Our research is focused on the immune system of Atlantic salmon and how this species combat viral infections. The knowledge contributes to an increased understanding of the evolution of the immune system and to improved vaccines for the aquaculture industry. Farming fish in dense populations in the open sea inevitably leads to outbreaks of infectious diseases. While vaccination has brought most bacterial diseases in Norwegian salmon farming under control, virus diseases are still a major threat having impacts on fish welfare and economy. At present the main viral diseases are salmon pancreas disease (PD), heart- and skeletal muscle inflammation (HSMI), cardiomyopathy syndrome (CMS) and infectious salmon anemia (ISA). Salmon virus vaccines have so far shown relatively low protective effect. A major goal of the research group is to contribute to improved virus vaccines for the aquaculture industry. To achieve this, we study the innate and adaptive immune systems of Atlantic salmon and how they inhibit virus infection. Study of immune mechanisms are also translated into investigation of secretable exosomes from salmon leucocytes. These exosomes are nanometer-sized vesicles oftenly secreted during immune pathology and they contain different RNAs together with MHC I and II molecules. The research group is also responsible for the Aquamedicine study programme.
Forbasert kontra lysstimulert smoltifisering av Atlantisk laks (Salmo salar L.)
Effekten av smoltifiseringsregime p smoltutvikling og immunstatus i ferskvann, samt vekst og mottakelighet for infeksiøs lakseanemi (ILA) etter sjøsetting.
Forfatter: Bjørn Ellingsen
Veiledere: Even Jørgensen, Ingvill Jensen
Humorale immunresponser i bukhule og systemiske organer hos Atlantisk laks (Salmo salar L.) – sammenligning av levende og inaktivert salmonid alphavirus 3 administrert i bukhulen
Forfatter: Mathias Abrahamsen
Veiledere: Jorunn Jørgensen, Ingvill Jensen
Karakterisering av humoral immunrespons mot salmonid alfavirus 3 i atlantisk laks (Salmo salar L.).
Kartlegging av IgM antistoffsekrerende B-celleresponser i bukhule og systemiske organer.
Forfatter: Morten B Styrvold
Veiledere: Jorunn Jørgensen, Ingvill Jensen
The distribution, expression pattern and effects of TNF superfamily members BAFF and APRIL in Atlantic salmon (Salmo salar L.) and salmon derived leukocytes
Forfatter: Mikael Fjeld Vold
Veiledere: Jorunn Jørgensen, Michelle Ma Penaranda, Ingvill Jensen
Effekt av interferon, poly I:C og virussmitte på induksjon av nyoppdagede Mx-gener hos atlantisk laks (Salmo salar L.)
Forfatter: Jørgensen, Lars Gaute
Veileder: Børre Robertsen
Produksjonsindusert stress på rogn (Salmo salar L.) og effekter på immun- og stressrelaterte gener etter smitte av plommesekkyngel med Yersinia ruckeri
Forfatter: Per Kristian Sætre
Veiledere: Hanne Johnsen, Helge Tveiten, Lill Heidi Johansen, Ingvill Jensen.
Toll-lignende reseptor 9 og 21 i atlantisk laks (Salmo salar L.) - Samme ligand, unik eller felles uttrykksprofil?
Forfatter: Louise Rebekka Ingebrigtsen
Veiledere: Jorunn Jørgensen, Ingvill Jensen
Transkripsjonsfaktorer som markør for modning av B-celler i Atlantisk laks (Salmo salar L.)
Effekt av TLR-ligander på utrykk av Pax5 in vitro og in vivo
Forfatter: Håvard Ingulfsvann Hagen
Veiledere: Veiledere: Jorunn Jørgensen, Ingvill Jensen
Vaksinering og smoltifisering av atlantisk laks
Betydning av sjøvannstoleranse for mottakelighet for infeksiøs pankreas nekrose (IPN)
Forfatter: Mathias Overrein
Veiledere: Ingvill Jensen, Tore Seternes, Børge Nilsen Fredriksen.
HPI-axis and heat shock protein (HSP) gene transcripts, and their responsiveness to stress in Atlantic salmon embryos and larvae.
