Transcriptomic Profiling of the Adaptive and Innate Immune Responses of Atlantic Salmon to Renibacterium salmoninarum Infection.

Bacterial Kidney Disease (BKD), which is caused by a Gram-positive, intracellular bacterial pathogen (Renibacterium salmoninarum), affects salmonids including Atlantic salmon (Salmo salar). However, the transcriptome response of Atlantic salmon to BKD remained unknown before the current study. We used a 44K salmonid microarray platform to characterise the global gene expression response of Atlantic salmon to BKD. Fish (~54 g) were injected with a dose of R. salmoninarum (H-2 strain, 2 × 108 CFU per fish) or sterile medium (control), and then head kidney samples were collected at 13 days post-infection/injection (dpi). Firstly, infection levels of individuals were determined through quantifying the R. salmoninarum level by RNA-based TaqMan qPCR assays. Thereafter, based on the qPCR results for infection level, fish (n = 5) that showed no (control), higher (H-BKD), or lower (L-BKD) infection level at 13 dpi were subjected to microarray analyses. We identified 6,766 and 7,729 differentially expressed probes in the H-BKD and L-BKD groups, respectively. There were 357 probes responsive to the infection level (H-BKD vs. L-BKD). Several adaptive and innate immune processes were dysregulated in R. salmoninarum-infected Atlantic salmon. Adaptive immune pathways associated with lymphocyte differentiation and activation (e.g., lymphocyte chemotaxis, T-cell activation, and immunoglobulin secretion), as well as antigen-presenting cell functions, were shown to be differentially regulated in response to BKD. The infection level-responsive transcripts were related to several mechanisms such as the JAK-STAT signalling pathway, B-cell differentiation and interleukin-1 responses. Sixty-five microarray-identified transcripts were subjected to qPCR validation, and they showed the same fold-change direction as microarray results. The qPCR-validated transcripts studied herein play putative roles in various immune processes including pathogen recognition (e.g., tlr5), antibacterial activity (e.g., hamp and camp), regulation of immune responses (e.g., tnfrsf11b and socs1), T-/B-cell differentiation (e.g., ccl4, irf1 and ccr5), T-cell functions (e.g., rnf144a, il13ra1b and tnfrsf6b), and antigen-presenting cell functions (e.g., fcgr1). The present study revealed diverse immune mechanisms dysregulated by R. salmoninarum in Atlantic salmon, and enhanced the current understanding of Atlantic salmon response to BKD. The identified biomarker genes can be used for future studies on improving the resistance of Atlantic salmon to BKD.

Full characterization and transcript expression profiling of the interferon regulatory factor (IRF) gene family in Atlantic cod (Gadus morhua).

Atlantic cod (Gadus morhua) represents a unique immune system among teleost fish, making it a species of interest for immunological studies, and especially for investigating the evolutionary history of immune gene families. The interferon regulatory factor (IRF) gene family encodes transcription factors which function in the interferon pathway, but also in areas including leukocyte differentiation, cell growth, autoimmunity, and development. We previously characterized several IRF family members in Atlantic cod (Irf4a, Irf4b, Irf7, Irf8, and two Irf10 splice variants) at the cDNA and putative amino acid levels, and in the current study we took advantage of the new and improved Atlantic cod genome assembly in combination with rapid amplification of cDNA ends (RACE) to characterize the remaining family members (i.e. Irf3, Irf5, Irf6, Irf9, and two Irf2 splice variants). Real-time quantitative PCR (QPCR) was used to investigate constitutive expression of all IRF transcripts during embryonic development, suggesting several putative maternal transcripts, and potential stage-specific roles. QPCR studies also showed 11 of 13 transcripts were responsive to stimulation with poly(I:C), while 6 of 13 transcripts were responsive to lipopolysaccharide (LPS) in Atlantic cod head kidney macrophages, indicating roles for cod IRF family members in both antiviral and antibacterial responses. This study is the first to investigate expression of the complete IRF family in Atlantic cod, and suggests potential novel roles for several of these transcription factors within immunity as well as in early development of this species.

Characterization and Transcript Expression Analyses of Atlantic Cod Viperin.

