Zebra Fish Lacking Adaptive Immunity Acquire an Antiviral Alert State Characterized by Upregulated Gene Expression of Apoptosis, Multigene Families, and Interferon-Related Genes

Abstract

To investigate fish innate immunity, we have conducted organ and cell immune-related transcriptomic as well as immunohistologic analysis in mutant zebra fish (Danio rerio) lacking adaptive immunity (rag1^-/-) at different developmental stages (egg, larvae, and adult), before and after infection with spring viremia carp virus (SVCV). The results revealed that, compared to immunocompetent zebra fish (rag1^+/+ ), rag1^-/- acquired increased resistance to SVCV with age, correlating with elevated transcript levels of immune genes in skin/fins and lymphoid organs (head kidney and spleen). Gene sets corresponding to apoptotic functions, immune-related multigene families, and interferon-related genes were constitutively upregulated in uninfected adult rag1^-/- zebra fish. Overexpression of activated CASPASE-3 in different tissues before and after infection with SVCV further confirmed increased apoptotic function in rag1^-/- zebra fish. Concurrently, staining of different tissue samples with a pan-leukocyte antibody marker showed abundant leukocyte infiltrations in SVCV-infected rag1^-/- fish, coinciding with increased transcript expression of genes related to NK-cells and macrophages, suggesting that these genes played a key role in the enhanced immune response of rag1^-/- zebra fish to SVCV lethal infection. Overall, we present evidence that indicates that rag1^-/- zebra fish acquire an antiviral alert state while they reach adulthood in the absence of adaptive immunity. This antiviral state was characterized by (i) a more rapid response to viral infection, which resulted in increased survival, (ii) the involvement of NK-cell- and macrophage-mediated transcript responses rather than B- and/or T-cell dependent cells, and (iii) enhanced apoptosis, described here for the first time, as well as the similar modulation of multigene family/interferon-related genes previously associated to fish that survived lethal viral infections. From this and other studies, it might be concluded that some of the characteristics of mammalian trained immunity are present in lower vertebrates.

Keywords: antiviral alert state; multigene families and apoptosis in resistance to viral infections; spring viremia carp viral infections; trained immunity NK/macrophages in fish; zebra fish rag1−/− adaptive deficient mutants.

Figure 1

Selected gene sets (sGS) were downregulated in rag1^−/− egg embryos but were similar in early hatched larvae and both showed high mortalities when infected by spring viremia carp virus (SVCV). (A) Expression folds in embryo eggs 1 day after fertilization (n = 60). Reverse transcriptase and quantitative polymerase chain reaction data were first normalized by the formula, expression of each gene/expression of ef1a. Differential folds were then calculated by the formula, normalized expression of each rag1^−/− gene/normalized expression of each rag1^+/+ gene. Tbk1 fold was lower than 2⁻⁶ (not shown). Tnfa and nklysin were not done. Red dashed line, onefold boundary. *Statistically different from onefold with p < 0.05 by the t-test. (B) Expression folds in hatched larvae 3 days after fertilization (n = 45) calculated as in (A). (C) Kaplan–Meier survival curves of naïve rag1^−/− (n = 90) and rag^+/+ (n = 90) recently hatched larvae after bath infection in 10⁴ pfu of SVCV per ml (n = 2) at 22°C (optimal replication for SVCV). Differences between rag1^−/− and rag^+/+ survival curves of SVCV-infected larvae as determined by the Gehan–Breslow–Wilcoxon test were significant with p < 0.05 (*). Solid lines, rag1^−/− larvae. Dashed lines, rag^+/+ larvae.

Figure 2

Naïve adult rag1^−/− zebra fish are more resistant to lethal spring viremia carp virus (SVCV) infection than rag1^+/+ zebra fish. (A) Kaplan–Meier survival curves of naïve rag1^−/− (n = 84) and rag^+/+ (n = 100) adult zebra fish after exposure to a lethal dose of 10⁴ pfu/ml of SVCV under the same challenge conditions as in panel (A). *Statistically significant differences between survival curves between rag1^−/− and rag^+/+ as determined by the Gehan–Breslow–Wilcoxon test with p < 0.05 (n = 2). Solid lines, rag1^−/− zebra fish larvae. Dashed lines, rag^+/+ zebra fish larvae. (B) SVCV titers from pooled whole zebra fish (n = 4 zebra fish per genotype) 3 days after SVCV infection as determined by plaque forming unit (pfu) assays.

Figure 3

Differential expression in skin/fins of adult zebra fish before and after spring viremia carp virus (SVCV) infection. The transcript differential expression folds of rag1^−/−/rag1^+/+ adult zebra fish in skin/fins were calculated as in Figure 1: (A) before infection and (B) 3 days after SVCV infection. Means and SDs were represented (n = 4–6 zebra fish). Red dashed line, onefold boundary. *Statistically different from onefold with p < 0.05 by the t-test.

Figure 4

Active CASPASE-3 staining in different tissues from uninfected and spring viremia carp virus (SVCV)-infected rag1^−/− and rag1^+/+ adult zebra fish. Tissue sections from (A) uninfected and (B) SVCV-infected rag1^−/− and rag1^+/+ adult zebra fish were stained by anti-human active CASPASE-3 antibody. Dark-stained areas in all rag1^−/− tissues indicate the presence of active CASPASE-3. Images are representative of at least two independent experiments. Horizontal red line, 30 μm.

Figure 5

Leukocyte infiltration in muscle and skin tissues in rag1^−/− and rag1^+/+ adult zebra fish. Histological sections from muscle and skin tissues of rag1^−/− and rag1^+/+ were stained with an antibody anti-l-plastin, a pan-leukocyte marker (dark-stained areas). Red arrows, examples of l-plastin stained cells. Images are representative of at least two independent experiments. Horizontal red line, 30 μm.