Identification and functional characterization of fish IL-17 receptors suggest important roles in the response to nodavirus infection.

Th17 is a lymphocyte T helper (Th) subpopulation relevant in the control and regulation of the immune response characterized by the production of interleukin (IL)-17. This crucial cytokine family acts through their binding to the IL-17 receptors (IL-17R), having up to six members. Although the biology of fish Th17 is well-recognized, the molecular and functional characterization of IL-17 and IL-17R has been limited. Thus, our aim was to identify and characterize the IL-17R repertory and regulation in the two main Mediterranean cultured fish species, the gilthead seabream (Sparus aurata) and the European sea bass (Dicentrarchus labrax). Our in silico results showed the clear identification of six members in each fish species, from IL-17RA to IL-17RE-like, with well-conserved gene structure and protein domains with their human orthologues. All of them showed wide and constitutive transcription in naïve tissues but with highest levels in mucosal tissues, namely skin, gill or intestine. In leucocytes, T mitogens showed the strongest up-regulation in most of the il17 receptors though il17ra resulted in inhibition by most stimulants. Interestingly, in vivo nodavirus infection resulted in alterations on the transcription of il17 receptors. While nodavirus infection led to some increments in the il17ra, il17rb, il17rc and il17rd transcripts in the susceptible European sea bass, many down-regulations were observed in the resistant gilthead seabream. Our data identify the presence and conservation of six coding IL-17R in gilthead seabream and European sea bass as well as their differential regulation in vitro and upon nodavirus infection. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-024-00225-1.

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

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.

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

Cell-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.