Proteome reference map of the skin mucus of Atlantic cod (Gadus morhua) revealing immune competent molecules

The skin mucosal proteome of Atlantic cod (Gadus morhua) was mapped using a 2D PAGE, LC-MS/MS coupled approach. Mucosal proteins from naive fish were identified primarily by similarity searches across various cod EST databases. The identified proteins were clustered into 8 groups based on gene ontology classification for biological process. Most of the proteins identified from the gel are hitherto unreported for cod. Galectin-1, mannan binding lectin (MBL), serpins, cystatin B, cyclophilin A, FK-506 binding protein, proteasome subunits (alpha-3 and -7), ubiquitin, and g-type lysozyme are considered immune competent molecules. Five of the aforementioned proteins were cloned and their tissue distribution was analysed by RT-PCR.     

Intraperitoneal vaccination of Atlantic cod, Gadus morhua with heat-killed Listonella anguillarum enhances serum antibacterial activity and expression of immune response genes

Serum-mediated reduction in bacterial count and expression of a number of immune response genes in the blood of Atlantic cod, Gadus morhua were investigated following intraperitoneal vaccination with heat-killed Listonella (Vibrio) anguillarum. Blood was collected from the caudal vein of both vaccinated and non-vaccinated (PBS-injected) fish at 0, 1, 3, 7 and 10 days post-vaccination (dpv). Serum protein concentration and antibacterial activity of the serum samples were determined. Whole blood was used for semi-quantitative RT-PCR of immune-related genes. Total serum protein was not significantly different between the vaccinated and non-vaccinated groups. Sera from the vaccinated fish significantly reduced L. anguillarum count on 3 dpv, with reductions of at least 2 log colony forming units per ml (CFU/ml) relative to the non-vaccinated fish. Expression of antibacterial genes, bactericidal/permeability-increasing protein/lipopolysaccharide-binding protein (BPI/LBP), g-type lysozyme and transferrin was significantly upregulated in the vaccinated fish, with maximum expression within 7 dpv. Cytotoxic-related and cell-mediated immunity genes such as, apolipoprotein A-I and the non-specific cytotoxic cell receptor protein (NCCRP-1) had maximum expression at 3 and 7 dpv, respectively. Significant upregulation in expression of pro-inflammatory cytokines, IL-1 beta and IL-8 was also observed in the vaccinated fish at 1 dpv. The upregulation of immune response genes following vaccination provides valuable information in the understanding of immune mechanisms against vibriosis in Atlantic cod particularly on the acute phase response during bacterial infection.     

Response to vaccination of Atlantic cod (Gadus morhua L.) progenies from families with different estimated family breeding values for vibriosis resistance

The purpose of the study was to elucidate whether responses to vibriosis vaccination and gene expressions in parts of the innate immune system were different in families of Atlantic cod (Gadus morhua). The fish were progenies of families with differences in estimated breeding values (EBV) for vibriosis resistance. Families of coastal cod (CC) and northeast Arctic cod (AC) responded well to vaccination with a relative percent survival of 72–95. No correlation between response to vaccination and vibriosis resistance were found (p = 0.146). The AC family with medium low (M) resistance had significant (p ≤ 0.019) lowest mortality among all the unvaccinated fish but the CC-M family. Further, when comparing the vaccinated fish the AC family with very high (VH) resistance had significant (p ≤ 0.004) higher mortality than all except the CC-VL and CC-H families.Parts of the innate immune response were studied by measuring the gene expression of innate immune genes 2 and 4 days post dip vaccination. Vaccinated fish from two families had a weak but significant higher innate immune response compared to control fish of the same family. In vaccinated fish, the gene expression of interleukin (IL) 1b, IL-10, IL-12p40 and hepcidin were significant up-regulated. While, no measureable activations of interferon gamma (IFNγ), IL-8, cathelicidin, LBP/BPI and G-type lysozyme were found.     

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.     

