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1.
Fish Shellfish Immunol ; 117: 320-327, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34246784

RESUMO

The polymeric immunoglobulin receptor (pIgR) plays an important role in mediating the transcytosis of polymeric immunoglobulins (pIgs) to protect organisms against pathogen invasion. Here, a polyclonal antibody against grass carp (Ctenopharyngodon idellus) recombinant pIgR was developed by immunizing New Zealand white rabbit, and the responses of pIgR, IgM and IgZ were analyzed after bath immunization and intraperitoneal administration with Flavobacterium columnare. The results showed that pIgR transcription level was similar to IgM and IgZ, but pIgR rose much faster and peaked earlier than IgM and IgZ; the pIgR mRNA levels were higher in the skin and spleen for both immunized groups, while IgM and IgZ mRNA expression were higher in skin, gills, and intestines in bath immersion group, or spleen and head kidney in intraperitoneal immunization group. ELISA revealed that the IgM, IgZ and pIgR protein levels were up-regulated in skin mucus, gill mucus, gut mucus and bile, reaching a higher peak level earlier in skin mucus and gill mucus in bath immersion group, but a higher peak level in bile in injection group. Moreover, secretory component molecules were detected in grass carp's skin, gill and intestine mucus and bile, but not in serum, which molecular mass was near the theoretical mass obtained from the sequence of grass carp pIgR. These results demonstrated that bath and intraperitoneal immunization up-regulated pIgR and secretory Ig expression in secretions, which provided more insights into the role of pIgR in immunity and offer insight into ways of protecting teleost against pathogen invasion.


Assuntos
Carpas/imunologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/imunologia , Infecções por Flavobacteriaceae/imunologia , Flavobacterium , Imunoglobulinas/imunologia , Animais , Bile/imunologia , Carpas/microbiologia , Infecções por Flavobacteriaceae/veterinária , Brânquias/imunologia , Muco/imunologia , Coelhos , Proteínas Recombinantes/imunologia , Pele/imunologia
2.
Front Immunol ; 12: 633324, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34262558

RESUMO

In the process of microbial invasion, the inflammation reaction is induced to eliminate the pathogen. However, un-controlled or un-resolved inflammation can lead to tissue damage and death of the host. MicroRNAs (miRNAs) are the signaling regulators that prevent the uncontrolled progress of an inflammatory response. Our previous work strongly indicated that miR-142a-3p is related to the immune regulation in grass carp. In the present study, we found that the expression of miR-142a-3p was down-regulated after infection by Aeromonas hydrophila. tnfaip2 and glut3 were confirmed as be the target genes of miR-142a-3p, which were confirmed by expression correlation analysis, gene overexpression, and dual luciferase reporter assay. The miR-142a-3p can reduce cell viability and stimulate cell apoptosis by targeting tnfaip2 and glut3. In addition, miR-142a-3p also regulates macrophage polarization induced by A. hydrophila. Our results suggest that miR-142a-3p has multiple functions in host antibacterial immune response. Our research provides further understanding of the molecular mechanisms between miRNAs and their target genes, and provides a new insights for the development of pro-resolution strategies for the treatment of complex inflammatory diseases in fish.


Assuntos
Apoptose/genética , Carpas/genética , Citocinas/genética , Transportador de Glucose Tipo 3/genética , Infecções por Bactérias Gram-Negativas/veterinária , Macrófagos/fisiologia , MicroRNAs/genética , Aeromonas hydrophila/imunologia , Aeromonas hydrophila/patogenicidade , Animais , Carpas/imunologia , Carpas/microbiologia , Células Cultivadas , Citocinas/classificação , Infecções por Bactérias Gram-Negativas/imunologia , Inflamação/imunologia , Rim/citologia , Rim/microbiologia , Macrófagos do Fígado/microbiologia , Ativação de Macrófagos , MicroRNAs/classificação , Transdução de Sinais
3.
Fish Shellfish Immunol ; 116: 98-106, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34252543

RESUMO

Medicinal herbs are used for growth promotion, disease control and other health benefits in aquaculture industry. Here, we examined the effect of dietary laurel-leaf cistus (Cistus laurifolius) ethanolic extract on growth performance, digestive enzyme activity, haematological profile and nonspecific immune responses in common carp (Cyprinus carpio). In addition, resistance against Aeromonas hydrophila infection was examined. Common carp was fed diets containing 0 (Control), 0.1 (CL0.1), 0.5 (CL0.5) and 1 (CL1) g kg-1 laurel-leaf cistus extract for 45 days. After 30 days, superoxide anion production (SAP) increased in CL0.1 and CL0.5 fish groups and at the end of the study all experimental fish groups had higher SAP compared to that of the control (P ˂ 0.05). Lysozyme activity (LA) was elevated in CL0.5 and CL1 treated groups on 30th day (P < 0.05), and this increase was only observed in C0.1 fish group at the end of study compared to control (P ˂ 0.05). Myeloperoxidase activity was significantly increased in CL0.5 and CL1 fish groups at the end of study. IL-1ßgene expression was significantly increased in treated fish in a dose-depended manner. Similar results were observed for transcription of IL-6 and IL-8 (P < 0.05). Anti-inflammatory cytokines, IL-10 and TGF-ß were highly up-regulated in the intestine and head kidney of CL treated fish groups compared to control (P < 0.05). At the end of experiment, significantly higher final body weight, weight gain, and specific growth rate were obtained in CL0.1 treated fish group compared to control. However, growth was negatively affected in CL1 fish group (P < 0.05). CL1 fish group had also a significantly higher FCR. Amylase activity was significantly increased in all experimental fish groups compared to control (P ˂ 0.05). Trypsin activity was decreased in CL0.1 and CL1 fish groups (P ˂ 0.05). WBC and RBC were significantly increased (P ˂ 0.05) in CL0.5 and CL1 fish groups, whereas haemoglobin, haematocrit, mean cell, mean cell haemoglobin contents were no significantly changed among control and treatment groups. Result of challenge test with A. hydrophila exhibited that survival rate in all treatment groups was significantly higher than that of control. These findings demonstrated that laurel-leaf cistus at 0.1 g kg-1 can be a suitable candidate for growth promotion, immune system induction and infection control in fish.


