Gene expression analyses of immune responses in Atlantic salmon during early stages of infection by salmon louse (Lepeophtheirus salmonis) revealed bi-phasic responses coinciding with the copepod-chalimus transition

The salmon louse (Lepeophtheirus salmonis Krøyer), an ectoparasitic copepod with a complex life cycle causes significant losses in salmon aquaculture. Pesticide treatments against the parasite raise environmental concerns and their efficacy is gradually decreasing. Improvement of fish resistance to lice, through biological control methods, needs better understanding of the protective mechanisms. We used a 21 k oligonucleotide microarray and RT-qPCR to examine the time-course of immune gene expression changes in salmon skin, spleen, and head kidney during the first 15 days after challenge, which encompassed the copepod and chalimus stages of lice development. Results Large scale and highly complex transcriptome responses were found already one day after infection (dpi). Many genes showed bi-phasic expression profiles with abrupt changes between 5 and 10 dpi (the copepod-chalimus transitions); the greatest fluctuations (up- and down-regulation) were seen in a large group of secretory splenic proteases with unknown roles. Rapid sensing was witnessed with induction of genes involved in innate immunity including lectins and enzymes of eicosanoid metabolism in skin and acute phase proteins in spleen. Transient (1-5 dpi) increase of T-cell receptor alpha, CD4-1, and possible regulators of lymphocyte differentiation suggested recruitment of T-cells of unidentified lineage to the skin. After 5 dpi the magnitude of transcriptomic responses decreased markedly in skin. Up-regulation of matrix metalloproteinases in all studied organs suggested establishment of a chronic inflammatory status. Up-regulation of putative lymphocyte G0/G1 switch proteins in spleen at 5 dpi, immunoglobulins at 15 dpi; and increase of IgM and IgT transcripts in skin indicated an onset of adaptive humoral immune responses, whereas MHCI appeared to be down-regulated. Conclusions Atlantic salmon develops rapid local and systemic reactions to L. salmonis, which, however, do not result in substantial level of protection. The dramatic changes observed after 5 dpi can be associated with metamorphosis of copepod, immune modulation by the parasite, or transition from innate to adaptive immune responses

Adaptive immune responses at mucosal surfaces of teleost fish

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Differential Modulation of igT and igM upon Parasitic, Bacterial, Viral, and Dietary challenges in a Perciform Fish

16 páginas, 8 figuras, 2 tablas.-- This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these termsThree different immunoglobulin (Ig) isotypes can be found in teleost fish, IgM, IgD, and the teleost-specific IgT. IgM is considered to have a systemic activity, and IgT is attributed a mucosal role, similar to mammalian IgA. In this study, the complete sequence of gilthead sea bream IgM and IgT in their membrane (m) and soluble (s) forms are described for the first time in a perciform fish. Their constitutive gene expression is analyzed in different tissues, and their regulation upon viral, bacterial, parasitic, mucosal vaccination and dietary challenges are studied. GCB IgM and IgT have the prototypical structure when compared to other fish Igs. The constitutive expression of sIgM was the highest overall in all tissues, whereas mIgT expression was highest in mucosal tissues, such as gills and intestine. IgM and IgT were differentially regulated upon infection. IgT was highly upregulated locally upon infection with the intestinal parasite Enteromyxum leei or systemically after Nodavirus infection. Long-term intestinal parasitic infections increased the serum titer of both isotypes. Mucosal vaccination against Photobacterium damselae subsp. piscicida finely regulated the Ig response inducing a systemic increase of IgM titers in serum and a local IgT response in skin mucus when animals were exposed to the pathogen by bath challenge. Interestingly, plant-based diets inhibit IgT upregulation upon intestinal parasitic challenge, which was related to a worse disease outcome. All these results corroborate the mucosal role of IgT and emphasize the importance of a finely tuned regulation of Ig isotypes upon infection, which could be of special interest in vaccination studiesThis work has been carried out with financial support from by the Spanish MINECO under projects AGL2013-48560-R to JP-S and AS-B and AGL2014-51773-C3-3-R to EG-C. Additional funding was provided by the European Union, through the Horizon 2020 research and innovation program under grant agreement 634429 (ParaFishControl) and through the 7th Framework Programme for Research and Technological Development (FP7) under grant 311993 (TARGETFISH). Additional support was provided by Generalitat Valenciana (PROMETEOII/2014/085). MCP was supported by the Spanish grant Formación Postdoctoral 2013 (FPDI-2013-15741). We acknowledge support of the publication fee by the CSIC Open Access Support Initiative through its Unit of Information Resources for Research (URICI).Peer reviewe