The stress and metabolic responses of juvenile Atlantic cod Gadus morhua L. to an acute thermal challenge

Survival, oxygen consumption (     ), total plasma cortisol and glucose levels and gill heat-shock protein 70 (hsp70) expression were measured in 10 and 50 g juvenile Atlantic cod Gadus morhua during an acute temperature increase (2° C h⁻¹) to their critical thermal maximum. Ninety three per cent of the fish in both size classes survived to 24° C; however, mortality was 100% within 15 min of reaching this temperature. The     for both size classes increased significantly with temperature, reaching peak values at 22° C that were c. 2·8-fold those of control (10° C) fish. Resting plasma cortisol and glucose levels were lower in 10 g as compared to 50 g fish. Plasma glucose levels were highly variable in both size classes, and significant increases were only seen at >22° C for the 10 g fish. In contrast, plasma cortisol showed an exponential increase with temperature starting at 16° C in both size classes, and reached maximum levels at 22° C that were 19-fold (10 g fish) and 35-fold (50 g fish) higher than their respective control groups. Both the constitutive (73 kDa) and inducible (72 kDa) isoforms of hsp70 were detected in both size classes using the widely utilized mouse monoclonal antibody. Expression of these isoforms, however, did not change when Atlantic cod were exposed to elevated temperature, and the 72 kDa isoform was not detected using salmonid-specific antibodies. These results indicate that juvenile Atlantic cod are very sensitive to acute increases in water temperature. In addition, they (1) show that     and plasma cortisol, but not plasma glucose or gill hsp 70 levels, are sensitive indicators of thermal stress in Atlantic cod and (2) support previous reports that the upper critical temperature for this species is 16° C.     

Interferonregulatoryfactor-8(IRF-8) regulates the expression of matrix metalloproteinase-13 (MMP-13) in chondrocytes

Low levels of inflammation-induced expression of matrix metalloproteinase (MMP) play a crucial role in articular cartilage matrix destruction in osteoarthritis (OA) patients. Interferon regulatory factor-8 (IRF-8), an important member in the IRF family, plays a key role in regulating the inflammation-related signaling pathway. The aim of this study is to investigate the physiological roles of IRF-8 in the pathological progression of OA. We found that IRF-8 was expressed in human primary chondrocytes. Interestingly, the expression of IRF-8 was upregulated in OA chondrocytes. In addition, IRF-8 was increased in response to interleukin-1β (IL-1β) treatment, mediated by the Janus kinase 2 (JAK2) pathway. Overexpression of IRF-8 in human chondrocytes by transduction with lentiviral-IRF-8 exacerbated IL-1β-induced expression of matrix metalloproteinase-13 (MMP-13) in human chondrocytes. In contrast, knockdown of IRF-8 inhibited IL-1β-induced expression of MMP-13. Importantly, IRF-8 could bind to the promoter of MMP-13 and stimulate its activity. Additionally, overexpression of IRF-8 exacerbated IL-1β-induced degradation of type II collagen. However, silencing IRF-8 abrogated the degradation of type II collagen. Taken together, our findings identified a novel function of IRF-8 in regulating articular cartilage matrix destruction by promoting the expression of MMP-13.     

Complex formation of the interferon (IFN) consensus sequence-binding protein with IRF-1 is essential for murine macrophage IFN-gamma-induced iNOS gene expression

This study describes the role of the interferon (IFN) consensus sequence-binding protein (ICSBP or IRF-8) in iNOS gene expression by murine macrophages. An ICSBP binding site in the iNOS promoter region (-923 to -913) was identified using an electrophoretic mobility shift assay and chromatin co-immunoprecipitation. Overexpression of ICSBP greatly enhanced IFN-gamma-induced iNOS promoter activation in RAW264.7 cells, and IFN-gamma-induced iNOS promoter activation was abolished in ICSBP-/- macrophages. Furthermore, transduction of retrovirus-ICSBP in ICSBP-/- macrophages rescued IFN-gamma-induced iNOS gene expression. However, transduction of retrovirus-ICSBP in the absence of IFN-gamma activation did not induce iNOS expression in either RAW264.7 cells or ICSBP-/- macrophages. Interestingly, ICSBP alone transduced into ICSBP-/- macrophages did not bind to IFN-stimulated response element site (-923 to -913) of the iNOS promoter region, although following activation with IFN-gamma, a DNA.protein complex was formed that contains ICSBP and IRF-1. Co-transduction of ICSBP with IRF-1 clearly induces nitric oxide production. In addition, interleukin-4 inhibits IFN-gamma-induced iNOS gene expression by attenuating the physical interaction of ICSBP with IRF-1. Complex formation of ICSBP with IRF-1 is essential for iNOS expression, and interleukin-4 attenuates the physical interaction of ICSBP with IRF-1 resulting in the inhibition of INOS gene expression.