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Background: Fish gut microbiota undergo dynamic changes under the influence of many factors and play an important role in the nutrition, immunity and development in fish. Although common carp (Cyprinus carpio L.) is an economically important freshwater fish, there are few reports on its gut microbiota changes at different early developmental stages. In the present study, the gut microbiota of common carp during the early developmental stages and its correlation with the feed and pond water flora were studied using the Illumina MiSeq sequencing platform., Results: The results showed that the gut microbiota of common carp underwent continuous and mild changes over the development process, and the pond water environment might provide bacterial resources and have a certain influence on the changes in the gut microbiota of common carp. However, host selection pressure played a more important role in shaping the gut microbiota. Although the gut microbiota was affected by many factors, the presence of core microbiota indicated that some bacterial species adapt to the gut microenvironment of common carp and played a role in its growth process., Conclusions: The dynamic changes of gut microbiota of carp in early development stage were related to the feed, water environment and host selection. The results of this study provide a theoretical basis for healthy farming and disease prevention of common carp., (© 2024. The Author(s).)

Stimulator of interferon genes (STING), as an imperative adaptor protein in innate immune, responds to nucleic acid from invading pathogens to build antiviral responses in host cells. Aberrant activation of STING may trigger tissue damage and autoimmune diseases. Given the decisive role in initiating innate immune response, the activity of STING is intricately governed by several posttranslational modifications, including phosphorylation and ubiquitination. Here, we cloned and characterized a novel RNF122 homolog from common carp (named CcRNF122L). Expression analysis disclosed that the expression of CcRNF122L is up-regulated under spring viremia of carp virus (SVCV) stimulation in vivo and in vitro. Overexpression of CcRNF122L hampers SVCV- or poly(I:C)-mediated the expression of IFN-1 and ISGs in a dose-dependent way. Mechanistically, CcRNF122L interacts with STING and promotes the polyubiquitylation of STING. This polyubiquitylation event inhibits the aggregation of STING and the subsequent recruitment of TBK1 and IRF3 to the signaling complex. Additionally, the deletion of the TM domain abolishes the negative regulatory function of CcRNF122L. Collectively, our discoveries unveil a mechanism that governs the STING function and the precise adjustment of the innate immune response in teleost., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