Forfatter: Renate Andersen
Veiledere: Helge Tveiten, Hanne Johnsen, Ingvill Jensen. Oppgaven ble utført ved Nofima.
Interferon som adjuvant i DNA-vaksine mot ILA-virus hos atlantisk laks (Salmo salar L). Kinetikk i antistoffrespons, effekt på drap av celler som uttrykker antigen og effekt på vekst
Forfatter: Bratland, Lisa Charlotte
Veileder: Børre Robertsen
Effekt av interferon-plasmider på ekspresjon av antiviralt Mx-protein i pankreas og beskyttelse mot SAV3-infeksjon hos atlantisk laks (Salmo salar L.)
Forfatter: Jenssen, Iris
Veileder: Børre Robertsen
Hjertehelse som risikofaktor for slaktedødelighet i Nord-Norge.
Forfatter: Synne Karoline Jerman Bjørstad
Veiledere Harald Takle, Nofima og Ingvill Jensen UiT. Samarbeidsprosjekt med Lerøy Aurora.
Antiviralt forsvar i hjertet hos atlantisk laks (Salmo salar)
Uttrykk av type I interferoner in vivo og i primære cellekulturer in vitro.
Forfatter: Astrid-Elisabeth Christiansen Hanssen
Veiledere: Ingvill Jensen & Børre Robertsen.
SOCS-proteiner i laksens antivirale forsvar - til nytte for virus eller vert?
Forfatter: Lisbeth Lindenskov Joensen
Veileder Jorunn Jørgensen
SAV Ag/CpG/polyI:C formulated vaccination potentiate protective immune responses in Atlantic salmon
Forfatter: Hanna Leena Thim
Veileder Jorunn B. Jørgensen
Studier av hjerte og skjelettmuskelbetennelse i Atlantisk laks
Effekter av infeksiøs pankreas nekrose virus og vaksinasjon på utviklingen av HSMB in vivo, og etablering av primærkultur av hjerteceller for dyrking av piscine reovirus (PRV) in vitro.
Forfatter: Ingrid Moan
Veileder Ingvill Jensen UiT og Lill-Heidi Johansen Nofima
Betydning av stress ved badevaksinering av lakseyngel mot yersiniose (Yersinia ruckeri).
Utførte oppgaven ved Universitetet i Nordland og ved Pharmaq.
Forfatter: Anniken Malene Frantzen Sørflaten
Veiledere Ingvill Jensen UiT, Martin Iversen UiN og Børge Nilsen Fredriksen Pharmaq.
Swain, Jaya Kumari; Carpio, Yamila; Johansen, Lill-Heidi; Velazquez, Janet; Hernandez, Liz; Leal, Yeny; Kumar, Ajey; Estrada, Mario Pablo. Impact of a candidate vaccine on the dynamics of salmon lice (Lepeophtheirus salmonis) infestation and immune response in Atlantic salmon (Salmo salar L.). PLOS ONE, 2020 doi:
Jenberie Shiferaw, Peñaranda Ma. Michelle D., Thim Hanna L., Styrvold Morten Bay, Strandskog Guro, Jørgensen Jorunn B., Jensen Ingvill. Salmonid Alphavirus Subtype 3 Induces Prolonged Local B Cell Responses in Atlantic Salmon (Salmo salar) After Intraperitoneal Infection. Frontiers in Immunology. Volume 11 2020. 1682 ISSN 1664-3224 doi: 10.3389/fimmu.2020.01682 (read)
Dennis Berbulla Bela-ong, Linn Greiner-Tollersrud, Yorick Andreas van der Wal, Ingvill Jensen, Ole Morten Seternes, Jorunn B. Jørgensen. Infection and microbial molecular motifs modulate transcription of the interferon-inducible gene ifit5 in a teleost fish. Developmental & Comparative Immunology. Volume 111 2020. 103746 ISSN 0145-305X doi: 10.1016/j.dci.2020.103746. (read)
Nepal A, Wolfson DL, Ahluwalia BS, Jensen I, Jørgensen J, Iliev DB. Intracellular distribution and transcriptional regulation of Atlantic salmon (Salmo salar) Rab5c, 7a and 27a homologs by immune stimuli. Fish Shellfish Immunol. 2020 Apr;99:119-129. doi: 10.1016/j.fsi.2020.01.058. Epub 2020 Jan 31. PubMed PMID: 32014587. (read)
Svenning S, Gondek-Wyrozemska AT, van der Wal YA, Robertsen B, Jensen I, Jørgensen JB, Edholm ES. Microbial Danger Signals Control Transcriptional Induction of Distinct MHC Class I L Lineage Genes in Atlantic Salmon. Front Immunol. 2019 Oct 11;10:2425. doi: 10.3389/fimmu.2019.02425. PMID: 31681311; PMCID: PMC6797598.