Assuntos
Carpas , Cistus , Doenças dos Peixes/prevenção & controle , Infecções por Bactérias Gram-Negativas/prevenção & controle , Imunidade Inata/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Extratos Vegetais/farmacologia , Aeromonas hydrophila , Amilases/metabolismo , Animais , Contagem de Células Sanguíneas , Carpas/sangue , Carpas/genética , Carpas/imunologia , Carpas/metabolismo , Citocinas/genética , Etanol/química , Infecções por Bactérias Gram-Negativas/veterinária , Rim Cefálico/citologia , Rim Cefálico/imunologia , Lipase/metabolismo , Muramidase/imunologia , Folhas de Planta/química , Solventes/química , Superóxidos/imunologia , Tripsina/metabolismo
4.
Fish Shellfish Immunol ; 116: 150-160, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34265416

RESUMO

As a tyrosine phosphatase, Src homology 2-containing protein tyrosine phosphatase 2 (SHP2) serves as an inhibitor in PI3K-Akt pathway. In mammals, SHP2 can phosphorylate GSK3ß at Y216 site to control the expression of IFN. So far, the multiple functions of SHP2 have been reported in mammals. However, little is known about fish SHP2. In this study, we cloned and identified a grass carp (Ctenopharyngodon idellus) SHP2 gene (CiSHP2, MT373151). SHP2 is conserved among different vertebrates by amino acid sequences alignment and the phylogenetic tree analysis. CiSHP2 shared the closest homology with Danio rerio SHP2. Simultaneously, SHP2 was also tested in grass carp tissues and CIK (C. idellus kidney) cells. We found that it responded to poly I:C stimulation. CiSHP2 was located in the cytoplasm just as the same as those of mammals. Interestingly, it inhibited the phosphorylation level of GSK3ß in a non-contact manner. Meanwhile CiGSK3ß interacted with and directly phosphorylated CiTBK1. In addition, we found that CiSHP2 also reduced the phosphorylation level of CiTBK1 by CiGSK3ß, and then it depressed the expression of IFN I via GSK3ß-TBK1 axis. These results suggested that CiSHP2 was involved in CiGSK3ß and CiTBK1 activity but not regulated their transcriptional level. At the same time, we also found that CiSHP2 also influenced the activity of CiIRF3. Therefore, fish SHP2 inhibited IFN I expression through blocking GSK3ß-TBK1 signal axis.


Assuntos
Carpas/imunologia , Proteínas de Peixes/imunologia , Glicogênio Sintase Quinase 3 beta/imunologia , Interferon Tipo I/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/imunologia , Sequência de Aminoácidos , Animais , Carpas/genética , Linhagem Celular , Proteínas de Peixes/genética , Fosforilação , Filogenia , Poli I-C/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética
5.
Vet Res ; 52(1): 88, 2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34130754

RESUMO

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.


Assuntos
Carpas/genética , Carpas/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Aeromonas hydrophila , Animais , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Infecções por Bactérias Gram-Negativas , Interferons/genética , Filogenia , Poli I-C , Análise de Sequência de DNA/veterinária , Transdução de Sinais , Receptores Toll-Like/química
6.
Fish Shellfish Immunol ; 116: 1-11, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34174452

RESUMO

NK-lysin, an effector of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), not only exhibits cytotoxic effect in fish cells, but also participates in the immune defense against pathogenic infection. In this study, ORF sequences of RCC-NK-lysin, WCC-NK-lysin and WR-NK-lysin were 369 bp. Tissue-specific analysis revealed that the highest expressions of RCC-NK-lysin and WCC-NK-lysin were observed in gill, while the peaked level of WR-NK-lysin mRNA was observed in spleen. A. hydrophila infection sharply increased RCC-NK-lysin, WCC-NK-lysin and WR-NK-lysin mRNA expression in liver, trunk kidney and spleen. In addition, elevated levels of NK-lysin mRNA were observed in cultured fin cell lines of red crucian carp (RCC), white crucian carp (WCC) and their hybrid offspring (WR) after Lipopolysaccharide (LPS) challenge. RCC-NK-lysin, WCC-NK-lysin and WR-NK-lysin exerted regulatory roles in inducing ROS generation, modulating mitochondrial membrane potential, decreasing fish cell viability and antagonizing survival signalings, respectively. RCC/WCC/WR-NK-lysin-overexpressing fish could up-regulate expressions of inflammatory cytokines and decrease bacterial loads in spleen. These results indicated that NK-lysin in hybrid fish contained close sequence similarity to those of its parents, possessing the capacities of cytotoxicity and immune defense against bacterial infection.


