Marit Rode

A role for scavenger receptors in phagocytosis of protein-coated particles in rainbow trout head kidney macrophages.

In macrophages of higher vertebrates, Fc receptors and receptors for complement and other serum factors, are generally known to enhance the phagocytic process. In lower vertebrates like salmonid fishes, none of these or other phagocytic receptors have been thoroughly characterized. The purpose of this study was to elucidate to what extent these and other receptors are involved in the process of phagocytosis in rainbow trout (Oncorhynchus mykiss) head kidney macrophages. We used tosyl activated, paramagnetic dynabeads (2.8 microm in diameter), specifically coated with 125I labeled Atlantic salmon (Salmo salar) IgM or bovine serum albumin (BSA) as phagocytic probes. The effect of complement opsonization was also investigated by incubating the beads in serum. Our results indicate that neither the Fc- nor the complement-receptor(s) were important for phagocytosis of these beads. Our data support the idea that scavenger receptors are involved in phagocytosis in rainbow trout head kidney macrophages, as the use of a competitive scavenger receptor ligand extensively decreased degradation of the labeled protein coat on the beads.

CpG-induced secretion of MHCIIβ and exosomes from salmon (Salmo salar) APCs

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.

Transcriptome profiling of immune responses to cardiomyopathy syndrome (CMS) in Atlantic salmon

BackgroundCardiomyopathy syndrome (CMS) is a disease associated with severe myocarditis primarily in adult farmed Atlantic salmon (Salmo salar L.), caused by a double-stranded RNA virus named piscine myocarditis virus (PMCV) with structural similarities to the Totiviridae family. Here we present the first characterisation of host immune responses to CMS assessed by microarray transcriptome profiling.ResultsUnvaccinated farmed Atlantic salmon post-smolts were infected by intraperitoneal injection of PMCV and developed cardiac pathology consistent with CMS. From analysis of heart samples at several time points and different tissues at early and clinical stages by oligonucleotide microarrays (SIQ2.0 chip), six gene sets representing a broad range of immune responses were identified, showing significant temporal and spatial regulation. Histopathological examination of cardiac tissue showed myocardial lesions from 6 weeks post infection (wpi) that peaked at 8-9 wpi and was followed by a recovery. Viral RNA was detected in all organs from 4 wpi suggesting a broad tissue tropism. High correlation between viral load and cardiac histopathology score suggested that cytopathic effect of infection was a major determinant of the myocardial changes. Strong and systemic induction of antiviral and IFN-dependent genes from 2 wpi that levelled off during infection, was followed by a biphasic activation of pathways for B cells and MHC antigen presentation, both peaking at clinical pathology. This was preceded by a distinct cardiac activation of complement at 6 wpi, suggesting a complement-dependent activation of humoral Ab-responses. Peak of cardiac pathology and viral load coincided with cardiac-specific upregulation of T cell response genes and splenic induction of complement genes. Preceding the reduction in viral load and pathology, these responses were probably important for viral clearance and recovery.ConclusionsBy comparative analysis of gene expression, histology and viral load, the temporal and spatial regulation of immune responses were characterised and novel immune genes identified, ultimately leading to a more complete understanding of host-virus responses and pathology and protection in Atlantic salmon during CMS.

Characterization of a novel calicivirus causing systemic infection in atlantic salmon (Salmo salar L.): proposal for a new genus of caliciviridae.

The Caliciviridae is a family of viruses infecting humans, a wide range of animals, birds and marine fish and mammals, resulting in a wide spectrum of diseases. We describe the identification and genetic characterization of a novel calicivirus replicating in Atlantic salmon. The virus has a high prevalence in farmed salmon and is found in fish suffering from several diseases and conditions and also in presumable healthy fish. A challenge and vaccination trial shows that the calicivirus replicates in Atlantic salmon and establishes a systemic infection, which can be reduced by vaccination with formalin-inactivated virus preparation. The virus, named Atlantic salmon calicivirus (ASCV), is found in two genetically distinct variants, a cell culture isolated and a variant sequenced directly from field material. The genomes are 7,4 kb and contain two open reading frames where typical conserved amino acid motifs and domains predict a gene order reminiscent of calicivirus genomes. Phylogenetic analysis performed on extracted capsid amino acid sequences segregated the two ASCV variants in a unique cluster sharing root with the branch of noroviruses infecting humans and the unassigned Tulane virus and St-Valérien like viruses, infecting rhesus monkey and pig, respectively, with relatively large distance to the marine calicivirus subgroup of vesiviruses. Based on the analyses presented, the ASCV is predicted to represent a new genus of Caliciviridae for which we propose the name Salovirus.

