Poly (I:C)-Induced microRNA-30b-5p Negatively Regulates the JAK/STAT Signaling Pathway to Mediate the Antiviral Immune Response in Silver Carp (Hypophthalmichthys molitrix) via Targeting CRFB5.

In aquaculture, viral diseases pose a significant threat and can lead to substantial economic losses. The primary defense against viral invasion is the innate immune system, with interferons (IFNs) playing a crucial role in mediating the immune response. With advancements in molecular biology, the role of non-coding RNA (ncRNA), particularly microRNAs (miRNAs), in gene expression has gained increasing attention. While the function of miRNAs in regulating the host immune response has been extensively studied, research on their immunomodulatory effects in teleost fish, including silver carp (Hyphthalmichthys molitrix), is limited. Therefore, this research aimed to investigate the immunomodulatory role of microRNA-30b-5p (miR-30b-5p) in the antiviral immune response of silver carp (Hypophthalmichthys molitrix) by targeting cytokine receptor family B5 (CRFB5) via the JAK/STAT signaling pathway. In this study, silver carp were stimulated with polyinosinic-polycytidylic acid (poly (I:C)), resulting in the identification of an up-regulated miRNA (miR-30b-5p). Through a dual luciferase assay, it was demonstrated that CRFB5, a receptor shared by fish type I interferon, is a novel target of miR-30b-5p. Furthermore, it was found that miR-30b-5p can suppress post-transcriptional CRFB5 expression. Importantly, this study revealed for the first time that miR-30b-5p negatively regulates the JAK/STAT signaling pathway, thereby mediating the antiviral immune response in silver carp by targeting CRFB5 and maintaining immune system stability. These findings not only contribute to the understanding of how miRNAs act as negative feedback regulators in teleost fish antiviral immunity but also suggest their potential therapeutic measures to prevent an excessive immune response.

Type II interferons (IFN-γ and IFN-γrel) activate downstream genes through various potential receptor combinations to exert antiviral functions in spotted sea bass (Lateolabrax maculatus).

Type II interferons (IFNs) exert antiviral functions by binding to receptors and activating downstream signaling pathways. However, our understanding of the antiviral functions and the receptor complex model of type II IFNs in teleost fish remains limited. In this study, we determined the functions of type II IFNs (LmIFN-γ and LmIFN-γrel) in Lateolabrax maculatus and assessed their antiviral ability mediated by their combination with different cytokine receptor family B members (LmCRFB6, LmCRFB13, and LmCRFB17). After infection with largemouth bass ulcer syndrome virus (LBUSV), the expression levels of LmIFNs and LmCRFBs increased significantly in vitro and in vivo. Incubation or injection with LmIFNs-His activated the expressions of LmISG15, LmMx, and LmIRF1. LmIFN-γ and LmIFN-γrel both bound to the extracellular domains of the three CRFBs via Pull-down. Furthermore, LmIFN-γ combined with LmCRFB6, LmCRFB6+LmCRFB13, and LmCRFB6+LmCRFB13+LmCRFB17 and LmIFN-γrel combined with all combinations containing LmCRFB17 induced the transcription of downstream genes and reduced the number of LBUSV copies. Therefore, type II IFNs (LmIFN-γ and LmIFN-γrel) contribute to enhanced antiviral immunity in L. maculatus and that ligand-receptor combinations effectively suppress virus replication. These findings provide a reference for future studies of the signal transduction mechanism of type II IFNs in teleost fish.

CRFB5a, a Subtype of Japanese Eel (Anguilla japonica) Type I IFN Receptor, Regulates Host Antiviral and Antimicrobial Functions through Activation of IRF3/IRF7 and LEAP2.

IFNAR1, one of the type I IFN receptors, is crucial to mammalian host defense against viral invasion. However, largely unknown is the immunological role of the fish teleost protein IFNAR1, also known as CRFB5. We have successfully cloned the whole cDNA of the Japanese eel's (Anguilla japonica) CRFB5a homolog, AjCRFB5a. The two fibronectin-3 domains and the transmembrane region (238-260 aa) of AjCRFB5a are normally present, and it shares a three-dimensional structure with zebrafish, Asian arowana, and humans. According to expression analyses, AjCRFB5a is highly expressed in all tissues found, particularly the liver and intestine. In vivo, Aeromonas hydrophila, LPS, and the viral mimic poly I:C all dramatically increased AjCRFB5a expression in the liver. Japanese eel liver cells were reported to express AjCRFB5a more strongly in vitro after being exposed to Aeromonas hydrophila or being stimulated with poly I: C. The membranes of Japanese eel liver cells contained EGFP-AjCRFB5a proteins, some of which were condensed, according to the results of fluorescence microscopy. Luciferase reporter assays showed that AjCRFB5a overexpression strongly increased the expression of immune-related genes in Japanese eel liver cells, such as IFN1, IFN2, IFN3, IFN4, IRF3, IRF5, and IRF7 of the type I IFN signaling pathway, as well as one of the essential antimicrobial peptides LEAP2, in addition to significantly inducing human IFN-promoter activities in HEK293 cells. Additionally, RNA interference (RNAi) data demonstrated that knocking down AjCRFB5a caused all eight of those genes to drastically lower their expression in Japanese eel liver cells, as well as to variable degrees in the kidney, spleen, liver, and intestine. Our findings together showed that AjCRFB5a participates in the host immune response to bacterial infection by inducing antimicrobial peptides mediated by LEAP2 and favorably modulates host antiviral immune responses by activating IRF3 and IRF7-driven type I IFN signaling pathways.