Assuntos
Aeromonas hydrophila , Carpas/imunologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Proteolipídeos/imunologia , Nadadeiras de Animais/citologia , Animais , Carpas/genética , Sobrevivência Celular , Células Cultivadas , Quimera , Doenças dos Peixes/genética , Doenças dos Peixes/microbiologia , Proteínas de Peixes/genética , Expressão Gênica , Infecções por Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/veterinária , Rim/metabolismo , Lipopolissacarídeos/farmacologia , Fígado/metabolismo , Potencial da Membrana Mitocondrial , Proteolipídeos/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Baço/metabolismo , Baço/microbiologia
7.
Fish Shellfish Immunol ; 115: 43-57, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33992768

RESUMO

IL-20 is a pleiotropic cytokine that belongs to the IL-10 family and plays an important biological role in tissue homeostasis and regulation of host immune defenses. IL-20 homologues have recently been discovered in fish, but their functions have not been studied. In this study, an IL-20 like (IL-20L) cytokine was cloned in grass carp (Ctenopharyngodon idella) and its bioactivities were investigated. Expression analysis showed that the CiIL-20L gene was constitutively expressed in tissues with the highest expression detected in the head kidney. It was upregulated in the head kidney after infection with Flavobactrium columnare (F. cloumnare) and grass carp reovirus II (GCRV II). The recombinant CiIL-20L produced in E. coli cells was shown to be effective in inducing the expression of Th cytokine genes (IFN-γ, IL-4/13A, IL-4/13B and IL-10), macrophage marker genes (arginase 2, IRF4, KLF4 and SOCS3) and inflammatory genes (IL-1ß, IL-6, IL-8 and TNFα) in the head kidney leukocytes when stimulated at 12 h. Long term culture (6 days) of head kidney macrophages in the presence of CiIL-20L leads to high expression of IRF4, TGFß1 and arginase 2. Our data suggest that IL-20 may play regulatory roles in promoting Th responses, macrophage differentiation and inflammation.


Assuntos
Carpas/genética , Carpas/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Interleucinas/genética , Interleucinas/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Infecções por Flavobacteriaceae/imunologia , Infecções por Flavobacteriaceae/veterinária , Flavobacterium/fisiologia , Perfilação da Expressão Gênica/veterinária , Interleucinas/química , Filogenia , Reoviridae/fisiologia , Infecções por Reoviridae/imunologia , Infecções por Reoviridae/veterinária , Alinhamento de Sequência/veterinária
8.
Fish Shellfish Immunol ; 115: 14-21, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34015480

RESUMO

The heavy use of prophylactic antibiotics in aquaculture leads to elevated antibiotic residues, posing a huge hidden danger in aquaculture products and other natural aquatic environments. Therefore, this study aims to isolate probiotics that can replace antibiotics from the gut of grass carp for disease control. Bacillus velezensis B8 was isolated from the gut of grass carp and showed broad-spectrum antimicrobial activity against several fish pathogenic bacteria, including Aeromonas hydrophilis, Aeromonas veronii, Vibrio parahaemolyticus, Escherichia coli, Edwardsiella tarda and Vibrio mimicus. The safety evaluation showed that the strain B8 was non-toxic to grass carp, had no hemolytic activity, and was sensitive to most antibiotics. In vitro study indicated that strain B8 was viable at pH 2-7, had weak tolerance to 0.1% (w/v) bile salt, and could grow at 10°C-40 °C. The grass carps were fed with diets containing 0 (control), 107, and 109 cfu/g of strain B8 for 4 weeks. Various immune parameters were measured at 1, 2, 3, and 4 weeks of post-feeding. The results of non-specific immunoassay showed that diets supplemented with B8 significantly increased alkaline phosphatase (AKP) and superoxide dismutase (SOD) activity in serum samples (p < 0.05). The expression levels of immune-related genes in the kidney and spleen of grass carp were measured. Among them, the expression levels of IgM and TNF-α both in spleen and kidney were significantly increased after 3 and 4 weeks of post-feeding (p < 0.05). The expression of IgD and MHCI in kidney was significantly upregulated in high-dose groups after 2 and 3 weeks of feeding, respectively (p < 0.05). In addition, after 7 days of challenging with A. veronii, the high-dose group and low-dose group had 48% and 53% survival compared to 25% survival for the control group. These results suggest that B. velezensis B8 has the potential to be developed into a microecological preparation for the alternatives of antibiotics in aquaculture.


