Mechanism of Chinese sturgeon IFN-γ inhibition on Mycobacterium marinum (Acipenser sinensis).

IFN-γ plays a crucial role in both innate and adaptive immune responses and is a typical Th1 cytokine that promotes Th1 response and activates macrophages. When macrophages were incubated with IFN-γ, their phagocytosis ratio against Mycobacterium marinum increased significantly, as observed under fluorescence microscopy. The macrophages engulfed a large number of M. marinum. The proliferative ability of macrophages treated with IFN-γ was significantly weaker on the 4th and 7th day after phagocytosis and subsequent re-infection with marine chlamydia (P < 0.001). This suggests that IFN-γ enhances the phagocytosis and killing ability of macrophages against M. marinum. IFN-γ protein also significantly increased the production of reactive oxygen species (H2O2) and nitric oxide (NO) by macrophages. Additionally, the expression levels of toll-like receptor 2 (tlr2) and caspase 8 (casp8) were significantly higher in macrophages after IFN-γ incubation compared to direct infection after 12 h of M. marinum stimulation. Apoptosis was also observed to a higher degree in IFN-γ incubated macrophage. Moreover, mRNA expression of major histocompatibility complex (MHC) molecules produced by macrophages after IFN-γ incubation was significantly higher than direct infection. This indicates that IFN-γ enhances antigen presentation by upregulating MHC expression. It also upregulates tlr2 and casp8 expression through the TLR2 signaling pathway to induce apoptosis in macrophages. The pro-inflammatory cytokine showed an initial increase followed by a decline, suggesting that IFN-γ enhances the immune response of macrophages against M. marinum infection. On the other hand, the anti-inflammatory cytokine showed a delayed increase, significantly reducing the expression of pro-inflammatory cytokines. The expression of both cytokines balanced each other and together regulated the inflammatory reaction against M. marinum infection.

Functional characterization of group â ¡ interferon, IFNf in the acipenseriform fish, Chinese sturgeon (Acipenser sinensis).

Teleost fish possess a diversity of type Ⅰ interferons (IFNs) repertoire, which play a crucial role in antiviral and antimicrobial immune responses. In our previous study, IFNe1-3 and IFNb were identified and cloned from Chinese sturgeon (Acipenser sinensis), an acipenseriform fish. However, the absence of Chinese sturgeon genome data has left the question of whether there are other type Ⅰ IFN members in this species unresolved. In this study, we have identified and characterized a novel IFN, IFNf in Chinese sturgeon (AsIFNf). Bioinformatics analysis revealed that the AsIFNf contains a unique disulfide bond (2 cysteines) located in the second exon and fifth exon region, distinguishing it from other reported teleost type I IFNs. Meanwhile, qPCR results showed that AsIFNf mRNA was detectable in all examined tissues and up-regulated in the spleen or kidney in response to poly I: C, Citrobacter freundii, and Spring Viremia of Carp Virus (SVCV), but not by LPS. Furthermore, compared to recombinant AsIFNe2 protein (rAsIFNe2), rAsIFNf exhibited a stronger protective effect on Chinese sturgeon fin cells against SVCV and also induced higher expression of antiviral genes Mx and viperin. Importantly, AsIFNf displayed characteristics similar to antimicrobial peptides (AMPs) with a positive charge and demonstrated a broad spectrum of antimicrobial activity in vitro. These findings provide a theoretical foundation for understanding the primitive structure and function of interferon, as well as deepening our comprehension of the innate immune system and disease defense in the endangered Chinese sturgeon.

Pathogenicity of Plesiomonas shigelloides and Citrobacter freundii isolated from the endangered Chinese sturgeon (Acipenser sinensis).

Chinese sturgeon (Acipenser sinensis) is an indigenous species of China and is listed as a critically endangered species. Recently, second filial generations of Chinese sturgeon in the Yangtze River Fisheries Research Institute suffered from a severe disease. In this study, two kinds of pathogenic bacteria were isolated from diseased sturgeon and identified as Plesiomonas shigelloides and Citrobacter freundii, based on 16S rDNA gene sequence alignment analysis. Antimicrobial susceptibility testing showed that P. shigelloides was resistant to ampicillin, penicillin, midecamycin, oxacillin, and clindamycin; and sensitive to tocefatriaxone, piperacillin, cefoperazone, cefazolin, and ciprofloxacin. C. freundii was resistant to ampicillin, penicillin, midecamycin, oxacillin, and clindamycin; and sensitive to chloramphenicol, cefuroxime, norfloxacin, ciprofloxacin, and ceftazidime. The median lethal dose (LD50) values of P. shigelloides and C. freundii were 4.50 × 103 colony forming units (CFU)/g and 3.20 × 103 CFU/g, respectively. Clinical symptoms of challenged sturgeons were the same as those of naturally infected sturgeons. Histopathological examination disclosed severe damage in the viscera of P. shigelloides and C. freundii-infected sturgeons. This is the first report suggesting that P. shigelloides infection is associated with mortality of Chinese sturgeon. The results of this study revealed the pathogenesis and severe pathogenicity of P. shigelloides and C. freundii in cultured Chinese sturgeon, and offer insights into the prevention and treatment of bacterial infection caused by P. shigelloides and C. freundii in cultured sturgeons.