NLRP3 inflammasome can be activated by a variety of stimuli and plays an important role in protecting host from pathogen invasion and maintaining homeostasis. However, the activation mechanism of NLRP3 inflammasome in fish is still unclear. In the present study, the NLRP3 gene (CcNLRP3) was identified from common carp, which was 3069 bp in length and encoded a protein with five domains. Sequence analysis showed that NLRP3 was evolutionarily conserved, and CcNLRP3 was closely related to that in grass carp and zebrafish. Real-time PCR showed that CcNLRP3 was widely expressed in various immune-related tissues of healthy common carp, and significantly increased after stimulation with E. tarda, A. hydrophila and Cyprinus spring viremia virus (SVCV), suggesting that CcNLRP3 might be involved in the immune defense of common carp. The results of co-IP, spot formation, oligomerization and fluorescence localization showed that CcNLRP3 could interact with CcASC and assemble into inflammasome. The cytotoxicity assays showed that CcNLRP3 inflammasome was involved in the pyroptosis induced by CcGSDME. At the same time, CcNLRP3 could directly interact with CcCaspase-A/B and result in increased Caspase-B enzyme activity and LDH release, indicating that CcNLRP3 could also form inflammasome through ASC-independent pathway. Taken together, the results provide targets and theoretical basis for the prevention and control of infectious diseases in aquaculture., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Stimulator of interferon genes (STING) serve as an endoplasmic reticulum (ER) protein and modulates innate immune responses to viral contagion. Most investigations involving teleost STING antiviral immunity have examined DNA viruses. Therefore, fish STING signaling events against RNA viruses require additional exploration. Here, common carp STING (named CcSTING) was cloned and characterized. The bioinformatics analyses of CcSTING showed evolutionary conservations and were most closely related to other cyprinid STINGs. Immunofluorescence staining discovered that the CcSTING was chiefly placed in the cytoplasm, specifically within the ER. CcSTING was ubiquitously generated in all analyzed organs, with especially strong expression in the gills and head kidney. Spring viremia of carp virus (SVCV) stimulation and poly(I:C) infection induced the generation of CcSTING in immune-associated organs, as well as in peripheral blood leukocytes. Additional investigations revealed that CcSTING overexpression strongly suppressed SVCV replication in EPC cells. Mechanistically, CcSTING enhanced IFN-1 and ISGs expression following SVCV infection. CcSTING also substantially increased both IFN and NF-κB promoter luciferase activity via a dosage-dependent fashion. Lastly, CcSTING significantly up-regulated both TBK1 and p65 phosphorylation. Collectively, these findings demonstrated the critical role and underlying mechanism of fish STING in response to RNA virus., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Stimulator of interferon genes (STING) is an endoplasmic reticulum (ER)-associated protein that plays critical roles in innate immunity and pathogenesis of various diseases. To date, teleost STING against viral stimulation has been identified, whereas STING signaling events in fish against bacteria are not well understood. In the present study, the open reading frame (ORF) of STING from Asian swamp eel (Monopterus albus) was cloned (named MaSTING) and its roles in bacterial infection were investigated. Amino acid sequence alignment and phylogenetic analysis revealed that MaSTING had conserved structures with mammalian STING and shared the closest relationship with mandarin fish STING. Subcellular localization analysis showed that MaSTING distributed in the whole cytoplasm and mainly co-localized with ER. Expression pattern analysis found that MaSTING was constitutively expressed in all the examined tissues with the highest expression in the liver and spleen. Post stimulation with bacteria and various PAMPs, the expression of MaSTING was induced at indicated time points in the immune-related organs and isolated peripheral blood leucocytes. Furthermore, the mechanism underlying MaSTING against bacterial infection was further studied. The qPCR analysis showed that MaSTING overexpression promoted 2'3'-cGAMP induced the expression of IFN-1, ISG15, Viperin, Mx, IL-1β and TNF-α. Western blotting assay suggested that MaSTING significantly enhanced the phosphorylation of TANK-binding kinase 1 (TBK1) and p65. MaSTING also significantly increased the luciferase activity of IFN-1 and NF-κB promoters. Taken together, MaSTING is involved in host defense against bacterial infection by inducing the inflammatory response., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Gasdermin family proteins are important effector proteins mediating pyroptosis and play an important role in innate immune response. GSDME can be cleaved by inflammatory Caspases at specific sites, releasing an active form of N-terminal fragment that binds to the plasma membrane to form pores and release cellular contents. Here, two GSDME genes, CcGSDME-like (CcGSDME-L) and CcGSDMEa, were cloned from common carp. The sequence similarity of the two genes were very high and more similar to DrGSDMEa of zebrafish in evolution. The expression levels of CcGSDME-L and CcGSDMEa can respond to the stimulation of Edwardsiella tarda. The results of cytotoxicity assay showed that CcGSDMEs were cleaved by the activation of canonical CcNLRP1 inflammasome, leading to obvious pyroptosis characteristics and increased cytotoxicity. In EPC cells, three CcCaspases responded to intracellular LPS stimulation and induced significantly cytotoxicity. In order to clarify the molecular mechanism of CcGSDME-induced pyroptosis, the N-terminal of CcGSDME-L (CcGSDME-L-NT) was expressed in 293T cells, which showed strong cytotoxicity and obvious pyroptosis characteristics. Fluorescence localization assay showed that the CcGSDME-L-NT was expressed on cell membrane, and CcGSDMEa-NT was located on the cell membrane or some organelle membranes. These findings can enrich the knowledge of CcNLRP1 inflammasome and GSDMEs mediated pyroptosis in common carp, and provide basic data for the prevention and treatment of fish infectious diseases., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Tripartite motifs (TRIM) is a large family of E3 ubiquitin ligases that play an important role in ubiquitylation. TRIM proteins regulate a wide range of biological processes from cellular response to viral infection and are implicated in various pathologies, from Mendelian disease to cancer. Although the TRIM family has been identified and characterized in tetrapods, but the knowledge about common carp and other teleost species is limited. The genes and proteins in the TRIM family of common carp were analyzed for evolutionary relationships, characterization, and functional annotation. Phylogenetic analysis was used to elucidate the evolutionary relationship of TRIM protein among teleost and higher vertebrate species. The results show that the TRIM orthologs of highly distant vertebrates have conserved sequences and domain architectures. The pairwise distance was calculated among teleost species of TRIMs, and the result exhibits very few mismatches at aligned position thus, indicating that the members are not distant from each other. Furthermore, TRIM family of common carp clustered into six groups on the basis of phylogenetic analysis. Additionally, the analysis revealed conserved motifs and functional domains in the subfamily members. The difference in functional domains and motifs is attributed to the evolution of these groups from different ancestors, thus validating the accuracy of clusters in the phylogenetic tree. However, the intron-exon organization is not precisely similar, which suggests duplication of genes and complex alternative splicing. The percentage of secondary structural elements is comparable for members of the same group, but the tertiary conformation is varied and dominated by coiled-coil segments required for catalytic activity. Gene ontology analysis revealed that these proteins are mainly associated with the catalytic activity of ubiquitination, immune system, zinc ion binding, positive regulation of transcription, ligase activity, and cell cycle regulation. Moreover, the biological pathway analyses identified four KEGG and 22 Reactome pathways. The predicted pathways correspond to functional domains, and gene ontology which proposes that proteins with similar structures might perform the same functions.