Robertsen B, Greiner-Tollersrud L, Jørgensen LG. Analysis of the Atlantic salmon genome reveals a cluster of Mx genes that respond more strongly to IFN gamma than to type I IFN. Dev Comp Immunol. 2019;90:80-89. doi:10.1016/j.dci.2018.09.004
Jensen I, Overrein MC, Fredriksen BN, Strandskog G, Seternes T. Differences in smolt status affect the resistance of Atlantic salmon (Salmo salar L.) against infectious pancreatic necrosis, while vaccine-mediated protection is unaffected. J Fish Dis. 2019 Sep;42(9):1271-1282. doi: 10.1111/jfd.13049. Epub2019 Jun 11. PMID: 31211446.
Iliev DB, Lagos L, Thim HL, Jørgensen SM, Krasnov A, Jørgensen JB. CpGs Induce Differentiation of Atlantic Salmon Mononuclear Phagocytes Into Cells With Dendritic Morphology and a Proinflammatory Transcriptional Profile but an
Exhausted Allostimulatory Activity. Front Immunol. 2019 Mar 13;10:378. doi: 10.3389/fimmu.2019.00378. PMID: 30918507; PMCID: PMC6424866.
Peñaranda MMD, Jensen I, Tollersrud LG, Bruun JA, Jørgensen JB. 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. Front Immunol. 2019 Jan 29;10:37. doi: 10.3389/fimmu.2019.00037. PMID: 30761128; PMCID: PMC6362898.
Dahle MK, Jørgensen JB. Antiviral defense in salmonids - Mission made possible? Fish Shellfish Immunol. 2019 Apr;87:421-437. doi: 10.1016/j.fsi.2019.01.043. Epub 2019 Jan 30. PMID: 30708056.
Robertsen, Børre. The role of type I interferons in innate and adaptive immunity against viruses in Atlantic salmon. Developmental and Comparative Immunology 2018; Volum 80. ISSN 0145-305X.s 41 - 52.s doi: 10.1016/j.dci.2017.02.005.
Sobhkhez, M., Krasnov, A. & Robertsen, B. (2018). Transcriptome analyses of Atlantic salmon muscle genes induced by a DNA vaccine against salmonid alphavirus, the causative agent of salmon pancreas disease (PD). PLoS ONE, 13:e0204924(10). https://doi.org/10.1371/journal.pone.0204924
Iliev D, Strandskog G, Nepal A, Aspar A, Olsen R, Jørgensen J, Wolfson D, Ahluwalia BS, Handzhiyski J, Mironova R. Stimulation of exosome release by extracellular DNA is conserved across multiple cell types. FEBS J. 2018 Aug;285(16):3114-3133. doi: 10.1111/febs.14601. Epub 2018 Jul 12. PMID: 29953723.
Jenberie S, Thim HL, Sunyer JO, Skjødt K, Jensen I, Jørgensen JB. Profiling Atlantic salmon B cell populations: CpG-mediated TLR-ligation enhances IgM secretion and modulates immune gene expression. Sci Rep. 2018 Feb 23;8(1):3565. doi: 10.1038/s41598-018-21895-9. Erratum in: Sci Rep. 2018 Apr 19;8(1):6491. PMID: 29476080; PMCID: PMC5824956.
