Shan Nan Chen

Melanoma differentiation-associated gene 5 in zebrafish provoking higher interferon-promoter activity through signalling enhancing of its shorter splicing variant

Melanoma differentiation‐associated gene 5 (MDA5) is one of the three members in the retinoic acid‐inducible gene I‐like receptor (RLR) family, which are cytoplasmic pathogen recognition receptors recognizing intracellular viruses. In the present study, MDA5 and its spliced shorter forms, named as MDA5a and MDA5b, were identified in zebrafish. MDA5a and MDA5b can be up‐regulated in cell lines following the infection of a negative ssRNA virus, the spring viraemia of carp virus (SVCV), and an intracellular Gram‐negative bacterial pathogen Edwardsiella tarda, implying that the RLR may also be able to sense elements released from bacteria. The over‐expression of MDA5a and MDA5b in fish cells resulted in significant induction of type I interferon promoter activity and enabled the protection of transfected cells against SVCV infection. Furthermore, the shorter spliced form, MDA5b when co‐transfected with MDA5a or mitochondrial antiviral signalling protein (MAVS), induced a significantly higher level of interferon promoter activity, indicating that MDA5b may function as an enhancer in the interaction between MDA5 and MAVS.

Higher antiviral response of RIG-I through enhancing RIG-I/MAVS-mediated signaling by its long insertion variant in zebrafish.

As an intracellular pattern recognition receptor (PRR), the retinoic acid-inducible gene-I (RIG-I) is responsible for the recognition of cytosolic viral nucleic acids and the production of type I interferons (IFNs). In the present study, an insertion variant of RIG-I with 38 amino acids inserted in the N-terminal CARD2 domain, as well as the typical type, named as RIG-Ia and RIG-Ib respectively were identified in zebrafish. RIG-Ia and RIG-Ib were all up-regulated following the infection of a negative ssRNA virus, the Spring Viremia of Carp Virus (SVCV), and an intracellular Gram-negative bacterial pathogen Edwardsiella tarda, indicating the RLR may have a role in the recognition of both viruses and bacteria. The over-expression of RIG-Ib in cultured fish cells resulted in significant increase in type I IFN promoter activity, and in protection against SVCV infection, whereas the over-expression of RIG-Ia had no direct effect on IFN activation nor antiviral response. Furthermore, it was revealed that both RIG-Ia and RIG-Ib were associated with the downstream molecular mitochondrial antiviral signaling protein, MAVS, and interestingly RIG-Ia when co-transfected with RIG-Ib or MAVS, induced a significantly higher level of type I IFN promoter activity and the expression level of Mx and IRF7, implying that the RIG-Ia may function as an enhancer in the RIG-Ib/MAVS-mediated signaling pathway.

Retinoic acid‐inducible gene I (RIG‐I)‐like receptors (RLRs) in fish: current knowledge and future perspectives

Retinoic acid‐inducible gene I (RIG‐I) ‐like receptors (RLRs) are found conservatively present in teleost fish. All three members, RIG‐I, MDA5 and LGP2, together with the downstream molecules such as MITA, TRAF3 and TBK1, have been identified in a range of fish species. However, it is unexpected that RIG‐I has not been reported in fish of Acanthopterygii, and it would be important to clarify the presence and role of the RIG‐I gene in a broad range of taxa in Teleostei. RLRs in fish can be induced in vivo and in vitro by viral pathogens as well as synthetic dsRNA, poly(I:C), leading to the production of type I interferons (IFNs) and the expression of IFN‐stimulated genes (ISGs). Bacterial pathogens, such as Edwardsiella tarda, and their components, such as lipopolysaccharide are also found to induce the expression of RLRs, and whether such induction was mediated through the direct recognition by RLRs or through crosstalk with other pattern recognition receptors recognizing directly bacterial pathogen‐associated molecular patterns awaits to be investigated. On the other hand, RLR‐activated type I IFN production can be negatively regulated in fish by molecules, such as TBK‐1‐like protein and IRF10, which are found to negatively regulate RIG‐I and MAVS‐activated type I IFN production, and to block MITA or bind ISRE motifs, respectively. It is considered that the evolutionary occurrence of RLRs in fish, and their recognized ligands, especially those from their fish pathogens, as well as the mechanisms involved in the RLR signalling pathways, are of significant interest for further investigation.

