responses were prolonged up to nine weeks post-infection and positively correlated to the anti-SAV3 E2 and to neutralizing antibody responses in serum. For the systemic immune sites, virus-induced changes in B cell responses were more modest or decreased compared to controls in the same period. Collectively, data reported herein indicated that PerC could serve as a peripheral immunological site by providing a niche for prolonged maintenance of the ASC response in Atlantic salmon.

Detection of specific Atlantic salmon antibodies against salmonid alphavirus using a bead-based immunoassay.

Salmonid alphavirus (SAV) is the etiological cause of pancreas disease (PD) in Atlantic salmon (Salmo salar). Several vaccines against SAV are in use, but PD still cause significant mortality and concern in European aquaculture, raising the need for optimal tools to monitor SAV immunity. To monitor and control the distribution of PD in Norway, all salmonid farms are regularly screened for SAV by RT-qPCR. While the direct detection of SAV is helpful in the early stages of infection, serological methods could bring additional information on acquired SAV immunity in the later stages. Traditionally, SAV antibodies are monitored in neutralization assays, but they are time-consuming and cumbersome, thus alternative assays are warranted. Enzyme-linked immunosorbent assays (ELISAs) have not yet been successfully used for anti-SAV antibody detection in aquaculture. We aimed to develop a bead-based immunoassay for SAV-specific antibodies. By using detergent-treated SAV particles as antigens, we detected SAV-specific antibodies in plasma collected from both a SAV challenge trial and a field outbreak of PD. Increased levels of SAV-specific antibodies were seen after most fish had become negative for viral RNA. The bead-based assay is time saving compared to virus neutralization assays, and suitable for non-lethal testing due to low sample size requirements. We conclude that the bead-based immunoassay for SAV antibody detection is a promising diagnostic tool to complement SAV screening in aquaculture.

Infection and microbial molecular motifs modulate transcription of the interferon-inducible gene ifit5 in a teleost fish.

Interferon-induced proteins with tetratricopeptide repeats (IFITs) are involved in antiviral defense. Members of this protein family contain distinctive multiple structural motifs comprising tetratricopeptides that are tandemly arrayed or dispersed along the polypeptide. IFIT-encoding genes are upregulated by type I interferons (IFNs) and other stimuli. IFIT proteins inhibit virus replication by binding to and regulating the functions of cellular and viral RNA and proteins. In teleost fish, knowledge about genes and functions of IFITs is currently limited. In the present work, we describe an IFIT5 orthologue in Atlantic salmon (SsaIFIT5) with characteristic tetratricopeptide repeat motifs. We show here that the gene encoding SsaIFIT5 (SsaIfit5) was ubiquitously expressed in various salmon tissues, while bacterial and viral challenge of live fish and in vitro stimulation of cells with recombinant IFNs and pathogen mimics triggered its transcription. The profound expression in response to various immune stimulation could be ascribed to the identified IFN response elements and binding sites for various immune-relevant transcription factors in the putative promoter of the SsaIfit5 gene. Our results establish SsaIfit5 as an IFN-stimulated gene in A. salmon and strongly suggest a phylogenetically conserved role of the IFIT5 protein in antimicrobial responses in vertebrates.

Intracellular distribution and transcriptional regulation of Atlantic salmon (Salmo salar) Rab5c, 7a and 27a homologs by immune stimuli.

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.

Microbial Danger Signals Control Transcriptional Induction of Distinct MHC Class I L Lineage Genes in Atlantic Salmon.