Assuntos
Bacillus/química , Carpas/imunologia , Resistência à Doença , Doenças dos Peixes/imunologia , Infecções por Bactérias Gram-Negativas/veterinária , Imunidade Inata , Probióticos/metabolismo , Aeromonas veronii/fisiologia , Ração Animal/análise , Animais , Dieta/veterinária , Resistência à Doença/efeitos dos fármacos , Relação Dose-Resposta a Droga , Infecções por Bactérias Gram-Negativas/imunologia , Imunidade Inata/efeitos dos fármacos , Probióticos/administração & dosagem , Distribuição Aleatória
9.
BMC Vet Res ; 17(1): 159, 2021 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-33853603

RESUMO

BACKGROUND: Immunoglobulins (Igs) distributed among systemic immune tissues and mucosal immune tissues play important roles in protecting teleosts from infections in the pathogen-rich aquatic environment. Teleost IgZ/IgT subclasses with different tissue expression patterns may have different immune functions. RESULTS: In the present study, a novel secreted IgZ heavy chain gene was cloned and characterized in common carp (Cyprinus carpio). This gene exhibited a different tissue-specific expression profile than the reported genes IgZ1 and IgZ2. The obtained IgZ-like subclass gene designated CcIgZ3, had a complete open reading frame contained 1650 bp encoding a protein of 549 amino acid residues. Phylogenetic analysis revealed that CcIgZ3 was grouped with carp IgZ2 and was in the same branch as IgZ/IgT genes of other teleosts. Basal expression detection of the immunoglobulin heavy chain (IgH) in healthy adult common carp showed that CcIgZ3 transcripts were widely expressed in systemic immune tissues and mucosal-associated lymphoid tissues. CcIgZ3 was expressed at the highest levels in the head kidneys, gills, and gonads, followed by the spleen, hindgut, oral epithelium, liver, brain, muscle, foregut, and blood; it was expressed at a very low level in the skin. The transcript expression of CcIgZ3 in leukocytes isolated from peripheral blood cells was significantly higher than that in leukocytes isolated from the spleen. Different groups of common carp were infected with Aeromonas hydrophila via intraperitoneal injection or immersion. RT-qPCR analysis demonstrated that significant differences in CcIgZ3 mRNA levels existed between the immersion and injection groups in all the examined tissues, including the head kidney, spleen, liver, and hindgut; in particular, the CcIgZ3 mRNA level in the hindgut was higher in the immersion group than in the injection group. The different routes of A. hydrophila exposure in common carp had milder effects on the IgM response than on the CcIgZ3 response. Further study of the relative expression of the IgH gene during the development of common carp showed that the tissue-specific expression profile of CcIgZ3 was very different from those of other genes. RT-qPCR analysis demonstrated that the CcIgZ3 mRNA level increased gradually in common carp during the early larval development stage from 1 day post fertilization (dpf) to 31 dpf with a dynamic tendency similar to those of IgZ1 and IgZ2, and IgM was the dominant Ig with obviously elevated abundance. Analyses of the tissue-specific expression of IgHs in common carp at 65 dpf showed that CcIgZ3 was expressed at mucosal sites, including both the hindgut and gill; in contrast, IgZ1 was preferentially expressed in the hindgut, and IgZ2 was preferentially expressed in the gill. In addition to RT-qPCR analysis, in situ hybridization was performed to detect CcIgZ3-expressing cells and IgM-expressing cells. The results showed that CcIgZ3 and IgM transcripts were detectable in the spleens, gills, and hindguts of common carp at 65 dpf. CONCLUSIONS: These results reveal that CcIgZ3 gene transcripts are expressed in common carp during developmental stage not only in systemic tissues but also in mucosal tissues. CcIgZ3 expression can be induced in immune tissues by A. hydrophila challenge via immersion and intraperitoneal injection with significantly different expression profiles, which indicates that CcIgZ3 is involved in the antimicrobial immune response and might play an important role in gut mucosal immunity.


Assuntos
Carpas/imunologia , Doenças dos Peixes/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Aeromonas hydrophila/imunologia , Animais , Carpas/crescimento & desenvolvimento , Clonagem Molecular , Doenças dos Peixes/microbiologia , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Cadeias Pesadas de Imunoglobulinas/química , Larva/imunologia , Filogenia , Análise de Sequência de Proteína
10.
Front Immunol ; 12: 635015, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33717179