Viperin is a key antiviral effector in immune responses of vertebrates including the Atlantic cod (Gadus morhua). Using cloning, sequencing and gene expression analyses, we characterized the Atlantic cod viperin at the nucleotide and hypothetical amino acid levels, and its regulating factors were investigated. Atlantic cod viperin cDNA is 1,342 bp long, and its predicted protein contains 347 amino acids. Using in silico analyses, we showed that Atlantic cod viperin is composed of 5 exons, as in other vertebrate orthologs. In addition, the radical SAM domain and C-terminal sequences of the predicted Viperin protein are highly conserved among various species. As expected, Atlantic cod Viperin was most closely related to other teleost orthologs. Using computational modeling, we show that the Atlantic cod Viperin forms similar overall protein architecture compared to mammalian Viperins. qPCR revealed that viperin is a weakly expressed transcript during embryonic development of Atlantic cod. In adults, the highest constitutive expression of viperin transcript was found in blood compared with 18 other tissues. Using isolated macrophages and synthetic dsRNA (pIC) stimulation, we tested various immune inhibitors to determine the possible regulating pathways of Atlantic cod viperin. Atlantic cod viperin showed a comparable pIC induction to other well-known antiviral genes (e.g., interferon gamma and interferon-stimulated gene 15-1) in response to various immune inhibitors. The pIC induction of Atlantic cod viperin was significantly inhibited with 2-Aminopurine, Chloroquine, SB202190, and Ruxolitinib. Therefore, endosomal-TLR-mediated pIC recognition and signal transducers (i.e., PKR and p38 MAPK) downstream of the TLR-dependent pathway may activate the gene expression response of Atlantic cod viperin. Also, these results suggest that antiviral responses of Atlantic cod viperin may be transcriptionally regulated through the interferon-activated pathway.

Effects of Vitamin D2 (Ergocalciferol) and D3 (Cholecalciferol) on Atlantic Salmon (Salmo salar) Primary Macrophage Immune Response to Aeromonas salmonicida subsp. salmonicida Infection.

Vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol) are fat-soluble secosteroid hormones obtained from plant and animal sources, respectively. Fish incorporates vitamin D2 and D3 through the diet. In mammals, vitamin D forms are involved in mineral metabolism, cell growth, tissue differentiation, and antibacterial immune response. Vitamin D is an essential nutrient in aquafeeds for finfish. However, the influence of vitamin D on fish cell immunity has not yet been explored. Here, we examined the effects of vitamin D2 and vitamin D3 on Salmo salar primary macrophage immune response to A. salmonicida subspecies salmonicida infection under in vitro conditions. We determined that high concentrations of vitamin D2 (100,000 ng/ml) and D3 (10,000 ng/ml) affect the growth of A. salmonicida and decrease the viability of S. salar primary macrophages. In addition, we determined that primary macrophages pre-treated with a biologically relevant concentration of vitamin D3 for 24 h showed a decrease of A. salmonicida infection. In contrast, vitamin D2 did not influence the antibacterial activity of the S. salar macrophages infected with A. salmonicida. Vitamin D2 and D3 did not influence the expression of canonical genes related to innate immune response. On the other hand, we found that A. salmonicida up-regulated the expression of several canonical genes and suppressed the expression of leukocyte-derived chemotaxin 2 (lect-2) gene, involved in neutrophil recruitment. Primary macrophages pre-treated for 24 h with vitamin D3 counteracted this immune suppression and up-regulated the transcription of lect-2. Our results suggest that vitamin D3 affects A. salmonicida attachment to the S. salar primary macrophages, and as a consequence, the A. salmonicida invasion decreased. Moreover, our study shows that the positive effects of vitamin D3 on fish cell immunity seem to be related to the lect-2 innate immunity mechanisms. We did not identify positive effects of vitamin D2 on fish cell immunity. In conclusion, we determined that the inactive form of vitamin D3, cholecalciferol, induced anti-bacterial innate immunity pathways in Atlantic salmon primary macrophages, suggesting that its utilization as a component of a healthy aquafeed diet in Atlantic salmon could enhance the immune response against A. salmonicida.

Discovery of microRNAs associated with the antiviral immune response of Atlantic cod macrophages.

MicroRNAs (miRNAs) are known to play important immunoregulatory roles in teleosts, although miRNAs involved in the antiviral immune response of Atlantic cod (Gadus morhua) were previously uncharacterised. Using deep sequencing and qPCR, the present study was conducted to identify miRNAs responsive to the viral mimic, polyriboinosinic polyribocytidylic acid (pIC) in Atlantic cod macrophages. Macrophage samples isolated from Atlantic cod (n=3) and treated with pIC or phosphate buffered saline (PBS control) for 24 and 72h were used for miRNA profiling. Following deep sequencing, DESeq2 analyses identified four (miR-731-3p, miR-125b-3-3p, miR-150-3p and miR-462-3p) and two (miR-2188-3p and miR-462-3p) significantly differentially expressed miRNAs at 24 and 72h post-stimulation (HPS), respectively. Sequencing-identified miRNAs were subjected to qPCR validation using a larger number of biological replicates (n=6) exposed to pIC or PBS over time (i.e. 12, 24, 48 and 72 HPS). As in sequencing, miR-731-3p, miR-462-3p and miR-2188-3p showed significant up-regulation by pIC. The sequencing results were not qPCR-validated for miR-125b-3-3p and miR-150-3p as up- and down-regulated miRNAs at 24 HPS, respectively; however, qPCR results showed significant up-regulation in response to pIC stimulation at later time points (i.e. 48 and/or 72 HPS). We also used qPCR to assess the expression of other miRNAs that were previously shown as immune responsive in other vertebrates. qPCR results at 48 and/or 72 HPS revealed that miR-128-3-5p, miR-214-1-5p and miR-451-3p were induced by pIC, whereas miR-30b-3p and miR-199-1-3p expression were repressed in response to pIC. The present study identified ten pIC-stimulated miRNAs, suggesting them as important in antiviral immune responses of Atlantic cod macrophages. Some pIC-responsive miRNAs identified in this study were predicted to target putative immune-related genes of Atlantic cod (e.g. miR-30b-3p targeting herc4), although the regulatory functions of these miRNAs need to be validated by future studies.