Host-microbiota interactions and responses to grass carp reovirus infection in Ctenopharyngodon idellus.

Gut microbiota could facilitate host to defense diseases, but fish-microbiota interactions during viral infection and the underlying mechanism are poorly understood. We examined interactions and responses of gut microbiota to grass carp reovirus (GCRV) infection in Ctenopharyngodon idellus, which is the most important aquaculture fish worldwide. We found that GCRV infection group with serious haemorrhagic symptoms (G7s) showed considerably different gut microbiota, especially with an abnormally high abundance of gram-negative anaerobic Cetobacterium somerae. It also showed the lowest (p < 0.05) alpha-diversity but with much higher ecological process of homogenizing dispersal (28.8%), confirming a dysbiosis of the gut microbiota after viral infection. Interestingly, signaling pathways of NOD-like receptors (NLRs), toll-like receptors (TLRs), and lipopolysaccharide (LPS) stimulation genes were significantly (q-value < 0.01) enriched in G7s, which also significantly (p < 0.01) correlated with the core gut microbial genera of Cetobacterium and Acinetobacter. The results suggested that an expansion of C. somerae initiated by GCRV could aggravate host inflammatory reactions through the LPS-related NLRs and TLRs pathways. This study advances our understanding of the interplay between fish immunity and gut microbiota challenged by viruses; it also sheds new insights for ecological defense of fish diseases with the help of gut microbiota.

Molecular characterization and expression analysis of Asian swamp eel (Monopterus albus) CXC chemokine receptor (CXCR) 1a, CXCR1b, CXCR2, CXCR3a, CXCR3b, and CXCR4 after bacteria and poly I:C challenge.

The CXC chemokine receptors (CXCRs) play critical roles in innate and adaptive immune systems. In this study, six Asian swamp eel (Monopterus albus) CXCRs (MaCXCR1-4) were identified and their molecular characterization and expression patterns were analyzed. The open reading frames (ORFs) of MaCXCR1a, MaCXCR1b, MaCXCR2, MaCXCR3a, MaCXCR3b, and MaCXCR4 were 1074 bp (base pairs), 1080 bp, 1125 bp, 1146 bp, 1083 bp, and 1140 bp, and encoded proteins of 357 aa (amino acids), 359 aa, 374 aa, 381 aa, 360 aa, and 379 aa, respectively. All these CXCRs have seven conserved transmembrane domains and four cysteines (with the exception of MaCXCR3b). Multiple sequence alignment revealed that the MaCXCRs possess a typical G-protein receptor family 1 signature and a DRY motif. There are also one to four potential N-glycosylation sites in the extracellular regions of the MaCXCRs, mainly distributed in the N-terminus and extracellular hydrophilic loop (ECL) 2 region. Phylogenetic analysis demonstrated that the MaCXCRs were clustered together with homologous proteins from other fish. Taken together with the amino acid identity and similarity analysis, these results suggested that the MaCXCRs are conserved with other homologous genes, in which CXCR4 is more conserved than CXCR1-3. The MaCXCRs loci showed conserved synteny among teleost fish, and we found that human CXCR1 shares a common ancestor with fish CXCR1a. MaCXCRs were constitutively expressed in a wide range of tissues (especially in immune-related tissues) with different expression levels, suggesting that the MaCXCRs have different roles in un-stimulated tissues, and may play vital roles under normal conditions. MaCXCRs showed different fold changes in the spleen after Aeromonas veronii and polyinosinic-polycytidylic acid (poly I:C) challenge, which suggested that MaCXCR1a and MaCXCR3a have longer antiviral activities compared with their antibacterial functions, and that MaCXCR1b possesses stronger antiviral than antibacterial activity. MaCXCR4 may play vital roles during bacterial and viral infection; however, MaCXCR2 has relatively small effect in antibacterial and antiviral responses. The differential responses of these genes to bacteria and poly I:C implied the differences in the mechanisms of defense against viruses and bacteria.