Jenberie S, Thim HL, Sunyer JO, Skjødt K, Jensen I, Jørgensen JB. Author Correction: Profiling Atlantic salmon B cell populations: CpG-mediated TLR- ligation enhances IgM secretion and modulates immune gene expression. Sci Rep. 2018 Apr 19;8(1):6491. doi: 10.1038/s41598-018-24843-9. Erratum for: Sci Rep. 2018 Feb 23;8(1):3565. PMID: 29670159; PMCID: PMC5906671.
Sobhkhez M, Joensen LL, Tollersrud LG, Strandskog G, Thim HL, Jørgensen JB. A conserved inhibitory role of suppressor of cytokine signaling 1 (SOCS1) in salmon antiviral immunity. Dev Comp Immunol. 2017 Feb;67:66-76. doi: 10.1016/j.dci.2016.11.001. Epub 2016 Nov 3. PMID: 27818171.
Sobhkhez, Mehrdad; Krasnov, Aleksei; Chang, Chia Jung; Robertsen, Børre. Transcriptome analysis of plasmid-induced genes sheds light on the role of type I IFN as adjuvant in DNA vaccine against infectious salmon anemia virus. PLOS ONE 2017; Volum 12:e0188456 (11). ISSN 1932-6203.s 1 - 20.s doi: 10.1371/journal.pone.0188456.
Li, Chun; Greiner-Tollersrud, Linn; Robertsen, Børre. Infectious salmon anemia virus segment 7 ORF1 and segment 8 ORF2 proteins inhibit IRF mediated activation of the Atlantic salmon IFNa1 promoter. Fish and Shellfish Immunology 2016; Volum 52. ISSN 1050-4648.s 258 - 262.s doi: 10.1016/j.fsi.2016.03.038.
Bakkemo, Kathrine Ryvold; Mikkelsen, Helene; Johansen, Audny; Robertsen, Børre; Seppola, Marit. Francisella noatunensis subsp. noatunensis invades, survives and replicates in Atlantic cod cells. Diseases of Aquatic Organisms 2016; Volum 121 (2). ISSN 0177-5103.s 149 - 159.s doi: 10.3354/dao03043.
Chang, Chia Jung; Jenssen, Iris; Robertsen, Børre. Protection of Atlantic salmon against salmonid alphavirus infection by type I interferons IFNa, IFNb and IFNc. Fish and Shellfish Immunology 2016; Volum 57. ISSN 1050-4648.s 35 - 40.s doi: 10.1016/j.fsi.2016.08.020.
Robertsen, Børre; Chang, Chia Jung; Bratland, Lisa Charlotte. IFN-adjuvanted DNA vaccine against infectious salmon anemia virus: Antibody kinetics and longevity of IFN expression. Fish and Shellfish Immunology 2016; Volum 54. ISSN 1050-4648.s 328 - 332.s doi: 10.1016/j.fsi.2016.04.027.
Johansen LH, Thim HL, Jørgensen SM, Afanasyev S, Strandskog G, Taksdal T, Fremmerlid K, McLoughlin M, Jørgensen JB, Krasnov A. Comparison of transcriptomic responses to pancreas disease (PD) and heart and skeletal muscle inflammation (HSMI) in heart of Atlantic salmon (Salmo salar L). Fish Shellfish Immunol. 2015 Oct;46(2):612-23. doi: 10.1016/j.fsi.2015.07.023. Epub 2015 Jul 29. PMID: 26232631.
Lauksund, Reidunn Silje; Greiner-Tollersrud, Linn; Chang, Chia Jung; Robertsen, Børre. Infectious pancreatic necrosis virus proteins VP2, VP3, VP4 and VP5 antagonize IFNa1 promoter activation while VP1 induces IFNa1. Virus Research 2015; Volum 196. ISSN 0168-1702.s 113 - 121.s doi: 10.1016/j.virusres.2014.11.018.
This meeting provides a venue for exciting interactions to generate new ideas and collaborations. We aim to foster interactions among established investigators, postdoctoral fellows and graduate students interested in immune function in fish. We also aim to present ongoing research into fish immunology and vaccinology to potential master students at the University of Tromsø - the Arctic University of Norway.
