Regulation of MHC expression in vivo. II. IFN-alpha/beta inducers and recombinant IFN-alpha modulate MHC antigen expression in mouse tissues

We examined the effect of type I IFN inducers and rIFN-alpha on MHC expression in mouse tissues in vivo. MHC expression was assessed in a radiolabeled mAb binding assay and by indirect immunoperoxidase staining of tissue sections. polyI:C, an inducer of IFN-alpha/beta, induced large increases in class I MHC in many tissues, with little effect on class II expression. In the kidney, which was studied in detail, polyI:C increased class I expression from day 1 to day 6, localized in glomeruli, tubules, and arterial endothelium. Renal class II MHC was less affected but tended to be decreased at days 3 to 6, corresponding to diminished staining of class II-positive interstitial cells. polyI:C increased renal class I MHC in nude mice and mice with severe combined immunodeficiency, and in mice treated with cyclosporine or mAb against IFN-gamma. The effects of influenza virus resembled those of polyI:C. However, a potent T cell stimulus, allogeneic ascites tumor cells, induced markedly different MHC changes, with massive and sustained increases in class I and II, presumably due to IFN-gamma release, which was inhibited by cyclosporine or by mAb against IFN-gamma. The effect of polyI:C was largely simulated by rIFN-alpha, whereas the effect of allogeneic cells was simulated by rIFN-gamma. Thus, rIFN-alpha and its inducers in vivo produce a sustained increase in renal class I expression in kidney and other tissues, sometimes with changes in class II expression. Such effects could be relevant to the immune modulatory actions of IFN, and to the immunologic consequences of viral infections.     

Expression kinetics of interferon and interferon-induced genes in Atlantic salmon (Salmo salar) following infection with infectious pancreatic necrosis virus and infectious salmon anaemia virus

Infectious pancreatic necrosis virus (IPNV) and infectious salmon anaemia virus (ISAV) are economically important pathogens of the salmonid aquaculture industry. Atlantic salmon were challenged by intraperitoneal injection (i.p.) with either virus followed by time-course sampling. Cohabiting fish in the IPNV challenge were also sampled. Kidney tissue was analysed using a TaqMan real-time PCR assay to measure the expression of a range of host immune genes in relation to the endogenous control, elongation factor 1 alpha (ELF). Host genes measured included Mx, type I and type II interferon (IFN), gammaIFN induced protein (gammaIP), interleukin-1 beta (IL-1beta) and tumour necrosis factor alpha (TNF-alpha). Viral levels were also measured. In i.p. injected fish, both viruses greatly induced expression of Mx, gammaIP, type I and type II IFN by day 6 post-infection, however only ISAV caused substantial mortality. Some differences between the expression kinetics produced by both viruses were noted. Infection with ISAV increased IL-1beta expression following day 6, but no effect was seen in fish infected with IPNV. Neither virus induced TNF-alpha expression. This study confirms the presence of both type I and type II IFN responses and their induced genes in Atlantic salmon upon infection with an orthomyxovirus and a birnavirus.     

Effect of double-stranded RNA and interferon on the antiviral activity of Atlantic salmon cells against infectious salmon anemia virus and infectious pancreatic necrosis virus

Type I interferons (IFN) establish an antiviral state in vertebrate cells by inducing expression of Mx and other antiviral proteins. We have studied the effect of Atlantic salmon interferon-like activity (AS-IFN) and poly I:C on the Mx protein expression and antiviral activity against infectious salmon anaemia virus (ISAV) and infectious pancreatic necrosis virus (IPNV) in the Atlantic salmon cell lines SHK-1 and TO. The double-stranded RNA poly I:C is an inducer of type I IFN in vertebrates. A cell cytotoxicity assay and measurements of virus yield were used to measure protection of cells against virus infection. Maximal induction of Mx protein in TO and SHK-1 cells occurred 48 h after poly I:C stimulation and 24 h after AS-IFN stimulation. TO cells pretreated with AS-IFN or poly I:C were protected from infection with IPNV 24 to 96 h after stimulation. Poly I:C or AS-IFN induced a minor protection against ISAV infection in SHK-1 cells, but no protection was induced against ISAV in TO cells. Western blot analysis showed that ISAV induced expression of Mx protein in TO and SHK-1 cells whereas IPNV did not induce Mx protein expression. These results suggest that ISAV and IPNV have very different sensitivities to IFN-induced antiviral activity and have developed different strategies to avoid the IFN-system of Atlantic salmon. Moreover, Atlantic salmon Mx protein appears not to inhibit replication of ISAV.     