RESUMO

An 8-week rearing trial was designed to appraise the dietary lysine levels on intestinal antioxidant capacity and immunity of grass carp fry. Six practical diets were prepared with graded levels of lysine (1.44, 1.79, 1.97, 2.44, 2.56 and 2.87% dry matter), and these diets were fed to grass carp fry. The results showed that the activities of intestinal antioxidant factors including catalase and glutathione peroxidase were markedly improved by the 2.44% dietary lysine compared with the control diet (1.44% dietary lysine) (P < 0.05). In terms of antioxidants, compared with the control diet, the 2.44% diet markedly upregulated the mRNA expression levels of target of rapamycin, S6 kinase1 and nuclear factor erythroid 2-related factor 2 pathway-related antioxidant genes, containing catalase and glutathione peroxidase 1α (P < 0.05) and downregulated the mRNA levels of Kelch-like ECH-associated protein 1 (P > 0.05). The mRNA levels of 4E-binding protein 2 showed the opposite trend compared with those of target of rapamycin, and the minimum value was observed in the group of 1.97% dietary lysine (P < 0.05). In terms of immunity, compared with the 1.44% diet, the 2.44% diet markedly suppressed the intestinal p38 mitogen-activated protein kinase and interferon γ2 mRNA levels (P < 0.05). Moreover, nuclear factor-kappa B p65, tumor necrosis factor α, interleukin 6, interleukin 8, and interleukin 15 mRNA levels all exhibited the same trend as p38 mitogen-activated protein kinase and interferon γ2; however, the difference among all the lysine treatments groups was not significant (P > 0.05). The anti-inflammatory cytokines transforming growth factor ß2 and interleukin 4/13B mRNA levels in the intestine were remarkably upregulated by high dietary lysine levels (2.56 and 2.87%) (P < 0.05), and when the dietary lysine level reached 2.44%, the interleukin 4/13A mRNA levels were strikingly increased (P < 0.05). Overall, the data suggested that 2.44% dietary lysine could strengthen the immune and antioxidant capacities of grass carp fry via activating the target of rapamycin (TOR) signaling pathway, and suppressing the p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway, which then improve the survival rate.


Assuntos
Ração Animal , Antioxidantes/metabolismo , Carpas/metabolismo , Citocinas/metabolismo , Proteínas de Peixes/metabolismo , Intestinos/enzimologia , Lisina/administração & dosagem , Serina-Treonina Quinases TOR/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Carpas/genética , Carpas/imunologia , Citocinas/genética , Proteínas de Peixes/genética , Regulação da Expressão Gênica , Intestinos/imunologia , Lisina/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética
11.
Fish Shellfish Immunol ; 111: 201-207, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33582280

RESUMO

Septicemia of grass carp is a systemic inflammatory reaction caused by bacterial infection. More and more evidences show that long non-coding RNAs (lncRNAs) can participate in the regulation of inflammatory response. In the present study, lncRNA-WAS and lncRNA-C8807 were confirmed to be involved in the inflammatory response following infection with Aeromonas hydrophila. LncRNA-WAS and lncRNA-C8807 could interact with miR-142a-3p. LncRNA-WAS and lncRNA-C8807 interact with miR-142a-3p to effect pro-inflammatory genes and NF-κB pathway. Our results provide a theoretical basis for studying the molecular mechanism underlying the regulation of inflammation by lncRNA in grass carp.


Assuntos
Carpas/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata , MicroRNAs/imunologia , RNA Longo não Codificante/imunologia , Aeromonas hydrophila/fisiologia , Animais , Doenças dos Peixes/genética , Doenças dos Peixes/microbiologia , Infecções por Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/veterinária , Inflamação/genética , Inflamação/imunologia , Inflamação/microbiologia , Inflamação/veterinária
12.
Artigo em Inglês | MEDLINE | ID: mdl-33610820

RESUMO

Nanoparticles can be toxic and put human and animals' life at risk. The present work was carried out to evaluate the possible immunosuppressive effects of water borne iron oxide nanoparticles (IONPs) and the FeCl3 on immune components of common carp (Cyprinus carpio). Fish were exposed to a series of chronic levels of 25%, 50%, and 75% of IONPs LC50 96 h concentration (referred to as control, NP1, NP2, and NP3 respectively) or FeCl3 (same concentrations as IONPs referred to as S1, S2, and S3 respectively) for 21 days. Results revealed alterations in blood parameters, where IONPs significantly decreased number of white blood cells at all concentrations. Glucose and cortisol increased in all exposed fish after 21 days, suggesting activation of the maintenance mechanism cascade against a chronic stressor. IONPs or FeCl3 significantly accumulated in liver tissue of exposed fish. Immune responses were remarkably decreased in serum and skin mucus in response to IONPs or FeCl3. These components were at lowest levels in fish exposed to the highest concentrations of IONPs and FeCl3. The findings of the present study suggested that IONPs is accumulated in fish liver and intestine, where they may exhibit immunotoxicity.


Assuntos
Bioacumulação , Carpas/imunologia , Compostos Férricos/toxicidade , Nanopartículas Magnéticas de Óxido de Ferro/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Imunidade , Intestinos/efeitos dos fármacos , Intestinos/imunologia , Fígado/efeitos dos fármacos , Fígado/imunologia
13.
J Immunol ; 206(6): 1337-1347, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33568398