EFFECT OF TEMPERATURE ON ENDOCYTOSIS AND INTRACELLULAR TRANSPORT IN THE CELL LINE SHK-1 DERIVED FROM SALMON HEAD KIDNEY

We have used a marker for fluid phase endocytosis, Horse Radish Peroxidase (HRP), and a ligand for the scavenger receptor, dinitrophenylated human serum albumin, (DNP-HSA), to study endocytosis, intracellular transport and degradation in a newly developed cell line from salmon head kidney, SHK-1. The influence of changing temperature on these processes has been studied. The rate of internalization in these cells increased from 0.006 μl/106 cells/hr at 4°C, to 0.01 μl/106 cells/hr, and 0.021 μl/106 cells/hr at 12 and 20°C, respectively. From these data, the activation energy for the internalization process was calculated to 40.3 ± 13 kJ/mol. In cells adapted to 20°C, both the transport to lysosomes and the degradation of a protein ligand came to an almost complete halt at 4°C, whereas some transport to and degradation in lysosomes took place at 12°C.

Comparison of Atlantic salmon individuals with different outcomes of cardiomyopathy syndrome (CMS)

BackgroundCardiomyopathy syndrome (CMS) is a severe disease of Atlantic salmon (Salmo salar L.) associated with significant economic losses in the aquaculture industry. CMS is diagnosed with a severe inflammation and degradation of myocardial tissue caused by a double-stranded RNA virus named piscine myocarditis virus (PMCV), with structural similarities to the Totiviridae family. In the present study we characterized individual host responses and genomic determinants of different disease outcomes.ResultsFrom time course studies of experimentally infected Atlantic salmon post-smolts, fish exhibited different outcomes of infection and disease. High responder (HR) fish were characterized with sustained and increased viral load and pathology in heart tissue. Low responder (LR) fish showed declining viral load from 6–10 weeks post infection (wpi) and absence of pathology. Global gene expression (SIQ2.0 oligonucleotide microarray) in HR and LR hearts during infection was compared, in order to characterize differences in the host response and to identify genes with expression patterns that could explain or predict the different outcomes of disease. Virus-responsive genes involved in early antiviral and innate immune responses were upregulated equally in LR and HR at the first stage (2–4 wpi), reflecting the initial increase in virus replication. Repression of heart muscle development was identified by gene ontology enrichment analyses, indicating the early onset of pathology. By six weeks both responder groups had comparable viral load, while increased pathology was observed in HR fish. This was reflected by induced expression of genes implicated in apoptosis and cell death mechanisms, presumably related to lymphocyte regulation and survival. In contrast, LR fish showed earlier activation of NK cell-mediated cytotoxicity and NOD-like receptor signaling pathways. At the late stage of infection, increased pathology and viral load in HR was accompanied by a broad activation of genes involved in adaptive immunity and particularly T cell responses, probably reflecting the increased infiltration and homing of virus-specific T cells to the infected heart. This was in sharp contrast to LR fish, where recovery and reduced viral load was associated with a significantly reduced transcription of adaptive immunity genes and activation of genes involved in energy metabolism.ConclusionsIn contrast to LR, a stronger and sustained expression of genes involved in adaptive immune responses in heart tissue of HR at the late stage of disease probably reflected the increased lymphocyte infiltration and pathological outcome. In addition to controlled adaptive immunity and activation of genes involved in cardiac energy metabolism in LR at the late stage, recovery of this group could also be related to an earlier activation of NOD-like receptor signaling and NK cell-mediated cytotoxicity pathways.