Publications by authors named "Geert F Wiegertjes"

77 Publications

Adherent Intestinal Cells From Atlantic Salmon Show Phagocytic Ability and Express Macrophage-Specific Genes.

Front Cell Dev Biol 2020 15;8:580848. Epub 2020 Oct 15.

Faculty of Biosciences and Aquaculture, Nord University, Bodø, Norway.

Our knowledge of the intestinal immune system of fish is rather limited compared to mammals. Very little is known about the immune cells including the phagocytic cells in fish intestine. Hence, employing imaging flow cytometry and RNA sequencing, we studied adherent cells isolated from healthy Atlantic salmon. Phagocytic activity and selected gene expression of adherent cells from the distal intestine (adherent intestinal cells, or AIC) were compared with those from head kidney (adherent kidney cells, or AKC). Phagocytic activity of the two cell types was assessed based on the uptake of BioParticles. AIC showed phagocytic ability but the phagocytes were of different morphology compared to AKC. Transcriptomic analysis revealed that AIC expressed genes associated with macrophages, T cells, and endothelial cells. Heatmap analysis of selected genes indicated that the adherent cells from the two organs had apparently higher expression of macrophage-related genes. We believe that the adherent intestinal cells have phagocytic characteristics and high expression of genes commonly associated with macrophages. We envisage the possibilities for future studies on enriched populations of adherent intestinal cells.
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http://dx.doi.org/10.3389/fcell.2020.580848DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7593592PMC
October 2020

Transcriptome sequencing supports a conservation of macrophage polarization in fish.

Sci Rep 2020 08 10;10(1):13470. Epub 2020 Aug 10.

Aquaculture and Fisheries Group, Wageningen University and Research, De Elst 1, 6708 WD, Wageningen, The Netherlands.

Mammalian macrophages can adopt polarization states that, depending on the exact stimuli present in their extracellular environment, can lead to very different functions. Although these different polarization states have been shown primarily for macrophages of humans and mice, it is likely that polarized macrophages with corresponding phenotypes exist across mammals. Evidence of functional conservation in macrophages from teleost fish suggests that the same, or at least comparable polarization states should also be present in teleosts. However, corresponding transcriptional profiles of marker genes have not been reported thus far. In this study we confirm that macrophages from common carp can polarize into M1- and M2 phenotypes with conserved functions and corresponding transcriptional profiles compared to mammalian macrophages. Carp M1 macrophages show increased production of nitric oxide and a transcriptional profile with increased pro-inflammatory cytokines and mediators, including il6, il12 and saa. Carp M2 macrophages show increased arginase activity and a transcriptional profile with increased anti-inflammatory mediators, including cyr61, timp2b and tgm2b. Our RNA sequencing approach allowed us to list, in an unbiased manner, markers discriminating between M1 and M2 macrophages of teleost fish. We discuss the importance of our findings for the evaluation of immunostimulants for aquaculture and for the identification of gene targets to generate transgenic zebrafish for detailed studies on M1 and M2 macrophages. Above all, we discuss the striking degree of evolutionary conservation of macrophage polarization in a lower vertebrate.
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http://dx.doi.org/10.1038/s41598-020-70248-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7418020PMC
August 2020

Lymphoid Tissue in Teleost Gills: Variations on a Theme.

Biology (Basel) 2020 Jun 15;9(6). Epub 2020 Jun 15.

Aquaculture and Fisheries Group, Department of Animal Sciences, Wageningen University & Research, 6700 AH Wageningen, The Netherlands.

In bony fish, the gill filaments are essential for gas exchanges, but also are vulnerable to infection by water-borne microorganisms. Omnipresent across fish, gill-associated lymphoid tissues (GIALT) regulate interactions with local microbiota and halt infection by pathogens. A special GIALT structure has recently been found in Salmonids, the interbranchial lymphoid tissue (ILT). However, the structural variation of GIALT across bony fish remains largely unknown. Here, we show how this critical zone of interaction evolved across fishes. By labeling a conserved T-cell epitope on tissue sections, we find that several basal groups of teleosts possess typical ILT, while modern teleosts have lymphoepithelium of different shape and size at the base of primary gill filaments. Within Cypriniformes, neither body size variation between two related species, zebrafish and common carp, nor morphotype variation, did have a drastic effect on the structure of ILT. Thereby this study is the first to describe the presence of ILT in zebrafish. The ILT variability across fish orders seems to represent different evolutionary solutions to balancing trade-offs between multiple adaptations of jaws and pharyngeal region, and immune responses. Our data point to a wide structural variation in gill immunity between basal groups and modern teleosts.
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http://dx.doi.org/10.3390/biology9060127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7344468PMC
June 2020

Fish Macrophages Show Distinct Metabolic Signatures Upon Polarization.

Front Immunol 2020 25;11:152. Epub 2020 Feb 25.

Aquaculture and Fisheries Group, Wageningen University & Research, Wageningen, Netherlands.

Macrophages play important roles in conditions ranging from host immune defense to tissue regeneration and polarize their functional phenotype accordingly. Next to differences in the use of L-arginine and the production of different cytokines, inflammatory M1 macrophages and anti-inflammatory M2 macrophages are also metabolically distinct. In mammals, M1 macrophages show metabolic reprogramming toward glycolysis, while M2 macrophages rely on oxidative phosphorylation to generate energy. The presence of polarized functional immune phenotypes conserved from mammals to fish led us to hypothesize that a similar metabolic reprogramming in polarized macrophages exists in carp. We studied mitochondrial function of M1 and M2 carp macrophages under basal and stressed conditions to determine oxidative capacity by real-time measurements of oxygen consumption and glycolytic capacity by measuring lactate-based acidification. In M1 macrophages, we found increased nitric oxide production and expression in addition to altered oxidative phosphorylation and glycolysis. In M2 macrophages, we found increased arginase activity, and both oxidative phosphorylation and glycolysis were similar to control macrophages. These results indicate that M1 and M2 carp macrophages show distinct metabolic signatures and indicate that metabolic reprogramming may occur in carp M1 macrophages. This immunometabolic reprogramming likely supports the inflammatory phenotype of polarized macrophages in teleost fish such as carp, similar to what has been shown in mammals.
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http://dx.doi.org/10.3389/fimmu.2020.00152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052297PMC
March 2021

Imaging Flow Cytometry Protocols for Examining Phagocytosis of Microplastics and Bioparticles by Immune Cells of Aquatic Animals.