With climate change, pollution and diseases, both wildlife and domesticated fish species used globally in aquaculture face severe challenges. The study of fish immunology provides unique insight into immunobiology, medicine and evolution and the potential for novel therapeutics for the aquaculture industry.
Here we highlight research into various aspects of fish immunology and vaccinology, including antigen presenting molecules, immune type receptors, B cells, development of immune repertoires, CRISPR/Cas9 gene editing and the immune dialogue between the hosts and pathogens.
Date and Time: October 9th 2020, 8:45 – 15:45
Location: Room 1.007, ILP-building UiT
Digital: available upon request
09:15-10:00 MHC in teleosts and salmonids in particular. UNNI GRIMHOLT (Norwegian veterinary Institute)
10:00-10:15 Effect of two different RNA viral challenges on non-classical MHC class I expression in Atlantic salmon. AGATA TERESA WYROZEMSKA (University of Tromsø)
10:15-10:30 Exploring the interplay between the interferon response and non-classical MHC class I L-lineage induction pathways in Atlantic salmon. MARYAM IMAM (University of Tromsø)
10:45-11:30 Piscine ortoreovirus (PRV) and salmon gill poxvirus (SGPV). Host responses and protection. MARIA K. DAHLE (Norwegian veterinary Institute/ University of Tromsø)
11:30-11:45 Novel-immune type receptors (NITRs) in Atlantic salmon. STEINGRIM SVENNING. (University of Tromsø)
11:45-12:00 Standardized IMGT® nomenclature of Salmonidae TRA/D genes: genomic organization, gene segment usage and potential for invariant T cells.
EVA-STINA EDHOLM (University of Tromsø)
13:00-13:15 BAFF is a critical B cell survival factor that plays additional roles in distinct peripheral blood B cell subset/s of Atlantic salmon. MA MICHELLE DEMOGINA PEÑARANDA (University of Tromsø)
13:15-13:30 B cell responses to salmonid alphavirus in Atlantic salmon with a renewed interest on the local PerC responses. SHIFERAW JENBERIE (University of Tromsø)
13:30-14:15 Effect on louse fecundity and immune response in Atlantic salmon in response to a candidate vaccine. JAYA KUMARI SWAIN (University of Tromsø)
14:15-14:30 WAFFELS AND COFFE
14:30-15:15 Intramuscular vaccination of Atlantic lumpfish (Cyclopterus lumpus L.) induces inflammatory reactions and local immunoglobulin M production at the vaccine administration. TORE SETTERNES (University of Tromsø)
15:15-15:30 Optimization of CRISPR/Cas protocols for precise genome editing in salmon cell lines. MIROSLAV BOBRIK (University of Tromsø)
15:30-15:45 Utilizing CRISPR in development of Immortalized cell-lines from Atlantic salmon immune organs. MEHRDAD SOBHKHEZ(University of Tromsø)
This meeting was financed and supported by the TROMSØ RESEARCH FOUNDATION (TFS)
(in order of appearance)
Effect of two different RNA viral challenges on non-classical MHC class I expression in Atlantic salmon
Agata T. Wyrozemska1, Maria K. Dahle1,2 and Eva-Stina Edholm1
1 The Norwegian College of Fishery Science, Faculty of Biosciences, Fisheries and Economics, UiT - The Arctic University of Norway, N-9037 Tromsø, Norway
2 Department of Fish Health, Norwegian Veterinary Institute, Oslo, Norway
Major histocompatibility complex (MHC) molecules play an important role in adaptive immune response by presenting peptides to conventional T cells. In addition to a single classical MHC class I gene (Sasa-UBA) the genome of Atlantic salmon contains several highly divergent non-classical MHC class I lineages. Their function is largely unknown. Recently we have reported that transcriptional regulation of distinct non-classical MHC class I L lineage genes was rapidly and transiently upregulated in response to Salmonid Alphavirus 3 (SAV3) infection, indicating that these molecules play a role in antiviral immunity. To gain further insight into the induction and expression kinetics of non-classical MHC class I genes, two long-term in vivo viral trials using either piscine orthoreovirus subtype 1 (PRV-1) or SAV3 as challenging factors were performed. Infected fish and respective control groups were subsampled and multiple organs were collected over 10 (PRV-1) and 12 (SAV3) weeks. Results from heart and spleen showed that unique sets of non-classical L lineage genes are differentially upregulated in response to PRV-1 and SAV3, suggesting that non-classical MHC class I L-lineage play different roles during the respective viral challenges. For better comprehension of the different induction potential among the various L-lineage genes, L lineage promoter regions were examined in multiple Salmonidae species using Northern pike (Exous lucius) as the group outer branch. Distinct combinations of interferon response elements and numerous other promoter specific elements proved to be conserved within each gene throughout the species analysed. Furthermore, both L lineage domain sequences’ conservation and secondary structure were analysed for each gene giving results that may help to explain the expression dynamics and function of these genes.
