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Background: Interferon (IFN) regulatory factors (IRFs) is a kind of transcription factors, which play an important role in regulating the expression of type I IFN and related genes. In mammals, IRF6 is not relevant with IFN expression, while zebrafish IRF6 was reported to be a positive regulator of IFN expression and could be phosphorylated by both MyD88 and TBK1. However, the role of IRF6 in the immune response and IFN transcription of common carp is unknown. Results: In the present study, the cDNA of IRF6 gene (CcIRF6) was cloned from common carp using RACE technique, with a total length of 1905 bp, encoding 471 amino acid residues, which possesses two functional domains of DBD and IAD. Similarity analysis showed that CcIRF6 had more than 50% similarity with IRFs of other vertebrates, and had the highest similarity with grass carp and zebrafish, among which the DBD domain was much more conserved. The phylogenetic analysis showed that CcIRF6 is in the branch of Osteichthyes and has the closest relationship with grass carp. In healthy common carp, the CcIRF6 was expressed in all the examined tissues, with the highest level in the oral epithelium, and the lowest level in the head kidney. After intraperitoneal injection of poly(I:C) or Aeromonas hydrophila, the expression of CcIRF6 increased in spleen, head kidney, foregut and hindgut of common carp. Moreover, poly(I:C), LPS, PGN and flagellin induced the expression of CcIRF6 in peripheral leukocytes and head kidney leukocytes of common carp in vitro. In EPC cells, CcIRF6 inhibited the expression of some IFN-related genes and pro-inflammatory cytokines, and dual luciferase reporter assay showed that CcIRF6 reduced the activity of IFN and NF-κB reporter genes. Conclusions: The present study suggests that CcIRF6 is involved in the antiviral and antibacterial immune response of common carp, and negatively regulate the expression of IFN and NF-κB signalling pathways, which provides a theoretical basis for the study and prevention of fish disease pathogenesis. [ABSTRACT FROM AUTHOR]

Background: The interferon (IFN) regulatory factors (IRFs) were originally identified as transcription factors playing critical roles in the regulation of IFN-related genes in the signal pathway. In mammals, IRF4 plays a vital role in both the innate and adaptive immune system. This study aims to reveal the molecular characterization, phylogenetic analysis, expression profiles and the regulatory role in the IFN and NF-κB signalling pathways of IRF4 in common carp (Cyprinus carpio. L) (abbreviation, ccIRF4). Results: Here, ccIRF4 was identified and characterized, it contained a DNA binding domain (DBD) which possess five tryptophans and an IRF-associated domain (IAD). The predicted protein sequence of the ccIRF4 showed higher identities with grass carp (Ctenopharyngodon idella) and zebrafish (Danio rerio). Phylogenetic analysis suggested that ccIRF4 has the closest relationship with zebrafish IRF4. Quantitative real-time PCR analysis showed that ccIRF4 was constitutively expressed in all investigated tissues with the highest expression level in the gonad. Polyinosinic:polycytidylic acid (poly I:C) stimulation up-regulated the ccIRF4 expressions in the liver, spleen, head kidney, skin, foregut and hindgut. Upon Aeromonas hydrophila injection, the expression level of ccIRF4 was up-regulated in all tissues with the exception of spleen. In addition, ccIRF4 was induced by lipopolysaccharide (LPS), peptidoglycan (PGN) and Flagellin in head kidney leukocytes (HKLs). Overexpression of the ccIRF4 gene in epithelioma papulosum cyprini cells (EPC) down regulated the expressions of IFN-related genes and proinflammatory factors. Dual-luciferase reporter assay revealed that ccIRF4 decreased the activation of NF-κB through MyD88. Conclusions: These results indicate that ccIRF4 participates in both antiviral and antibacterial immune response and negatively regulates the IFN and NF-κB response. Overall, our study on ccIRF4 provides more new insights into the innate immune system of common carp as well as a theoretical basis for investigating the pathogenesis and prevention of fish disease. [ABSTRACT FROM AUTHOR]

Background: Interferon regulatory factor 2 (IRF2) is an important transcription factor, which can regulate the IFN response and plays a role in antiviral innate immunity in teleost. Results: In the present study, the full-length cDNA sequence of IRF2 (CcIRF2) was characterized in common carp (Cyprinus carpio L.), which encoded a protein containing a conserved DNA-binding domain (DBD) and an IRF-associated domain (IAD). Phylogenetic analysis showed that CcIRF2 was most closely related with IRF2 of Ctenopharyngodon idella. CcIRF2 transcripts were detectable in all examined tissues, with higher expression in the gills, spleen and brain. CcIRF2 expression was upregulated in immune-related tissues of common carp upon polyinosinic:polycytidylic acid (poly (I:C)) and Aeromonas hydrophila stimulation and induced by poly (I:C), lipopolysaccharide (LPS), peptidoglycan (PGN) and flagellin in the peripheral blood leucocytes (PBLs) and head kidney leukocytes (HKLs). In addition, overexpression of CcIRF2 decreased the expression of IFN and IFN-stimulated genes (ISGs), and a dual-luciferase reporter assay revealed that CcIRF2 could increase the activation of NF-κB. Conclusions: These results indicate that CcIRF2 participates in antiviral and antibacterial immune response and negatively regulates the IFN response, which provide a new insight into the regulation of IFN system in common carp, and are helpful for the prevention and control of infectious diseases in carp farming. [ABSTRACT FROM AUTHOR]