Sequence analysis and characterization of type I interferon and type II interferon from the critically endangered sturgeon species, A. dabryanus and A. sinensis.

In the present study, we identify three type I interferon (IFN) genes (Ad/AsIFNe1-3) and a type II IFN gene (Ad/AsIFNγ) from the Dabry's sturgeon (Acipenser dabryanus) and the Chinese sturgeon (Acipenser sinensis). Sequence analysis revealed that Ad/AsIFNe1-3 and Ad/AsIFNγ contain several conserved characteristics, including signal peptides, interferon alpha, beta, and delta (IFabd) domains, and N-glycosylation sites. Ad/AsIFNe1-3 belongs to the type I IFN group I subgroup, possessing two conserved cysteines residues (C1 and C3), and Ad/AsIFNγ contained a conserved nuclear localization sequence (NLS) motif. Ad/AsIFNe1-3 and Ad/AsIFNγ contain signature motifs indicative of their corresponding IFN group. The Ad/AsIFNe1-3 and Ad/AsIFNγ genes were found to consist of 5 exons/4 introns and 4 exons/3 introns, respectively. These IFNs were separated by four phase 0 introns (type I IFN) and three phase 0 introns (type II IFN). The sequences of IFNe1-3 and IFNγ from the Dabry's sturgeon and the Chinese sturgeon were closely aligned, suggested that these two species are closely related. Phylogenetic analysis revealed that Ad/AsIFNe1-3 and Ad/AsIFNγ clustered together with the corresponding homologous proteins from other fish species. AdIFNe1-3 were found to be high expressed in early embryonic development, suggesting that AdIFNe1-3 might indicate maternal transmission, while AdIFNγ may not mediate embryonic development. Tissue distribution analysis revealed that AdIFNe1-3 and AdIFNγ carry out biological functions in immune and non-immune tissues compartments. AdIFNe1-3 and AdIFNγ can be stimulated by polyinosinic-polycytidylic acid (poly I:C) and lipopolysaccharides (LPS). AdIFNe1-3 have stronger antiviral activity than AdIFNγ, and AdIFNγ has a stronger antibacterial activity than AdIFNe1-3. The differential responses of these genes to poly I:C and LPS suggest differences in the mechanisms of defense against viruses and bacteria.

Sequence characterization and expression pattern analysis of six kinds of IL-17 family genes in the Asian swamp eel (Monopterus albus).

Interleukin-17 (IL-17) is an important cytokine that plays a critical role in the inflammatory response and host defense against extracellular pathogens. In the present study, six novel IL-17 family genes (MaIL-17) were identified by analyzing Asian swamp eel (Monopterus albus) genome. Sequence analysis revealed that the MaIL-17 family genes shared similar features, comprising a signal peptide, an IL-17 superfamily region, and four conserved cysteines. Phylogenetic analysis showed that the MaIL-17 genes were clustered together with their corresponding IL-17 genes from other species. The similarity and identity of all IL-17 family genes indicated that the MaIL-17 genes are conserved among teleosts, while Ma-IL-17D is more conserved than the other Ma-IL-17s. Except for MaIL-17A/F3 and MaIL-17D, all MaIL-17s shared the same genomic structure as the genes from other fish, namely three exons and two introns. The MaIL-17s showed conserved synteny among fish, and we found that the MaIL-17D locus has a more conserved syntenic relationship with the loci from other fish and humans. These results demonstrated that MaIL-17D and human IL-17D might have evolved from a common ancestral gene and subsequently diverged. The analysis of swamp eel reference genes revealed that EEF1A1 (encoding eukaryotic translation elongation factor 1 alpha 1) was an ideal reference gene for accurate real-time qRT-PCR normalization in the swamp eel. The MaIL-17 genes are widely distributed throughout tissues, suggesting that MaIL-17s carry out their biological functions in immune and non-immune tissues compartments. The transcript of Ma-IL17s exhibited different fold changes in head kidney cells in response to Aeromonas veronii phorbol 12-myristate 13-acetate (PMA) and polyinosinic:polycytidylic acid (poly I:C) challenge, showing that MaIL-17A/F1 has stronger antiviral activities compared with other MaIL-17 family genes, and that MaIL-17A/F3 and MaIL-17A/F2 possess stronger effects against extracellular pathogens compared with the others; however, MaIL-17C2 and MaIL-17D may play vital roles during pathogen infection. The differential immune responses of these genes to Aeromonas veronii, PMA and poly I:C implied distinct mechanisms of host defense against extracellular pathogens.