Key-words: Atlantic salmon, viral challenge, non-classical MHC class I, L lineage
Exploring the interplay between the interferon response and non-classical MHC class I L-lineage induction pathways in Atlantic salmon (Salmo salar)
Maryam Imam*1, Eva-Stina Edholm1
1The Norwegian College of Fishery Science, Faculty of Biosciences, Fisheries & Economics, University of Tromsø, The Arctic University of Norway, N-9037 Tromsø, Norway.
*Corresponding author: Maryam.Imam@uit.no
Non-classical MHC class I (Ib) genes are non-polymorphic and encode for proteins typically expressed with restricted patterns. Like mammals, fish have both classical and non-classical MHC class I genes. Apart from the classical U-lineage gene (SasaUBA), teleost fish also possess diverged non-classical lineages namely Z, L, S, P and H with unknown functions. Previous work have shown that non-classical MHC class I L-lineage genes have distinct tissue-specific and inducible expression patterns. Among the six L lineage genes, three genes in particular lia, lga and lha are differentially regulated in response to various immunological stimuli and disease states. However, the mode of their upregulation is unexplored. In the present study, we investigated non-classical MHC class I L -lineage gene expression in response to type I (IFNa and IFNc) and type II (IFN gamma) interferon stimulation. Further, we explored the molecular pathways associated with non-classical MHC class I L lineage regulation. Pronephros origin SSP9 cultured cell line stimulated with either recombinant type I or type II IFN expressed differential pattern of non-classical MHC class I L-Lineage genes as determined by quantitative reverse transcriptase PCR. Lia was significantly upregulated in response to both type I and Type II IFN with a peak expression at 48 hours that declined by 72 hours. Similar to lia, lga response to IFN gamma peaks at 48 hours (50-fold change) followed by a decline at 72 hours. However, the response to IFNa or IFNc stimulation was negligable. Expression levels of lha and lda remained unchanged. Thus, concurrent upregulation of lia and lga was favored following IFN gamma stimulation. Janus kinase–signal transducer and activator of transcription (JAK-STAT) activated upon cytokine stimulation facilitates several cellular reactions by expressing several genes that modulate the immune response. Inhibiting phosphorylation of JAK protein (using JAK Inhibitor) blocks the docking site for the STAT and therefore inhibits its translocation to the nucleus where it acts as a transcription factor and thereby regulate the gene expression. In the present work, adding JAK inhibitor in IFN gamma stimulated SSP9 cells, resulted in a downregulation of lia and lga along with Mx1/2 and Mx8 genes. Our future work will focus on developing transgenic SSP9 cells lines deficient in key IFN pathway genes to delineate the role(s) of the various non-classical MHC I L lineage genes in Atlantic salmon immunity.