RESUMO

Type I IFNs (IFN-Is) play pivotal roles in host defense against viral infections but remain enigmatic against bacterial pathogens. In this study, we recombinantly expressed and purified intact grass carp (Ctenopharyngodon idella) IFNφ1 (gcIFNφ1), a teleost IFN-I. gcIFNφ1 widely powerfully directly kills both Gram-negative and Gram-positive bacteria in a dose-dependent manner. gcIFNφ1 binds to LPS or peptidoglycan and provokes bacterial membrane depolarization and disruption, resulting in bacterial death. Furthermore, gcIFNφ1 can efficiently protect zebrafish against Aeromonas hydrophila infection and significantly reduce the bacterial loads in tissues by an infection model. In addition, we wonder whether antibacterial IFN-I members exist in other vertebrates. The amino acid compositions of representative IFN-Is with strong positive charges from Pisces, Amphibia, reptiles, Aves, and Mammalia demonstrate high similarities with those of 2237 reported cationic antimicrobial peptides in antimicrobial peptide database. Recombinant intact representative IFN-I members from the nonmammalian sect exhibit potent broad-spectrum robust bactericidal activity through bacterial membrane depolarization; in contrast, the bactericidal activity is very weak from mammalian IFN-Is. The findings display a broad-spectrum potent direct antimicrobial function for IFN-Is, to our knowledge previously unknown. The results highlight that IFN-Is are important and robust in host defense against bacterial pathogens, and unify direct antibacterial and indirect antiviral bifunction in nonmammalian jawed vertebrates.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Doenças dos Peixes/imunologia , Interferon Tipo I/metabolismo , Interferons/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Aeromonas hydrophila/imunologia , Aeromonas hydrophila/isolamento & purificação , Sequência de Aminoácidos , Animais , Carga Bacteriana , Carpas/genética , Carpas/imunologia , Carpas/metabolismo , Modelos Animais de Doenças , Doenças dos Peixes/microbiologia , Imunidade Inata , Interferon Tipo I/genética , Interferon Tipo I/isolamento & purificação , Interferons/genética , Interferons/isolamento & purificação , Testes de Sensibilidade Microbiana , Modelos Animais , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/imunologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/isolamento & purificação
14.
Fish Shellfish Immunol ; 111: 83-93, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33513437

RESUMO

Tumor necrosis factor receptor 1 (TNFR1) associated death domain protein (TRADD) is a pivotal adaptor in TNF signaling pathway and up-regulates MAVS/IFN signaling pathway in human and mammal. However, the role of TRADD in teleost fish remains obscure. To reveal the function of teleost TRADD in the innate immune response, the TRADD homologue (bcTRADD) of black carp (Mylopharyngodon piceus) has been cloned and the function of bcTRADD is investigated in this study, which shares similar functional domain to its mammalian counterpart. bcTRADD mRNA expression level increased in response to different stimuli, including LPS, poly (I:C) and virus infection in host cells. bcTRADD activated the transcriptional activity of NF-κB promoter in the reporter assay; however, showed hardly any effect on the transcriptional activity of IFN promoter. It was interesting that black carp mitochondria antiviral signaling protein (bcMAVS)-activated IFN promoter transcription were dramatically depressed by bcTRADD and the C-terminal death domain of bcTRADD was indispensable for its regulation of bcMAVS. Accordingly, the plaque assay result showed that EPC cells co-expressing bcMAVS and bcTRADD presented much attenuated antiviral activity than EPC cells expressing bcMAVS alone. Knockdown of bcTRADD slightly promoted the antiviral ability of the host cells against SVCV. The current data support the conclusion that bcTRADD suppresses MAVS-mediated antiviral signaling, which is different to its mammalian counterpart.


Assuntos
Carpas/genética , Carpas/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Proteína de Domínio de Morte Associada a Receptor de TNF/genética , Proteína de Domínio de Morte Associada a Receptor de TNF/imunologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Células HEK293 , Humanos , Lipopolissacarídeos/farmacologia , Filogenia , Poli I-C/farmacologia , Rhabdoviridae/fisiologia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/veterinária , Alinhamento de Sequência/veterinária , Proteína de Domínio de Morte Associada a Receptor de TNF/química
15.
Fish Shellfish Immunol ; 112: 168-178, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-32927052

RESUMO

Heme oxygenase (HO)-1, a rate-limiting enzyme in heme catabolism, results in the formation of equivalent amounts of biliverdin (BV), carbon monoxide (CO) and ferrous iron (Fe2+). Previous studies have revealed that HO-1 plays an important role in immune responses. However, the mechanism underlying the immune responses against bacterial infection of teleost HO-1 remains enigmatic. To decipher the mechanisms, we have cloned and characterized the HO-1 gene of grass carp (designated as GcHO-1) in this research. The results showed that the open reading frame (ORF) of GcHO-1 was 819 bp, which encoded a putative protein of 272 amino acids. The deduced amino acid sequence phylogenetically shared the highest identity with other teleosts, and contained two domains of heme-oxygenase and a single-pass transmembrane domain. The mRNA expressions of GcHO-1 in healthy grass carp have widely existed in examined tissues in the following order of spleen > head-kidney > middle head-kidney > intestines > liver > gills > heart > muscle > brain. Besides, the mRNA and protein transcription of GcHO-1 were both significantly up-regulated in the liver and head-kidney tissues after Staphylococcus aureus and Aeromonas hydrophila infection. In addition, overexpression of GcHO-1 in kidney cell line (CIK) cells of grass carp could reduce the expression of inflammatory cytokines (IL-1ß, IL-8, TNFα, CCL1 and IL-6). Herein, we demonstrate that GcHO-1 plays an anti-inflammatory role in innate immunity. Our results shed new light on the mechanisms underlying the antibacterial immunity of teleost.


