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1.
Fish Shellfish Immunol ; 107(Pt A): 156-162, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32961292

RESUMO

NF-κB is a typical transcription factor that regulates expression of various genes involved in inflammatory and immune responses. Therefore, it is essential that NF-κB signaling tightly regulated to maintain immune balance. Compared with those of mammals, the regulatory mechanisms of NF-κB signaling is rarely reported in teleost fish. IκBα is a prominent negative feedback regulator in the NF-κB signaling system. In this study, we determined that IRF11 enhances the inhibitory effect of IκBα on NF-κB activation in teleost fish. Overexpression of IRF11 can inhibit IκBα degradation, whereas its knockdown has the opposite effect of IκBα. Our study further indicates that IκBα was regulated via ubiquitin-proteasome degradation pathway, IRF11 inhibits IκBα in ubiquitin-proteasome degradation. This study provides a novel evidence on the regulation of innate immune signaling pathways in teleost fish and thus provides new insights into the regulatory mechanisms in mammals.


Assuntos
Proteínas de Peixes/genética , Inibidor de NF-kappaB alfa/genética , NF-kappa B/genética , Perciformes/genética , Perciformes/imunologia , Transdução de Sinais/imunologia , Fatores de Transcrição/genética , Animais , Proteínas de Peixes/metabolismo , Inibidor de NF-kappaB alfa/metabolismo , NF-kappa B/metabolismo , Perciformes/metabolismo , Fatores de Transcrição/metabolismo
2.
Dev Comp Immunol ; 110: 103723, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32387555

RESUMO

The suppressor of cytokine signaling 3 (SOCS3), as a negative regulator in inferferon (IFN) signaling pathways in mammals, has a vital role in immune systems. However, studies on the function of SOCS3 in lower vertebrates are limited. In this study, we identified SOCS3a and fish-specific SOCS3b gene in miiuy croaker. Sequence analysis results showed that SOCS3a and SOCS3b were evolutionarily conservative in fish. Expression analysis indicated that miiuy croaker SOCS3a and SOCS3b (mmSOCS3a and mmSOCS3b) were expressed in all of the tested miiuy croaker tissues, thus revealing the potential ability to perceive poly (I:C) stimulation. Further functional experiments showed that mmSOCS3a and mmSOCS3b could inhibit the IFNγ- and IFNα-induced ISRE reporter activation, respectively. Accordingly, the investigation of mmSOCS3a and mmSOCS3b can provide insights into fish SOCS3 and a basis for future research on the SOCS family of fish immune systems.


Assuntos
Proteínas de Peixes/genética , Perciformes/genética , Proteína 3 Supressora da Sinalização de Citocinas/genética , Animais , Clonagem Molecular , Evolução Molecular , Humanos , Imunidade Inata , Interferon-alfa/metabolismo , Interferon gama/metabolismo , Camundongos , Perciformes/imunologia , Filogenia , Poli I-C/imunologia , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais
3.
Front Immunol ; 11: 606, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32373114

RESUMO

MyD88 is a conserved intracellular adaptor, which plays an important role in the innate immune system. MyD88 transmits signals for downstream of toll-like and IL-1 receptors to activate NF-κB signaling pathway, which is tightly controlled in the immune response to maintain immune intensity and immune homeostasis at different stages. NF-κB signaling pathway has been extensively studied in mammals, but regulatory molecular mechanism is still unclear in teleost fish. We determined that IRF3 and IRF8 can regulate MyD88-mediated NF-κB signaling pathway in fish. Interestingly, MyD88 is precisely regulated by IRF3 and IRF8 through the same mechanism but in completely opposite ways. IRF3 promotes MyD88-mediated NF-κB signaling pathway, whereas IRF8 inhibits the signaling pathway. MyD88 is regulated via ubiquitin-proteasome degradation, whereas IRF3 or IRF8 inhibited or promoted MyD88 degradation in this pathway. Specifically, in the early stage of lipopolysaccharide (LPS) stimulation or Vibrio infection, up-regulation of IRF3 and down-regulation of IRF8 eventually increased MyD88 expression to activate the NF-κB signaling pathway to trigger immune response. In the late stage of stimulation, down-regulated IRF3 and up-regulated IRF8 synergistically regulate the expression of MyD88 to a normal level, thus maintaining the immune balance of homeostasis and preventing serious damage from persistent over-immunization. This study presents information on Myd88-NF-κB signaling pathway in teleost fish and provides new insights into its regulatory mechanism in fish immune system.