Functional characterization of interferon regulatory factor 5 and its role in the innate antiviral immune response.

In mammals, type I interferons (IFNs) are primarily regulated by transcription factors of the IFN regulatory (IRF) family. Interferon regulatory factor 5 (IRF-5) plays pivotal roles in antiviral and inflammatory responses. In the present study, we found that zebrafish (Danio rerio) IRF5 is a key player in the regulation of the expression of type I IFN and its antiviral immune response. IRF5 was upregulated in zebrafish embryonic fibroblast cells (ZF4) when challenged with grass carp reovirus (GCRV). Moreover, the expression profiles of Mx, IFN, Viperin, and IRF7, but not IRF3, were upregulated by overexpression of IRF5 in Epithelioma papulosum cyprinid cells (EPCs). Luciferase assays revealed that the activation of the IFNϕ1 promoter was stimulated by overexpression of IRF5 and IRF5-△IAD (IRF5 lacking the IRF-associated domain), respectively. However, overexpression of IRF5 or IRF5-△IAD inhibited the activity of the IFNϕ3 promoter. IRF5-△DBD (lacking the DNA-binding domain) had no influence in the activation of the IFNϕ1 and IFNϕ3 promoters. Furthermore, the determination of the cytopathic effect (CPE) numbers and viral titers revealed that the viral concentration was reduced by ectopic expression of IRF5 in EPC cells. Ectopic expression of IRF5 in EPC cells could protect cells from GCRV and significantly inhibited GCRV virus replication. These data indicated that IRF5 could limit viral replication through an IFN-dependent pathway.

Transcriptome analysis of the critically endangered Dabry's sturgeon (Acipenser dabryanus) head kidney response to Aeromonas hydrophila.

Dabry's sturgeon (Acipenser dabryanus), as a living fossil, is considered a critically endangered aquatic animal in China. To date, the immune system of this species remains largely unknown, with limited available sequence information. In addition, increasing incidence of bacterial pathogenic diseases has been reported. Hence, the present study aimed to characterize comprehensively transcriptome profile of the head kidney from Dabry's sturgeon infected with Aeromonas hydrophila using Illumina platform. Over 42 million high-quality reads were obtained and de novo assembled into a final set of 195240 unique transcript fragments (unigenes), with an average length of 564 bp. Approximately 41702 unigenes were annotated in the NR NCBI database. Dabry's sturgeon unigenes had the highest number of hits with 14365 (34.45%) to Lepisosteus oculatus. The 195240 unigenes were assigned to three Gene Ontology (GO) categories: biological process, cellular component, and molecular function. Among them, 27770 unigenes were clustered into 26 Eukaryotic Orthologous Group (KOG) functional categories, and 36031 unigenes were mapped to 335 known Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. After A. hydrophila administration, 1728 differentially expressed unigenes were identified, including 980 upregulated and 748 downregulated unigenes. Further KEGG enrichment analysis of these unigenes identified 16 immune-related pathways, including the Toll-like receptor signaling pathway, chemokine signaling pathway, complement and coagulation pathway, RIG-I-like receptor signaling pathway, and NOD-like receptor signaling pathway. 20 DEGs were selected and their expression patterns are largely consistent with the transcriptome profile analysis, which clearly validated the reliability of the DEGs in transcriptome analysis. This work revealed novel gene expression patterns of Dabry's sturgeon host defense and contributes to a better understanding of the immune system and defense mechanisms of Dabry's sturgeon in response to bacterial infection. The results provide valuable references for studies in sturgeons that lack complete genomic sequences, and could also be helpful for the analyzing evolution among cartilaginous and teleost fish.

Molecular characterization of three toll-like receptors (TLR21, TLR22, and TLR25) from a primitive ray-finned fish Dabry's sturgeon (Acipenser dabryanus).

