Structural and Functional Characterization of a Fish Type I Subgroup d IFN Reveals Its Binding to Receptors.

Teleost fish type I IFNs and the associated receptors from the cytokine receptor family B (CRFB) are characterized by remarkable diversity and complexity. How the fish type I IFNs bind to their receptors is still not fully understood. In this study, we demonstrate that CRFB1 and CRFB5 constitute the receptor pair through which type I subgroup d IFN (IFNd) from large yellow croaker, Larimichthys crocea, activates the conserved JAK-STAT signaling pathway as a part of the antiviral response. Our data suggest that L. crocea IFNd (LcIFNd) has a higher binding affinity with L. crocea CRFB5 (LcCRFB5) than with LcCRFB1. Furthermore, we report the crystal structure of LcIFNd at a 1.49-Å resolution and construct structural models of LcIFNd in binary complexes with predicted structures of extracellular regions of LcCRFB1 and LcCRFB5, respectively. Despite striking similarities in overall architectures of LcIFNd and its ortholog human IFN-ω, the receptor binding patterns between LcIFNd and its receptors show that teleost and mammalian type I IFNs may have differentially selected helices that bind to their homologous receptors. Correspondingly, key residues mediating binding of LcIFNd to LcCRFB1 and LcCRFB5 are largely distinct from the receptor-interacting residues in other fish and mammalian type I IFNs. Our findings reveal a ligand/receptor complex binding mechanism of IFNd in teleost fish, thus providing new insights into the function and evolution of type I IFNs.

Astragaloside IV inhibits epithelial-mesenchymal transition and pulmonary fibrosis via lncRNA-ATB/miR-200c/ZEB1 signaling pathway.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disease involving multiple factors and genes. Astragaloside IV (ASV) is one of the main bioactive ingredients extracted from the root of Astragalus membranaceus, which plays an important role in anti-inflammatory, antioxidant and improve cardiopulmonary function. Epithelial-mesenchymal transition (EMT) is a key driver of the process of pulmonary fibrosis, and Zinc finger E-box-binding homeobox 1 (ZEB1) can promote pulmonary fibrosis in an EMT-dependent manner. Here, we found that ASV effectively inhibited the ZEB1 and EMT in both bleomycin (BLM)-induced rat pulmonary fibrosis and TGF-ß1-treated A549 cells. To further elucidate the molecular mechanisms underlying effects of ASV in IPF, we explored the truth using bioinformatics, plasmid construction, immunofluorescence staining, western blotting and other experiments. Dual luciferase reporter assay and bioinformatics proved that miR-200c not only acts as an upstream regulatory miRNA of ZEB1 but also has binding sites for the lncRNA-ATB. In A549 cell-based EMT models, ASV reduced the expression of lncRNA-ATB and upregulated miR-200c. Furthermore, overexpression of lncRNA-ATB and silencing of miR-200c reversed the down-regulation of ZEB1 and the inhibition of EMT processes by ASV. In addition, the intervention of ASV prevented lncRNA-ATB as a ceRNA from regulating the expression of ZEB1 through sponging miR-200c. Taken together, the results showed that ASV inhibited the EMT process through the lncRNA-ATB/miR-200c/ZEB1 signaling pathway, which provides a novel approach to the treatment of IPF.

Molecular and Structural Basis of Receptor Binding and Signaling of a Fish Type I IFN with Three Disulfide Bonds.

In mammals, type I IFNs, which commonly contain one or two disulfide bonds, activate the JAK-STAT signaling pathway through binding to the common cell surface receptor formed by IFN-α/ß receptor (IFNAR)1 and IFNAR2 subunits. Although type I IFNs are also known to be essential for antiviral defense in teleost fish, very little is known about mechanisms underlying the recognition of fish type I IFNs by associated receptors. In this study, we demonstrate that a type I IFN of large yellow croaker Larimichthys crocea (LcIFNi), belonging to a new subgroup of fish type I IFNs, triggers antiviral response via the conserved JAK-STAT pathway through stable binding with a heterodimeric receptor comprising subunits LcCRFB5 and LcCRFB2. LcIFNi binds to LcCRFB5 with a much higher affinity than to LcCRFB2. Furthermore, we determined the crystal structure of LcIFNi at a 1.39 Å resolution. The high-resolution structure is, to our knowledge, the first reported structure of a type I IFN with three disulfide bonds, all of which were found to be indispensable for folding and stability of LcIFNi. Using structural analysis, mutagenesis, and biochemical assays, we identified key LcIFNi residues involved in receptor interaction and proposed a structural model of LcIFNi bound to the LcCRFB2-LcCRFB5 receptor. The results show that LcIFNi-LcCRFB2 exhibits a similar binding pattern to human IFN-ω-IFNAR2, whereas the binding pattern of LcIFNi-LcCRFB5 is quite different from that of IFN-ω-IFNAR1. Altogether, our findings reveal the structural basis for receptor interaction and signaling of a type I IFN with three disulfide bonds and provide new insights into the mechanisms underlying type I IFN recognition in teleosts.

