The Peculiar Characteristics of Fish Type I Interferons

Acknowledgments This work was supported by INRA, by Institut Pasteur, and by the “Projet TEFOR—Investissement d’avenir”—ANR-II-INBS-0014.Peer reviewedPublisher PD

FTR83, a member of the large fish-specific finTRIM family, triggers IFN pathway and counters viral infection

Publisher Copyright: © 2017 Langevin, Aleksejeva, Houel, Briolat, Torhy, Lunazzi, Levraud and Boudinot.Tripartite motif (TRIM) proteins are involved in various cellular functions and constitute key factors of the antiviral innate immune response. TRIM proteins can bind viral particles directly, sending them to degradation by the proteasome, or ubiquitinate signaling molecules leading to upregulation of innate immunity. TRIM proteins are present in across metazoans but are particularly numerous in vertebrates where genes comprising a B30.2 domain have been often duplicated. In fish, a TRIM subset named finTRIM is highly diversified, with large gene numbers and clear signatures of positive selection in the B30.2 domain suggesting they may be involved in antiviral mechanisms. finTRIM provides a beautiful model to investigate the primordial implication of B30.2 TRIM subsets in the arsenal of vertebrate antiviral defenses. We show here that ftr83, a zebrafish fintrim gene mainly expressed in the gills, skin and pharynx, encodes a protein affording a potent antiviral activity. In vitro, overexpression of FTR83, but not of its close relative FTR82, induced IFN and IFN-stimulated gene expression and afforded protection against different enveloped and non-enveloped RNA viruses. The kinetics of IFN induction paralleled the development of the antiviral activity, which was abolished by a dominant negative IRF3 mutant. In the context of a viral infection, FTR83 potentiated the IFN response. Expression of chimeric proteins in which the B30.2 domain of FTR83 and the non-protective FTR82 had been exchanged, showed that IFN upregulation and antiviral activity requires both the Ring/BBox/Coiled coil domain (supporting E3 ubiquitin ligase) and the B30.2 domain of FTR83. Finally, loss of function experiments in zebrafish embryos confirms that ftr83 mediates antiviral activity in vivo. Our results show that a member of the largest TRIM subset observed in fish upregulates type I IFN response and afford protection against viral infections, supporting that TRIMs are key antiviral factors across vertebrates.publishersversionPeer reviewe

A large new subset of TRIM genes highly diversified by duplication and positive selection in teleost fish

<p>Abstract</p> <p>Background</p> <p>In mammals, the members of the tripartite motif (TRIM) protein family are involved in various cellular processes including innate immunity against viral infection. Viruses exert strong selective pressures on the defense system. Accordingly, antiviral TRIMs have diversified highly through gene expansion, positive selection and alternative splicing. Characterizing immune TRIMs in other vertebrates may enlighten their complex evolution.</p> <p>Results</p> <p>We describe here a large new subfamily of TRIMs in teleosts, called finTRIMs, identified in rainbow trout as virus-induced transcripts. FinTRIMs are formed of nearly identical RING/B-box regions and C-termini of variable length; the long variants include a B30.2 domain. The zebrafish genome harbors a striking diversity of finTRIMs, with 84 genes distributed in clusters on different chromosomes. A phylogenetic analysis revealed different subsets suggesting lineage-specific diversification events. Accordingly, the number of <it>fintrim </it>genes varies greatly among fish species. Conserved syntenies were observed only for the oldest <it>fintrims</it>. The closest mammalian relatives are <it>trim16 </it>and <it>trim25</it>, but they are not true orthologs. The B30.2 domain of zebrafish finTRIMs evolved under strong positive selection. The positions under positive selection are remarkably congruent in finTRIMs and in mammalian antiviral TRIM5α, concentrated within a viral recognition motif in mammals. The B30.2 domains most closely related to finTRIM are found among NOD-like receptors (NLR), indicating that the evolution of TRIMs and NLRs was intertwined by exon shuffling.</p> <p>Conclusion</p> <p>The diversity, evolution, and features of finTRIMs suggest an important role in fish innate immunity; this would make them the first TRIMs involved in immunity identified outside mammals.</p

Whole-Body Analysis of a Viral Infection: Vascular Endothelium is a Primary Target of Infectious Hematopoietic Necrosis Virus in Zebrafish Larvae

The progression of viral infections is notoriously difficult to follow in whole organisms. The small, transparent zebrafish larva constitutes a valuable system to study how pathogens spread. We describe here the course of infection of zebrafish early larvae with a heat-adapted variant of the Infectious Hematopoietic Necrosis Virus (IHNV), a rhabdovirus that represents an important threat to the salmonid culture industry. When incubated at 24°C, a permissive temperature for virus replication, larvae infected by intravenous injection died within three to four days. Macroscopic signs of infection followed a highly predictable course, with a slowdown then arrest of blood flow despite continuing heartbeat, followed by a loss of reactivity to touch and ultimately by death. Using whole-mount in situ hybridization, patterns of infection were imaged in whole larvae. The first infected cells were detectable as early as 6 hours post infection, and a steady increase in infected cell number and staining intensity occurred with time. Venous endothelium appeared as a primary target of infection, as could be confirmed in fli1:GFP transgenic larvae by live imaging and immunohistochemistry. Disruption of the first vessels took place before arrest of blood circulation, and hemorrhages could be observed in various places. Our data suggest that infection spread from the damaged vessels to underlying tissue. By shifting infected fish to a temperature of 28°C that is non-permissive for viral propagation, it was possible to establish when virus-generated damage became irreversible. This stage was reached many hours before any detectable induction of the host response. Zebrafish larvae infected with IHNV constitute a vertebrate model of an hemorrhagic viral disease. This tractable system will allow the in vivo dissection of host-virus interactions at the whole organism scale, a feature unrivalled by other vertebrate models

Single-cell transcriptional analysis reveals ILC-like cells in zebrafish.