Dabry's sturgeon (Acipenser dabryanus) is a useful model for the study of fish evolution, as it is one of the most primitive actinopterygian species. However, studies of the immune system of this fish are limited. Here, we identified three toll-like receptors (adaTLR21, adaTLR22, and adaTLR25) from Dabry's sturgeon. The three sturgeon TLRs had characteristic TLR features, including a signal peptide, several leucine rich repeat (LRR) domains, a transmembrane domain, and a Toll/interleukin-1 receptor (TIR) domain. Although the predicted amino acid sequences encoded by the sturgeon adaTLR21, adaTLR22, and adaTLR25 had somewhat low levels of sequence identity and similarity with TLRs from other fish species, the three sturgeon TLRs fell in well-supported clades with other teleost TLRs in our neighbor-joining phylogenetic tree. Real-time quantitative PCR showed that the three sturgeon TLRs were ubiquitously expressed in all examined tissues from healthy adult sturgeon, but that their expression patterns varied greatly among the different tissues. The three sturgeon TLRs were also expressed across all embryonic developmental stages that were examined, but their expression levels differed between developmental stages. All three TLRs were upregulated in head-kidney primary leucocytes following lipopolysaccharide (LPS) and polyinosinic: polycytidylic acid (polyI:C) stimulation. To the best of our knowledge, this is the first characterization of these three TLRs in Darby's sturgeon. Our results provide a framework for further studies of TLR ligand specificity and signaling pathways in sturgeon, and increase our understanding of the functional evolution of TLRs in vertebrates.

Evolution, expression, and characterisation of liver-expressed antimicrobial peptide genes in ancient chondrostean sturgeons.

Liver-expressed antimicrobial peptide 2 (leap-2) is an evolutionarily ancient molecule that acts as the key component in vertebrate innate immunity against invading pathogens. Leap-2 has been identified and characterised in several teleosts, but not yet in chondrosteans. Herein, the complete coding sequences of leap-2b and leap-2c were identified from expressed sequence tags (ESTs) isolated from Dabry's sturgeon (Acipenser dabryanus) and Chinese sturgeon (A. sinensis), designated as adleap-2b, adleap-2c, asleap-2b, and asleap-2c, respectively. Adleap-2b and adleap-2c sequences share 98% and 100% sequence identity with asleap-2b, and asleap-2c, respectively. Sequence alignment revealed that all four genes contain four cysteine residues, conserved in all fish leap-2 homologs, that form two disulfide bonds. Comparative analysis of the exon-intron structure revealed a three exon/two intron structure for that leap-2 genes in animals, but intron 1 is much longer in sturgeons than in other species. The adleap-2c gene was expressed mainly in the liver of Dabry's sturgeon, and transcription of adleap-2c was significantly up-regulated (p < 0.05) in the liver and midkidney in response to Aeromonas hydrophila challenge. These results suggest adleap-2c may contribute to the defence against pathogenic bacterial invasion. The findings further our understanding of the function of adleap-2c and the molecular mechanism of innate immunity in chondrosteans.

Characterization and expression analysis of g- and c-type lysozymes in Dabry's sturgeon (Acipenser dabryanus).

Dabry's sturgeon (Acipenser dabryanus) is mainly distributed in the upper Yangtze River. Although extensively farmed, little information is available on its innate immune system. In this study, we conducted de novo transcriptome assembly of the head kidney to create a comprehensive dataset for A. dabryanus. A total of 51,324,686 high quality reads were obtained from head kidney cDNA library by the Illumina sequencing platform and 131,261 unigenes were determined to contain complete ORFs. The complete coding sequences of g- and c-type lysozymes were identified from unigenes, and designated as ADLysG and ADLysC. Aeromonas hydrophila infection of Dabry's sturgeon caused a significant increase (P < 0.05) in blood for both lysozyme types, confirming their active defensive role against bacterial infections. This research provides the first characterization of these enzymes in an ancestral chondrostean. These data suggest that ADLysG and ADLysC have the potential for immune defense system against bacterial infection.

Expression pattern analysis of IRF4 and its related genes revealed the functional differentiation of IRF4 paralogues in teleost.

In mammals, interferon regulatory factor 4 (IRF4) plays an important role in the process of development and differentiation of B cells, T cells and dendritic cells. It can regulate immune pathway through IRF5, MyD88, IL21, PGC1α, and NOD2. In the present study, we investigated the expression pattern of IRF4 paralogues and these related genes for the first time in teleosts. The results showed that these genes were all expressed predominantly in known immune tissues while IRF5 was also relatively highly expressed in muscle. IRF4b, IL21, MyD88, IRF5 and NOD2 showed maternal expression in the oocyte and the higher expression of IRF4a, Mx and PGC1α before hatching might be involved in the embryonic innate defense system. Zebrafish embryonic fibroblast (ZF4) cells were infected with GCRV and SVCV. During GCRV infection, the expression of Mx was significantly up-regulated from 3 h to 24 h, reaching the highest level at 12 h (101.5-fold over the controls, P < 0.001). And the expression of IRF4a was significantly up-regulated from 3 h to 48 h, reaching the highest level at 12 h (13.75-fold over the controls, P < 0.001). While the expression of IRF4b was only slightly up-regulated at 12 h and 24 h (3.39-fold, 1.93-fold) above control levels, respectively. Whereas the expression of Mx was significantly up-regulated during SVCV infection from 1 h to 48 h, reaching the highest level at 24 h (11.49-fold over the controls, P < 0.001). IRF4a transcripts were significantly up-regulated from 6 h to 24 h, reaching the highest level at 24 h (41-fold over the controls, P < 0.01). IRF4b only showed a slightly up-regulation by SVCV at 24 h (3.2-fold over the controls, P < 0.01). IRF4a and IRF4b displayed a distinct tissue expression pattern, embryonic stages expression and inducible expression in vivo and in vitro, suggesting that IRF4 paralogues might play different roles in immune system.