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., (© 2022. The Author(s).)

Inflammatory Caspases are key effectors of the inflammasomes and play an important role in innate immune response. However, there are few studies on the homologs of inflammatory Caspases in bony fish. In the present study, three inflammatory Caspase genes were cloned from common carp and named CcCaspase-A1, CcCaspase-A2 and CcCaspase-B. Nucleotide sequences alignment revealed that the three Caspases were very similar in structure, which contained a PYD domain in the N-terminal, and a CASc domain in the C-terminal. In the phylogenetic tree, CcCaspase-A1 and CcCaspase-A2 were close to the Caspase-A of grass carp, and CcCaspase-B was close to the DrCaspase-B of zebrafish. In healthy common carp, the expression levels of CcCaspase-A1 and CcCaspase-A2 were the highest in the gills, and CcCaspase-B was the highest in the spleen. After immune stimulation with Edwardsiella tarda or Aeromonas hydrophila, the expression levels of all CcCaspases increased significantly. The fluorescence localization assays showed that all these CcCaspases were expressed in the cytoplasm, and were involved in the assembly of CcNLRP1 inflammasome. These results suggest that the inflammatory CcCaspases play a key role in immune response of common carp against bacterial infection, which may enrich the knowledge of inflammasome in fish, and provide basic data for the prevention and treatment of fish infectious diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The common carp ( Cyprinus carpio L.) is an important farmed species worldwide. Mucosal-associated lymphoid tissues play an essential role in the fight against pathogen infection. Spring viremia of carp virus (SVCV) poses a serious threat to the common carp aquaculture industry. Understanding the molecular mechanisms driving mucosal immune responses to SVCV infection is critical. In this study, the mucosal tissues (gills, foregut and hindgut) were collected from normal and infected fishes for transcriptome analysis. A total of 932,378,600 clean reads were obtained, of which approximately 80% were successfully mapped to the common carp genome. 577, 1,054 and 1,014 differential expressed genes (DEGs) were identified in the gills, foregut and hindgut, respectively. A quantitative polymerase chain reaction assay indicated that the DEGs expression in the foregut following SVCV infection was consistent with the transcriptome results. Among them, two key genes of the retinoic acid-inducible gene I (RIG-I)-like receptor family, melanoma-differentiation-associated gene 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2) (i.e., Cc MDA5 and Cc LGP2), underwent further analysis. Overexpression of Cc MDA5 or Cc LGP2 increased phosphorylation of TANK-binding kinase 1 and interferon regulatory factor 3 and the expression of interferon-1 ( ifn-1 ), myxovirus resistance ( mx ), viperin and interferon-stimulated gene 15 ( isg15 ), and inhibited SVCV replication in epithelioma papulosum cyprini cells. Furthermore, Cc LGP2 significantly upregulated the Cc MDA5-induced ifn-1 mRNA expression and the activation of the ifn-1 promoter. Finally, confocal microscopy and coimmunoprecipitation experiments revealed that Cc LGP2 colocalizes and interacts with Cc MDA5 via the C-terminal regulatory domain. This study provides essential gene resources for understanding the fish immune response to SVCV infection and sheds light on the potential role of fish LGP2 in the MDA5 regulation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Liu, Niu, Qi, Li, Yang and Shan.)