Front Immunol 2020 18;11:203. Epub 2020 Feb 18.

Faculty of Biosciences and Aquaculture, Nord University, Bodø, Norway.

Imaging flow cytometry (IFC) is a powerful tool which combines flow cytometry with digital microscopy to generate quantitative high-throughput imaging data. Despite various advantages of IFC over standard flow cytometry, widespread adoption of this technology for studies in aquatic sciences is limited, probably due to the relatively high equipment cost, complexity of image analysis-based data interpretation and lack of core facilities with trained personnel. Here, we describe the application of IFC to examine phagocytosis of particles including microplastics by cells from aquatic animals. For this purpose, we studied (1) live/dead cell assays and identification of cell types, (2) phagocytosis of degradable and non-degradable particles by Atlantic salmon head kidney cells and (3) the effect of incubation temperature on phagocytosis of degradable particles in three aquatic animals-Atlantic salmon, Nile tilapia, and blue mussel. The usefulness of the developed method was assessed by evaluating the effect of incubation temperature on phagocytosis. Our studies demonstrate that IFC provides significant benefits over standard flow cytometry in phagocytosis measurement by allowing integration of morphometric parameters, especially while identifying cell populations and distinguishing between different types of fluorescent particles and detecting their localization.
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http://dx.doi.org/10.3389/fimmu.2020.00203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7039858PMC
February 2021

Feed, Microbiota, and Gut Immunity: Using the Zebrafish Model to Understand Fish Health.

Front Immunol 2020 5;11:114. Epub 2020 Feb 5.

Cell Biology and Immunology Group, Wageningen University and Research, Wageningen, Netherlands.

Aquafeed companies aim to provide solutions to the various challenges related to nutrition and health in aquaculture. Solutions to promote feed efficiency and growth, as well as improving the fish health or protect the fish gut from inflammation may include dietary additives such as prebiotics and probiotics. The general assumption is that feed additives can alter the fish microbiota which, in turn, interacts with the host immune system. However, the exact mechanisms by which feed influences host-microbe-immune interactions in fish still remain largely unexplored. Zebrafish rapidly have become a well-recognized animal model to study host-microbe-immune interactions because of the diverse set of research tools available for these small cyprinids. Genome editing technologies can create specific gene-deficient zebrafish that may contribute to our understanding of immune functions. Zebrafish larvae are optically transparent, which allows for imaging of specific (immune) cell populations in whole transgenic organisms. Germ-free individuals can be reared to study host-microbe interactions. Altogether, these unique zebrafish features may help shed light on the mechanisms by which feed influences host-microbe-immune interactions and ultimately fish health. In this review, we first describe the anatomy and function of the zebrafish gut: the main surface where feed influences host-microbe-immune interactions. Then, we further describe what is currently known about the molecular pathways that underlie this interaction in the zebrafish gut. Finally, we summarize and critically review most of the recent research on prebiotics and probiotics in relation to alterations of zebrafish microbiota and immune responses. We discuss the advantages and disadvantages of the zebrafish as an animal model for other fish species to study feed effects on host-microbe-immune interactions.
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http://dx.doi.org/10.3389/fimmu.2020.00114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7014991PMC
March 2021

Different transcriptional response between susceptible and resistant common carp (Cyprinus carpio) fish hints on the mechanism of CyHV-3 disease resistance.

BMC Genomics 2019 Dec 26;20(1):1019. Epub 2019 Dec 26.

Department of Animal Sciences, RH Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel.

Background: Infectious disease outbreaks form major setbacks to aquaculture production and to further development of this important sector. Cyprinid herpes virus-3 (CyHV-3) is a dsDNA virus widely hampering production of common carp (Cyprinus carpio), one of the most farmed fish species worldwide. Genetically disease resistant strains are highly sought after as a sustainable solution to this problem. To study the genetic basis and cellular pathways underlying disease resistance, RNA-Seq was used to characterize transcriptional responses of susceptible and resistant fish at day 4 after CyHV-3 infection.

Results: In susceptible fish, over four times more differentially expressed genes were up-regulated between day 0 and 4 compared to resistant fish. Susceptible and resistant fish responded distinctively to infection as only 55 (9%) of the up-regulated genes were shared by these two fish types. Susceptible fish elicited a typical anti-viral response, involving interferon and interferon responsive genes, earlier than resistant fish did. Furthermore, chemokine profiles indicated that the two fish types elicited different cellular immunity responses. A comparative phylogenetic approach assisted in chemokine copies annotation pointing to different orthologous copies common to bony-fishes and even carp-specific paralogs that were differentially regulated and contributed to the different response of these two fish types. Susceptible fish up-regulated more ccl19 chemokines, which attract T-cells and macrophages, the anti-viral role of which is established, whereas resistant fish up-regulated more cxcl8/il8 chemokines, which attract neutrophils, the antiviral role of which is unfamiliar.

Conclusions: Taken together, by pointing out transcriptional differences between susceptible and resistant fish in response to CyHV-3 infection, this study unraveled possible genes and pathways that take part in disease resistance mechanisms in fish and thus, enhances our understanding of fish immunogenetics and supports the development of sustainable and safe aquaculture.
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http://dx.doi.org/10.1186/s12864-019-6391-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6933926PMC
December 2019

Visualizing trypanosomes in a vertebrate host reveals novel swimming behaviours, adaptations and attachment mechanisms.