Assuntos
Carpas/genética , Carpas/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Heme Oxigenase-1/genética , Heme Oxigenase-1/imunologia , Imunidade Inata/genética , Aeromonas hydrophila/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Doenças dos Peixes/microbiologia , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/veterinária , Heme Oxigenase-1/química , Filogenia , Alinhamento de Sequência/veterinária , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/fisiologia
16.
J Fish Biol ; 98(1): 208-218, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33000466

RESUMO

Selenium, as an essential trace element, interferes through selenoproteins in many physiological processes of plants and mammals. Its antiviral activity has recently attracted much attention because selenium improves the antiviral capacity of animal cells against a few viruses relevant to human diseases. In this study, the red elemental selenium was purified from the fermentative culture of Herbaspirillum camelliae WT00C and then used to culture epithelioma papulosum cyprinid (EPC) cells or feed crucian carp and zebrafish. Finally, its antiviral effects were investigated at the cell level and living fishes after spring viraemia of carp virus infection. At the cell level, 5, 10 and 20 µg ml-1 red elemental selenium significantly induced the expression of interferon (IFN) and ISG15 genes in EPC cells. The viral TCID50 (50% tissue culture infective dose) values in the EPC cells incubated with 5, 10 and 20 µg ml-1 red elemental selenium were significantly less than those of the control. More expression of IFN and ISG15 genes and less TCID50 values indicate that red elemental selenium indeed improves the antiviral capability of EPC cells. In the crucian carp fed with the food containing 5 and 10 µg g-1 red elemental selenium, IFN expressions showed 13- and 39-fold increases at the 16th day of post-injection, and its expression was dependent on selenium concentrations. Meanwhile, no fish death occurred in all the experimental groups. In the zebrafish fed with the red worm containing 5 µg g-1 red elemental selenium, IFN and Mx expressions and survival rate were significantly higher than those of the control. The results of this study show that red elemental selenium indeed improves the antiviral activity of fish. The antiviral effects of selenium mainly come from its immune regulation through its incorporation into selenoproteins. The optimum level of selenium contributes to improving fish immunity, whereas excess selenium causes excessive immune and inflammatory responses.


Assuntos
Carpas/imunologia , Doenças dos Peixes/tratamento farmacológico , Doenças dos Peixes/imunologia , Infecções por Rhabdoviridae/veterinária , Selênio/farmacologia , Viremia/veterinária , Peixe-Zebra/imunologia , Adjuvantes Imunológicos/farmacologia , Adjuvantes Imunológicos/uso terapêutico , Animais , Antivirais/farmacologia , Carcinoma , Carpas/virologia , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Interferons/genética , Rhabdoviridae , Infecções por Rhabdoviridae/tratamento farmacológico , Infecções por Rhabdoviridae/imunologia , Selênio/uso terapêutico , Viremia/tratamento farmacológico , Viremia/imunologia , Peixe-Zebra/virologia
17.
Fish Shellfish Immunol ; 109: 12-19, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33285165

RESUMO

Sustainable aquaculture arises as key to increase food production in the coming years. However, the sector still faces many challenges such as the exposure of the cultured animals to pesticide-contaminated water. Pesticides used in agriculture can reach aquaculture systems either directly (integrated-agriculture aquaculture practices) or indirectly (soil leakage) and cause a broad range of ecotoxicological effects on cultured fish and shellfish. Here, we studied how glyphosate affects several haematological, biochemical, and immune parameters in common carp (Cyprinus carpio) fingerlings, the fourth most important cultured fish species worldwide. We also evaluated the potential of dietary supplementation with black seed (Nigella sativa, 0.25, 0.5 and 1%) to lower glyphosate-associated toxicity. Our results showed that 14-day sub-lethal exposure of common carp fingerlings to glyphosate increases oxidative stress, decreases antioxidant defences, affects several metabolic pathways, and induced immune depression. Furthermore, we showed that fish fed with N. sativa-enriched diets at 0.25, 0.5 and 1% for 60 days coped better with glyphosate exposure than control fish and displayed more stable levels of biochemical serum parameters (total protein, albumin, triglycerides, low-density lipoprotein LDL), cholesterol and high-density lipoprotein HDL), higher levels of immune defences (lysozyme and immunoglobulin) and higher antioxidant enzymes (superoxide dismutase SOD, glutathione peroxidase GPx) than control fish. Fish fed with all enriched diets also displayed lower lipid peroxidation (malondialdehyde MDA), lower metabolic enzymes (alanine aminotransferase ALT, aspartate aminotransferase AST and alkaline phosphatase ALP) levels in blood serum and lower cortisol levels than control fish. Altogether, our results show that dietary inclusion of black seed can be used as a sustainable bio-remediation strategy, mitigating many of the negative effects of glyphosate exposure in fish.