Common carp (Cyprinus carpio L.) is one of the most widely cultivated fish in China. Spring viraemia of carp virus (SVCV) is a highly pathogenic virus and has often caused excessive losses in carp pond fisheries. Innate immune play important roles against virus infection. To better understand the immune response of common carp against SVCV infection, transcriptome analysis was performed using the Illumina Novaseq 6000 platform. It was showed that a total of 3953 differentially expressed unigenes were identified, and the RLR signaling pathway were significantly enriched after SVCV infection. Subsequently, the role of RLR signaling pathway in SVCV infection was studied. The results showed that common carp RIG-I (CcRIG-I) and TRIM25 (CcTRIM25) significantly decreased the replication of SVCV by inducing the phosphorylation of TBK1, IRF3 and p65 and the expression of ifn-1, viperin, isg15 and mx. Further studies illustrated that CcTRIM25 could positive regulate CcRIG-I mediated downstream signaling pathway. Finally, the mechanism of CcTRIM25 promoting CcRIG-I-mediated signaling was investigated. CcTRIM25 could interact with the caspase activation and recruitment domain (CARD) of CcRIG-I and promoted K63-linked polyubiquitination of CcRIG-I. Altogether, the study revealed a mechanism of CcTRIM25 regulating CcRIG-I mediated immune response in SVCV infection., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Mannose receptor (MR), as a member of the C-type lectin (CLEC) family, plays an important role in the internalize pathogen-associated ligands and activate immune response. In the present study, MR was identified and characterized from Asian swamp eel (Monopterus albus) (namely MaMR). The open reading frame of MaMR was 4311 bp in length encoding 1437 amino acids of a ∼162.308 kDa protein, including a cysteine-rich (CR) domain, a fibronectin type II (FNII) domain, eight C-type lectin-like domains (CTLDs), a transmembrane domain and a short cytoplasmic domain. Phylogenetic analysis indicated that MaMR shared the highest similarity with that of Paralichthys olivaceus. The expression of MaMR was found in all the examined tissues, with the highest expression in the spleen and kidney. After injection with Edwardsiella tarda, the transcript level of MaMR was initially reduced and then significantly elevated in the liver, spleen, foregut and hindgut. In the isolated peripheral blood leukocytes, the expression of MaMR was significantly induced post stimulated with LPS and LTA. Then the MaMR-CTLD4-8 recombinant protein was purified. Bacterial agglutination and binding assay showed that rMaMR-CTLD4-8 could bind with both Gram-positive and Gram-negative bacteria and agglutinate bacteria in the presence of calcium in vitro. Further analysis revealed that MaMR and TLR2 coordinately induced the expression of TRAF6 and promoted the phosphorylation level of p65, leading to the expression of proinflammatory cytokines il-1β and tnf-α in EPC cells. Taken together, these results reveal that MaMR plays an important role in the immune response of fish to pathogen infections., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