Elife 2019 09 24;8. Epub 2019 Sep 24.

Department of Animal Sciences, Cell Biology and Immunology Group, Wageningen University & Research, Wageningen, Netherlands.

Trypanosomes are important disease agents of humans, livestock and cold-blooded species, including fish. The cellular morphology of trypanosomes is central to their motility, adaptation to the host's environments and pathogenesis. However, visualizing the behaviour of trypanosomes resident in a live vertebrate host has remained unexplored. In this study, we describe an infection model of zebrafish () with . By combining high spatio-temporal resolution microscopy with the transparency of live zebrafish, we describe in detail the swimming behaviour of trypanosomes in blood and tissues of a vertebrate host. Besides the conventional tumbling and directional swimming, can change direction through a 'whip-like' motion or by swimming backward. Further, the posterior end can act as an anchoring site in vivo. To our knowledge, this is the first report of a vertebrate infection model that allows detailed imaging of trypanosome swimming behaviour in vivo in a natural host environment.
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http://dx.doi.org/10.7554/eLife.48388DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6759355PMC
September 2019

Corrigendum: Exposure to Antibiotics Affects Saponin Immersion-Induced Immune Stimulation and Shift in Microbial Composition in Zebrafish Larvae.

Front Microbiol 2019 3;10:1242. Epub 2019 Jun 3.

Cell Biology and Immunology, Animal Sciences Group, Wageningen University and Research, Wageningen, Netherlands.

[This corrects the article DOI: 10.3389/fmicb.2018.02588.].
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http://dx.doi.org/10.3389/fmicb.2019.01242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6557216PMC
June 2019

Evidence of Trained Immunity in a Fish: Conserved Features in Carp Macrophages.

J Immunol 2019 07 24;203(1):216-224. Epub 2019 May 24.

Cell Biology and Immunology Group, Wageningen University & Research, 6708 WD Wageningen, the Netherlands; and

Trained immunity is a form of innate immune memory best described in mice and humans. Clear evidence of the evolutionary conservation of trained immunity in teleost fish is lacking. Given the evolutionary position of teleosts as early vertebrates with a fully developed immune system, we hypothesize that teleost myeloid cells show features of trained immunity common to those observed in mammalian macrophages. These would at least include the ability of fish macrophages to mount heightened responses to a secondary stimulus in a nonspecific manner. We established an in vitro model to study trained immunity in fish by adapting a well-described culture system of head kidney-derived macrophages of common carp. A soluble NOD-specific ligand and a soluble β-glucan were used to train carp macrophages, after which cells were rested for 6 d prior to exposure to a secondary stimulus. Unstimulated trained macrophages displayed evidence of metabolic reprogramming as well as heightened phagocytosis and increased expression of the inflammatory cytokines and Stimulated trained macrophages showed heightened production of reactive oxygen and nitrogen species as compared with the corresponding stimulated but untrained cells. We discuss the value of our findings for future studies on trained immunity in teleost fish.
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http://dx.doi.org/10.4049/jimmunol.1900137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6571502PMC
July 2019

The kinetics of cellular and humoral immune responses of common carp to presporogonic development of the myxozoan Sphaerospora molnari.

Parasit Vectors 2019 May 6;12(1):208. Epub 2019 May 6.

Institute of Parasitology, Biology, Centre of the Czech Academy of Sciences, České Budějovice, Czech Republic.

Background: Sphaerospora molnari is a myxozoan parasite causing skin and gill sphaerosporosis in common carp (Cyprinus carpio) in central Europe. For most myxozoans, little is known about the early development and the expansion of the infection in the fish host, prior to spore formation. A major reason for this lack of information is the absence of laboratory model organisms, whose life-cycle stages are available throughout the year.

Results: We have established a laboratory infection model for early proliferative stages of myxozoans, based on separation and intraperitoneal injection of motile and dividing S. molnari stages isolated from the blood of carp. In the present study we characterize the kinetics of the presporogonic development of S. molnari, while analyzing cellular host responses, cytokine and systemic immunoglobulin expression, over a 63-day period. Our study shows activation of innate immune responses followed by B cell-mediated immune responses. We observed rapid parasite efflux from the peritoneal cavity (< 40 hours), an initial covert infection period with a moderate proinflammatory response for about 1-2 weeks, followed by a period of parasite multiplication in the blood which peaked at 28 days post-infection (dpi) and was associated with a massive lymphocyte response. Our data further revealed a switch to a massive anti-inflammatory response (up to 1456-fold expression of il-10), a strong increase in the expression of IgM transcripts and increased number of IgM B lymphocytes, which produce specific antibodies for the elimination of most of the parasites from the fish at 35 dpi. However, despite the presence of these antibodies, S. molnari invades the liver 42 dpi, where an increase in parasite cell number and indistinguishable outer cell membranes are indicative of effective exploitation and disguise mechanisms. From 49 dpi onwards, the acute infection changes to a chronic one, with low parasite numbers remaining in the fish.

Conclusions: To our knowledge, this is the first time myxozoan early development and immune modulation mechanisms have been analyzed along with innate and adaptive immune responses of its fish host, in a controlled laboratory system. Our study adds important information on host-parasite interaction and co-evolutionary adaptation of early metazoans (Cnidaria) with basic vertebrate (fish) immune systems and the evolution of host adaptation and parasite immune evasion strategies.
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http://dx.doi.org/10.1186/s13071-019-3462-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6501462PMC
May 2019

Studies Into β-Glucan Recognition in Fish Suggests a Key Role for the C-Type Lectin Pathway.

Front Immunol 2019 26;10:280. Epub 2019 Feb 26.

Cell Biology and Immunology Group, Wageningen University & Research, Wageningen, Netherlands.