Keywords – lia, IFN gamma inhibitor, non-classical MHC class I
BAFF is a critical B cell survival factor that plays additional roles in distinct peripheral blood B cell subset/s of Atlantic salmon
Ma. Michelle D. Peñaranda, Mikael Fjeld Wold, Shiferaw Jenberie, Ingvill Jensen, and Jorunn B. Jørgensen
Fish Immunology and Vaccinology Group, The Norwegian College of Fishery Science, UiT The Arctic University of Norway, N-9037 Tromsø, Norway
B cell surviving factor (BAFF) is a member of the TNF- superfamily known to play important roles in B cell maturation, survival, and differentiation. Little is still known about teleost BAFF expression and function in B cells to date. To address this gap, we investigated this cytokine ligand in Atlantic salmon (Salmo salar L.). Constitutive expression of BAFF gene was profiled in naïve fish tissues and relevant cell subpopulations, as well as the changes in gene expression following viral challenge. Highest basal level of BAFF transcripts was found in the spleen, a major lymphoid organ. Consistent with that of mammals, macrophage-like cells (MLCs) had the highest constitutive BAFF gene expression among the leukocyte subpopulations examined. Significant up-regulation of BAFF transcripts was also found in fish infected with salmonid alphavirus 3 (SAV3), with the highest induction observed in the target organs of the virus, implying a potential involvement of this cytokine ligand in the immune response of salmon to pathogens. In vitro functional assays using recombinant AsBAFF (rAsBAFF) protein were also performed to measure the effect of the soluble form of this cytokine ligand in the heterogeneous B cell population from major lymphoid compartments: head kidney (HK), spleen (Sp), and peripheral blood (PB). Cell apoptosis assays resulted to a higher number of viable IgM+ B cell following pre-treatment with rAsBAFF in all leukocyte cultures examined. Interestingly, rAsBAFF- associated increase in IgM-secreting cell population and IgM+ proliferation was only observed in PB, but not in HK and Sp cultures. Data from these functional studies suggest that BAFF is critical in the survival of Atlantic salmon IgM+ B cells, with as-yet unidentified PB B cell subpopulation being the most affected by this TNFSF ligand. Overall, our results support the idea that AsBAFF, similar to their mammalian counterparts, are important for B cell homeostasis and responses in Atlantic salmon. The recognition of AsBAFF as an important teleost B cell survival factor may help in future fish vaccination strategies.
B cell responses to salmonid alphavirus in Atlantic salmon with a renewed interest on the local PerC responses
Shiferaw Jenberie1, Henriette Nordli1, Guro Strandskog1, Linn Greiner-Tollersrud1, Ma. Michelle D. Peñaranda1, Jorunn B. Jørgensen1 and Ingvill Jensen1*
1Norwegian College of Fishery Science, Faculty of Biosciences, Fisheries and Economics, University of Tromsø - The Arctic University of Norway, Tromsø, Norway.
In fish, intraperitoneally administered vaccines have a long and well-documented reputation inducing a superior protection over other platforms. However, it is only recently that the peritoneal cavity (PerC) and its adipose tissue (AT) in teleost species have been described to have an active immunological role against intraperitoneal (ip)-injected antigens. In the current study, aiming at providing insight on Ag-specific antibody secreting cells (ASCs) response in the PerC, we analyzed PerC washes and PerC ATs after ip injection of wild-type SAV3 (WtSAV3) or inactivated SAV (InSAV) with a booster injection at 6 wpi to only the InSAV group. We found that PerC AT retained ip injected WtSAV3 for over 6 wpi. Whereas both WtSAV3 and InSAV induced Ag-specific ASCs response in the PerC, a higher frequency was seen in WtSAV3 group at 13 wpi. Within the WtSAV3 group, PerC had a higher frequency of Ag-specific ASCs compared to HK and spleen at 13 wpi suggesting on their local importance. A booster injection of InSAV was not accompanied by increased Ag-specific ASCs. Analysis of AT from WtSAV3 infected fish revealed a higher transcription of key immune genes at 6 wpi compared with control and InSAV. Taken together, this work shows, for the first time in teleost species, the presence of Ag-specific ASCs in the PerC and further suggests on a role the PerC could possibly play in orchestrating the overall immune response against ip-injected antigens.