In the present study, interferon (IFN) regulatory factor (IRF) 9 gene (irf9) was identified and characterized in common carp Cyprinus carpio. The predicted protein sequence of Irf9 contains a DNA binding domain (DBD) that possess five tryptophans, an IRF association domain (IAD) and two nuclear localisation signals (NLS). Alignment of Irf9 of C. carpio with the corresponding Irf9 proteins of other species showed that the DBD is more highly conserved than the IAD. The putative Irf9 protein sequence of C. carpio shares higher identities with teleosts (53.8–82.3%) and lower identities with mammals (30.2–31.0%). Phylogenetic studies of the putative amino‐acid sequence of IRF9 based on the neighbour‐joining method showed that Irf9 of C. carpio has the closest relationship with the grass carp Ctenopharyngodon idella. Tissue distribution analysis showed that irf9 transcripts were detectable in all examined tissues with the highest expression in the skin and the lowest expression in the head kidney. Poly I:C and Aeromonas hydrophila stimulation up‐regulated irf9 expression in the spleen, head kidney, foregut and hindgut at different time intervals. In addition, irf9 was induced by Poly I:C and lipopolysaccharides (LPS) in vitro. These results indicate that Irf9 participates in antiviral and antibacterial immunity. Transfection of irf9 up‐regulated the expression of cytokines, including type I IFN, protein kinase R (PKR), interferon‐stimulated gene (ISG)15 and tumour necrosis factor (TNF)α in epithelioma papulosum cyprini cells (EPC) upon poly I:C and LPS stimulation. A dual‐luciferase reporter assay revealed that Irf9 has no effect on NF‐κB activation. The present study on Irf9 provides new insights into the IFN system of C. carpio and a valuable experimental platform for future studies on the immune system of fish. [ABSTRACT FROM AUTHOR]

Toll-interacting protein (Tollip) is a mediator involved in the TLRs signaling pathway which is critical for innate immune response. In the present study, a full-length Tollip cDNA was first cloned from common carp ( CcTollip), which was 1284 bp in length, containing an open reading frame of 831 bp encoding a peptide of 276 amino acids. Multiple sequence alignment showed that the CcTollip shared the highest similarity with that of grass carp and zebrafish. Phylogenetically, the CcTollip clustered together well with their piscine family members. Quantitative real-time PCR analysis indicated that CcTollip was widely expressed in all tissues tested and showed up-regulation with challenges of Vibrio anguillarum and poly(I:C), suggesting that CcTollip was activated by V. anguillarum and poly(I:C). These data indicated that CcTollip might play an important role in immune response to bacterial and viral invasion. [ABSTRACT FROM AUTHOR]

Background: Interferon (IFN) regulatory factors (IRFs), as transcriptional regulatory factors, play important roles in regulating the expression of type I IFN and IFN- stimulated genes (ISGs) in innate immune responses. In addition, they participate in cell growth and development and regulate oncogenesis. Results: In the present study, the cDNA sequence of IRF10 in common carp (Cyprinus carpio L.) was characterized (abbreviation, CcIRF10). The predicted protein sequence of CcIRF10 shared 52.7–89.2% identity with other teleost IRF10s and contained a DNA-binding domain (DBD), a nuclear localization signal (NLS) and an IRF-associated domain (IAD). Phylogenetic analysis showed that CcIRF10 had the closest relationship with IRF10 of Ctenopharyngodon idella. CcIRF10 transcripts were detectable in all examined tissues, with the highest expression in the gonad and the lowest expression in the head kidney. CcIRF10 expression was upregulated in the spleen, head kidney, foregut and hindgut upon polyinosinic:polycytidylic acid (poly I:C) and Aeromonas hydrophila stimulation and induced by poly I:C, lipopolysaccharide (LPS) and peptidoglycan (PGN) in peripheral blood leucocytes (PBLs) and head kidney leukocytes (HKLs) of C. carpio. In addition, overexpression of CcIRF10 was able to decrease the expression of the IFN and IFN-stimulated genes PKR and ISG15. Conclusions: These results indicate that CcIRF10 participates in antiviral and antibacterial immunity and negatively regulates the IFN response, which provides new insights into the IFN system of C. carpio. [ABSTRACT FROM AUTHOR]