Immune-modulatory effects of β-glucans are generally considered beneficial to fish health. Despite the frequent application of β-glucans in aquaculture practice, the exact receptors and downstream signalling remains to be described for fish. In mammals, Dectin-1 is a member of the C-type lectin receptor (CLR) family and the best-described receptor for β-glucans. In fish genomes, no clear homologue of Dectin-1 could be identified so far. Yet, in previous studies we could activate carp macrophages with curdlan, considered a Dectin-1-specific β-(1,3)-glucan ligand in mammals. It was therefore proposed that immune-modulatory effects of β-glucan in carp macrophages could be triggered by a member of the CLR family activating the classical CLR signalling pathway, different from Dectin-1. In the current study, we used primary macrophages of common carp to examine immune modulation by β-glucans using transcriptome analysis of RNA isolated 6 h after stimulation with two different β-glucan preparations. Pathway analysis of differentially expressed genes (DEGs) showed that both β-glucans regulate a comparable signalling pathway typical of CLR activation. Carp genome analysis identified 239 genes encoding for proteins with at least one C-type Lectin Domains (CTLD). Narrowing the search for candidate β-glucan receptors, based on the presence of a conserved glucan-binding motif, identified 13 genes encoding a WxH sugar-binding motif in their CTLD. These genes, however, were not expressed in macrophages. Instead, among the β-glucan-stimulated DEGs, a total of six CTLD-encoding genes were significantly regulated, all of which were down-regulated in carp macrophages. Several candidates had a protein architecture similar to Dectin-1, therefore potential conservation of synteny of the mammalian region was investigated by mining the zebrafish genome. Partial conservation of synteny with a region on the zebrafish chromosome 16 highlighted two genes as candidate β-glucan receptor. Altogether, the regulation of a gene expression profile typical of a signalling pathway associated with CLR activation and, the identification of several candidate β-glucan receptors, suggest that immune-modulatory effects of β-glucan in carp macrophages could be a result of signalling mediated by a member of the CLR family.
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http://dx.doi.org/10.3389/fimmu.2019.00280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6400144PMC
September 2020

Paralogs of Common Carp Granulocyte Colony-Stimulating Factor (G-CSF) Have Different Functions Regarding Development, Trafficking and Activation of Neutrophils.

Front Immunol 2019 19;10:255. Epub 2019 Feb 19.

Laboratory of Comparative Immunology, Department of Veterinary Medicine, Nihon University, Fujisawa, Japan.

Mammalian granulocyte colony-stimulating factor (G-CSF; CSF3) is a primary cytokine that promotes the development, mobilization, and activation of neutrophils and their precursors. Teleosts have been reported to possess two paralogs as a likely result of the teleost-wide whole genome duplication (WGD) event, but functional divergence of G-CSF paralogs remains poorly understood. Common carp are an allotetraploid species owing to an additional WGD event in the carp lineage and here, we report on genomic synteny, sequence similarity, and phylogeny of four common carp G-CSF paralogs ( and ; and ). and show differential and relatively high gene expression levels, while and show low basal gene expression levels in most tissues. All paralogs are expressed higher in macrophages than in other leukocyte sub-types and are highly up-regulated by treatment of macrophages with mitogens. Recombinant G-CSFa1 and G-CSFb1 both promoted the proliferation of kidney hematopoietic cells, while only G-CSFb1 induced the differentiation of kidney cells along the neutrophil-lineage. Colony-forming unit assays revealed that G-CSFb1 alone stimulates the formation of CFU-G colonies from head- and trunk-kidney whereas the combination of G-CSFa1 and G-CSFb1 stimulates the formation of both CFU-G and CFU-GM colonies. Recombinant G-CSFa1 and G-CSFb1 also exhibit chemotactic activity against kidney neutrophils and up-regulation of mRNA expression was highest in neutrophils after G-CSFb1 stimulation. Furthermore, G-CSFb1 more than G-CSFa1 induced priming of kidney neutrophils through up-regulation of a NADPH-oxidase component p47 . administration of G-CSF paralogs increased the number of circulating blood neutrophils of carp. Our findings demonstrate that gene duplications in teleosts can lead to functional divergence between paralogs and shed light on the sub-functionalization of G-CSF paralogs in cyprinid fish.
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http://dx.doi.org/10.3389/fimmu.2019.00255DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6389648PMC
October 2020

Exposure to Antibiotics Affects Saponin Immersion-Induced Immune Stimulation and Shift in Microbial Composition in Zebrafish Larvae.

Front Microbiol 2018 29;9:2588. Epub 2018 Oct 29.

Cell Biology and Immunology, Animal Sciences Group, Wageningen University and Research, Wageningen, Netherlands.

In the last decades, pollution of the environment by large scale use of antibiotics in agriculture and human medicine have led to increased antimicrobial resistance in both the environment and the host animal microbiome. Disturbances in the host microbiome can result in impaired immunity and reduced resilience of aquaculture species. Here, we investigated whether environmentally measured levels of the commonly used antibiotics ciprofloxacin and oxytetracycline influences the host microbiome and susceptibility toward saponin-induced immune stimulation in larval zebrafish. Firstly, neutrophil and macrophage reporter zebrafish larvae were exposed to different concentrations of soy saponin by immersion. A dose-dependent increase in neutrophil presence in the intestinal area was observed together with increased expression of immune genes and . To investigate the effect of antibiotics, larval zebrafish were immersed in ciprofloxacin or oxytetracycline in the presence or absence of a low dose of saponin. imaging revealed that antibiotic treatment did not reduce the number of neutrophils that were recruited to the intestinal area upon saponin exposure, although it did tend to lower pro-inflammatory cytokine levels. Microbial sequencing of whole larvae revealed that exposure to a low dose of saponin already shifted the microbial composition. The combination of oxytetracycline and saponin significantly increased α-diversity compared to the controls. In conclusion, the current study provides evidence that the combination of low levels of antibiotics with low levels of anti-nutritional factors (saponin) can induce inflammatory phenotypes and can modify the microbiota, which might lead to altered disease susceptibility.
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http://dx.doi.org/10.3389/fmicb.2018.02588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6215861PMC
October 2018

Pichia pastoris yeast as a vehicle for oral vaccination of larval and adult teleosts.