NOD2 in zebrafish functions in antibacterial and also antiviral responses via NF-κB, and also MDA5, RIG-I and MAVS

NOD2/RIPK2 signalling plays essential role in the modulation of innate and adaptive immunity in mammals. In this study, NOD2 was functionally characterized in zebrafish (Danio rerio), and its interaction with a receptor-interaction protein, RIPK2, and RLRs such as MDA5 and RIG-I, as well as the adaptor, MAVS was revealed in fish innate immunity. The expression of NOD2 and RIPK2 in ZF4 cells has been constitutive and can be induced by the infection of Edwardsiella tarda and SVCV. The NOD2 can sense MDP in PGN from Gram-negative and -positive bacteria. It is further revealed that the NOD2 and RIPK2 can activate NF-κB and IFN promoters, inducing significantly antiviral defense against SVCV infection. As observed in the reduced bacterial burden in RIPK2 overexpressed cells, RIPK2 also has a role in inhibiting the bacterial replication. The overexpression of NOD2 in zebrafish embryos resulted in the increase of immune gene expression, especially those encoding PRRs and cytokines involved in antiviral response such as MDA5, RIG-I, and type I IFNs, etc. Luciferase reporter assays and co-immunoprecipitation assays demonstrated that zebrafish NOD2 is associated with MDA5 and RIG-I in signalling pathway. In addition, it is further demonstrated that RIPK2 and MAVS in combination with NOD2 have an enhanced role in NOD2-mediated NF-κB and type I IFN activation. It is concluded that teleost fish NOD2 can not only sense MDP for activating innate immunity as reported in mammals, but can also interact with other PRRs to form a network in antiviral innate response.

IFN-γ in turtle: Conservation in sequence and signalling and role in inhibiting iridovirus replication in Chinese soft-shelled turtle Pelodiscus sinensis

The IFN-γ gene was identified in a turtle, the Chinese soft-shelled turtle, Pelodiscus sinensis, with its genome consisting of 4 exons and 3 introns. The deduced amino acid sequence of this gene contains a signal peptide, an IFN-γ family signature motif (130)IQRKAVNELFPT, an NLS motif (155)KRKR and three potential N-glycosylation sites. As revealed by real-time quantitative PCR, the gene was constitutively expressed in all tested organs/tissues, with higher level observed in blood, intestine and thymus. An induced expression of IFN-γ at mRNA level was observed in peripheral blood leucocytes (PBLs) in response to in vitro stimulation of LPS and PolyI:C. The overexpression of IFN-γ in the Chinese soft-shelled turtle artery (STA) cell line resulted in the increase in the expression of transcriptional regulators, such as IRF1, IRF7 and STAT1, and antiviral genes, such as Mx, PKR, implying possibly the existence of a conserved signalling network and role for IFN-γ in the turtle. Furthermore, the infection of soft-shelled turtle iridovirus (STIV) in the cell line transfected with IFN-γ may cause the cell death as demonstrated with the elevated lactate dehydrogenase (LDH) level and cell mortality. However, the mechanism involved in the antiviral activity may require further investigation.

IFN-γ and its receptors in a reptile reveal the evolutionary conservation of type II IFNs in vertebrates.

In this study, interferon gamma (IFN-γ) and interferon gamma receptor (IFN-γR) genes have been identified in non-avian reptile, the North American green anole lizard (Anolis carolinensis). Like their counterparts from other jawed vertebrates, lizard IFN-γ, IFN-γR1 and IFN-γR2 show conserved features in genomic organizations, gene loci and protein sequences. The IFN-γ gene has the full cDNA sequence of 936 bp, with 522 bp open reading frame (ORF) encoding 174 amino acids, and has the genomic organization of four exons and three introns as observed in IFN-γ genes of other classes of vertebrates. The receptors, IFN-γR1 and IFN-γR2 have the ORF of 1278 and 984 bp, coding for 425 and 327 aa, respectively, with the genome organization of seven exons and six introns. In the gene loci of IFN-γ, DYRK2, IL22, IL26 and MDM1 are found with conserved synteny in vertebrates, and similar genes adjacent to IFN-γR1 and IFN-γR2 were also found. These receptors also contain conserved motifs, such as the membrane-proximal region and the C-terminal five residue motif in IFN-γR1, and intracellular conservative sequence in IFN-γR2, which have been confirmed to mediate down-stream JAK-STAT signaling pathway in mammals. IFN-γ and its receptors, IFN-γR1 and IFN-γR2 were constitutively expressed in organs/tissues examined in the lizard, and up-regulated expression of IFN-γ was observed in organs/tissues examined following the poly(I:C) stimulation, suggesting its antiviral role in lizards. The conserved features of IFN-γ and its receptors, IFN-γR1 and IFN-γR2, in gene organization and gene locus as well as in functional domain or motif may imply that the function of type II IFN system is evolutionarily conserved in the green anole lizard, as observed in other classes of vertebrates.

