Manipulated C5aR1 over/down-expression associates with IL-6 expression during bacterial inflammation in half-smooth tongue sole (Cynoglossus semilaevis).

The complement component 5a/complement component 5 receptor 1 (C5a/C5aR1) pathway plays a crucial role in the onset and development of inflammation, but relevant studies in fish are lacking. In this study, we successfully characterized the relationship between half-smooth tongue sole (Cynoglossus semilaevis) C5aR1 (CsC5aR1) and bacterial inflammation. First, we showed that the overexpression of CsC5aR1 significantly increased bacterial pathological damage in the liver and intestine, whereas inhibition attenuated the damage. The in vitro experiments suggested that CsC5aR1 was able to positively regulate the phagocytic activity and respiratory burst of tongue sole macrophages. In terms of both transcriptional and translational levels, overexpression/inhibition of CsC5aR1 was followed by a highly consistent up-regulation/decrease of its downstream canonical inflammatory factor interleukin-6 (CsIL-6). Furthermore, we stimulated macrophages by lipopolysaccharide (LPS) and lipoteichoic acid (LTA) and found a broad-spectrum response to bacterial infections by the C5a/C5aR1 complement pathway together with the downstream inflammatory factor CsIL-6. Subsequently, we directly elucidated that CsIL-6 is an indicator of C5a/C5aR1-mediated inflammation at different infection concentrations, different infectious bacteria (Vibrio anguillarum and Mycobacterium marinum), and different detection levels. These results might provide a new inflammation bio-marker for early warning of bacteria-induced hyperinflammation leading to fish mortality and a promising target for the treatment of bacterial inflammation in teleost.

Isolation and characterization of monocyte/macrophage from peripheral blood of half smooth tongue sole (Cynoglossus semilaevis).

In the present study, the peripheral blood cells of half smooth tongue sole (Cynoglossus semilaevis) were examined by blood smear under the light microscopy. The proportion of main types of blood cells are as following: erythrocyte occupied the majority (92.3%), followed by thrombocyte (4.15%), granulocyte (1.7%), lymphocyte (1.5%) and monocyte (0.3%), respectively. Meanwhile, the isolation method of monocytes was established, by density gradient centrifugation to isolate mononuclear leukocytes of peripheral blood. In primary culture, the monocytes were adhered to the bottom of the flask without feeder cells and separated easily with suspended leukocytes in the medium in 3 h. After suspended leukocytes were removed, the monocytes multiplied rapidly with the two doubly during the 24 h, then the cells proliferated and kept stable until 48 h. When co-cultured with suspended leukocytes after three days, the monocytes could derive to typical macrophages, of which the size enlarged significantly and showed various forms such as like fried eggs, and giant irregular shape with pseudopod because cells fusion or deformation occurred until macrophages died in about two weeks. Monocytes showed strong respiratory burst activity after treated with Phorbol ester PMA and challenged by bacteria respectively. In addition, macrophage of half smooth tongue sole had typical macrophage features such as phagocytic capability, positive esterase activity, and the considerable expression of M-CSFR, MHC-II, IL-6, IL-10, TNF and arginase genes. That arginase expression in macrophages (3d and 5d after differentiation) was upregulated fluctuant suggest that the cultivation was mixture of alternatively activated type macrophage (M2) in the majority while the classically activated type (M1) win the minority. Furthermore, MHC-Ⅱ, M-CSFR and IL-6 were significantly induced following LPS challenge. Collectively, the present study will be useful for the study on half smooth tongue sole immune systems and immune function.

Expression Profile Analysis of miR-221 and miR-222 in Different Tissues and Head Kidney Cells of Cynoglossus semilaevis, Following Pathogen Infection.