Fish Shellfish Immunol 2019 Feb 27;85:52-60. Epub 2018 Jul 27.

Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, 08193, Bellaterra, Spain. Electronic address:

Oral vaccination is of major interest because it can be used for mass vaccination of fish of various size and age. Given that their administration is relatively easy and stress-free, oral vaccines have both economic and animal welfare benefits. Yet, mostly due to their limited efficacy, only very few oral vaccines are available to aquaculture industry. Here we present a method for oral vaccine delivery based on the yeast Pichia pastoris. We could express a model antigen, green fluorescent protein (GFP), in this yeast and subsequently show delivery of the GFP protein to the intestine of juvenile flounder or adult carp and trout. We tested this approach in several commercially-relevant fish species, from juvenile to adult stage. To test the oral delivery of antigen to larval fish, the GFP-expressing Pichia pastoris was first fed to planktonic crustacean Daphnia or rotifers that served as 'bioencapsulation vehicles' and afterwards, fed to flounder larvae. Again, we could show delivery of intact GFP protein to the intestine. In rainbow trout, the orally-administered GFP-expressing yeast elicited a rapid local innate immune response in the intestine and a subsequent systemic response in the spleen. Our results show that Pichia pastoris is a good vehicle for oral antigen delivery and that it can be used in non-encapsulated form for older fish or in bioencapsulated form for larval fish. We discuss the immunomodulatory properties of the yeast itself, and its potential to enhance local immune responses and act as an adjuvant.
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http://dx.doi.org/10.1016/j.fsi.2018.07.033DOI Listing
February 2019

Intra-muscular and oral vaccination using a Koi Herpesvirus ORF25 DNA vaccine does not confer protection in common carp (Cyprinus carpio L.).

Fish Shellfish Immunol 2019 Feb 19;85:90-98. Epub 2018 Mar 19.

Cell Biology and Immunology Group, Wageningen University, The Netherlands. Electronic address:

Koi Herpes Virus (KHV or Cyprinid Herpesvirus 3, CyHV-3) is among the most threatening pathogens affecting common carp production as well as the highly valuable ornamental koi carp. To date, no effective commercial vaccine is available for worldwide use. A previous study reported that three intramuscular injections with an ORF25-based DNA vaccine, led to the generation of neutralizing antibodies and conferred significant protection against an intraperitoneal challenge with KHV. In the present study, we set out to optimize an ORF25-based DNA vaccination protocol that required fewer injections and would confer protection upon a challenge that better resembled the natural route of infection. To this end, ORF25 was cloned in pcDNA3 either as a soluble protein or as a full-length transmembrane GFP-fusion protein. We tested our ORF25-based DNA vaccines in multiple vaccination trials using different doses, vaccination routes (i.m. injection and oral gavage) and challenge methods (bath and cohabitation). Furthermore, we analysed local and systemic responses to the i.m. injected DNA vaccine through histological and RT-qPCR analysis. We observed a strong protection when fish received three injections of either of the two DNA vaccines. However, this protection was observed only after bath challenge and not after cohabitation challenge. Furthermore, protection was insufficient when fish received one injection only, or received the plasmid orally. The importance of choosing a challenge model that best reflects the natural route of infection and the possibility to include additional antigens in future DNA vaccination strategies against KHV will be discussed.
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http://dx.doi.org/10.1016/j.fsi.2018.03.037DOI Listing
February 2019

Exploring fish microbial communities to mitigate emerging diseases in aquaculture.

FEMS Microbiol Ecol 2018 01;94(1)

Department of Microbial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Droevendaalsesteeg 10, Wageningen 6708PB, The Netherlands.

Aquaculture is the fastest growing animal food sector worldwide and expected to further increase to feed the growing human population. However, existing and (re-)emerging diseases are hampering fish and shellfish cultivation and yield. For many diseases, vaccination protocols are not in place and the excessive use of antibiotics and other chemicals is of substantial concern. A more sustainable disease control strategy to protect fish and shellfish from (re-)emerging diseases could be achieved by introduction or augmentation of beneficial microbes. To establish and maintain a 'healthy' fish microbiome, a fundamental understanding of the diversity and temporal-spatial dynamics of fish-associated microbial communities and their impact on growth and health of their aquatic hosts is required. This review describes insights in the diversity and functions of the fish bacterial communities elucidated with next-generation sequencing and discusses the potential of the microbes to mitigate (re-)emerging diseases in aquaculture.
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http://dx.doi.org/10.1093/femsec/fix161DOI Listing
January 2018

Intramuscular DNA Vaccination of Juvenile Carp against Spring Viremia of Carp Virus Induces Full Protection and Establishes a Virus-Specific B and T Cell Response.

Front Immunol 2017 24;8:1340. Epub 2017 Oct 24.

Cell Biology and Immunology Group, Department of Animal Sciences, Wageningen University, Wageningen, Netherlands.

Although spring viremia of carp virus (SVCV) can cause high mortalities in common carp, a commercial vaccine is not available for worldwide use. Here, we report a DNA vaccine based on the expression of the SVCV glycoprotein (G) which, when injected in the muscle even at a single low dose of 0.1 µg DNA/g of fish, confers up to 100% protection against a subsequent bath challenge with SVCV. Importantly, to best validate vaccine efficacy, we also optimized a reliable bath challenge model closely mimicking a natural infection, based on a prolonged exposure of carp to SVCV at 15°C. Using this optimized bath challenge, we showed a strong age-dependent susceptibility of carp to SVCV, with high susceptibility at young age (3 months) and a full resistance at 9 months. We visualized local expression of the G protein and associated early inflammatory response by immunohistochemistry and described changes in the gene expression of pro-inflammatory cytokines, chemokines, and antiviral genes in the muscle of vaccinated fish. Adaptive immune responses were investigated by analyzing neutralizing titers against SVCV in the serum of vaccinated fish and the proliferation capacity of peripheral SVCV-specific T cells. We show significantly higher serum neutralizing titers and the presence of SVCV-specific T cells in the blood of vaccinated fish, which proliferated upon stimulation with SVCV. Altogether, this is the first study reporting on a protective DNA vaccine against SVCV in carp and the first to provide a detailed characterization of local innate as well as systemic adaptive immune responses elicited upon DNA vaccination that suggest a role not only of B cells but also of T cells in the protection conferred by the SVCV-G DNA vaccine.
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http://dx.doi.org/10.3389/fimmu.2017.01340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5660689PMC
October 2017

Genomic and transcriptomic approaches to study immunology in cyprinids: What is next?