Innate lymphoid cells (ILCs) are important mediators of the immune response and homeostasis in barrier tissues of mammals. However, the existence and function of ILCs in other vertebrates are poorly understood. Here, we use single-cell RNA sequencing to generate a comprehensive atlas of zebrafish lymphocytes during tissue homeostasis and after immune challenge. We profiled 14,080 individual cells from the gut of wild-type zebrafish, as well as of rag1-deficient zebrafish that lack T and B cells, and discovered populations of ILC-like cells. We uncovered a rorc-positive subset of ILCs that could express cytokines associated with type 1, 2, and 3 responses upon immune challenge. Specifically, these ILC-like cells expressed il22 and tnfa after exposure to inactivated bacteria or il13 after exposure to helminth extract. Cytokine-producing ILC-like cells express a specific repertoire of novel immune-type receptors, likely involved in recognition of environmental cues. We identified additional novel markers of zebrafish ILCs and generated a cloud repository for their in-depth exploration.The study was supported by Cancer Research UK grant number C45041/A14953 (to A.C. and E.I.A.), European Research Council project 677501 – ZF_Blood (to A.C. and P.M.S.), EMBO Long-Term Fellowship ALTF-807-2015 (to P.P.H), ANR grant 17-CE15-0017-01 – ZF-ILC (to P.P.H) and ANR-16-CE20-0002-03 (to J.-P.L), H2020-MSCA-IF-2015 grant 708128 – ZF-ILC (to P.P.H), ANR-10-LABX-73 (‘revive’ to P. Herbomel) and a core support grant from the Wellcome Trust and MRC to the Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute

The zebrafish as a new model for the in vivo study of Shigella flexneri interaction with phagocytes and bacterial autophagy.

Autophagy, an ancient and highly conserved intracellular degradation process, is viewed as a critical component of innate immunity because of its ability to deliver cytosolic bacteria to the lysosome. However, the role of bacterial autophagy in vivo remains poorly understood. The zebrafish (Danio rerio) has emerged as a vertebrate model for the study of infections because it is optically accessible at the larval stages when the innate immune system is already functional. Here, we have characterized the susceptibility of zebrafish larvae to Shigella flexneri, a paradigm for bacterial autophagy, and have used this model to study Shigella-phagocyte interactions in vivo. Depending on the dose, S. flexneri injected in zebrafish larvae were either cleared in a few days or resulted in a progressive and ultimately fatal infection. Using high resolution live imaging, we found that S. flexneri were rapidly engulfed by macrophages and neutrophils; moreover we discovered a scavenger role for neutrophils in eliminating infected dead macrophages and non-immune cell types that failed to control Shigella infection. We observed that intracellular S. flexneri could escape to the cytosol, induce septin caging and be targeted to autophagy in vivo. Depletion of p62 (sequestosome 1 or SQSTM1), an adaptor protein critical for bacterial autophagy in vitro, significantly increased bacterial burden and host susceptibility to infection. These results show the zebrafish larva as a new model for the study of S. flexneri interaction with phagocytes, and the manipulation of autophagy for anti-bacterial therapy in vivo

Origin and Evolution of TRIM Proteins: New Insights from the Complete TRIM Repertoire of Zebrafish and Pufferfish

Tripartite motif proteins (TRIM) constitute a large family of proteins containing a RING-Bbox-Coiled Coil motif followed by different C-terminal domains. Involved in ubiquitination, TRIM proteins participate in many cellular processes including antiviral immunity. The TRIM family is ancient and has been greatly diversified in vertebrates and especially in fish. We analyzed the complete sets of trim genes of the large zebrafish genome and of the compact pufferfish genome. Both contain three large multigene subsets - adding the hsl5/trim35-like genes (hltr) to the ftr and the btr that we previously described - all containing a B30.2 domain that evolved under positive selection. These subsets are conserved among teleosts. By contrast, most human trim genes of the other classes have only one or two orthologues in fish. Loss or gain of C-terminal exons generated proteins with different domain organizations; either by the deletion of the ancestral domain or, remarkably, by the acquisition of a new C-terminal domain. Our survey of fish trim genes in fish identifies subsets with different evolutionary dynamics. trims encoding RBCC-B30.2 proteins show the same evolutionary trends in fish and tetrapods: they evolve fast, often under positive selection, and they duplicate to create multigenic families. We could identify new combinations of domains, which epitomize how new trim classes appear by domain insertion or exon shuffling. Notably, we found that a cyclophilin-A domain replaces the B30.2 domain of a zebrafish fintrim gene, as reported in the macaque and owl monkey antiretroviral TRIM5α. Finally, trim genes encoding RBCC-B30.2 proteins are preferentially located in the vicinity of MHC or MHC gene paralogues, which suggests that such trim genes may have been part of the ancestral MHC

Le système immunitaire des poissons téléostéens

National audienceTeleosts, the modern branch of bony fishes, make up the richest group among Vertebrates with more than 20.000 species displaying considerable diversity and found in all aquatic biota. The basal position of fish in vertebrate phylogeny makes them very attractive for genomic and functional comparative studies, especially of the immune system. Although the rapid evolution of proteins involved in immunity and the tumultuous history of fish genomes often obscure the identification of orthologous genes, these studies have demonstrated that the essential components of the mammalian immune system (macrophages, B and T lymphocytes whose receptors undergo V(D)J recombination, MHC, CDs, cytokines, interferon pathway...) are present in fish. The discovery of fundamental immune mechanisms in fish uncovers the primordial vertebrate immune repertoire ; while some unique adaptations also illustrate how a group undergoing adaptative radiation may innovate, drawing on the available genomic resources, in response to specific constraints