IRF1 and IRF2 act as positive regulators in antiviral response of large yellow croaker (Larimichthys crocea) by induction of distinct subgroups of type I IFNs.

Interferon regulatory factors (IRFs) are crucial transcription factors involved in transcriptional regulation of type I interferons (IFNs) and IFN-stimulated genes (ISGs) against viral infection. In teleost fish, eleven IRFs have been found, however, understanding of their roles in the antiviral response remains limited. In the previous study, IRF1 (LcIRF1) and IRF2 (LcIRF2) genes were cloned from large yellow croaker (Larimichthys crocea). Here, we further characterized their function in the antiviral response. LcIRF1 and LcIRF2 were constitutively expressed in primary head kidney monocytes/macrophages (PKMs), lymphocytes (PKLs), granulocytes (PKGs) and large yellow croaker head kidney (LYCK) cell line, and significantly upregulated in PKMs and LYCK cells after stimulation with poly (I:C). LcIRF1 could induce promoter activities of three large yellow croaker type I IFNs, IFNc, IFNd and IFNh, while LcIRF2 could only induce those of IFNd and IFNh, and inhibit IFNc promoter activity. Correspondingly, overexpression of LcIRF1 in LYCK cells increased expression of all three IFNs (IFNc, IFNd and IFNh), while that of LcIRF2 only upregulated the expression levels of IFNd and IFNh, and inhibited expression of IFNc, although both LcIRF1and LcIRF2 induced expression of IFN-stimulated genes (ISGs), MxA, PKR and Viperin. Additionally, both LcIRF1 and LcIRF2 inhibited the Spring Viremia of Carp Virus (SVCV) replication in epithelioma papulosum cyprinid (EPC) cells, thus showing antiviral activity. Taken together, these results indicated that both LcIRF1 and LcIRF2 play positive roles in regulating the antiviral response of large yellow croaker by induction of distinct subgroups of type I IFNs.

Molecular characterization of the interferon regulatory factor (IRF) family and functional analysis of IRF11 in the large yellow croaker (Larimichthys crocea).

Interferon regulatory factors (IRFs) are a family of transcription factors involved in regulating interferon (IFN) responses and immune cell development. A total of 11 IRFs have been identified in teleost fish. Here, a complete repertoire of 11 IRFs (LcIRFs) in the large yellow croaker (Larimichthys crocea) was characterized with the addition of five newly identified members, LcIRF2, LcIRF5, LcIRF6, LcIRF10, and LcIRF11. These five LcIRFs possess a DNA-binding domain (DBD) at the N-terminal that contains five to six conserved tryptophan residues and an IRF-association domain (IAD) or IAD2 at the C-terminal that is responsible for interaction with other IRFs or co-modulators. Phylogenetic analysis showed that the 11 LcIRFs were divided into four clades including the IRF1 subfamily, IRF3 subfamily, IRF4 subfamily, and IRF5 subfamily. These are evolutionarily related to their respective counterparts in other fish species. The 11 LcIRFs were constitutively expressed in all examined tissues, although at different expression levels. Upon polyinosinic: polycytidylic acid (poly (I:C)) stimulation, the expression of all 11 LcIRFs was significantly induced in the head kidney and reached the highest levels at 6 h post-stimulation (except LcIRF4). LcIRF1, LcIRF3, LcIRF7, LcIRF8, and LcIRF10 were more strongly induced by poly (I:C) than the other LcIRFs. Significant induction of all LcIRFs was observed in the spleen, with LcIRF2, LcIRF5, LcIRF6, LcIRF7, LcIRF9, and LcIRF11 reaching their highest levels at 48 h LcIRF3 and LcIRF11 showed a stronger response to poly (I:C) in the spleen than the other LcIRFs. In addition, LcIRF1, LcIRF3, LcIRF7, LcIRF9, LcIRF10, and LcIRF11 were significantly induced by Vibro alginolyticus in both the spleen and the head kidney, with LcIRF1 strongly induced. Thus, LcIRFs exhibited differential inducible expression patterns in response to different stimuli in different tissues, suggesting that LcIRFs have different functions in the regulation of immune responses. Furthermore, overexpression of LcIRF11 activated the promoters of LcIFNc, LcIFNd, and LcIFNh, and differentially induced the expression levels of LcIFNs and IFN-stimulated genes (ISGs). Overexpression of LcIRF11 in epithelioma papulosum cyprinid (EPC) cells inhibited the replication of viral genes after infection of spring viremia of carp virus (SVCV). These data suggested that LcIRF11 may function as a positive regulator in regulating the cellular antiviral response through induction of type I IFN expression. Taken together, the present study reported molecular characterization and expression analysis of 11 IRFs in the large yellow croaker, and investigated the role of LcIRF11 in the antiviral response, which laid a good foundation for further study on the evolution and functional characterization of fish IRFs.