Dev Comp Immunol 2017 10 28;75:48-62. Epub 2017 Feb 28.

Cell Biology and Immunology Group, Wageningen Institute of Animal Sciences, Wageningen University, PO Box 338, 6700 AH, Wageningen, The Netherlands. Electronic address:

Accelerated by the introduction of Next-Generation Sequencing (NGS), a number of genomes of cyprinid fish species have been drafted, leading to a highly valuable collective resource of comparative genome information on cyprinids (Cyprinidae). In addition, NGS-based transcriptome analyses of different developmental stages, organs, or cell types, increasingly contribute to the understanding of complex physiological processes, including immune responses. Cyprinids are a highly interesting family because they comprise one of the most-diversified families of teleosts and because of their variation in ploidy level, with diploid, triploid, tetraploid, hexaploid and sometimes even octoploid species. The wealth of data obtained from NGS technologies provides both challenges and opportunities for immunological research, which will be discussed here. Correct interpretation of ploidy effects on immune responses requires knowledge of the degree of functional divergence between duplicated genes, which can differ even between closely-related cyprinid fish species. We summarize NGS-based progress in analysing immune responses and discuss the importance of respecting the presence of (multiple) duplicated gene sequences when performing transcriptome analyses for detailed understanding of complex physiological processes. Progressively, advances in NGS technology are providing workable methods to further elucidate the implications of gene duplication events and functional divergence of duplicates genes and proteins involved in immune responses in cyprinids. We conclude with discussing how future applications of NGS technologies and analysis methods could enhance immunological research and understanding.
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http://dx.doi.org/10.1016/j.dci.2017.02.022DOI Listing
October 2017

Transcriptome Sequence of the Bloodstream Form of , a Hematozoic Parasite of Fish Transmitted by Leeches.

Genome Announc 2017 Mar 2;5(9). Epub 2017 Mar 2.

Department of Plant Sciences, University of Oxford, Oxford, United Kingdom

Here, we report a transcriptome sequence of isolated from its natural host, the common carp, The transcriptome allows an analysis of abundant cell surface proteins and acts as a comparator for understanding the evolution and pathogenicity of other , including several that infect humans.
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http://dx.doi.org/10.1128/genomeA.01712-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5334587PMC
March 2017

Conserved Fever Pathways across Vertebrates: A Herpesvirus Expressed Decoy TNF-α Receptor Delays Behavioral Fever in Fish.

Cell Host Microbe 2017 Feb;21(2):244-253

Immunology-Vaccinology, FARAH, Faculty of Veterinary Medicine, University of Liège, Liège 4000, Belgium. Electronic address:

Both endotherms and ectotherms (e.g., fish) increase their body temperature to limit pathogen infection. Ectotherms do so by moving to warmer places, hence the term "behavioral fever." We studied the manifestation of behavioral fever in the common carp infected by cyprinid herpesvirus 3, a native carp pathogen. Carp maintained at 24°C died from the infection, whereas those housed in multi-chamber tanks encompassing a 24°C-32°C gradient migrated transiently to the warmest compartment and survived as a consequence. Behavioral fever manifested only at advanced stages of infection. Consistent with this, expression of CyHV-3 ORF12, encoding a soluble decoy receptor for TNF-α, delayed the manifestation of behavioral fever and promoted CyHV-3 replication in the context of a temperature gradient. Injection of anti-TNF-α neutralizing antibodies suppressed behavioral fever, and decreased fish survival in response to infection. This study provides a unique example of how viruses have evolved to alter host behavior to increase fitness.
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http://dx.doi.org/10.1016/j.chom.2017.01.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5301049PMC
February 2017

Carp Il10a and Il10b exert identical biological activities in vitro, but are differentially regulated in vivo.

Dev Comp Immunol 2017 02 29;67:350-360. Epub 2016 Aug 29.

Cell Biology and Immunology Group, Department of Animal Sciences, Wageningen University, 6708 WD, Wageningen, The Netherlands. Electronic address:

We recently reported on the functional characterization of carp Il10. We showed that carp Il10 is able to downregulate proinflammatory activities by carp phagocytes and promote B cell proliferation, differentiation and antibody production as well as proliferation of memory T cells. Taking advantage of the recent annotation of the carp genome, we completed the sequence of a second il10 paralogue, named il10b, the presence of which was expected owing to the recent (8 million years ago) fourth round of whole genome duplication that occurred in common carp. In the present study we closely compared the two Il10 paralogues and show that Il10a and Il10b have almost identical gene structure, synteny, protein sequence as well as bioactivity on phagocytes. Although the two il10 paralogues show a large overlap in tissue expression, il10b has a low constitutive expression and is highly upregulated upon infection, whereas il10a is higher expressed under basal conditions but its gene expression remains constant during viral and parasitic infections. This differential regulation is most likely due to the observed differences in their promoter regions. Altogether our results demonstrate that gene duplication in carp, although recent, led to sub-functionalization and expression divergence rather than functional redundancy of the Il10 paralogues, yet with very similar protein sequences.
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http://dx.doi.org/10.1016/j.dci.2016.08.016DOI Listing
February 2017

Molecular and functional characterization of Toll-like receptor (Tlr)1 and Tlr2 in common carp (Cyprinus carpio).