Bioinformatics and expression analysis of finTRIM genes in grass carp, Ctenopharyngodon idella.

The tripartite motifs (TRIMs) constitute a large family of proteins containing a Really Interesting New Gene (RING) domain, a B-box domain and coiled-coil region followed by different C-terminal domains. TRIM proteins play multiple roles in various cellular processes, including cell growth, differentiation, apoptosis and antiviral immunity. Fish novel large multigene TRIM genes (finTRIM/ftr) appear only in teleosts and play a vital role in antiviral responses. Phylogenetic analysis revealed the existence of different subsets of novel fish TRIM 14 genes (finTRIM14/ftr14), ftr51, ftr67, ftr72, ftr82, ftr83, and ftr99 in grass carp (Ctenopharyngodon idella), suggesting lineage-specific diversification events. Therefore, the number of finTRIM genes varies greatly among species. The ftr genes in grass carp, which are closely related to zebrafish and possess various evolutionary branches, have evolved faster than human TRIMs. The predicted protein domains were almost identical RING zinc finger domains, with the exception of ftr72, the B-box domain (excluding ftr67, ftr82, ftr83), and the B30.2 domain, which evolved under positive selection (with the exception of ftr67, and ftr72). The genes were predominantly expressed in the spleen, gill and head kidney. These findings indicate that the ftr genes in grass carp are involved diverse cellular processes, including innate immune responses.

Analysis of the expression patterns of the novel large multigene TRIM gene family (finTRIM) in zebrafish.

Tripartite motif (TRIM) proteins are receiving increased research interest because of their roles in a wide range of cellular biological processes in innate immunity. In zebrafish (Danio rerio), the functions of the finTRIM (ftr) family are unclear. In the present study, we investigated the expression pattern of ftr12, ftr51, ftr67, ftr82, ftr83, and ftr84 in zebrafish for the first time. The results showed that ftr12, ftr67, and ftr84 are maternally expressed in the oocyte and highly expressed at the early stage (0-4 hpf) of embryo (P < 0.05), suggesting their involvement in the embryonic innate defense system. The ftr82 gene was highly expressed at 8 hpf (P < 0.05), which implied that the embryos could synthesize their own immunity-related mRNAs. However, ftr51 and ftr83 were highest at 8 hpf (2.33 and 51.53 relative to ß-actin respectively) and might mediate embryonic development. The expression levels of ftr12, ftr51, and ftr67 were highest in the gill, intestines, and liver, respectively. Ftr82, ftr83, and ftr84 were predominantly expressed in the kidney, suggesting that these finTRIMs might play roles in both immunity and non-immunity-related tissue compartments. Zebrafish embryonic fibroblast (ZF4) cells were infected with Grass carp reovirus (GCRV) and Spring viremia of carp virus (SVCV). During GCRV infection, the expression of ftr12 was significantly upregulated from 12 h to 24 h; and ftr51 and ftr67 increased from 3 h to 12 h. The expressions of ftr82, ftr83, and ftr84 were only upregulated at 12 h, 12 h, and 24 h, respectively. All of these genes were significantly downregulated at 48 h (P < 0.05). Challenge with SVCV upregulated the expressions of ftr12 and ftr51 at 12 h and 48 h (P < 0.05), respectively, and ftr67 reached its highest expression level at 3 h. ftr82 showed only a slight upregulation at 6 h and 48 h, and ftr83 and ftr84 were consecutively increased, reaching their highest levels at 12 h (P < 0.05). Meanwhile, ftr67 and ftr83 were significantly downregulated at 48 h (P < 0.05). Our research demonstrated that ftr12, ftr51, ftr67, ftr82, ftr83, and ftr84 probably have important roles in innate immune responses and in non-immunity-related tissues.

Identification of the salmonid IL-17A/F1a/b, IL-17A/F2b, IL-17A/F3 and IL-17N genes and analysis of their expression following in vitro stimulation and infection.