Characterization and function of a group II type I interferon in the perciform fish, large yellow croaker (Larimichthys crocea).

Teleost fish possess two groups of type I interferons (IFNs) with two (group I IFNs) or four (group II IFNs) conserved cysteines, which are further classified into seven subgroups. In our previous study, two group I type I IFNs, LcIFNd and LcIFNh (a new subgroup member), were identified in the perciform fish, large yellow croaker (Larimichthys crocea). Here, we identified a group II type I IFN, LcIFNc, in this species. The deduced LcIFNc contained six cysteines, four of which are highly conserved (C1: C28, C2:C53, C3: C130, and C4:C159) in the fish group II type I IFNs, and a typical type I IFN signature motif was also found in it. Phylogenetic analysis indicated that LcIFNc belongs to the IFNc subgroup of fish group II type I IFNs. LcIFNc was constitutively expressed in all examined tissues, and was rapidly up-regulated in spleen and head kidney by poly(I:C) and Aeromonas hydrophila. Recombinant LcIFNc protein (rLcIFNc) could increase the expression of antiviral genes, Mx1, PKR and ISG15, in large yellow croaker peripheral blood leukocytes (PBLs). The rLcIFNc also exhibited obvious antiviral activity based on less cytopathic effect (CPE) and decreased expression levels of several viral genes in the rLcIFNc-treated grouper spleen (GS) cells following Singapore grouper iridovirus (SGIV) infection. Additionally, rLcIFNc was able to induce the expression of LcIFNc, as well as LcIFNd and LcIFNh in the PBLs and primary head kidney cells (HKCs) from large yellow croaker. These results therefore indicated that LcIFNc not only had antiviral activity, but also mediated the regulation of type I IFN response.

An improved genome assembly for Larimichthys crocea reveals hepcidin gene expansion with diversified regulation and function.

Larimichthys crocea (large yellow croaker) is a type of perciform fish well known for its peculiar physiological properties and economic value. Here, we constructed an improved version of the L. crocea genome assembly, which contained 26,100 protein-coding genes. Twenty-four pseudo-chromosomes of L. crocea were also reconstructed, comprising 90% of the genome assembly. This improved assembly revealed several expansions in gene families associated with olfactory detection, detoxification, and innate immunity. Specifically, six hepcidin genes (LcHamps) were identified in L. crocea, possibly resulting from lineage-specific gene duplication. All LcHamps possessed similar genomic structures and functional domains, but varied substantially with respect to expression pattern, transcriptional regulation, and biological function. LcHamp1 was associated specifically with iron metabolism, while LcHamp2s were functionally diverse, involving in antibacterial activity, antiviral activity, and regulation of intracellular iron metabolism. This functional diversity among gene copies may have allowed L. crocea to adapt to diverse environmental conditions.

Induction of type I interferons in response to bacterial stimuli in large yellow croaker Larimichthys crocea.

In addition to the crucial roles in coordinating antiviral immune responses, type I interferons (IFNs) also play a role in the host immunity against bacterial pathogens. Our previous study identified two type I IFNs from large yellow croaker Larimichthys croaea(Lc), LcIFNd and LcIFNh, and showed their strong induction by poly(I:C) and antiviral activities. In the present study, both LcIFNd and LcIFNh were found to be rapidly induced in head kidney and spleen by mixed bacteria of Vibrio alginolyticus, Vibrio parahaemolyticus, and Aeromonas hydrophila. In the head kidney primary cells (HKCs), expression of these two LcIFN genes was increased by peptidoglycan (PGN) from Bacillus subtilis and lipopolysaccharide (LPS) from Escherichia coli. Consistently, Lc IFN-regulatory factor (LcIRF) 3 and LcIRF7, two key transcription factors of type I IFN expression, were also induced by these three bacteria, PGN, and LPS. These observations strongly suggested that large yellow croaker type I IFNs are involved in the immune response against bacterial infection. Luciferase assays showed that promoters of both LcIFNd and LcIFNh were activated by PGN, LPS, and genomic DNA of A. hydrophila, and A. hydrophila DNA was more potent than PGN and LPS in activating LcIFNd and LcIFNh promoters. Furthermore, the induction of LcIFNd promoter by these bacterial stimuli was further enhanced by the overexpression of LcIRF7 or LcIRF7 along with LcIRF3, while that of LcIFNh promoter was increased following the overexpression of LcIRF3 alone, suggesting that the induction of these two large yellow croaker IFNs by bacterial stimuli may be regulated via distinct manners. These results therefore revealed novel aspects of the functional regulation of teleost type I IFNs.