Optimization of CRISPR/Cas protocols for precise genome editing in salmon cell lines
Miroslav Bobrik1×, Mehrdad Sobhkhez2, Greta K. Johansen3, Eva Stina Edholm2, Ole-Morten Seternes1
1 Department of Pharmacy, Faculty of Health Sciences, UiT - The Arctic University of Norway, N-9037 Tromsø, Norway
2 The Norwegian College of Fishery Science, Faculty of Biosciences, Fisheries and Economics, UiT - The Arctic University of Norway, N-9037 Tromsø, Norway
3 Department of Chemistry, Faculty of Science and Technology, UiT - The Arctic University of Norway, N-9037 Tromsø, Norway
Salmonids have a combined economic and scientific importance that is unique among fish. Understanding key immunological defenses in salmon is essential for the development of prophylactic strategies including vaccines and the breeding of healthy fish. Advancement and implementation of CRISPR/Cas9 technology to marine vertebrates could be a landmark breakthrough in fish immunology and disease prevention. However, the current CRISPR/Cas9 gene editing approaches, while encouraging in terms of feasibility, are highly ineffective. In fact, <40% of injected embryos have Cas9 introduced mutations with <10% of fish showing a phenotypical change. One likely explanation for this sub-optimal effect is the mismatch in optimal temperature between the activity of the Cas9 nuclease and optimal salmon embryo viability at 8°C resulting in significantly reduced Cas9 enzymatic activity. In order to achieve improvements of CRISP/Cas9 based genome editing in salmon several optimization strategies were implemented focusing on i ) guide RNA design and ii ) transfection optimization Firstly, the guide RNA (gRNA) design was validated by a two-plasmid system where homology directed repair quantifies cleaving efficiency. Second, as transfection efficiency of salmon cell lines can be low, we set out to optimize the transfection protocol for the head-kidney derived Atl. salmon cell line SSP9. The efficiency of GFP-Cas9 protein coupled with specific gRNA introduction into SSP9 cells was determined by fluorescent activated cell sorting (FACS) and GFP positive cells were sorted. Many transfection strategies were tested with varying success. Electroporation of ribonucleoprotein complexes (RNP) showed the highest transfection efficiency compared to other tested methods. Furthermore, in addition to generate Knock-Out (KO) salmon cell lines, a three-component approach will be used to attempt Knock-In (KI) of an epitope tag to key genes of interest. Due to a lack of functioning antibodies in salmon, this KI strategy could help with the study of many hard to detect proteins. Strategies to increase KI efficiency, such as modified ends and nanoparticles stabilization provide a feasible option for maximizing a KI efficiency.
Utilizing CRISPR in development of Immortalized cell-lines from Atlantic salmon immune organs
Mehrdad Sobhkhez1, Miroslav Bobrik2 and Eva-Stina Edholm1
1The Norwegian College of Fishery Science, Faculty of Biosciences, Fisheries and Economics, UiT - The Arctic University of Norway
2Department of Pharmacy, Faculty of Health Sciences, UiT - The Arctic University of Norway
Some of the major challenges in fish research are lack of necessary tools like antibodies, clonal fish (except for a few species) as well as very limited range of available cell-line. Use of cell-line is especially important with regard to the Three R’s of animal research: Replacement, Reduction and Refinement as many experiments such as preliminary studies regarding organism’s immune response to stimulants and pathogens, among others, could be performed in cell-lines. This would reduce number of fish used in research and lessen their stress and discomfort. However, access to fish cell-line in general and Atlantic salmon cell-lines in particular are limited and not all cells are easily transfected, infected nor do they express all of the molecules of interest in a study. Most importantly, to the authors’ knowledge no true immune cell-line (T-cell etc.) is available for Atlantic salmon to date. Numerus studies in mammalian models have shown Tumor Protein 53 (p53) to be involved in DNA-repair, growth arrest, apoptosis and secession of cell division. Additionally in naturally immortalized cells like tumor cells, often p53 is mutated or its signaling pathway is altered. These facts make p53 an interesting target in cell-line development. Main goal of this project is to produce immortalized cell-line by using CRISPR-Cas gene editing to knock out p53 in cells derived from Atlantic salmon’s major immune organs. The project involves: Transfection optimization, P53 specific RNA-guide design and transfection of RNA-Cas9 complex and evaluation of result. The available results will be presented in the meeting.
Key words: CRISPR, P53, Cell-line