Assuntos
Fator Regulador 3 de Interferon/fisiologia , Fatores Reguladores de Interferon/fisiologia , Fator 88 de Diferenciação Mieloide/fisiologia , NF-kappa B/fisiologia , Perciformes/imunologia , Animais , Células Cultivadas , Células HEK293 , Humanos , Lipopolissacarídeos/farmacologia , Fator 88 de Diferenciação Mieloide/análise , Complexo de Endopeptidases do Proteassoma/fisiologia , Transdução de Sinais/fisiologia , Ubiquitinação , Vibrioses/imunologia
4.
Dev Comp Immunol ; 106: 103632, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31987876

RESUMO

Tightly regulation of NF-κB signaling is essential to innate and adaptive immune responses, but its regulatory mechanism remains unclear in various organisms, especially teleost fish. In this study, we reported that IRF3 attenuates the inhibitory effect of IκBα on NF-κB activation in teleost fish. Overexpression of IRF3 can promote IκBα degradation, whereas its knockdown can relieve degradation of IκBα. IRF3 promoted the degradation of IκBα protein, but this effect could be inhibited by MG132 treatment. IRF3 is crucial for the polyubiquitination and proteasomal degradation of IκBα. Our findings indicate that IRF3 regulates NF-κB pathway by targeting IκBα for ubiquitination and degradation. This study provides novel evidence on the regulation of innate immune signaling pathways in teleost fish and thus provides new insights into the regulatory mechanisms in mammals.


Assuntos
Cyprinidae/imunologia , Células Epiteliais/imunologia , Proteínas de Peixes/genética , Fator Regulador 3 de Interferon/genética , Inibidor de NF-kappaB alfa/metabolismo , NF-kappa B/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Linhagem Celular , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Fator Regulador 3 de Interferon/metabolismo , Proteólise , Transdução de Sinais , Ubiquitinação
5.
Fish Shellfish Immunol ; 80: 109-114, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29857132

RESUMO

Toll-like receptors (TLRs) play the key role in host defense of invasion of pathogens, not only in the innate immunity, but also in adaptive immunity. There are significant varieties and distinct features in fish TLRs, the TLR5 subfamily have two members (TLR5M and TLR5S). However, the exact role of TLR5 was lack of research in fish. In this study, a soluble form of TLR5 (TLR5S) was identified in miiuy croaker. The bioinformatics analysis showed that miiuy croaker TLR5S lacked the transmembrane domain and TIR domain. In other words, mmiTLR5S only has leucine-rich repeats (LRRs) domain, it is one of differences between TLR5M and TLR5S. Comparative genomic analysis showed that TLR5S might have happened an evolution between species. Expression analysis showed that mmiTLR5S was expressed in all tested miiuy croaker tissues and the mmiTLR5S expressions were significantly upregulated at 12 h in liver and kidney after Vibrio harveyi infection. Further functional experiments showed that NF-кB can be actived by mmiTLR5S, TLR5S might be an indispensable role in organism immune response. In short, the study of mmiTLR5S enriches the information of TLR5S and lays the foundation for future research on teleost TLRs system.


Assuntos
Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perciformes/genética , Perciformes/imunologia , Receptor 5 Toll-Like/genética , Receptor 5 Toll-Like/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar/genética , Evolução Molecular , Doenças dos Peixes , Genômica , Células HEK293 , Humanos , Rim/imunologia , Fígado/imunologia , Fases de Leitura Aberta , Filogenia , Alinhamento de Sequência , Análise de Sequência de DNA , Vibrio , Vibrioses/veterinária
6.
Front Immunol ; 9: 867, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29755465

RESUMO

NF-κB signaling is tightly regulated and essential to innate and adaptive immune responses, its regulatory mechanism remains unclear in various organisms, especially teleosts. In this study, we reported that IRF3 can negatively regulate TRIF-mediated NF-κB signaling pathway. Overexpression of IRF3 can inhibit TRIF-mediated NF-κB signaling pathway. However, knockdown of IRF3 had an opposite effect. IRF3 can promote the degradation of TRIF protein in mammal and fish cells, but this effect could be inhibited by MG132 treatment. Furthermore, we found that the inhibitory effect of IRF3 primary depended on its IRF association domain domain. IRF3 is crucial for the polyubiquitination and proteasomal degradation of TRIF. Our findings indicate that IRF3 negatively regulates TLR-mediated NF-κB signaling pathway by targeting TRIF for ubiquitination and degradation. This study provides a novel evidence on the negative regulation of innate immune signaling pathways in teleost fish and thus might provide new insights into the regulatory mechanisms in mammals.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas de Peixes/imunologia , Fator Regulador 3 de Interferon/metabolismo , Perciformes/imunologia , Transdução de Sinais/imunologia , Proteínas Adaptadoras de Transporte Vesicular/imunologia , Animais , Proteínas de Peixes/metabolismo , Imunidade Inata , Fator Regulador 3 de Interferon/imunologia , Leupeptinas/farmacologia , NF-kappa B/metabolismo , Perciformes/metabolismo , Proteólise/efeitos dos fármacos , Receptores Toll-Like/metabolismo , Ubiquitinação/imunologia
7.
Infect Immun ; 86(6)2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29632247