This study identifies four new IL-17A/F isoforms in salmonids, as well as IL-17N. IL-17A/F1 and IL-17A/F2 are each represented by two paralogues, with a predicted pseudogene of IL-17N also apparent in the salmonid genome. Analysis of the sequences and genes of the known IL-17A/F and IL-17N molecules suggests that IL-17N is a member within the IL-17A/F subfamily. Analysis of factors that modulated the expression of these genes showed that PHA and PMA were good inducers of salmon IL-17A/F1a and IL-17A/F2a, with rIL-21 a potent stimulator of IL-17A/F1a and IL-17A/F3. The potential involvement of these isoforms during responses post-vaccination and infection was also studied. In unvaccinated control fish, Yersinia ruckeri infection resulted in a marked up-regulation of IL-17A/F1a and IL-17N in the spleen and head kidney and IL-17A/F2a and IL-17A/F3 in the spleen. In the vaccinated fish, only one significant increase was seen relative to control fish, of IL-17A/F2a in the gills, whether the fish were challenged with Y. ruckeri or given the saline placebo. It was also apparent in the gills and head kidney that the level of IL-17A/F1b remained elevated in the Y. ruckeri-challenged fish at a time when it had decreased in saline-injected fish. The relative importance of these isoforms for disease resistance remains to be determined.

Two types of TNF-α exist in teleost fish: phylogeny, expression, and bioactivity analysis of type-II TNF-α3 in rainbow trout Oncorhynchus mykiss.

TNF-α is a cytokine involved in systemic inflammation and regulation of immune cells. It is produced chiefly by activated macrophages as a membrane or secreted form. In rainbow trout, two TNF-α molecules were described previously. In this article, we report a third TNF-α (TNF-α3) that has only low identities to known trout molecules. Phylogenetic tree and synteny analyses of trout and other fish species suggest that two types (named I and II) of TNF-α exist in teleost fish. The fish type-II TNF-α has a short stalk that may impact on its enzymatic release or restrict it to a membrane-bound form. The constitutive expression of trout TNF-α3 was generally lower than the other two genes in tissues and cell lines, with the exception of the macrophage RTS-11 cell line, in which expression was higher. Expression of all three TNF-α isoforms could be modulated by crude LPS, peptidoglycan, polyinosinic:polycytidylic acid, and rIFN-γ in cell lines and primary macrophages, as well as by bacterial and viral infections. TNF-α3 is the most responsive gene at early time points post-LPS stimulation and can be highly induced by the T cell-stimulant PHA, suggesting it is a particularly important TNF-α isoform. rTNF-α3 produced in CHO cells was bioactive in different cell lines and primary macrophages. In the latter, it induced the expression of proinflammatory cytokines (IL-1ß, IL-6, IL-8, IL-17C, and TNF-αs), negative regulators (SOCS1-3, TGF-ß1b), antimicrobial peptides (cathelicidin-1 and hepcidin), and the macrophage growth factor IL-34, verifying its key role in the inflammatory cytokine network and macrophage biology of fish.

Transcriptome Sequencing Reveals the Antiviral Innate Immunity by IFN-γ in Chinese Sturgeon Macrophages.

To further study the biological function of interferon-gamma (IFN-γ) in the Chinese sturgeon (Acipenser sinensis), we conducted a transcriptome analysis of primary macrophages induced by IFN-γ using Illumina sequencing technology. We obtained 88,879 unigenes, with a total length of 93,919,393 bp, and an average length of 1,057bp. We identified 8,490 differentially expressed genes (DEGs) between the untreated and IFN-γ-treated macrophages, with 4,599 upregulated and 3,891 downregulated. Gene ontology (GO) analysis showed that 4,044 DEGs were enriched in the biological, cellular components, and molecular function categories. Kyoto Encyclopedia of Genes and Genomes (KEGG) identified 278 immunity-related pathways enriched for the DEGs. According to the GO enrichment results, eight key immunity-related genes were screened for verification using qPCR. Results indicate that IFN-γ can activate macrophage Interferon Regulatory Factors (IRFs) and type I interferon (IFN-I), activate RIG-I-like and Toll-like receptor-related pathways, and improve the antiviral ability of macrophages in Chinese sturgeon.

Comprehensive comparison of thirteen kinds of cytokine receptors from the endangered fish Chinese sturgeon (Acipenser sinensis).

