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

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

CD4 homologue in sea bass (Dicentrarchus labrax): molecular characterization and structural analysis

CD4 is a transmembrane glycoprotein fundamental for cell-mediated immunity. Its action as a T cell coreceptor increases the avidity of association between a T cell and an antigen-presenting cell by interacting with portions of the complex between MHC class II and TR molecules. In this paper we report the cDNA cloning, expression and structural analysis of a CD4 homologue from sea bass (Dicentrarchus labrax). The sea bass CD4 cDNA consists of 2071 bp that translates in one reading frame to give the entire molecule containing 480 amino acids. The analysis of the sequence shows the presence of four putative Ig-like domains and that some fundamental structural features, like a disulphide bond in domain D2 and the CXC signalling motif in the cytoplasmic tail, are conserved from sea bass to mammals. Real-time PCR analysis showed that very high levels of CD4 mRNA transcripts are present in thymus, followed by gut and gills. In vitro stimulation of head kidney leukocytes with LPS and PHA-L gave an increase of CD4 mRNA levels after 4 h and a decrease after 24 h. Homology modelling has been applied to create a 3D model of sea bass CD4 and to investigate its interaction with sea bass MHC-II. The analysis of the 3D complex between sea bass CD4 and sea bass MHC-II suggests that the absence of a disulfide bond in the CD4 D1 domain could make this molecule more flexible, inducing a different conformation and affecting the binding and the way of interaction between CD4 and MHC-II. Our results will add new insights into the sea bass T cell immune responses and will help in the identification of T cell subsets in teleost fishes to better understand the evolution of cell-mediated immunity from fish to mammals.L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com