Molecular characterization of nineteen cytokine receptor family B (CRFB) members, CRFB1, CRFB2, CRFB4-17, with three CRFB9 and two CRFB14 in a cyprinid fish, the blunt snout bream Megalobrama amblycephala.

The class II cytokine receptor family members are receptors of class 2 helical cytokines in mammals, and are named cytokine receptor family B (CRFB) in fish. In zebrafish, sixteen members, including CRFB1, CRFB2 and CRFB4-17 were reported. With the availability of genome sequence, a total of nineteen CRFBs was identified in the blunt snout bream (Megalobrama amblycephala), including CRFB1, CRFB2, CRFB4-17 with the presence of three CRFB9 isoforms, and two CRFB14 isoforms. These CRFB molecules contain well conserved features, such as fibronectin type III (FNIII) domain, transmembrane and intracellular domains as other class II cytokine receptors, and are phylogenetically grouped into thirteen clades with their homologues from other species of fish. The CRFB genes were constitutively expressed in organs/tissues examined in the fish. The finding of more CRFB members in the bream may provide clues to understand possible receptor-ligand interaction and their diversity from an evolutionary point of view.

Four type I IFNs, IFNa1, IFNa2, IFNb, IFNc, and their receptor usage in an osteoglossomorph fish, the Asian arowana, Scleropages formosus.

In fish, type I IFNs are classified into three groups, i.e. Group I, Group II and Group III, which are further divided into seven subgroups according to the number of conservative cysteines, phylogenetic relationship, and probably their receptor complexes. In the present study, four type I IFNs and four cytokine receptor family B members (CRFBs) were identified in the Asian arowana, Scleropages formosus, an ancient species in the Osteoglossomorpha with commercial and conservation values. According to multiple sequence alignment and phylogenetic relationship, the four type I IFNs are named as IFNa1, IFNa2, IFNb and IFNc, with the former two belonging to Group I, and the latter two to Group II. The four receptors are named as CRFB1, CRFB2, CRFB5a and CRFB5b. The IFNs and their possible receptor genes are widely expressed in examined organs/tissues, and are induced following the stimulation of polyinosinic polycytidylic acid (polyI:C) in vivo. It was found that IFNa1, IFNa2, IFNb and IFNc use preferentially the receptor complexes, CRFB1 and CRFB5b, CRFB1 and CRFB5b, CRFB2 and CRFB5a, and CRFB2 and CRFB5b, respectively, indicating the evolutionary diversification in the interaction of type I IFNs and their receptors in this ancient fish species, S. formosus.

Identification of type I IFNs and their receptors in a cyprinid fish, the topmouth culter Culter alburnus.

In fish, type I IFNs are classified into three groups, i.e. group one, group two and group three, and further separated into seven subgroups based on the number of conserved cysteines and phylogenetic relationships. In the present study, four type I IFNs, named as IFNϕ1, IFNϕ2, IFNϕ3, IFNϕ4, as reported in zebrafish, were identified in a cyprinid, the topmouth culter, Culter alburnus, a species introduced recently into China's aquaculture. These IFNs may be classified as IFNa, IFNc, IFNc and IFNd in a recent nomenclature, with IFNa and IFNd having two cysteines in group one, and IFNc four cysteines in group two. These IFNs, together with their possible receptors, IFNϕ1, IFNϕ2, IFNϕ3, IFNϕ4, and CRFB1, CRFB2 and CRFB5 have an open reading frame (ORF) of 540, 552, 567, 516 bp, and 1572, 1392, 1125 bp, respectively. These IFNs have high amino acid sequence identities, being 91.1-93.6% and 66.9-77.3%, with those in grass carp and zebrafish, respectively, and are expressed constitutively in organs/tissues examined in the fish. The expression of these IFNs can be further induced following poly (I:C) stimulation. However, the possible function of these IFNs and their signalling pathway are of interest for further research.