Half-smooth tongue sole (Cynoglossus semilaevis) is an important marine commercial fish species in China, which suffers from widespread disease outbreaks. Recently, in this regard, our group identified immune-related microRNAs (miRNAs) of C. semilaevis following Vibrio anguillarum infection. Furthermore, miRNA microarray was utilized to characterize the immune roles of important miRNA candidates in response to bacterial infection. Therefore, in the present study, we characterized miR-221 and miR-222 and profiled their expression after challenge. Here, miR-221 and miR-222 precursors were predicted to have a typical hairpin structure. Both miRNAs were expressed in a broad range of tissues in C. semilaevis, while miR-221 and miR-222 were significantly differentially expressed in the immune tissues of C. semilaevis among three small RNA libraries [control group (CG), bacteria-challenged fish without obvious symptoms of infection (NOSG), and bacteria-challenged fish with obvious symptoms of infection (HOSG)]. In order to further characterize and understand the immune response of miR-221 and miR-222, therefore, we profiled miR-221 and miR-222 expression in selected immune tissues after challenge with V. anguillarum. Both miR-221 and miR-222 were upregulated in the liver and spleen, while different expression patterns were observed in the head kidney. In addition, in half-smooth tongue sole head kidney cell line after challenge with lipopolysaccharide (LPS), polyinosinic:polycytidylic acid (poly I:C), peptidoglycan (PGN), and red-spotted grouper nervous necrosis virus (RGNNV), both miR-221 and miR-222 showed significant difference in expression response to pathogen. Meanwhile, the target gene of miR-221 and miR-222 was predicted, which indicated that tumor necrosis factor receptor-associated factor 6 (TRAF6) and interleukin-1 beta (IL-1ß) were the target genes of miR-221 and miR-222, respectively. Collectively, these findings indicated that miR-221 and miR-222 have putative roles in innate immune response during C. semilaevis exposure to pathogens. Our findings could expand the knowledge of immune function of C. semilaevis miRNA and guide future studies on C. semilaevis immunity.

Complement factor I from flatfish half-smooth tongue (Cynoglossus semilaevis) exhibited anti-microbial activities.

Complement factor I (Cfi) is a soluble serine protease which plays a crucial role in the modulation of complement cascades. In the presence of substrate modulating cofactors (such as complement factor H, C4bp, CR1, etc), Cfi cleaves and inactivates C3b and C4b, thereby controlling the complement-mediated processes. In this study, we sequenced and characterized Cfi gene from Cynoglossus Semilaevis (designated as CsCfi) for the first time. The full-length cDNA of CsCfi was 2230 bp in length, including a 98 bp 5'-untranslated region (UTR), a 164 bp 3'-UTR and a 1968 bp open reading frame (ORF). It encoded a polypeptide of 656 amino acids, with a molecular mass of 72.28 kDa and an isoelectric point of 7.71. A signal peptide was defined at N-terminus, resulting in a 626-residue mature protein. Multiple sequence alignment revealed that Cfi proteins were well conserved with the typical modular architecture and identical active sites throughout the vertebrates, which suggested the conserved function of Cfi. Phylogenetic analysis indicated that CsCfi and the homologous Cfi sequences from teleosts clustered into a clade, separating from another clade from the cartilaginous fish and other vertebrates. Tissue expression profile analysis by quantitative real-time PCR (qRT-PCR) showed that CsCfi mRNA constitutively expressed in all tested tissues, with the predominant expression in liver and the lowest in stomach. Temporal expression levels of CsCfi after challenging with Vibrio anguillarum showed different expression patterns in intestine, spleen, skin, blood, head kidney and liver. The recombinant CsCfi (rCsCfi) protein showed broad-spectrum antimicrobial activities against the Gram-positive bacteria Staphylococcus aureus and the Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa and Shewanella putrefaciens. The research revealed that CsCfi plays an important role in C. Semilaevis immunity.

Channel catfish (Ictalurus punctatus) protein disulphide isomerase, PDIA6: molecular characterization and expression regulated by bacteria and virus inoculation.

Protein disulfide isomerases (PDIs) are thought to aid protein folding and assembly by catalyzing formation and shuffling of cysteine disulfide bonds in the endoplasmic reticulum (ER). Currently, increasing evidence suggests PDIs play an important role in host cell invasion and they are relevant targets for the host immune response. However the roles of specific PDIs in teleosts are little known. Here, we characterized the Protein disulfide isomerase family A, member 6 (PDIA6) from channel catfish, Ictalurus punctatus (named as ccPDIA6). The catfish ccPDIA6 gene was homologous to those of other vertebrate species with 13 exons and 12 introns. The consensus full-length ccPDIA6 cDNA contained an ORF of 1320 bp encoding a putative protein of 439 amino acids. It had a 19 amino acid signal peptide and two active thioredoxin-like domains. Sequence of phylogenic analysis and multiple alignments showed that ccPDIA6 was conserved throughout vertebrate evolution. Southern blot analysis suggested the presence of one copy of the ccPDIA6 gene in the catfish genome. Tissue distribution shows that ccPDIA6 was expressed in all examined tissues at the mRNA level. When using the aquatic zoonotic pathogens such as Edwardsiella tara, Streptococcus iniae, and channel catfish reovirus （CCRV） to challenge channel catfish, ccPDIA6 expression was significant changed in immune-related tissues such as head kidney, intestine, liver and spleen. The results suggested that ccPDIA6 might play an important role in the immunity of channel catfish. This is the first report that the PDI gene may be involved in fish host defense against pathogen infection.

Identification and expression analysis of goose-type lysozyme in half-smooth tongue sole (Cynoglossus semilaevis).