NLRP1 (NLR family pyrin domain containing 1) is the first member of NOD-like receptors (NLRs) which can form inflammasome and play critical roles in innate immunity and pathogenesis of various diseases. To date, many NLRs and inflammasome-related genes have been identified in teleost, however, the activation of NLRP1 inflammasome is only found in zebrafish, and the activator of fish NLRP1 is unclear. In the present study, the activation of CcNLRP1 inflammasome and its function in innate immune defence of common carp was investigated. The expression of CcNLRP1 was induced in immune-related tissues of common carp upon challenge with Edwardsiella tarda and Aeromonas hydrophila. The colocalization of CcNLRP1 and CcASC, ASC oligomerization, and interaction between CcNLRP1 CARD and CcASC was observed in 293T, Hela and EPC cells, suggesting that the CcNLRP1 inflammasome was activated in common carp. Furthermore, we found that MDP may be the specific ligand of CcNLRP1, which can activate the CcNLRP1 inflammasome. Taken together, the present study identifies a new inflammasome in common carp, and is beneficial to the control of infectious diseases in carp farming., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Toll-like receptors (TLRs) are a class of pattern recognition receptors (PRRs) that play a critical role in innate immune responses against pathogens. In the present study, a fish-specific TLR14 was identified and characterized from Monopterus albus (named MaTLR14), which consisted of a 2658 bp open reading frame encoding a protein of 885 amino acids. Phylogenetic analysis revealed that MaTLR14 belong to the TLR1 subfamily and shared the highest similarity to Paralichthys olivaceus TLR14. Immunohistochemistry assay showed that MaTLR14 mainly located in intestinal epithelial cells of hindgut. Immunofluorescence revealed that MaTLR14 largely localized to the intracellular region and partially co-localized with cell membrane of HeLa cells. The expression levels of MaTLR14 were upregulated in the liver, spleen, foregut and hindgut post infection with Aeromonas hydrophila. When stimulated with LPS and Flagellin, the MaTLR14 expression was elevated in isolated peripheral blood leukocytes. Further studies showed that recombinant MaTLR14-LRR could bind to both the gram-negative and gram-positive bacteria and cause agglutination. Subsequently, the signaling pathway of MaTLR14 was investigated. Confocal microscopy and co-immunoprecipitation assay demonstrated that MaTLR14 recruited MyD88 as adaptor. When overexpressed, MaTLR14 augmented the expression of TRAF6 and phosphorylation of ERK and p65, activated NF-κB and AP-1 and elicited the expression of il-6 and tnf-α. Collectively, MaTLR14 plays an important role in the microorganism recognition and signaling transduction., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., (© 2021. The Author(s).)

TRIF, an important adaptor downstream of Toll-like receptor signaling, plays a critical role in the innate immune response. In this study, the full-length coding sequence of TRIF from common carp ( Cyprinus carpio L.) was cloned and characterized. Bioinformatics analysis showed that common carp TRIF exhibited a conserved TIR domain and had the closest relationship with grass carp TRIF. Expression analysis revealed that TRIF was constitutively expressed in the examined tissues of common carp, with the highest expression in the spleen and the lowest expression in the head kidney, and could be upregulated under Aeromonas hydrophila and poly(I:C) stimulation in vivo and under poly(I:C), LPS, PGN, flagellin, and Pam3CSK4 stimulation in vitro . Laser confocal microscopy showed that common carp TRIF colocalized with the Golgi apparatus. A luciferase reporter assay showed that carp TRIF elicited the activity of ifn-1 and nf-κb through the C-terminal domain. Additionally, crystal violet staining and qPCR assays revealed that carp TRIF inhibited the replication of SVCV in epithelioma papulosum cyprini (EPC) cells. Then, the signaling downstream of carp TRIF was investigated. Coimmunoprecipitation and Western blotting analysis demonstrated that carp TRIF interacted with TBK1 and augmented the expression of TRAF6 and phosphorylation of TBK1. Overexpression of carp TRIF significantly enhanced the expression of interferon-stimulated genes and inflammatory cytokines. Furthermore, flow cytometric (FCM) analysis suggested that carp TRIF induced apoptosis through the activation of caspase-8. In summary, our study indicated that TRIF plays an essential role in the innate immune responses of common carp against bacterial and viral infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Liu, Liu, Song, Qi, Li, Yang and Shan.)

Toll-like receptor 19 (Tlr19) is a fish-specific TLR that plays a critical role in innate immunity. In the present study, we aimed to identify tlr19 from common carp (Cyprinus carpio L.) and explored its expression profile, localization, adaptor, and signaling pathways. A novel tlr19 cDNA sequence (Cctlr19) was identified in common carp. Phylogenetic analysis revealed that CcTlr19 was most closely related to Danio rerio Tlr19. Subcellular localization analysis indicates that CcTlr19 was synthesized in the free ribosome and then transported to early endosomes. Cctlr19 was constitutively expressed in all the examined tissues, with the highest expression in the brain. After poly(I:C) and Aeromonas hydrophila injection, the expression of Cctlr19 was significantly upregulated in immune-related organs. In addition, the expression of Cctlr19 was upregulated in head kidney leukocytes (HKL) upon stimulation with different ligands. Immunofluorescence and luciferase analyses indicate that CcTlr19 recruited TRIF as an adaptor. Furthermore, CcTlr19 can activate the expression of ifn-1 and viperin. Taken together, these findings lay the foundation for future research to investigate the mechanisms underlying fish tlr19.