Receptor complex and signalling pathway of the two type II IFNs, IFN-γ and IFN-γrel in mandarin fish or the so-called Chinese perch Siniperca chuatsi.

IFN-γ, as the sole member of mammalian type II IFN, is a multifunctional cytokine which exerts its effects through two distinct IFN-γ receptors, IFNGR1 and IFNGR2. However, in teleost fish, another IFN-γ homologous gene, namely IFN-γ related gene (IFN-γrel), has been identified. Although IFN-γ and IFN-γrel genes have been described in some fish species, many important aspects remain poorly understood in relation with their signalling and function. In the present study, IFN-γ and IFN-γrel, as well as their receptors, cytokine receptor family B (CRFB) 17, CRFB13, two of which are homologous to IFNGR1 in mammals, and CRFB6, homolomous to IFNGR2, have been characterized in mandarin fish, Siniperca chuatsi. It was revealed that the two type IFN members exhibit antiviral activity, and IFN-γ transduces downstream signalling through CRFB13 and CRFB6, while IFN-γrel interacts with CRFB17 to activate downstream signalling. Moreover, IFN-γ and IFN-γrel have been shown to exert antiviral biological activity in a STAT1-dependent manner. Intracellular domain analysis of CRFB17 and CRFB13 demonstrated that the Y386 tyrosine residue of CRFB13 is required for the activation of the IFN-γ-mediated biologic response, and the Y324 and Y370 residues in CRFB17 are required to activate IFN-γrel signalling.

Functional, signalling and transcriptional differences of three distinct type I IFNs in a perciform fish, the mandarin fish Siniperca chuatsi.

Teleost fish are unique in having type I and type II interferons (IFNs) only, and the type I IFNs are classified into Group one and Group two based on the presence of two or four cysteines respectively, and are further classified into seven subgroups. In the present study, three distinct type I IFNs, IFNc, IFNd and IFNh, have been identified in the genome sequences of a perciform fish, the mandarin fish Siniperca chuatsi. These IFNs are induced following the stimulation of Polyinosinic polycytidylic acid (poly(I:C)) and Resiquimod (R848) either in vivo or in vitro. But, the infectious spleen and kidney necrosis virus (ISKNV) infection caused a delayed response of IFNs, which may be resulted from the viral inhibition of type I IFN production and related signalling. The three receptor subunits, cytokine receptor family B 1 (CRFB1), CRFB2 and CRFB5 are also expressed in a similar manner as observed for the IFNs, and IFNc, IFNd and IFNh use preferentially the receptor complex, CRFB2 and CRFB5, CRFB1 and CRFB5, CRFB1 and CRFB5 respectively for their effective signalling in the induction of IFN-stimulated genes (ISGs). Moreover, the IFNs are able to induce their own expression, and also the IRF3 and IRF7 expression, leading to the amplification of IFN cascade. It is further revealed that these three IFNs are transcribed differently by IRF7 and IRF3. The composition, function, signalling and transcription of type I IFNs have been investigated in detail in a teleost fish.

Bioinformatics analysis of organizational and expressional characterizations of the IFNs, IRFs and CRFBs in grass carp Ctenopharyngodon idella.

Interferons (IFNs) play crucial roles in the immune response of defense against viral infection and bacteria invasion. In the present study, we systematically identified and characterized the IFNs, their regulatory factors (Interferon Regulatory Factors, IRFs) and receptors (Cytokine Receptor Family B, CRFBs) in grass carp (Ctenopharyngodon idella). Grass carp IFNs can be classified into type I IFN (IFN-I) and type II IFN (IFN-II) like other teleosts. IFN-I consist of two groups with two (group I) or four (group II) cysteines in the mature peptide and can be further divided into three subgroups (IFN-a, -c and -d), containing four members: IFN1, IFN2, IFN3, IFN4 in grass carp. IFN-II contain two members, IFNγ2 with the similarity to mammalian IFNγ and a cyprinid specific IFNγ1 (IFNγ-rel) molecule. mRNA expression analyses of IFNs discovered that IFN1 and IFN-II were sustainably expressed in many tissues, while other IFN members were transiently expressed in specific tissues and time points. In the immune response, IFN transcriptions are primarily regulated through multiple IRFs after grass carp reovirus (GCRV) challenge. IRF family possess thirteen members in grass carp, which can be further divided into four subfamilies (IRF-1, -3, -4 and -5 subfamily), each of them plays different roles in the innate and adaptive immunity via various signaling pathways to interact with IFNs (mainly IFN-I). IFNs have to bind receptors (CRFBs) to perform their functions. CRFBs as IFN receptors contain six members in grass carp. The structure and expression characterizations of IFNs, IRFs and CRFBs were analyzed using bioinformatics tools. These results might provide basic data for the further functional research of IFN system, and deeply understand fish immune mechanisms against virus infection.

