Analysis of the expression and modulation of selected immune-related gene transcripts in the DLEC cell line from European sea bass (Dicentrarchus labrax)

Cell lines have been established from different fish species especially for virus isolation and for studying cell-pathogen interactions, and therefore are of interest in aquaculture. In this paper, we have investigated the presence and the regulation of some immune genes in the DLEC (Dicentrarchus labrax embryonic cells) cell line from European sea bass (Dicentrarchus labrax L.) to preliminary elucidate their action. The basal expression of the selected genes (interleukin- 1β (IL-1β), cyclooxygenase-2 (COX-2), transforming growth factor-β (TGF-β), CD8-α, major histocompatibility complex II-β (MHC II-β), interferon (IFN) and Mx protein (Mx)) have been investigated and, successively, their modulation have been studied both after stimulation with different mitogen agents and after a transfection with a sequence codifying for the coat protein of a fish nervous necrosis virus (NNV). The results have evidenced that the inflammatory molecules (IL-1β, COX-2, TGF-β), constitutively expressed by the DLEC cell line, are not up-regulated by the stimulation with lipopolysaccharide (LPS) from E. coli, whether the expression of the T-cell marker transcripts (CD8-α, MHC II-β) is influenced by the action of a lectin from Phaseolus vulgaris (PHA-L). Finally, the expression of the coat NNV protein in the DLEC cell line, after the transfection, led to an high up-regulation of IFN and Mx gene transcripts. These data suggest that the DLEC cell line recognize specific pathogen-associated molecular patterns (PAMPs) and, therefore, could be useful for studying T-cell pathways and viral responses in sea bass avoiding the use of live test animals

Analysis of the expression and modulation of selected immune-related gene transcripts in the DLEC cell line from European sea bass (Dicentrarchus labrax)

Cell lines have been established from different fish species especially for virus isolation and for studying cell-pathogen interactions, and therefore are of interest in aquaculture. In this paper, we have investigated the presence and the regulation of some immune genes in the DLEC (Dicentrarchus labrax embryonic cells) cell line from European sea bass (Dicentrarchus labrax L.) to preliminary elucidate their action. The basal expression of the selected genes (interleukin- 1β (IL-1β), cyclooxygenase-2 (COX-2), transforming growth factor-β (TGF-β), CD8-α, major histocompatibility complex II-β (MHC II-β), interferon (IFN) and Mx protein (Mx)) have been investigated and, successively, their modulation have been studied both after stimulation with different mitogen agents and after a transfection with a sequence codifying for the coat protein of a fish nervous necrosis virus (NNV). The results have evidenced that the inflammatory molecules (IL-1β, COX-2, TGF-β), constitutively expressed by the DLEC cell line, are not up-regulated by the stimulation with lipopolysaccharide (LPS) from E. coli, whether the expression of the T-cell marker transcripts (CD8-α, MHC II-β) is influenced by the action of a lectin from Phaseolus vulgaris (PHA-L). Finally, the expression of the coat NNV protein in the DLEC cell line, after the transfection, led to an high up-regulation of IFN and Mx gene transcripts. These data suggest that the DLEC cell line recognize specific pathogen-associated molecular patterns (PAMPs) and, therefore, could be useful for studying T-cell pathways and viral responses in sea bass avoiding the use of live test animals

Transcriptional profile and serological studies of the European sea bass immune response against betanodavirus infections