The interferon receptor system in teleost fish is more complex than that in mammals. In the present study, we identified 13 cytokine receptor genes (10 interferon receptor genes and 3 IL10R2-like genes) from Chinese sturgeon (Acipenser sinensis) using RNA-sequencing. Sequence analysis indicated that these receptors had conserved domains, including signal peptides, FNⅢ, and transmembrane domains. Phylogenetic analysis suggested that they belonged to the cytokine receptor family. In the present study, we named them IFNAR1-like (CRFB5a, CRFB5b), IFNAR2-like (CRFB3a, CRFB3b), IFNGR1-like (IFNGR1), IFNGR2-like (CRFB6a, CRFB6b/IFNGR2-1, CRFB6c/IFNGR2-2, CRFB6d/IFNGR2-3, CRFB6e/IFNGR2-4) and IL10R2-like (CRFB4a, CRFB4b, CRFB4c), respectively. Constitutive expression analysis revealed that these receptor genes had potential functions in immune and non-immune tissue compartments. After stimulating with Poly (I:C), the expression fold changes of CRFB3a, CRFB4a, CRFB4b, CRFB5b, and CRFB6e/IFNGR2-4 in Chinese sturgeon were higher than those of other receptor genes, which revealed that these five genes had important functions in the immune process to resist virus invasion in the host. After stimulating with IFN gamma, the expression fold changes of CRFB3a, CRFB4a, and CRFB6b/IFNGR2-1 were higher than those other receptor genes. Based on other teleost fish interferon receptor models, we speculated that IFNAR1-like (CRFB5a, CRFB5b) and IFNAR2-like (CRFB3a, CRFB3b), comprised Chinese sturgeon type Ⅰ IFN receptors; and IFNGR1-like (IFNGR1) and IFNGR2-like (CRFB6/IFNGR2) comprised Chinese sturgeon type Ⅱ IFN receptors.

FoxO3 Modulates LPS-Activated Hepatic Inflammation in Turbot (Scophthalmus maximus L.).

In mammals, forkhead box O3 (foxo3) plays important roles in liver immune system. The foxo3 can regulate cell cycle, DNA repair, hypoxia, apoptosis and so on. However, as such an important transcription factor, few studies on foxo3 in fish have been reported. The present study characterized the foxo3 in turbot (Scophthalmus maximus L.). Lipopolysaccharide (LPS) incubated in vitro (hepatocytes) and injected in vivo (turbot liver) were used to construct inflammatory models. The foxo3 was interfered and overexpressed to investigate its functions in liver inflammation. The open reading frame (ORF) of foxo3 was 1998 bp (base pair), encoding 665 amino acids. Sequence analysis showed that foxo3 of turbot was highly homologous to other fishes. Tissue distribution analysis revealed that the highest expression of foxo3 was in muscle. Immunofluorescence result showed that foxo3 was expressed in cytoplasm and nucleus. Knockdown of foxo3 significantly increased mRNA levels of tumor necrosis factor-α (tnf-α), interleukin-1ß (il-1ß), interleukin-6 (il-6), myeloid-differentiation factor 88 (myd88), cd83, toll-like receptor 2 (tlr-2) and protein level of c-Jun N-terminal kinase (JNK) in sifoxo3 + LPS (siRNA of foxo3+ LPS) group compared with NC + LPS (negative control + LPS) group in turbot hepatocytes. Overexpressed foxo3 significantly decreased mRNA levels of tnf-α, il-6, nuclear transcription factor-kappa B (nf-κb), cd83, tlr-2 and the protein level of JNK in vitro. In vivo analysis, foxo3 knockdown significantly increased levels of GOT in serum after LPS injection compared with NC+LPS group. Overexpressed foxo3 significantly decreased levels of GPT and GOT in pcDNA3.1-foxo3+LPS group compared with pcDNA3.1+LPS group in vivo. Foxo3 knockdown significantly increased mRNA levels of tnf-α, il-1ß, il-6, nf-κb, myd88 and protein level of JNK in vivo in sifoxo3+LPS group compared with NC+LPS group in turbot liver. Overexpressed foxo3 significantly decreased mRNA levels of il-1ß, il-6, myd88, cd83, jnk and protein level of JNK in pcDNA3.1-foxo3+LPS group compared with pcDNA3.1+LPS group in turbot liver. The results indicated that foxo3 might modulate LPS-activated hepatic inflammation in turbot by decreasing the proinflammatory cytokines, the levels of GOT and GPT as well as activating JNK/caspase-3 and tlr-2/myd88/nf-κb pathways. Taken together, these findings indicated that FoxO3 may play important roles in liver immune responses to LPS in turbot and the research of FoxO3 in liver immunity enriches the studies on immune regulation, and provides theoretical basis and molecular targets for solving liver inflammation and liver injury in fish.