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

Pablo García-Valtanen,Beatriz Novoa,Alicia Martínez-López,Azucena López-Muñoz,Antonio Figueras,Julio Coll,Amparo Estepa,Regla M Medina-Gali,Víctoriano Mulero,Melissa Bello-Perez,María Del Mar Ortega-Villaizán,Monica Varela

doi:10.3389/fimmu.2017.00121

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.

Highlights

Immunity against infections in vertebrate species includes both innate and adaptive responses
Our resulting selected gene set included the genes of inflammatory cytokines, innate immune transcription factors and effector molecules, antimicrobial peptides, and molecules involved in adaptive immune responses
The results showed that in uninfected rag1−/− fish several apoptosis-related modulated MultiPath Genes (mMPG) were regulated, being fas, faslg, hsp90, casp7, and hspb the most upregulated (Table 3 gray rows, column A, genes labeled with +), which confirms the importance of apoptosis for the survival of this genotype

Summary

Introduction

Immunity against infections in vertebrate species includes both innate (early and unspecific) and adaptive (late and specific) responses. Recent research conducted in mammal species has revealed that cells of the innate immune system can be primed so that upon a secondary immune challenge they are capable of eliciting more efficient immune responses, a characteristic previously attributed only to the adaptive arm of the immune system. These types of innate responses have been termed trained immunity [5,6,7,8,9,10]. Such non-specific vaccine strategies will be most practical for farmed fish vaccination because with short lifespans, heterologous vaccines would represent a more cost-effective alternative than traditional vaccines

Methods

Results

Conclusion