European sea bass is highly affected by outbreaks of viral nervous necrosis disease, provoked by the nervous necrosis virus (NNV). This virus displays a positive-sense RNA genome composed of two segments, RNA1, encoding the viral polymerase; and RNA2, encoding the capsid protein. Two genotypes of NNV have been detected in sea bass, although showing different virulence: RGNNV is highly virulent, whereas SJNNV replicates in sea bass brain without causing clinical signs. In the present work, the comparative analysis of the European sea bass immune response against both genotypes has been performed. The immune response has been evaluated in brain and head kidney of infected sea bass by relative real-time PCR of genes involved in the type I interferon system, and genes related to inflammatory and adaptive responses. In addition, a serological study, consisting of the ELISA quantification of IgM in sera, was also performed. The transcription analyses point out the importance of IFN system to control betanodavirus infections. The results obtained showed a strong induction of ifn-I, mxA, isg15 and isg12 in both organs analysed, especially in response to RGNNV. However, the response was quicker in head kidney of SJNNV inoculated sea bass, suggesting that this genotype induces a more rapid systemic response. Regarding the inflammatory response, RGNNV triggered a strong transcription of proinflammatory genes in brain, which provides evidences about the importance of the inflammatory process in NNV infection. Thus, the massive inflammatory process may be responsible for the eventual damage in nervous tissues, which would lead to fish dead. Finally, the high values of tr-g and mhc-b mRNA recorded in brain and the high IgM titer in sera, which was higher in SJNNV-inoculated fish, suggest that the adaptive response constitutes another important factor in the sea bass immune response against betanodaviruses, both at systemic and at local level.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. This study has been supported by the project AGL2017-84644-R (MINECO/ AEI/FEDER, UE). P. Moreno was supported by a fellowship from Ministerio de Educacio n, Cultura y Deporte (FPU12/00265, Spanish Government)

Molecular cloning, differential expression and 3D structural analysis of the MHC class-II β chain from sea bass (Dicentrarchus labrax L.)

The major histocompatibility complex class I and II molecules (MHC-I and MHC-II) play a pivotal role in vertebrate immune response to antigenic peptides. In this paper we report the cloning and sequencing of the MHC class II b chain from sea bass (Dicentrarchus labrax L.). The six obtained cDNA sequences (designated as Dila-DAB) code for 250 amino acids, with a predicted 21 amino acid signal peptide and contain a 28 bp 50-UTR and a 478 bp 30-UTR. A multiple alignment of the predicted translation of the Dila-DAB sequences was assembled together with other fish and mammalian sequences and it showed the conservation of most amino acid residues characteristic of the MHC class II b chain structure. The highest basal Dila-DAB expression was found in gills, followed by gut and thymus, lower mRNA levels were found in spleen, peripheral blood leucocytes (PBL) and liver. Stimulation of head kidney leukocytes with LPS for 4 h showed very little difference in the Dila-DAB expression, but after 24 h the Dila-DAB level decreased to a large extent and the difference was statistically significant. Stimulation of head kidney leukocytes with different concentrations of rIL-1b (ranging from 0 to 100 ng/ml) resulted in a dose-dependent reduction of the Dila-DAB expression. Moreover, two 3D Dila-DAB*0101 homology models were obtained based on crystallographic mouse MHC-II structures complexed with D10 T-cell antigen receptor or human CD4; features and differences between the models were evaluated and discussed. Taken together these results are of interest as MHC-II structure and function, molecular polymorphism and differential gene expression are in correlation with disease resistance to virus and bacteria in teleost fish.L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com/This work was supported by the European Commission within the project IMAQUANIM (EC contract number FOOD-CT-2005-007103)

Early treatment with Lactobacillus delbrueckii strain induces rise in intestinal T cells and granulocytes and modulates immune related genes of larval Dicentrarchus labrax (L.)

Lactobacillus delbrueckii ssp. delbrueckii (AS13B), isolated from the gut of adult Dicentrarchus labrax, was administered live to developing sea bass using rotifers and Artemia as live carriers. Immune-related gene transcripts were quantified in post-larvae at day 70 post-hatch (ph) and histology, electron microscopy and immunocytochemistry of the intestinal tissue were performed at day 74 ph. Since the probiotic was orally administered the studies were focused on intestinal immunity. In treated fish gut integrity was unaffected, while the density of T-cells and acidophilic granulocytes in the intestinal mucosa was significantly higher than in controls. Probiotic-induced increases in intestinal T-cells and total body TcR-beta transcripts are first reported in fish. Significantly lower IL-1beta transcripts and a trend towards lower IL-10, Cox-2 and TGF-beta transcription were found in the treated group. Evidence is provided that early feeding with probiotic-supplemented diet stimulated the larval gut immune system and lowered transcription of key pro-inflammatory genes.L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com