Intelectin (ITLN) is a type of glycan-binding lectin involved in many physiological processes and some human diseases. Here we report a common carp intelectin (cITLN). Like other orthologs, cITLN also contains a conserved fibrinogen-related domain (FReD) and a unique intelectin domain, expresses in all the tissues tested with the highest level in the hindgut, and responds to bacterial challenge in the acute phase. We also expressed cITLN in Escherichia coli (E. coli) system, and the purified recombinant cITLN could neither affect the surface of bacteria nor inhibit the growth of bacteria, but it can agglutinate both gram-positive and gram-negative bacteria in a calcium-dependent manner. The cITLN's ability of agglutination of gram-positive bacteria is stronger than that of gram-negative bacteria. This is probably because recombinant cITLN could binding peptidoglycan (PGN) with a higher degree to lipopolysaccharide (LPS). Our results of cITLN provided new insight into the function of intelectin in the intestinal mucosal immunity., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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.

p65 is an important subunit of the transcription factor NF-κB in the regulation of immune response. In the present study, the p65 cDNA was identified from common carp (Cyprinus carpio L.) (named Ccp65). Phylogenetic analysis revealed that Ccp65 located in the same clade as piscine p65 and exhibited closest relationship to that of Ctenopharyngodon idella. Ccp65 was constitutively expressed in all the examined tissues. Aeromonas hydrophila and poly(I:C) can induce the expression of Ccp65 in the designated tissues and the Ccp65 expression was up-regulated in HKLs following LPS and poly(I:C) stimulation. In addition, the nuclear localization signal (NLS) and C-terminal domain are the important elements of Ccp65. Immunofluorescence assay revealed that the nuclear localization signal deletion mutation of Ccp65 (Ccp65ΔNLS) failed to translocate to the nucleus even though stimulation with poly(I:C) or LPS, and the C-terminal domain deletion mutation of Ccp65 (Ccp65ΔC) did not up-regulate the luciferase activity. Furthermore, Ccp65 can induce the expression of il-1β and tnf-α. And LPS and poly(I:C) inducing the expression of il-1β and tnf-α, is dependent on the Ccp65. Taken altogether, these findings lay the foundations for future research to investigate the mechanisms underlying fish p65., (Copyright © 2019. Published by Elsevier Ltd.)

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., (© 2019 The Fisheries Society of the British Isles.)

Background: Although teleost fish developed acquired immunity firstly in evolution, innate immunity is still very important for them. Innate immunity depends on pattern recognition receptors (PRRs) to distinguish "self" and "non-self", Peptidoglycan (PGN) recognition protein (PGRP) is one of the receptors and it can bind to multiple components of bacterial envelope., Results: We report the cloning and expression analysis of two PGRPs (Ccpgrp5 and Ccpgrp6) from common carp (Cyprinus carpio L). The Ccpgrp5 gene encodes a protein of 199 amino acid (aa) with PGRP domain, Ami_2 domain and four Zn 2+ binding sites required for amidase activity, but without signal peptide and transmembrane domain. The Ccpgrp6 gene encodes a protein of 446 aa with PGRP domain, Ami_2 domain, signal peptide, five Zn 2+ binding sites required for amidase activity and two sites for N-glycosylation. The phylogenetic analysis revealed that the CcPGRP5 and CcPGRP6 are closely related to Ctenopharyngodon idella and Danio rerio. Ccpgrp5 and Ccpgrp6 were expressed in all tissues examined including liver, spleen, muscle, oral epithelium, head kidney, gill, skin, gonad, brain, foregut and hindgut and showed different distribution characteristics. During the embryonic and early larval developmental stages of common carp, Ccpgrp6 was detected to be highly expressed at 10 days post fertilization(dpf) and 36 dpf, while Ccpgrp5 were hardly detected using Real-time quantitative PCR. After being challenged with Aeromonas hydrophila, Ccpgrp5 in adult common carp was induced and up-regulated in all the tissues, especially in gill and spleen, but not in head kidney, while Ccpgrp6 was up-regulated in all the tissues, especially in liver, head kidney and gill. The varied expression profiling of Ccpgrp5 and Ccpgrp6 indicated they had different roles in the host immune response., Conclusions: These results indicated the two PGRPs, especially Ccpgrp6, played an important role in the immune defense of common carp during larva development and against Aeromonas hydrophila, providing insight to further exploration of protecting fish against bacteria infectious disease.

