Analysis of the long-lived responses induced by immunostimulants and their effects on a viral infection in zebrafish (Danio rerio). Analysis of the long-lived responses induced by immunostimulants and their effects on a viral infection in zebrafish (Danio rerio)

In the last years, the innate immune response has gained importance since evidences indicate that, after an adequate priming protocol, it is possible to obtain some prolonged and enhanced immune response. Nevertheless, several factors, such as timing and method of administration of the immunostimulants need to be carefully considered. An inappropriate protocol can transform the treatments into a double-edged sword for the teleost immune system, resulting in a stressful and immunosuppressive status. In this work, we analyzed the long-term effect of different stimuli (β-glucans, lipopolysaccharide and Polyinosinic:polycytidylic acid) on the transcriptome modulation induced by Spring Viraemia Carp Virus (SVCV) in adult zebrafish (Danio rerio) and on the mortalities caused by this infection. At 35 days post-immunostimulation the transcriptome was found to be highly altered compared to the control fish, and these stimuli also conditioned the response to SVCV challenge, especially in the case of β-glucans. No protection against SVCV was found with any of the stimuli and even non-significant higher mortalities were observed, especially with β-glucans. However, at short-term (a pre-stimulation with β-glucan and infection after 7 days) a slight protection was observed after infection. The transcriptome response in zebrafish kidney at 35 days post-treatment with β-glucans revealed a significant response associated to stress and immunosuppression. The identification of genes differentially expressed before and after the infection seem to indicate a high energy cost of the immunostimulation prolonged in time that could explain the lack of protection against the SVCV. The differential response to stress, alterations in the lipid metabolism, the tryptophan-kynurenine pathway and the interferon-gamma signaling seem to be some of the mechanisms involved in this particular response, which means the end of the trained immunity and the beginning of a stressful status characterized by immunosuppression. Overall design: Four groups composed of 92 adult zebrafish/each were intraperitoneally (i.p.) injected with 20 µl of one of the following treatments: β-glucans (1 mg/ml), LPS (0.75 mg/ml), Poly(I:C) (1 mg/ml) and the control group with phosphate-buffered saline (PBS). After 35 days, half of the individuals were i.p. infected with 20 µl of a SVCV suspension (3 × 102 TDCI50/ml) and the remaining fish, which served as uninfected control, were inoculated with viral medium (MEM + 2% FBS + Primocin). For microarray hybridization, a total of 16 fish from each treatment (β-glucans-control, LPS-control, Poly(I:C)-control, β-glucans-SVCV, LPS-SVCV and Poly(I:C)-SVCV) were sacrificed at 24h post-infection and the kidney was removed, obtaining 4 pooled biological replicates (four fish/replicate).