De novo assembly of Sockeye salmon kidney transcriptomes reveal a limited early response to piscine reovirus with or without infectious hematopoietic necrosis virus superinfection.

BACKGROUND: Piscine reovirus (PRV) has been associated with the serious disease known as Heart and Skeletal Muscle Inflammation (HSMI) in cultured Atlantic salmon Salmo salar in Norway. PRV is also prevalent in wild and farmed salmon without overt disease manifestations, suggesting multifactorial triggers or PRV variant-specific factors are required to initiate disease. In this study, we explore the head kidney transcriptome of Sockeye salmon Oncorhynchus nerka during early PRV infection to identify host responses in the absence of disease in hopes of elucidating mechanisms by which PRV may directly alter host functions and contribute to the development of a disease state. We further investigate the role of PRV as a coinfecting agent following superinfection with infectious hematopoietic necrosis virus (IHNV) - a highly pathogenic rhabdovirus endemic to the west coast of North America. RESULTS: Challenge of Sockeye salmon with PRV resulted in high quantities of viral transcripts to become present in the blood and kidney of infected fish without manifestations of disease. De novo transcriptome assembly of over 2.3 billion paired RNA-seq reads from the head kidneys of 36 fish identified more than 320,000 putative unigenes, of which less than 20 were suggested to be differentially expressed in response to PRV at either 2 or 3 weeks post challenge by DESeq2 and edgeR analysis. Of these, only one, Ependymin, was confirmed to be differentially expressed by qPCR in an expanded sample set. In contrast, IHNV induced substantial transcriptional changes (differential expression of > 20,000 unigenes) which included transcripts involved in antiviral and inflammatory response pathways. Prior infection with PRV had no significant effect on host responses to superinfecting IHNV, nor did host responses initiated by IHNV exposure influence increasing PRV loads. CONCLUSIONS: PRV does not substantially alter the head kidney transcriptome of Sockeye salmon during early (2 to 3 week) infection and dissemination in a period of significant increasing viral load, nor does the presence of PRV change the host transcriptional response to an IHNV superinfection. Further, concurrent infections of PRV and IHNV do not appear to significantly influence the infectivity or severity of IHNV associated disease, or conversely, PRV load.

Characterization and expression analyses of five interferon regulatory factor transcripts (Irf4a, Irf4b, Irf7, Irf8, Irf10) in Atlantic cod (Gadus morhua).

The interferon regulatory factor (IRF) family of genes encodes a group of transcription factors that have important roles not only in regulating the expression of Type I interferons (IFNs) and other genes in the IFN pathway, but also in growth, development and the regulation of oncogenesis. In this study, several IRF family members (Irf4a, Irf4b, Irf7, Irf8, Irf10) in Atlantic cod (Gadus morhua) were characterized at the cDNA and putative amino acid levels, allowing for phylogenetic analysis of these proteins in teleost fish, as well as the development of gene-specific primers used in RT-PCR and quantitative PCR (QPCR) analyses. Two Atlantic cod Irf10 splice variants were identified and their presence confirmed by sequencing of the Irf10 genomic region. RT-PCR showed that Irf7, Irf8 and both Irf10 transcripts were expressed in all 15 cod tissues tested, while Irf4a and Irf4b were absent in some tissues. QPCR analysis of spleen expression expanded upon this, and upon previous work. All IRF transcripts in the study were responsive to stimulation by the viral mimic poly(I:C), and all except Irf4a were responsive to exposure to formalin-killed Aeromonas salmonicida (ASAL). These IRF genes, thus, are likely important in the cod immune response to both viral and bacterial infections. Increased temperature (10 °C to 16 °C) was also observed to modulate the antibacterial responses of all IRF transcripts, and the antiviral responses of Irf4b and Irf10-v2. This research supports earlier studies which reported that elevated temperature modulates the expression of many immune genes in Atlantic cod.