Antigen processing and presentation by major histocompatibility complex (MHC) molecules is a cornerstone in vertebrate immunity. Like mammals, teleosts possess both classical MHC class I and multiple families of divergent MHC class I genes. However, while certain mammalian MHC class I-like molecules have proven to be integral in immune regulation against a broad array of pathogens, the biological relevance of the different MHC class I lineages in fish remains elusive. This work focuses on MHC class I L lineage genes and reveals unique regulatory patterns of six genes (Sasa-lia, Sasa-lda, Sasa-lca, Sasa-lga, Sasa-lha, and Sasa-lfa) in antimicrobial immunity of Atlantic salmon (Salmo salar L.). Using two separate in vivo challenge models with different kinetics and immune pathologies combined with in vitro stimulation using viral and bacterial TLR ligands, we show that de novo synthesis of different L lineage genes is distinctly regulated in response to various microbial stimuli. Prior to the onset of classical MHC class I gene expression, lia was rapidly and systemically induced in vivo by the single-stranded (ss) RNA virus salmonid alpha virus 3 (SAV3) but not in response to the intracellular bacterium Piscirickettsia salmonis. In contrast, lga expression was upregulated in response to both viral and bacterial stimuli. A role for distinct MHC class I L-lineage genes in anti-microbial immunity in salmon was further substantiated by a marked upregulation of lia and lga gene expression in response to type I IFNa stimulation in vitro. Comparably, lha showed no transcriptional induction in response to IFNa stimulation but was strongly induced in response to a variety of viral and bacterial TLR ligands. In sharp contrast, lda showed no response to viral or bacterial challenge. Similarly, induction of lca, which is predominantly expressed in primary and secondary lymphoid tissues, was marginal with the exception of a strong and transient upregulation in pancreas following SAV3 challenge Together, these findings suggest that certain Atlantic salmon MHC class I L lineage genes play important and divergent roles in early anti-microbial response and that their regulation, in response to different activation signals, represents a system for selectively promoting the expression of distinct non-classical MHC class I genes in response to different types of immune challenges.

CpGs Induce Differentiation of Atlantic Salmon Mononuclear Phagocytes Into Cells With Dendritic Morphology and a Proinflammatory Transcriptional Profile but an Exhausted Allostimulatory Activity.

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.

Antiviral defense in salmonids - Mission made possible?

Viral diseases represent one of the major threats for salmonid aquaculture. Survival from viral infections are highly dependent on host innate antiviral immune defense, where interferons are of crucial importance. Neutralizing antibodies and T cell effector mechanisms mediate long-term antiviral protection. Despite an immune cell repertoire comparable to higher vertebrates, farmed fish often fail to mount optimal antiviral protection. In the quest to multiply and spread, viruses utilize a variety of strategies to evade or escape the host immune system. Understanding the specific interplay between viruses and host immunity at depth is crucial for developing successful vaccination and treatment strategies in mammals. However, this knowledge base is still limited for pathogenic fish viruses. Here, we have focused on five RNA viruses with major impact on salmonid aquaculture: Salmonid alphavirus, Infectious salmon anemia virus, Infectious pancreatic necrosis virus, Piscine orthoreovirus and Piscine myocarditis virus. This review explore the protective immune responses that salmonids mount to these viruses and the existing knowledge on how the viruses counteract and/or bypass the immune response, including their IFN antagonizing effects and their mechanisms to establish persisting infections.

Profiling the Atlantic Salmon IgM+ B Cell Surface Proteome: Novel Information on Teleost Fish B Cell Protein Repertoire and Identification of Potential B Cell Markers.

Fish immunology research is at a pivotal point with the increasing availability of functional immunoassays and major advances in omics approaches. However, studies on fish B cells and their distinct subsets remain a challenge due to the limited availability of differentially expressed surface markers. To address this constraint, cell surface proteome of Atlantic salmon IgM+ B cells were analyzed by mass spectrometry and compared to surface proteins detected from two adherent salmon head kidney cell lines, ASK and SSP-9. Out of 21 cluster of differentiation (CD) molecules identified on salmon IgM+ B cells, CD22 and CD79A were shortlisted as potential markers based on the reported B cell-specific surface expression of their mammalian homologs. Subsequent RT-qPCR analyses of flow cytometry-sorted subpopulations from head kidney leukocytes confirmed that both cd22 and cd79a genes were highly expressed in IgM+ lymphoid cells but were observed in barely detectable levels in IgM- non-lymphoid suspension and adherent cells. Similarly, significantly high cd22 and cd79a mRNA levels were observed in IgM+ or IgT+ lymphoid cells from the spleen and peritoneal cavity, but not in their corresponding IgM- IgT- non-lymphoid fractions. This suggests that the B cell restrictive expression of CD22 and CD79A extend down to the transcription level, which was consistent across different lymphoid compartments and immunoglobulin isotypes, thus strongly supporting the potential of CD22 and CD79A as pan-B cell markers for salmon. In addition, this study provides novel information on the salmon B cell surface protein repertoire, as well as insights on B cell evolution. Further investigation of the identified salmon CD molecules, including development of immunological tools for detection, will help advance our understanding of the dynamics of salmon B cell responses such as during infection, vaccination, or immunostimulation.

