Cytotoxic activity and gene expression during in vitro adaptive cell-mediated cytotoxicity of head-kidney cells from betanodavirus-infected European sea bass

© 2023. The authors. This document is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc /4.0/ This document is the accepted version of a published work that appeared in final form in Developmental and Comparative ImmunologyCell-mediated cytotoxicity (CMC) is essential in eradicating virus-infected cells, involving CD8+ T lymphocytes (CTLs) and natural killer (NK) cells, through the activation of different pathways. This immune response is well-studied in mammals but scarcely in teleost fish. Our aim was to investigate the adaptive CMC using head-kidney (HK) cells from European sea bass infected at different times with nodavirus (NNV), as effector cells, and the European sea bass brain cell line (DLB-1) infected with different NNV genotypes, as target cells. Results showed low and unaltered innate cytotoxic activity through the infection time. However, adaptive CMC against RGNNV and SJNNV/RGNNV-infected target cells increased from 7 to 30 days post-infection, peaking at 15 days, demonstrating the specificity of the cytotoxic activity and suggesting the involvement of CTLs. At transcriptomic level, we observed up-regulation of genes related to T cell activation, perforin/granzyme and Fas/FasL effector pathways as well as apoptotic cell death. Further studies are necessary to understand the adaptive role of European sea bass CTLs in the elimination of NNV-infected cells

Regulation and distribution of European sea bass perforins point to their role in the adaptive cytotoxic response against NNV

© 2023. The authors. This document is made available under the CC-BY 4.0 license http://creativecommons.org/licenses/by /4.0/ This document is the accepted version of a published work that appeared in final form in Fish and Shellfish Immunology.Cell-mediated cytotoxicity is a complex immune mechanism that involves the release of several killing molecules, being perforin (PRF) one of the most important effector players. Perforin is synthesized by T lymphocytes and natural killer cells in mammals and responsible for the formation of pores on the target cell membrane during the killing process. Although perforin has been extensively studied in higher vertebrates, this knowledge is very limited in fish. Therefore, in this study we have identified four prf genes in European sea bass (Dicentrarchus labrax) and evaluated their mRNA levels. All sea bass prf genes showed the typical and conserved domains of its human orthologue and were closely clustered by the phylogenetic analysis. In addition, all genes showed constitutive and ubiquitous tissular expression, being prf1.9 gene the most highly expressed in immune tissues. Subsequently, in vitro stimulation of head-kidney (HK) cells with phytohemagglutinin, a T-cell activator, showed an increase of all prf gene levels, except for prf1.3 gene. European sea bass HK cells increased the transcription of prf1.2 and prf1.9 during the innate cell-mediated cytotoxic activity against xenogeneic target cells. In addition, sea bass infected with nodavirus (NNV) showed a similar expression pattern of all prf in HK and brain at 15 days post-infection, except for prf1.3 gene and in the gonad. Finally, the use of a polyclonal antibody against PRF1.9 showed an increase of positive cells in HK, brain and gonad from NNV-infected fish. Taken together, the data seem to indicate that all prf genes, except prf1.3, appear to be involved in the European sea bass immunity, and probably in the cell-mediated cytotoxic response, with PRF1.9 playing the most important role against nodavirus. The involvement of the PRFs and the CMC activity in the vertical transmission success of the virus is also discussed

Hepcidin and dicentracin peptides show preventive antiviral applications against NNV infection in European sea bass through immunomodulatory roles

© 2024. The authors. This document is made available under the CC-BY-NC 4.0 license http://creativecommons.org/licenses/by-nc /4.0/ This document is the published version of a published work that appeared in final form in Aquaculture.Aquaculture is an extremely prosperous market threatened by pathogen outbreaks, including viruses as nodavirus (NNV), which infect fish species with special interest in trading such as European sea bass. Antimicrobial peptides (AMPs) might constitute potential antiviral agents, which had been previously evaluated in fish with positive prospects, based on their properties as immunomodulators or directly killing pathogens. In this line, we aimed to evaluate this dual role by administering two European sea bass synthetic AMPs (Hamp or Dic) prior to NNV infection. Both treatments conferred partial protection against NNV though viral replication and load were not affected. Both AMPs elicited, prior to infection, AMP response and leukocyte mobilization whilst downregulated pro-inflammatory markers. Upon infection, Hamp and Dic peptides abrogated the inflammatory response provoked by NNV as well as avoid NNV-induced disturbance of the leucocyte distribution in the brain, mainly neutrophils, macrophages and CD8+ T cells. This study points that preventive applications of synthetic Hamp and Dic peptides exert their antiviral actions through the immunomodulatory role and not by a direct action of the antimicrobial on NNV. This work opens the door to the use of AMPs as potential prophylactic tools against NNV as well as immunostimulant in fish farm

Molecular characterization of the T cell costimulatory receptors 1 CD28 and CTLA4 in the European sea bass

For the activation of T cells, it is necessary the specific recognition of the peptide by the T cell receptors (TCR) in the surface of antigen-presenting cells (APCs) and additional signals delivered by costimulatory receptors. In fish, knowledge about the presence of these costimulatory signals is limited and functional evidence almost absent. Thus, in this study, we have identified the stimulatory CD28 and the inhibitory cytotoxic T-lymphocyte-associated protein 4 (CTLA4) coreceptors in the European sea bass (Dicentrarchus labrax), and evaluated their transcription. In parallel, the transcription encoding for the T cell markers CD8 and CD4 was also evaluated. Both coreceptors showed the canonical architecture including a signal peptide, an immunoglobulin domain, a transmembrane region and a cytosolic tail. Protein predictions and phylogenetic tree identify them as true mammalian orthologues of CD28 and CTLA4. We found these genes constitutively expressed in all studied organs of European sea bass with high expression in lymphoid organs (thymus, spleen and head-kidney) and liver. The molecular expression pattern of these genes was up-regulated in head-kidney leucocytes stimulated with T mitogens as concanavalin A and phytohemagglutinin (PHA), but not with the B cell mitogen lipopolysaccharide (LPS). Fish challenged with nodavirus (NNV) evidenced a differential and opposing regulation of the cd28 and ctla4 transcription levels in the brain, the target organ for viral replication, and head-kidney. While cd28 transcription tends to decrease over the infection time in both organs the expression of the ctla4 gene tends to increase. Interestingly, the coreceptor expression is highly and significantly correlated to the transcription of the T cell markers. Our results highlight the important role of CD28 and CTLA4 as costimulatory receptors of T cells in European sea bass but further studies are deserved