Abstract Toll-like receptor 8 (Tlr8) is a member of intracellular TLRs family and play a critical role in the innate immunity. In the present study, we aimed to identify tlr8 from common carp (Cyprinus carpio L.), and explored its expression profile, localization, adaptor, and signaling pathways. A novel tlr8 cDNA sequence (Cctlr8) was identified from the carp, containing a signal peptide, a LRR-N-terminal (LRR-NT), 14 leucine-rich repeats, a LRR-C-terminal (LRR-CT), a transmembrane region and a TIR domain. Phylogenetic analysis revealed that Cc Tlr8 exhibited closest relationship to that of Ctenopharyngodon idella and Danio. rerio. Subcellular localization analysis indicated that Cc Tlr8 was localized to the endoplasmic reticulum in both HeLa cells and EPC cells. Quantitative Real-Time PCR analysis demonstrated that Cctlr8 was constitutively expressed in all the examined tissues, with the highest expression observed in the spleen. After poly (I:C) injection, the expression of Cctlr8 was significantly up-regulated in all the tested tissues. In addition, the expression of Cctlr8 was up-regulated in both PBLs and HKLs following poly (I:C) stimulation. The results of immuofluorescence and coimmunoprecipitation analysis indicated that Cc Tlr8 might recruit TIRAP as the adaptor. Furthermore, Luciferase reporter assays revealed that Cc Tlr8 could activate AP-1 in 293 T cells. Taken altogether, these findings lay the foundations for future research to investigate the mechanisms underlying fish tlr8. Highlights • Cc Tlr8 was localized to the endoplasmic reticulum. • The expression of Cctlr18 was up-regulated after challenged with poly (I:C). • Cc Tlr8 recruited TIRAP as the adaptor. • Cc Tlr8 activated AP-1 in 293 T cells. [ABSTRACT FROM AUTHOR]

Toll-like receptors are important pattern recognition receptors that can recognize pathogen-associated molecular patterns (PAMPs) and play a critical role in innate immunity. In the present study, tlr18 was identified from common carp ( Cyprinus carpio L.) (named Cctlr18 ). The deduced amino acid sequence contained only a signal peptide, eight LRR (leucine-rich repeat) motifs, a transmembrane region and a TIR (Toll/IL-1 receptor) domain. Phylogenetic analysis showed that Cc Tlr18 was most closely related to Ctenopharyngodon idella Tlr18. Quantitative real-time PCR analysis showed that Cctlr18 was constitutively expressed in all investigated tissues with the highest expression level in the skin and lowest expression in the gonad. After injection with inactivated Aeromonas hydrophila , Cctlr18 expression was significantly up-regulated in the head kidney, foregut, hindgut and skin. Moreover, significant up-regulation of Cctlr8 was observed in the spleen, head kidney, hindgut and skin after immersion with live A. hydrophila . In addition, the expression of Cctlr18 was up-regulated in PGN or flagellin-stimulated HKLs. Luciferase reporter assays showed that Cctlr18 activated NF-κB in 293 T cells and that NF-κB activity was enhanced in Cctlr18 and Ccmyd88 co-transfected cells. Furthermore, Cctlr18 could induce the expression of cytokines genes, including ifn , il-1β and il-10 , in EPC cells. The results suggested that Cctlr18 plays an important role in the immune response and provides basic information for investigating the mechanisms of fish tlr18 . [ABSTRACT FROM AUTHOR]

Abstract: Hepcidin is a cysteine-rich cationic antimicrobial peptide (AMP), which plays an important role in host innate immune system and iron regulation. A great many of hepcidin genes have been identified from vertebrates, including various fish species. Here we report the cloning and identification of a hepcidin cDNA from the liver of common carp (Cyprinus carpio L.). The full-length cDNA of the common carp hepcidin was 647 bp, which contained an ORF of 276 bp encoding a prepropeptide of 91 amino acid residues. The predicted preprohepcidin consisted of three domains: a signal peptide of 24 amino acids, a prodomain of 42 amino acids and a mature peptide of 25 amino acids, which containd eight cysteine residues at the identical conserved position. The genomic organization of common carp hepcidin was identified, which contained three exons and two introns, similarly to corresponding genes in mammals and other fish species. Sequence alignment and phylogenetic analysis showed that hepcidins were conserved in different species, and common carp hepcidin was type 1 hepcidin according to the phylogenetic tree, which had the highest identity with mud loach and zebrafish. Real-time PCR assay showed that the hepcidin gene was mostly expressed in liver, and expressed in all the other tested tissues of common carp in different levels. When challenged with pathogenic bacterium, Vibrio anguillarum, the expression level of common carp hepcidin was quickly up-regulated in liver, spleen, head kidney and hindgut, implying that hepcidin may be an important component of the innate immune system of common carp and involved in mucosal immune response against invading pathogens. [Copyright &y& Elsevier]