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 CcTlr8 exhibited closest relationship to that of Ctenopharyngodon idella and Danio. rerio. Subcellular localization analysis indicated that CcTlr8 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 CcTlr8 might recruit TIRAP as the adaptor. Furthermore, Luciferase reporter assays revealed that CcTlr8 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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are a large group of cytoplasmic pattern recognition receptors (PRRs), which play an important role in pathogen recognition and regulation of innate immune response. In fish, NLRs are divided into three distinct subfamilies: NLR-A resembling mammalian NODs, NLR-B resembling mammalian NALPs and fish-specific NLR-C. Presently, no data is available about the common carp NLR gene, and meanwhile the studies concerning fish NLR-C subfamily genes are relatively poor. In the present study, we cloned and characterized a novel NLRC gene (CcNLRC) from common carp. The full-length cDNA of CcNLRC was 3642 bp, with an ORF of 3078 bp encoding 1025 amino acids. CcNLRC appears to be unique to fish, consisting of a fish-specific NACHT associated (FISNA) domain, a NACHT domain, three LRR motifs and an extra B30.2 domain at C-terminus. Expression analysis revealed that CcNLRC was constitutively expressed in various healthy tissues, and during early developmental stages CcNLRC had two expression peaks (1 dpf and 24 dpf). In vivo stimulation with polyI:C and V. anguillarum showed significant up-regulation of CcNLRC expression in some immune-related tissues including liver, spleen, foregut, hindgut and skin. Additionally, in vitro study in common carp PBLs and HKLs stimulated with different ligands such as polyI:C, flagellin and PGN showed enhanced gene expression of CcNLRC. These results suggested that CcNLRC might play an important role in the innate immune defense of common carp against pathogen invasion., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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 CcTlr18 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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Common carp (Cyprinus carpio L.), one of the most economically valuable commercial farming fish species in China, is often infected by a variety of viruses. As the first line of defence against microbial pathogens, the innate immune system plays a crucial role in teleost fish, which are lower vertebrates. Interferon (IFN) regulatory factor 5 (IRF5) is a key molecule in antiviral immunity that regulating the expression of IFN and other pro-inflammatory cytokines. It is necessary to gain more insight into the common carp IFN system and the function of fish IRF5 in the antiviral and antibacterial response., Results: In the present study, we characterized the cDNA and genomic sequence of the IRF5 gene in common carp, and analysed tissue distribution and expression profile of this gene in response to polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccharides (LPS) treatment. The common carp IRF5 (ccIRF5) gene is 5790 bp in length and is composed of 9 exons and 8 introns. The open reading frame (ORF) of ccIRF5 is 1554 bp, and encodes 517 amino acid protein. The putative ccIRF5 protein shares identity (65.4-90.0 %) with other fish IRF5s and contains a DNA binding domain (DBD), a middle region (MR), an IRF-associated domain (IAD), a virus activated domain (VAD) and two nuclear localization signals (NLSs) similar to those found in vertebrate IRF5. Phylogenetic analysis clustered ccIRF5 into the IRF5 subfamily with other vertebrate IRF5 and IRF6 genes. Real-time PCR analysis revealed that ccIRF5 mRNA was expressed in all examined tissues of healthy carps, with high levels observed in the gills and the brain. After poly I:C challenge, expression levels of ccIRF5, tumour-necrosis factor α (ccTNFα) and two IFN stimulated genes [ISGs (ccISG5 and ccPKR)] were up-regulated in seven immune-related tissues (liver, spleen, head kidney, foregut, hindgut, skin and gills). Furthermore, all four genes were up-regulated in vitro upon poly I:C and LPS challenges., Conclusions: Our findings suggest that IRF5 might play an important role in regulating the antiviral and antibacterial response in fish. These results could provide a clue for preventing common carp infection by pathogenic microorganisms present in the aquatic environment.