Cloning, identification of the two cytokine receptor family B subunits CRFB1 and CRFB5 from grass carp (Ctenopharyngodon idella).

Similar to the mammalian counterparts, fish type I interferon (IFN) performs its potential biological activities via binding to the corresponding receptor on target cell membrane. Fish type I IFN receptor, a kind of enzyme-linked receptor, consists of two subunits and belongs to the class II cytokine receptor family B (CRFB). In the present study, we cloned and identified two putative grass carp (Ctenopharyngodon idella) type I interferon receptor subunits (termed CiCRFB1 and CiCRFB5) by homology cloning techniques. Phylogenetic tree analysis suggested that CiCRFB1 and CiCRFB5 shared highly homology to Danio rerio CRFB1 and CRFB5 respectively. CiCRFB1 and CiCRFB5 were up-regulated after the stimulation with Grass Carp Hemorrhagic Virus (GCHV) and Polyinosinic-polycytidylic acid (Poly I:C), indicating that they are related to the intracellular antiviral activity. In order to know more about the roles of CiCRFB1 and CiCRFB5 in the process, the extracellular domains of CiCRFB1 (CiCRFB1-EC) and CiCRFB5 (CiCRFB5-EC), as well as grass carp type I IFN (CiIFN) were expressed in Escherichia coli BL21, and purified by affinity chromatography with the Ni-NTA His-Bind resin. Cross-linking reactions were employed to analyze the affinity of the ligand (CiIFN) with the two putative receptor subunits (CiCRFB1-EC and CiCRFB5-EC). The result suggested the formation of (CiCRFB5)2 homodimer was more easily than that of (CiCRFB1)2 under the induction of CiIFN in vitro. However, CiIFN was inclined to bind to (CiCRFB1)2 homodimer. Interestingly, although CiIFN seemed unable to facilitate the formation of (CiCRFB1 + CiCRFB5) heterodimer in the absence of DSS cross linker, however it can bind to the heterodimer in the presence of DSS. This indicated that the homodimer and the heterodimer were the potential receptor for CiIFN.

Atlantic salmon possesses two clusters of type I interferon receptor genes on different chromosomes, which allows for a larger repertoire of interferon receptors than in zebrafish and mammals.

Mammalian type I interferons (IFNs) signal through a receptor composed of the IFNAR1 and IFNAR2 chains. In zebrafish two-cysteine IFNs utilize a receptor composed of CRFB1 and CRFB5, while four-cysteine IFNs signal through a receptor formed by CRFB2 and CRFB5. In the present work two CRFB clusters were identified in different chromosomes of Atlantic salmon. Genes of three CRFB5s, one CRFB1, one CRFB2 and the novel CRFB5x were identified, cloned and studied functionally. All CRFBs were expressed in 10 different organs, but the relative expression of CRFBs varied. Mx-reporter assay was used to study which CRFBs might be involved in receptors for salmon IFNa, IFNb and IFNc. The results of Mx-reporter assays suggest that IFNa signals through a receptor composed of CRFB1a as the long chain and either CRFB5a, CRFB5b or CRFB5c as the short chain; IFNc signals through a receptor with CRFB5a or CRFB5c as the short chain while IFNb may signal through a receptor with CRFB5x as a short chain. Taken together, the present work demonstrates that Atlantic salmon has a more diverse repertoire of type I IFN receptors compared to zebrafish or mammals.

The antiviral innate immune response in fish: evolution and conservation of the IFN system.

Innate immunity constitutes the first line of the host defense after pathogen invasion. Viruses trigger the expression of interferons (IFNs). These master antiviral cytokines induce in turn a large number of interferon-stimulated genes, which possess diverse effector and regulatory functions. The IFN system is conserved in all tetrapods as well as in fishes, but not in tunicates or in the lancelet, suggesting that it originated in early vertebrates. Viral diseases are an important concern of fish aquaculture, which is why fish viruses and antiviral responses have been studied mostly in species of commercial value, such as salmonids. More recently, there has been an interest in the use of more tractable model fish species, notably the zebrafish. Progress in genomics now makes it possible to get a relatively complete image of the genes involved in innate antiviral responses in fish. In this review, by comparing the IFN system between teleosts and mammals, we will focus on its evolution in vertebrates.