Molecular characterisation of key components of the mucosal immune system in Atlantic salmon (Salmo salar L) and transcriptome analysis of responses against the salmon louse (Lepeophtheirus salmonis)

Mucosal immunity in mammals is mediated mainly by secretory immunoglobulin A (SIgA), which is produced by IgA plasma cells commonly located in the lamina propria, and a transport system involving the polymeric Ig receptor (pIgR). In teleost fish, IgM plays some roles associated with mucosal defence. Very recent findings indicate that IgT, an antibody exclusive to teleosts, might have a special role in mucosal immune responses, and a possible pIgR counterpart has been identified. The salmon louse (Lepeophtheirus salmonis Krøyer), an ectoparasitic copepod targeting the skin (and the gill to a lesser extent), has been a major challenge to the aquaculture industry. While the first line of defence against this parasite is crucial, equally important, in the context of vaccine development is the generation of information on the adaptive immune system. Based on this line of reasoning, Atlantic salmon IgT and pIgR were selected as targets for further characterization in the present study. Three distinct IgT heavy chain () sub-variants, with an identity index of 76-80%, were described. The identity index between 1 and 1 (the first constant domains of the IgT and IgM heavy chains, respectively) in Atlantic salmon is 52%. It is plausible to assume that this relatively high similarity is a result of interactions with common light chains. The relative abundance of and transcripts in a series of tissues revealed an overall expression pattern of IgM >> IgT > IgD. Interestingly, challenge experiments with salmon louse showed 10 fold increase of IgM and IgT mRNA in skin samples, supporting the assumption that these antibodies are involved in mucosal immune responses. The search for pIgR homologues in Atlantic salmon resulted in two pIgR-like candidates: Salsal pIgR and Salsal pIgRL. Meanwhile, a comparative evaluation was made to a series of CD300-like molecules (CMRF-35 like molecules, CLM) reported to the databanks. Salsal pIgR and Salsal pIgRL were identified on the basis of similarity to homologous genes, and like the counterparts in other teleosts they are composed of two Ig superfamily (IgSF) V-like domains, a transmembrane region, a connecting peptide, and a cytoplasmic tail. Two CD300-like molecules in salmon (CLM1 and CLM7) also have the same domain structure, but their cytoplasmic region is predicted to contain putative immunoreceptor tyrosine based inhibition motifs (ITIM), which is a typical feature of CD300A and CD300F in humans. While Salsal pIgR and Salsal pIgRL were expressed in tissues of skin and gill respectively, their expression pattern is not restricted to mucosal tissues, but notably, their expression increased during infection with salmon louse. Further studies are needed to elucidate the transport mechanisms of mucosal antibodies in salmon. To investigate how the salmon louse evades the immune system of the host, temporal immune gene expression changes in skin, spleen, and head kidney of Atlantic salmon were analysed using microarray and quantitative real-time PCR (qPCR) during the first 15 days post infection (dpi). This window represents the copepodid and chalimus stages of lice development. Transcriptomic responses, recorded already at 1 dpi, were highly complex and large by scale. Many genes showed bi-phasic expression profiles with abrupt changes taking place between 5 and 10 dpi (the copepodid-chalimus transitions). Large group of secretory splenic proteases with unknown roles showed the greatest fluctuations: up-regulated 1-5 dpi and markedly down regulated afterwards. T cell related transcripts showed a short term (1-5 dpi) increase. After 5 dpi, the magnitude of transcriptomic responses decreased markedly in skin. The findings provided an insight into the time windows in the development of the parasite which are critical to the host and where modulation of the host immune system might occur