Evolution of IFN-λ in tetrapod vertebrates and its functional characterization in green anole lizard (Anolis carolinensis).

IFN-λ (IFNL), i.e. type III IFN genes were found in a conserved gene locus in tetrapod vertebrates. But, a unique locus containing IFNL was found in avian. In turtle and crocodile, IFNL genes were distributed in these two separate loci. As revealed in phylogenetic trees, IFN-λs in these two different loci and other amniotes were grouped into two different clades. The conservation in gene presence and gene locus was also observed for the receptors of IFN-λ, IFN-λR1 and IL-10RB in tetrapods. It is further revealed that in North American green anole lizard Anolis carolinensis, a single IFNL gene was situated collinearly in the conserved locus as in other tetrapods, together with its receptors IFN-λR1 and IL-10RB also identified in this study. The IFN-λ and its receptors were expressed in all examined organs/tissues, and their expression was stimulated following the injection of polyI:polyC. The ISREs in promoter of IFN-λ in lizard were responsible to IRF3 as demonstrated using luciferase report system, and IFN-λ in lizard functioned through the receptors, IFN-λR1 and IL-10RB, as the up-regulation of ISGs was observed in ligand-receptor transfected, and also in recombinant IFN-λ stimulated, cell lines. Taken together, it is concluded that the mechanisms involved in type III IFN ligand-receptor system, and in its signalling pathway and its down-stream genes may be conserved in green anole lizard, and may even be so in tetrapods from xenopus to human.

Intronless and intron-containing type I IFN genes coexist in amphibian Xenopus tropicalis: Insights into the origin and evolution of type I IFNs in vertebrates.

ABSTRACT Type I IFNs are considered to be the core IFN species in vertebrates because of their predominant antiviral effects. But, a puzzling question remains to be answered, as to how intronless type I IFN genes in amniotes might have evolved from intron‐containing type I IFN genes in fish and amphibians. In this study, intronless and intron‐containing type I IFNs were found in the amphibian model, Xenopus tropicalis, with a total of sixteen and five genes, respectively. The intronless IFNs can be divided into three subgroups, and the intron‐containing ones into two subgroups, implying that a retroposition event might have occurred in amphibians, resulting in the generation of intronless type I IFN genes. Two models were tentatively proposed to explain the evolution of type I IFNs in vertebrates: in model A, fish should possess the most primitive multi‐exon‐containing type I IFNs, and intronless type I IFN genes in amphibians are the ancestor of modern intronless type I IFNs in amniotes; in model B, intronless type I IFN genes in X. tropicalis may just represent an independent bifurcation in this species or probably in amphibians, and intronless type I IFN genes in amniotes may have arisen from another retroposition event occurred in a transition period even when reptiles were diverged from amphibians. It is considered that the model B can reflect the current knowledge on the occurrence of intronless and intron‐containing type I IFN genes in vertebrate lineages. This study thus contributes to a better understanding of the origin and evolution of type I IFNs in vertebrates, and of the occurrence of intronless I IFNs in higher vertebrates. Graphical abstract Figure. No Caption available. HighlightsIntronless and intron‐containing type I IFN genes coexist in Xenopus tropicalis.Intronless and intron‐containing IFNs phylogenetically divide into three and two subgroups respectively.Different gene expression patterns were observed among subgroups of IFNs.Retroposition might have occurred in amphibians with the generation of IFNi genes.Models were proposed to explain the evolution of type I IFNs in vertebrates.

