ABSTRACT
In mammals, IL-22 is considered as a critical cytokine regulating of immunity and homeostasis at barrier surfaces. Although IL-22 have been functional characterization in different species of fish, the studies about distinct responses of IL-22 in different organs/tissues/cell types is rather limited. Here, we identified and cloned IL-22 gene (named as Ec-IL-22) from grouper (Epinephelus coioides). Ec-IL-22 gene was detected in all orangs/tissues examined, and was induced in intestine, gill, spleen, head kidney, and primary head kidney/intestine leukocytes following the stimulation of LPS and poly (I:C), as well as Vibrio harveyi and Singapore grouper iridovirus infection (SGIV). In addition, the stimulation of DSS could induce the expression of Ec-IL-22 in intestine and primary leukocytes from intestine. Importantly, the treatment of recombinant Ec-IL-22 induced the mRNA level of proinflammatory cytokines in primary intestine/head kidney leukocytes. The present results improve the understanding of expression patterns and functional characteristics of fish IL-22 in different organs/tissues/cell types.
Subject(s)
Bass , DNA Virus Infections , Fish Diseases , Fish Proteins , Gene Expression Regulation , Interleukin-22 , Interleukins , Vibrio Infections , Vibrio , Animals , Fish Proteins/genetics , Fish Proteins/immunology , Fish Proteins/chemistry , Fish Diseases/immunology , Interleukins/genetics , Interleukins/immunology , Bass/immunology , Bass/genetics , Vibrio/physiology , DNA Virus Infections/immunology , DNA Virus Infections/veterinary , Gene Expression Regulation/immunology , Gene Expression Regulation/drug effects , Vibrio Infections/immunology , Vibrio Infections/veterinary , Amino Acid Sequence , Gene Expression Profiling/veterinary , Phylogeny , Sequence Alignment/veterinary , Immunity, Innate/genetics , Poly I-C/pharmacology , Lipopolysaccharides/pharmacology , Ranavirus/physiologyABSTRACT
The enteritis is a common disease in fish farming, but the pathogenesis is still not fully understood. The aim of the present study was to investigate the inducement of Dextran Sulfate Sodium Salt (DSS) intestinal inflammation on Orange-spotted grouper (Epinephelus coioides). The fish were challenged with 200 µl 3% DSS via oral irrigation and feeding, an appropriate dose based on the disease activity index of inflammation. The results indicated that the inflammatory responses induced by DSS were closely associated with the expression of pro-inflammatory cytokines including interleukin 1ß (IL-1ß), IL-8, IL16, IL-10 and tumor necrosis factor α (TNF-α), as well as NF-κB and myeloperoxidase (MPO) activity. At day5 after DSS treatment, the highest levels of all parameters were observed. Also, the severe intestinal lesions (intestinal villus fusion and shedding), strong inflammatory cell infiltration and microvillus effacement were seen through histological examination and SEM (scanning electronic microscopy) analysis. During the subsequent 18 days of the experimental period, the injured intestinal villi were gradually recovery. These data is beneficial to further investigate the pathogenesis of enteritis in farmed fish, which is helpful for the control of enteritis in aquaculture.
Subject(s)
Bass , Enteritis , Animals , Bass/metabolism , Dextran Sulfate/adverse effects , Inflammation , Enteritis/chemically induced , Enteritis/veterinary , Cytokines/metabolismABSTRACT
As a member of the γc family, interleukin 15 plays an important function in the immune response. In this study, we cloned an IL15 from Epinephelus coioides (named Ec-IL15). The open reading frame of Ec-IL15 is 528 bp, encoding 175 amino acids. Sequence alignment analysis showed that EcIL-15 has a conserved Pfam: IL15 domain and four cysteine residues. Subcellular localization studies have shown that Ec-IL15 is distributed in whole cells. In healthy groupers, Ec-IL15 was expressed in all 11 tissues tested and the highest in liver. After ConA, PHA, LPS and poly I:C stimulation, Ec-IL15 expression of HKLs was significantly upregulated. After V. harveyi infection, the expression of Ec-IL15 in 9 tissues was significantly upregulated and peaked within 48 h. In addition, recombinant Ec-IL15 protein can not only stimulate HKLs proliferation and cytokine expression, but also has the potential as an immune enhancer.
Subject(s)
Bass , Fish Diseases , Animals , Cloning, Molecular , Cysteine , Fish Proteins/chemistry , Interleukin-15/genetics , Lipopolysaccharides/pharmacology , Phylogeny , Poly I , VibrioABSTRACT
Natural killer lysin (Nklysin) is a small molecule antimicrobial peptide produced by natural killer cells and T lymphocytes and widely expressed in vertebrates. Homologues of Nklysin have been found in several fish, but only several of biological activity was identified. In this study, we characterized a Nklysin from grouper (Epinephelus coioides), and explored its expression pattern and biological function in bacterial infection. We also investigated the role of Nklysin in viral replication and maturation. The nklysin gene of grouper encodes a 169 amino acid, sharing 92.90% identity to H. septemfasciatus NKlysin protein, containing a saposin B domain and six well-conserved cysteine residues that necessary for antimicrobial activity by forming three intrachain disulfide bonds. Analysis of qRT-PCR revealed that nklysin gene widely expressed in all tested tissues with the higher expressions in spleen. After bacterial challenge, the nklysin gene expression significantly varied in different tissues. In addition, a large-scale of the recombinant Nklysin protein was secreted in Pichia pastoris strain GS115. The MIC assay showed that the Nklysin protein directly inhibited growth of several pathogens, including Proteus mirabilis, Bacillus subtilis, Salmonella typhi, Escherichia coli, Shigella sonnei and Streptococcus agalactiae. Further analysis showed the Nklysin protein over-expression might prevent viral genes transcriptions and replication in FHM cells. Our findings suggested that the Nklysin of grouper might be a potential agent for antibacterial and antiviral infection in the future.
Subject(s)
Bass , DNA Virus Infections , Fish Diseases , Animals , Bass/genetics , Bass/metabolism , Fish Proteins/chemistry , Antiviral Agents/pharmacology , Amino Acid Sequence , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/metabolism , Escherichia coli/genetics , Recombinant Proteins/genetics , Phylogeny , Gene Expression RegulationABSTRACT
Interleukin-15 (IL15) is a proinflammatory cytokine that could induce the production of inflammatory cytokines. In this study, the α chain of the IL15 receptor of Epinephelus coioides (Ec-IL15Rα), a natural regulator of IL15, was identified, and immune response functions of fish were determined and characterized. Ec-IL15Rα contains a 720 bp open reading frame that encodes 239 amino acids, including four typical conserved cysteine residues with a highly conserved sushi domain. Ec-IL15Rα is closely related to Epinephelus lanceolatus and is the most clustered with teleost. Subcellular localization studies showed that Ec-IL15Rα was situated in the cytoplasm and cell membrane. Ec-IL15Rα was detected in 11 tissues, with the highest expression in the liver and blood. Meanwhile, the Ec-IL15Rα transcriptional levels substantially increased in nine tissues after Vibrio harveyi infection. Ec-IL15Rα was significantly up-regulated in HKLs by ConA, PHA, LPS and poly I:C stimulation. In vitro analysis, the recombinant protein of rEc-IL15Rα stimulates HKL proliferation and IL1R, IL6R, IL10, and IL16 expression. Challenge experiments revealed that IL15Rα protein showed an increase of 6.67-10% survival protection rate after V. harveyi infection. This study provides a better understanding of the immune protection of IL15Rα in vertebrate fish.