Assuntos
Carpas/imunologia , Glicina/análogos & derivados , Imunidade Inata/efeitos dos fármacos , Nigella sativa/química , Estresse Oxidativo/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Ração Animal/análise , Animais , Dieta/veterinária , Suplementos Nutricionais/análise , Relação Dose-Resposta a Droga , Glicina/toxicidade , Herbicidas/toxicidade , Redes e Vias Metabólicas/efeitos dos fármacos
18.
Fish Shellfish Immunol ; 108: 127-133, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33253908

RESUMO

The present study aimed to investigate the potential effects of dietary marjoram extract on growth performance, health, and disease resistance in common carp, Cyprinus carpio. To this purpose, the fish were assigned into four treatments and fed by diets supplemented with 0 (control), 100, 200, and 400 mg marjoram extract kg-1 over eight weeks and then challenged with Aeromonas hydrophila. According to the results, 200 mg kg-1 dietary marjoram extract inclusion showed the highest final weight, weight gain, and specific growth rate, and lowest feed conversion ratio (FCR). White blood cell number, Red blood cells, hematocrit, hemoglobin, mean corpuscular volume and mean corpuscular hemoglobin were markedly increased particularly at 200 mg kg-1 treatment. Marjoram extract significantly increased plasma superoxide dismutase activity and decreased malondialdehyde level compared to the control treatment. Plasma complement and lysozyme activities and total immunoglobulin levels, mucosal complement, lysozyme and alkaline phosphatase activities and immunoglobulin levels were significantly increased compared to the control group. The lowest post-challenge survival rate was observed in the control treatment, whereas the highest value was related to the 200 mg kg-1 marjoram treatment. In conclusion, the present study demonstrated that, marjoram extract is a suitable feed supplements for common carp, as it stimulates the fish growth, antioxidant, and immune systems, which suppress the fish mortality during Aeromonas septicemia. According to the results, 200 mg marjoram extract kg-1 is recommended for carp feed formulation.


Assuntos
Adjuvantes Imunológicos/farmacologia , Carpas/imunologia , Doenças dos Peixes/imunologia , Imunidade Humoral , Imunidade nas Mucosas , Origanum/química , Extratos Vegetais/metabolismo , Aeromonas hydrophila/fisiologia , Ração Animal/análise , Animais , Antioxidantes/metabolismo , Carpas/sangue , Carpas/crescimento & desenvolvimento , Dieta/veterinária , Suplementos Nutricionais/análise , Resistência à Doença/imunologia , Relação Dose-Resposta a Droga , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/veterinária , Extratos Vegetais/administração & dosagem , Extratos Vegetais/farmacologia
19.
Front Immunol ; 11: 545302, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33193312

RESUMO

Viruses typically target host RIG-I-like receptors (RLRs), a group of key factors involved in interferon (IFN) production, to enhance viral infection. To date, though immune evasion methods to contradict IFN production have been characterized for a series of terrestrial viruses, the strategies employed by fish viruses remain unclear. Here, we report that all grass carp reovirus (GCRV) proteins encoded by segments S1 to S11 suppress mitochondrial antiviral signaling protein (MAVS)-mediated IFN expression. First, the GCRV viral proteins blunted the MAVS-induced expression of IFN, and impair MAVS antiviral capacity significantly. Interestingly, subsequent co-immunoprecipitation experiments demonstrated that all GCRV viral proteins interacted with several RLR cascades, especially with TANK-binding kinase 1 (TBK1) which was the downstream factor of MAVS. To further illustrate the mechanisms of these interactions between GCRV viral proteins and host RLRs, two of the viral proteins, NS79 (S4) and VP3 (S3), were selected as representative proteins for two distinguished mechanisms. The obtained data demonstrated that NS79 was phosphorylated by gcTBK1, leading to the reduction of host substrate gcIRF3/7 phosphorylation. On the other hand, VP3 degraded gcMAVS and the degradation was significantly reversed by 3-MA. The biological effects of both NS79 and VP3 were consistently found to be related to the suppression of IFN expression and the promotion of viral evasion. Our findings shed light on the special evasion mechanism utilized by fish virus through IFN regulation, which might differ between fish and mammals.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Carpas , Proteínas de Peixes/imunologia , Interferon gama/imunologia , Reoviridae/imunologia , Transdução de Sinais/imunologia , Animais , Carpas/imunologia , Carpas/virologia , Células HEK293 , Humanos , Proteínas Virais/imunologia
20.
Front Immunol ; 11: 586889, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33178219

RESUMO

Interleukin (IL)-22 plays an important role in regulating inflammation and clearance of infectious pathogens. IL-22 homologs have been discovered in fish, but the functions and sources of IL-22 have not been fully characterized. In this study, an IL-22 homolog was identified in grass carp and its bioactivities were investigated. The grass carp IL-22 was constitutively expressed in tissues, with the highest expression detected in the gills and hindgut. It was upregulated in the spleen after infection with Flavobacterium columnare and grass carp reovirus and in the primary head kidney and spleen leukocytes stimulated with LPS and IL-34. Conversely, it was downregulated by Th2 cytokines such as IL-4/13B and IL-10. The recombinant IL-22 produced in bacteria showed a stimulatory effect on the expression of inflammatory cytokines and STAT3 in the primary head kidney leukocytes and CIK cells. Moreover, the IL-22-positive cells were found to be induced in the hindgut and head kidney 24 h after infection by F. columnare. Our data suggest that IL-22 plays an important role in regulating mucosal and systemic immunity against bacterial and viral infection.


Assuntos
Carpas/imunologia , Proteínas de Peixes/imunologia , Interleucinas/imunologia , Animais
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