Role of zebrafish NLRC5 in antiviral response and transcriptional regulation of MHC related genes

ABSTRACT Intracellular NOD‐like receptors (NLRs) are emerging as critical regulators of innate and adaptive immune responses. Although the NLR family member NLRC5 is functionally defined, the role of NLRC5 in regulating innate immune signaling has been controversial in mammals, and is poorly understood in teleost fish. In the present study, we report the functional characterization of zebrafish NLRC5. The cloned NLRC5 consists of 6435 bp which encodes 1746 amino acids. The N‐terminal effector‐binding domain of zebrafish NLRC5 is absent which is different from all other human NLRs. Fluorescence microscopy showed that zebrafish NLRC5 is located throughout the entire cell. The higher expression of zebrafish NLRC5 in embryo than in larvae was observed, suggesting the action phase of NLRC5 in zebrafish ontogenetic stages. When the modulation of NLRC5 in pathogen infection was analyzed, it was found that zebrafish NLRC5 was upregulated by both bacterial and viral infection. Overexpression of zebrafish NLRC5 resulted in significant inhibition of SVCV replication in vivo and in vitro, but failed to activate interferon (IFN) promoters and type I IFN signaling pathway. Interestingly, NLRC5 overexpression could activate mhc2dab promoter, and induce the expression of MHC class II genes. All together, these results demonstrate that zebrafish NLRC5 is involved in IFN‐independent antiviral response, and also functions as a transcriptional regulator of MHC class II genes. HIGHLIGHTSZebrafish NLRC5 has a monopartite NLS, and localizes in the entire cell including the nucleus.Zebrafish NLRC5 was detected at the early stages of development with higher expression in embryo than in larvae.Zebrafish NLRC5 was induced by both bacterial and viral infection.Zebrafish NLRC5 is involved in IFN‐independent antiviral response.Zebrafish NLRC5 functions as a transcriptional regulator of MHC class II genes.

Two type II IFN members, IFN-γ and IFN-γ related (rel), regulate differentially IRF1 and IRF11 in zebrafish

&NA; Two members of type II IFNs have been identified in fish, i.e. an IFN‐&ggr; gene as in other vertebrates and a unique IFN‐&ggr; related (IFN‐&ggr; rel) gene being solely present in fish. However, the signalling pathways involved in the down‐stream signalling of type II IFNs in fish remains poorly described. In this study, the type II IFNs mediated IRF1 was investigated in zebrafish, and the true homologous gene of mammalian IRF1 in fish was revealed despite the report of so‐called IRF1a and IRF1b in zebrafish. As revealed in overexpression analysis, zebrafish IFN‐&ggr; had a higher induction ability than IFN‐&ggr; rel in relation with the expression of IRF1. IFN‐&ggr; stimulated the expression level of STAT1a and also STAT1b, but they had opposite trends with the increase of time; enhancement of STAT1a waned after 12 h post injection of plasmids; whereas STAT1b expression increased continuously. Zebrafish IRF1 gene promoter contained several putative transcription factor binding sites, including GAS and NF‐&kgr;B motifs. Luciferase assay revealed that the GAS site was essential in the IFN‐&ggr; triggered IRF1 expression. In contrast, IRF11 contained neither GAS nor NF‐&kgr;B elements, and did not respond to IFN‐&ggr; induction. It is considered that STAT1a and STAT1b are structurally and functionally similar to STAT1&agr; and STAT1&bgr; in mammal respectively, and that IRF11, although used to be nominated as IRF1a, is not the orthologue of mammalian IRF1, but IRF1b in zebrafish should be the orthologue. HighlightsIRF1 was conspicuously induced by IFN‐&ggr; in comparison with IFN‐&ggr; rel in zebrafish.GAS motif was indispensable to zebrafish IFN‐&ggr; triggered transcriptional activation of IRF1.Promoter activity and mRNA expression of IRF11 in zebrafish were not responsible to IFN‐&ggr; signalling.IRF1 and IRF11 in zebrafish showed difference in promoter elements, responses to IFN‐&ggr;, and organ/tissue expression.