Fish Shellfish Immunol 2016 Sep 29;56:70-83. Epub 2016 Jun 29.

Cell Biology and Immunology Group, Department of Animal Sciences, Wageningen University, PO Box 338, 6700 AH, Wageningen, The Netherlands. Electronic address:

Toll-like receptors (TLRs) are fundamental components of innate immunity that play significant roles in the defence against pathogen invasion. In this study, we present the molecular characterization of the full-length coding sequence of tlr1, tlr2a and tlr2b from common carp (Cyprinus carpio). Each is encoded within a single exon and contains a conserved number of leucine-rich repeats, a transmembrane region and an intracellular TIR domain for signalling. Indeed, sequence, phylogenetic and synteny analysis of carp tlr1, tlr2a and tlr2b support that these genes are orthologues of mammalian TLR1 and TLR2. The tlr genes are expressed in various immune organs and cell types. Furthermore, the carp sequences exhibited a good three-dimensional fit with the heterodimer structure of human TLR1-TLR2, including the potential to bind to the ligand Pam3CSK4. This supports the possible formation of carp Tlr1-Tlr2 heterodimers. However, we were unable to demonstrate Tlr1/Tlr2-mediated ligand binding in transfected cell lines through NF-κB activation, despite showing the expression and co-localization of Tlr1 and Tlr2. We discuss possible limitations when studying ligand-specific activation of NF-κB after expression of Tlr1 and/or Tlr2 in human but also fish cell lines and we propose alternative future strategies for studying ligand-binding properties of fish Tlrs.
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http://dx.doi.org/10.1016/j.fsi.2016.06.049DOI Listing
September 2016

Preface to the special issue: Intestinal immunity.

Dev Comp Immunol 2016 11;64

National Creative Research Initiative Center for Hologenomics, Seoul National University, Seoul, South Korea.

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http://dx.doi.org/10.1016/j.dci.2016.06.007DOI Listing
November 2016

Infectious Bronchitis Coronavirus Limits Interferon Production by Inducing a Host Shutoff That Requires Accessory Protein 5b.

J Virol 2016 08 27;90(16):7519-7528. Epub 2016 Jul 27.

Cell Biology and Immunology Group, Wageningen Institute of Animal Sciences, Wageningen University, Wageningen, The Netherlands

Unlabelled: During infection of their host cells, viruses often inhibit the production of host proteins, a process that is referred to as host shutoff. By doing this, viruses limit the production of antiviral proteins and increase production capacity for viral proteins. Coronaviruses from the genera Alphacoronavirus and Betacoronavirus, such as severe acute respiratory syndrome coronavirus (SARS-CoV), establish host shutoff via their nonstructural protein 1 (nsp1). The Gammacoronavirus and Deltacoronavirus genomes, however, do not encode nsp1, and it has been suggested that these viruses do not induce host shutoff. Here, we show that the Gammacoronavirus infectious bronchitis virus (IBV) does induce host shutoff, and we find that its accessory protein 5b is indispensable for this function. Importantly, we found that 5b-null viruses, unlike wild-type viruses, induce production of high concentrations of type I interferon protein in vitro, indicating that host shutoff by IBV plays an important role in antagonizing the host's innate immune response. Altogether, we demonstrate that 5b is a functional equivalent of nsp1, thereby answering the longstanding question of whether lack of nsp1 in gammacoronaviruses is compensated for by another viral protein. As such, our study is a significant step forward in the understanding of coronavirus biology and closes a gap in the understanding of some IBV virulence strategies.

Importance: Many viruses inhibit protein synthesis by their host cell to enhance virus replication and to antagonize antiviral defense mechanisms. This process is referred to as host shutoff. We studied gene expression and protein synthesis in chicken cells infected with the important poultry pathogen infectious bronchitis virus (IBV). We show that IBV inhibits synthesis of host proteins, including that of type I interferon, a key component of the antiviral response. The IBV-induced host shutoff, however, does not require degradation of host RNA. Furthermore, we demonstrate that accessory protein 5b of IBV plays a crucial role in the onset of host shutoff. Our findings suggest that inhibition of host protein synthesis is a common feature of coronaviruses and primarily serves to inhibit the antiviral response of the host.
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http://dx.doi.org/10.1128/JVI.00627-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4984617PMC
August 2016

Long-lived effects of administering β-glucans: Indications for trained immunity in fish.

Dev Comp Immunol 2016 11 2;64:93-102. Epub 2016 Mar 2.

Cell Biology and Immunology Group, Wageningen Institute of Animal Sciences, Wageningen University, PO Box 338, 6700 AH Wageningen, The Netherlands. Electronic address:

Over the past decades, it has become evident that immune-modulation of fish with β-glucans, using injection, dietary or even immersion routes of administration, has stimulating but presumed short-lived effects on both intestinal and systemic immunity and can increase protection against a subsequent pathogenic challenge. Although the exact effects can be variable depending on, among others, fish species and administration route, the immune-stimulating effects of β-glucans on the immune system of fish appear to be universal. This review provides a condensed update of the most recent literature describing the effects of β-glucans on the teleost fish immune system. We shortly discuss possible mechanisms influencing immune-stimulation by β-glucans, including microbial composition of the gut, receptor recognition and downstream signalling. Of interest, in mammalian monocytes, β-glucans are potent inducers of trained immunity. First, we screened the literature for indications of this phenomenon in fish. Criteria that we applied include indications for at least one out of three features considered characteristic of trained immunity; (i) providing protection against a secondary infection in a T- and B-lymphocyte independent manner, (ii) conferring increased resistance upon re-infection and, (iii) relying on key roles for innate immune cell types such as natural killer cells and macrophages. We conclude that several indications exist that support the notion that the innate immune system of teleost fish can be trained. Second, we screened the literature for indications of long-lived effects on innate immunity of fish after administering β-glucans, a criterion which could help to identify key roles for macrophages on resistance to infection. We discuss whether β-glucans, as well-known immune-stimulants, are able to train the immune system of fish and argue in favour of further studies designed to specifically investigate this phenomenon in fish.
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http://dx.doi.org/10.1016/j.dci.2016.03.003DOI Listing
November 2016

Polarization of immune responses in fish: The 'macrophages first' point of view.