Lysozymes are considered to be potent innate immune molecules against the invasion of bacterial pathogens. The goose-type lysozyme is one of the three major distinct lysozyme types identified in the animal kingdom including teleosts. In this report, we identified, sequenced, and characterized the goose-type lysozyme gene (CsGLys) from half-smooth tongue sole (Cynoglossus semilaevis). The full-length cDNA of CsGLys is 1191 bp in length from the transcription start site to polyadenylation site, including a 91 bp 5'-terminal untranslated region (UTR), a 452 bp 3'-terminal UTR and a 648 bp open reading frame (ORF) of encoding a polypeptide with 215 amino acids. The deduced amino acid sequence of CsGLys possesses a Goose Egg White Lysozyme (GEWL) domain with three conserved residues (E91, D104 and D121) essential for catalytic activity. The CsGLys gene consisting of 2535 bp, was similar to those of other teleost species such as Japanese flounder and large yellow croaker with five exons interrupted by four introns. The 5'-flanking region of CsGLys gene shows several transcriptional factor binding sites related to immune response. Tissue expression profile analysis by quantitative real-time reverse transcription PCR showed that CsGLys mRNA was constitutively expressed in all examined tissues with the predominant expression in skin and the weakest expression in heart. The expression of CsGLys after challenged with bacteria Vibrio anguillarum was up-regulated in blood, head kidney, liver and spleen at 12 h post-infection and it reached the peak level at the same time point with a 19.89-, 4.21-, 14.45- and 10.37-fold increase, respectively, while the CsGLys expression was down-regulated to lower level than the normal level in each tested tissues except in liver from the 48 h until 96 h. These results suggest that CsGLys might play an important role in half-smooth tongue sole host defense against the bacteria infection.

Molecular characterization and gene expression of the channel catfish ferritin H subunit after bacterial infection and iron treatment.

Ferritins are the major iron storage protein in the cytoplasm of cells, responsible for regulating levels of intracellular iron. Ferritin genes are widely distributed in both prokaryotes and eukaryotes. In mammals, ferritin molecules are composed of heavy- (H) and light- (L) chain subunits; amphibian genomes contain three ferritin-type genes (H; middle, M; and L subunits); and teleost genomes to date contain H and M subunits. The objective of this study was to characterize the ferritin H gene in channel catfish (Ictalurus punctatus) to determine its genomic organization and copy numbers, to determine its patterns of tissue expression, and to establish if it is involved in defense responses of catfish after bacterial infection. The catfish ferritin H gene was completely sequenced and characterized, using both mRNA and genomic DNA. Catfish ferritin H gene has a full-length mRNA sequence of 999 bp, an open reading frame of 534 bp, and 4,704 bp genomic DNA sequence. Catfish ferritin H has a 5 exon and 4 intron genetic organization, containing a long 5'-untranslated region, which shares high similarity with mammalian and zebrafish genes. Based on phylogenetic analyses, the catfish ferritin H gene is highly conserved throughout evolution. Southern blot analysis suggested that the ferritin H gene has only one copy in the catfish genome. The catfish ferritin H gene was widely expressed in various healthy tissues. The catfish ferritin H gene was significantly up-regulated in the liver after intraperitoneal injection of iron dextran and coinjection of Edwardsiella ictaluri and iron dextran treatment, suggesting its role in iron metabolism and immunity.

Molecular cloning and expression analysis of the myostatin gene in sea perch (Lateolabrax japonicus).

Myostatin (MSTN) is a member of the transforming growth factor-beta (TGF-beta) superfamily that functions as a negative regulator of skeletal muscle development and growth in mammals. However, few reports are available about the structure and function of MSTN in teleost. Here, the MSTN gene was cloned from sea perch (Lateolabrax japonicus) by homology cloning and genomic walking. In the 4873-bp genomic sequence, three exons, two introns, and 5' and 3' flanking sequences were identified. The sea perch MSTN gene encodes a 374-amino acid protein, including a signal peptide, conserved cysteine residues, and a RXXR proteolytic cleavage domain. Expression analysis of MSTN revealed that MSTN was highly expressed in eyes, brain, and muscle; intermediately in intestine; and weakly in gill, spleen, liver, and heart. It was demonstrated that MSTN mRNA was highly expressed in embryonic stem cell line (LJES1), but it was undetectable in several types of somatic cell lines from sea perch, including fibroblast-like cell, epithelioid cell, and lymphocyte-like cell. Further, it was demonstrated that the 5' flanking region of the MSTN gene can drive the expression of green fluorescent protein (GFP) reporter gene in LJES1 cells and transgenic zebrafish (Danio rerio). This is the first report on the expression profile of MSTN gene in various types of cell cultures.