Stimulation of exosome release by extracellular DNA is conserved across multiple cell types.

Exosomes are distinguished from other types of extracellular vesicles by their small and relatively uniform size (30-100 nm) and their composition which reflects their endo-lysosomal origin. Involvement of these extracellular organelles in intercellular communication and their implication in pathological conditions has fuelled intensive research on mammalian exosomes; however, currently, very little is known about exosomes in lower vertebrates. Here we show that, in primary cultures of head kidney leukocytes from Atlantic salmon (Salmo salar), phosphorothioate CpG oligodeoxynucleotides induce secretion of vesicles with characteristics very similar to these of mammalian exosomes. Further experiments revealed that the oligonucleotide-induced exosome secretion did not depend on the CpG motifs but it relied on the phosphorothioate modification of the internucleotide linkage. Exosome secretion was also induced by genomic bacterial and eukaryotic DNA in toll-like receptor 9-negative piscine and human cell lines demonstrating that this is a phylogenetically conserved phenomenon which does not depend on activation of immune signaling pathways. In addition to exosomes, stimulation with phosphorothioate oligonucleotides and genomic DNA induced secretion of LC3B-II, an autophagosome marker, which was associated with vesicles of diverse size and morphology, possibly derived from autophagosome-related intracellular compartments. Overall, this work reveals a previously unrecognized biological activity of phosphorothioate ODNs and genomic DNA - their capacity to induce secretion of exosomes and other types of extracellular vesicles. This finding might help shed light on the side effects of therapeutic phosphorothioate oligodeoxynucleotides and the biological activity of extracellular genomic DNA which is often upregulated in pathological conditions.

Author Correction: Profiling Atlantic salmon B cell populations: CpG-mediated TLR-ligation enhances IgM secretion and modulates immune gene expression.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Profiling Atlantic salmon B cell populations: CpG-mediated TLR-ligation enhances IgM secretion and modulates immune gene expression.

While TLR-activated pathways are key regulators of B cell responses in mammals, their impact on teleost B cells are scarcely addressed. Here, the potential of Atlantic salmon B cells to respond to TLR ligands was shown by demonstrating a constitutive expression of nucleic-acid sensing TLRs in magnetic sorted IgM+ cells. Of the two receptors recognizing CpG in teleosts, tlr9 was the dominating receptor with over ten-fold higher expression than tlr21. Upon CpG-stimulation, IgM secretion increased for head kidney (HK) and splenic IgM+ cells, while blood B cells were marginally affected. The results suggest that CpG directly affects salmon B cells to differentiate into antibody secreting cells (ASCs). IgM secretion was also detected in the non-treated controls, again with the highest levels in the HK derived population, signifying that persisting ASCs are present in this tissue. In all tissues, the IgM+ cells expressed high MHCII levels, suggesting antigen-presenting functions. Upon CpG-treatment the co-stimulatory molecules cd83 and cd40 were upregulated, while cd86 was down-regulated under the same conditions. Finally, ifna1 was upregulated upon CpG-stimulation in all tissues, while a restricted upregulation was evident for ifnb, proposing that salmon IgM+ B cells exhibit a type I IFN-response.

A conserved inhibitory role of suppressor of cytokine signaling 1 (SOCS1) in salmon antiviral immunity.

The SOCS proteins are regulators of JAK/STAT signaling. A number of viral infections has been associated with SOCS upregulation. Here we report that SOCS1 mRNA expression is up-regulated in salmon alphavirus (SAV3) infected TO cells, while infectious pancreatic necrosis virus infection has a negligible effect. SAV3 infected salmon showed increased SOCS1 mRNA levels in heart correlating with increased viral transcripts. Together, the in vitro and in vivo data suggests that SAV3-induced SOCS1 expression may affect the outcome of the virus infection. Using CHSE-214 cells overexpressing SOCS1 we revealed increased SAV3 replication compared to control, suggesting that SOCS1 suppress the antiviral capacity of the cells. In IFNa1-treated cells, the suppression of viral replication was partially rescued by SOCS1 overexpression. The increased viral replication in SOCS1 transgene cells was likely a result of impaired IFN-signaling and the reduced expression of interferon-stimulated genes in the transgene cells underscores this.