Mol Immunol 2016 Jan 21;69:146-56. Epub 2015 Oct 21.

Cell Biology and Immunology Group, Wageningen Institute of Animal Sciences, Wageningen University, P.O. Box 338, 6700 AH Wageningen, The Netherlands.

In this review, we support taking polarized immune responses in teleost fish from a 'macrophage first' point of view, a hypothesis that reverts the dichotomous T helper (TH)1 and TH2 driving forces by building on the idea of conservation of innate immune responses in lower vertebrates. It is plausible that the initial trigger for macrophage polarization into M1 (inflammation) or M2 (healing) could rely only on sensing microbial/parasite infection or other innate danger signals, without the influence of adaptive immunity. Given the long and ongoing debate on the presence/absence of a typical TH1 cytokine environment and, in particular, TH2 cytokine environment in fish immune responses, it stands out that the presence of macrophages with polarized phenotypes, alike M1 and M2, have been relatively easy to demonstrate for fish. We summarize in short present knowledge in teleost fish on those cytokines considered most critical to the dichotomous development of TH1/M1 and TH2/M2 polarization, in particular, but not exclusively, interferon-γ and interleukin (IL)-4/IL-13. We review, in more detail, polarization of fish immune responses taken from the macrophage point of view for which we adopted the simple nomenclature of M1 and M2. We discuss inducible nitric oxide synthase, or NOS-2, as a reliable M1 marker and arginase-2 as a reliable M2 marker for teleost fish and discuss the value of these macrophage markers for the generation of zebrafish reporter lines to study M1/M2 polarization in vivo.
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http://dx.doi.org/10.1016/j.molimm.2015.09.026DOI Listing
January 2016

Infectious Bronchitis Coronavirus Inhibits STAT1 Signaling and Requires Accessory Proteins for Resistance to Type I Interferon Activity.

J Virol 2015 Dec 23;89(23):12047-57. Epub 2015 Sep 23.

Cell Biology and Immunology Group, Wageningen Institute of Animal Sciences, Wageningen University, Wageningen, The Netherlands

Unlabelled: The innate immune response is the first line of defense against viruses, and type I interferon (IFN) is a critical component of this response. Similar to other viruses, the gammacoronavirus infectious bronchitis virus (IBV) has evolved under evolutionary pressure to evade and counteract the IFN response to enable its survival. Previously, we reported that IBV induces a delayed activation of the IFN response. In the present work, we describe the resistance of IBV to IFN and the potential role of accessory proteins herein. We show that IBV is fairly resistant to the antiviral state induced by IFN and identify that viral accessory protein 3a is involved in resistance to IFN, as its absence renders IBV less resistant to IFN treatment. In addition to this, we found that independently of its accessory proteins, IBV inhibits IFN-mediated phosphorylation and translocation of STAT1. In summary, we show that IBV uses multiple strategies to counteract the IFN response.

Importance: In the present study, we show that infectious bronchitis virus (IBV) is resistant to IFN treatment and identify a role for accessory protein 3a in the resistance against the type I IFN response. We also demonstrate that, in a time-dependent manner, IBV effectively interferes with IFN signaling and that its accessory proteins are dispensable for this activity. This study demonstrates that the gammacoronavirus IBV, similar to its mammalian counterparts, has evolved multiple strategies to efficiently counteract the IFN response of its avian host, and it identifies accessory protein 3a as multifaceted antagonist of the avian IFN system.
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http://dx.doi.org/10.1128/JVI.01057-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4645315PMC
December 2015

Cyprinid Herpesvirus 3 Il10 Inhibits Inflammatory Activities of Carp Macrophages and Promotes Proliferation of Igm+ B Cells and Memory T Cells in a Manner Similar to Carp Il10.

J Immunol 2015 Oct 14;195(8):3694-704. Epub 2015 Sep 14.

Cell Biology and Immunology Group, Department of Animal Sciences, Wageningen University, 6708WD Wageningen, the Netherlands; and

Cyprinid herpesvirus 3 (CyHV-3) is the causative agent of a lethal disease of carp and encodes for an Il10 homolog (ORF134). Our previous studies with a recombinant ORF134-deleted strain and the derived revertant strain suggested that cyprinid herpesvirus 3 Il10 (CyHV-3 Il10 [cyhv3Il10]) is not essential for viral replication in vitro, or virulence in vivo. In apparent contrast, cyhv3Il10 is one of the most abundant proteins of the CyHV-3 secretome and is structurally very similar to carp Il10 and also human IL10. To date, studies addressing the biological activity of cyhv3Il10 on cells of its natural host have not been performed. To address the apparent contradiction between the presence of a structurally conserved Il10 homolog in the genome of CyHV-3 and the lack of a clear phenotype in vivo using recombinant cyhv3Il10-deleted viruses, we used an in vitro approach to investigate in detail whether cyhv3Il10 exerts any biological activity on carp cells. In this study, we provide direct evidence that cyhv3Il10 is biologically active and, similarly to carp Il10, signals via a conserved Stat3 pathway modulating immune cells of its natural host, carp. In vitro, cyhv3Il10 deactivates phagocytes with a prominent effect on macrophages, while also promoting proliferation of Igm(+) B cells and memory T cells. Collectively, this study demonstrates a clear biological activity of cyhv3Il10 on cells of its natural host and indicates that cyhv3Il10 is a true viral ortholog of carp Il10. Furthermore, to our knowledge, this is the first report on biological activities of a nonmammalian viral Il10 homolog.
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http://dx.doi.org/10.4049/jimmunol.1500926DOI Listing
October 2015