Gene expression analyses of immune responses in Atlantic salmon during early stages of infection by salmon louse (<it>Lepeophtheirus salmonis</it>) revealed bi-phasic responses coinciding with the copepod-chalimus transition

Abstract Background The salmon louse (Lepeophtheirus salmonis Krøyer), an ectoparasitic copepod with a complex life cycle causes significant losses in salmon aquaculture. Pesticide treatments against the parasite raise environmental concerns and their efficacy is gradually decreasing. Improvement of fish resistance to lice, through biological control methods, needs better understanding of the protective mechanisms. We used a 21 k oligonucleotide microarray and RT-qPCR to examine the time-course of immune gene expression changes in salmon skin, spleen, and head kidney during the first 15 days after challenge, which encompassed the copepod and chalimus stages of lice development. Results Large scale and highly complex transcriptome responses were found already one day after infection (dpi). Many genes showed bi-phasic expression profiles with abrupt changes between 5 and 10 dpi (the copepod-chalimus transitions); the greatest fluctuations (up- and down-regulation) were seen in a large group of secretory splenic proteases with unknown roles. Rapid sensing was witnessed with induction of genes involved in innate immunity including lectins and enzymes of eicosanoid metabolism in skin and acute phase proteins in spleen. Transient (1-5 dpi) increase of T-cell receptor alpha, CD4-1, and possible regulators of lymphocyte differentiation suggested recruitment of T-cells of unidentified lineage to the skin. After 5 dpi the magnitude of transcriptomic responses decreased markedly in skin. Up-regulation of matrix metalloproteinases in all studied organs suggested establishment of a chronic inflammatory status. Up-regulation of putative lymphocyte G0/G1 switch proteins in spleen at 5 dpi, immunoglobulins at 15 dpi; and increase of IgM and IgT transcripts in skin indicated an onset of adaptive humoral immune responses, whereas MHCI appeared to be down-regulated. Conclusions Atlantic salmon develops rapid local and systemic reactions to L. salmonis, which, however, do not result in substantial level of protection. The dramatic changes observed after 5 dpi can be associated with metamorphosis of copepod, immune modulation by the parasite, or transition from innate to adaptive immune responses.</p

Characterization of Small, Mononuclear Blood Cells from Salmon Having High Phagocytic Capacity and Ability to Differentiate into Dendritic like Cells

Phagocytes are the principal component of the innate immune system, playing a key role in the clearance of foreign particles that include potential pathogens. In vertebrates, both neutrophils and mononuclear cells like monocytes, macrophages and dendritic cells are all professional phagocytes. In teleosts, B-lymphocytes also have potent phagocytic ability. We have isolated a population of small (<5 µm), mononuclear blood cells from Atlantic salmon (Salmo salar L.) not previously characterized. In order to identify them, we have performed morphological, gene expression, flow cytometry, cytochemical, ultrastructural and functional analyses. Interestingly, they highly express the gene encoding CD83, the most characteristic cell surface marker for dendritic cells in mammals, and MHC class II limited to professional antigen presenting cells. They did not express genes nor did they have cell markers for B-cells, T-cells, monocytes/macrophages or neutrophils as shown by qRT-PCR, flow cytometry and immunoblotting. A remarkable feature of these cells is their potent phagocytic capacity. Their oxygen-independent killing mechanism, as shown by intense acid phosphatase staining, is supported by lack of respiratory burst and myeloperoxidase activity and the acid phosphatase's sensitivity to tartrate. They show a high level of morphological plasticity, as, upon stimulation with mitogens, they change morphology and obtain branching protrusions similarly to dendritic cells. We suggest, based on our findings, that the small, round cells described here are progenitor cells with potential to differentiate into dendritic like cells, although we can not exclude the possibility that they represent a novel cell type