Production of rabbit antisera against recombinant MHC class II beta chain and identification of immunoreactive cells in Atlantic salmon (Salmo salar)

In the present work, rabbit antisera recognising the Atlantic salmon (Salmo salar) MHC class II beta chain polypeptide were produced and used in immunoblotting, immunohistochemistry and immunogold electron microscopy. A construct encoding the beta1 and beta2 domains fused to the E. coli protein thioredoxin was used to express the recombinant MHC class II beta chain. Immunoblotting revealed a band of approximately 30kDa in total protein samples from head kidney, spleen, gills, thymus and blood leukocytes, while being absent in muscle. The distribution of MHC class II positive cells was immunohistochemically demonstrated in Atlantic salmon epithelial and haemopoietic tissues. Ultrastructural demonstration of immunoreactive organelles in mid-kidney cells was performed by immunogold electron microscopy. The results indicate expression in lymphocytes, macrophages, epithelial cells and endritic-like cells. This is the first study to address morphological MHC class II expression in a fish species.     

Detection of infectious salmon anaemia virus (ISAV) by in situ hybridisation

An in situ hybridisation method was developed to detect infectious salmon anaemia virus (ISAV) in fixed tissues from Atlantic salmon Salmo salar L. Three DNA probes detected ISAV in heart, liver, kidney, spleen, caeca, and mid-gut from infected farmed Atlantic salmon obtained from a natural outbreak of ISA. The strongest signals were obtained using Probe S8, from Segment 8 of ISAV. Hybridisation was most prominent in the endothelial cells of heart tissue. The probes reacted specifically with ISAV; no hybridisation was evident in uninfected tissues from Atlantic salmon. Importantly, the probes did not cross react with the pathogens IHNV (haematopoietic necrosis virus), IPNV (infectious pancreatic necrosis virus), SPDV (salmon pancreas disease virus) and VHSV (viral haemorrhagic septicemia virus).     

Localised Infection of Atlantic Salmon Epithelial Cells by HPR0 Infectious Salmon Anaemia Virus

Infectious salmon anaemia (ISA) is an important, systemic viral disease of farmed Atlantic salmon, Salmo salar L. Endothelial cells are the main target cells for highly virulent HPR-deleted ISA virus (ISAV) types. Here we examine the pathogenesis of non-virulent ISAV HPR0 infections, presenting evidence of an epithelial tropism for this virus type, including actual infection and replication in the epithelial cells. Whereas all HPR0 RT-qPCR positive gills prepared for cryosection tested positive by immunohistochemistry (IHC) and immunofluorescent labelling, only 21% of HPR0 RT-qPCR positive formalin-fixed paraffin-embedded gills were IHC positive, suggesting different methodological sensitivities. Only specific epithelial cell staining was observed and no staining was observed in endothelial cells of positive gills. Furthermore, using an ISAV segment 7 RT-PCR assay, we demonstrated splicing of HPR0, suggesting initial activation of the replication machinery in the epithelial gill cells. Immunological responses were investigated by the expression of interferon-related genes (e.g. Mx and γIP) and by ELISA for presence of anti-ISAV antibodies on samples taken sequentially over several months during an episode of transient HPR0 infection. All fish revealed a variable, but increased expression of the immunological markers in comparison to normal healthy fish. Taken together, we conclude that HPR0 causes a localized epithelial infection of Atlantic salmon.     

IPNV modulation of pro and anti-inflammatory cytokine expression in Atlantic salmon might help the establishment of infection and persistence

IPNV is the agent of a well-characterized acute disease that produces a systemic infection and high mortality in farmed fish species and persistent infection in surviving fish after outbreaks. Because modulation of the host expression of pro and anti-inflammatory cytokines can help establish persistence, in this study, we examined the expression of IL-1β, IL-8, IFNα1 and IL-10 during acute and persistent IPNV infection of Atlantic salmon. Results showed that IPNV infection induces an increase of the IFNα1 and IL-10 mRNA levels in the spleen and head kidney (HK) of fish after acute experimental infection. Levels of the pro-inflammatory cytokines IL-1β and IL-8 did not rise in the spleen although an increase of IL-1β, but not of IL-8, was observed in head kidney. In carrier asymptomatic salmon, cytokine gene expression of IFNα1 in the spleen and IL-10 in head kidney were also significantly higher than expression in non-carrier fish. Interestingly, a decrease of IL-8 expression was also observed. IPNV infection of SHK-1, which is a macrophage-like cell line of salmon, also induced an increase of expression of the anti-inflammatory cytokine IL-10 with no effects on the expression of IL-1β and IL-8. The effects are induced by an unknown mechanism during viral infection because poly I:C and the viral genomic dsRNA showed the opposite effects on cytokine expression in SHK-1 cells. In summary, IPNV always induces up-regulation of the anti-inflammatory cytokine IL-10 in Atlantic salmon. As this is accompanied by a lack of induction of the pro-inflammatory cytokines IL-1β and IL-8, the anti-inflammatory milieu may explain the high frequency, prevalence and persistence of IPNV in salmon. Effects might be part of the viral mechanisms of immune evasion.