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.

Interferon regulatory factors (IRFs) are a family of transcription factors that mediate the transcriptional regulation of interferon (IFN) genes and IFN-inducible genes. In this study, IRF-1 gene is cloned from the common carp, Cyprinus carpio L., named CcIRF-1. The full-length cDNA of CcIRF-1 is 1427 bp, including an open reading frame of 861 bp encoding a protein of 286 amino acids. The putative CcIRF-1 is characterized by a conserved DNA-binding domain and includes a signature of six conserved tryptophan residues. The genomic sequence of CcIRF-1 is described, which consists of 9 exons and 8 introns. The sequence analysis shows that CcIRF-1 is clustered into IRF-1 subfamily, and has the closest relationship with the zebrafish IRF-1. CcIRF-1 is found constitutively expressed in different organs of healthy common carp. The main findings are that CcIRF-1 is up-regulated following stimulation with poly(I:C) in all tested tissues. Moreover, the downstream gene of IRF-1 - IFN is found to be correlated with the up-regulation of IRF-1 after injection with poly(I:C). Furthermore, we also isolate the peripheral blood leukocytes (PBLs) and find that there is a relevance between the expression profile of CcIRF-1 and IFN in poly(I:C) stimulated PBLs., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

LEAP-2 is a cysteine-rich cationic antimicrobial peptide (AMP) playing an important role in host innate immune system. LEAP-2 genes have been identified from higher vertebrates and several fish species. Here we report the cloning and identification of two LEAP-2 cDNA sequences from the liver of common carp (Cyprinus carpio L.). The LEAP-2A cDNA was 1325 bp long and contained an ORF of 279 bp encoding a protein of 92 amino acids. The LEAP-2B cDNA was 608 bp long and contained an ORF of 276 bp encoding a protein of 91 amino acids. Both LEAP-2 proteins consisted of 41 amino acid residues and shared four cysteines at the conserved positions in the predicted mature peptides, highly similar to LEAP-2 of other species. Sequence alignment showed that LEAP-2 amino acid sequences were well conserved in different species, and the phylogenetic relation of LEAP-2 was coincident with evolution of biological species. Expression analysis data revealed that LEAP-2A and LEAP-2B mRNAs were expressed in a wide range of common carp tissues including liver, spleen, head kidney, skin, gills, hindgut and foregut. When injected intraperitoneally with Vibrio anguillarum, the expression level of common carp LEAP-2A was quickly up-regulated in liver, spleen, head kidney, skin, gills, foregut and hindgut, however, the expression level of LEAP-2B was similarly up-regulated in spleen, skin, gills and hindgut but not in liver, head kidney and foregut. Our results showed that the LEAP-2A had a markedly high constitutive expression in skin, and the LEAP-2A and the LEAP-2B had a significantly high up-regulated expression after stimulus in skin. This differential expression of LEAP-2 in common carp suggests that it may play a key role in immune responses against invading pathogens and both LEAP-2 molecules may be involved in mucosal immunity., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

β-defensins are a group of cysteine-rich cationic antimicrobial peptides that play antibacterial and antiviral roles in immune systems of vertebrates. Here, we report the cloning and identification of a β-defensin 3 cDNA sequence from the common carp (Cyprinus carpio L.). Sequence alignment and phylogenetic analysis indicated that this β-defensin 3 belonged to the BD-2 group of fish. Real-time PCR showed that the β-defensin 3 mRNA was expressed in all the tissues of normal common carp that we examined and was highly expressed in the spleen and gills. When challenged with Vibrio anguillarum, the expression level of common carp β-defensin 3 mRNA was quickly upregulated in various tissues. Our results indicate that the β-defensin 3 showed markedly high constitutive expression in the gills, and significantly upregulated expression in the hindgut of the common carp after infection, suggesting it plays an important role in the innate and mucosal immunity of common carp.