RESUMO

Inflammation is the host self-protection mechanism to eliminate pathogen invasion. The excessive inflammatory response can result in uncontrolled inflammation, autoimmune diseases, or pathogen dissemination. Recent studies have widely shown that microRNAs (miRNAs) contribute to the regulation of inflammation in mammals by repressing gene expression at the posttranscriptional level. However, the miRNA-mediated mechanism in the inflammatory response in fish remains hazy. In the present study, the regulatory mechanism of the miR-216a-mediated inflammatory response in teleost fish was addressed. We found that the expression of miR-216a could be significantly upregulated in the miiuy croaker after challenge with Vibrio anguillarum and lipopolysaccharide. Bioinformatics predictions demonstrated a potential binding site of miR-216a in the 3' untranslated region of the p65 gene, and the result was further confirmed by luciferase assay. Moreover, both the mRNA and protein levels of p65 in macrophages were downregulated by miR-216a. Deletion mutant analysis of the miR-216a promoter showed that the Ap1 and Sp1 transcription factor binding sites are indispensable for the transcription of miR-216a. Further study revealed that overexpression of miR-216a suppresses inflammatory cytokine expression and negatively regulates NF-κB signaling, which inhibit an excessive inflammatory response. The collective results indicate that miR-216a plays a role as a negative regulator involved in modulating the bacterium-induced inflammatory response.


Assuntos
Citocinas/metabolismo , Inflamação/veterinária , MicroRNAs/metabolismo , NF-kappa B/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Perciformes , Animais , Citocinas/genética , Doenças dos Peixes/metabolismo , Regulação da Expressão Gênica/imunologia , Células HEK293 , Células HeLa , Humanos , Inflamação/metabolismo , Lipopolissacarídeos/toxicidade , MicroRNAs/genética , NF-kappa B/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Interferência de RNA
8.
Dev Comp Immunol ; 78: 124-131, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28962840

RESUMO

The suppressor of cytokine signaling 1 (SOCS1) is a crucial regulator in the immune systems of mammals, which functions classically as a negatively regulator in the IFN signaling pathways. However, data on functional characterization of SOCS1 in lower vertebrates is limited. In this study, we identified and characterized the full-length SOCS1a gene of miiuy croaker (Miichthys miiuy). The sequence analysis results showed that miiuy croaker SOCS1a (mmSOCS1a) shared some conserved motifs with other vertebrates. To further study the function of fish SOCS1, we identified mmSOCS1a and determined its potential ability to perceive poly(I:C) stimulation. Induction experiments with poly(I:C) indicated the significant expression levels of mmSOCS1a in liver and kidney. In addition, mmSOCS1a could inhibit poly(I:C)-induced or IFNs-induced ISRE reporter gene activation. In a word, we systematically and comprehensively analyzed evolution and function of mmSOCS1a, which will provide the basis for future research on fish SOCS family.


Assuntos
Proteínas de Peixes/genética , Rim/fisiologia , Fígado/fisiologia , Perciformes/imunologia , Proteína 1 Supressora da Sinalização de Citocina/genética , Animais , Evolução Biológica , Células Cultivadas , Clonagem Molecular , Citocinas/genética , Citocinas/metabolismo , Proteínas de Peixes/metabolismo , Interferons/metabolismo , Perciformes/genética , Filogenia , Poli I-C/imunologia , Transdução de Sinais , Proteína 1 Supressora da Sinalização de Citocina/metabolismo
9.
Dev Comp Immunol ; 78: 52-60, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28923593

RESUMO

MicroRNA-122 (miR-122) was originally identified in mouse and then lots of researches on miR-122 had been performed in mammals. However, the functional study of miR-122 were restricted in fish. In miiuy croaker, miR-122 is sensitive to poly(I:C) stimulation. In this study, a combination of bioinformatics and experimental techniques were used to investigate the functions of miR-122. DAK is a putative target gene of miR-122 which was predicted by bioinformatics, and further the luciferase reporter assays were used to confirm the target sites in DAK 3'untranslated region. The inhibiting effect of miR-122 mimics or pre-miR-122 on DAK presented the dose and time dependent manners, and the pre-miR-122 showed stronger inhibiting effect on DAK than the miR-122 mimics. Therefore, the miR-122 participate in regulating RIG-I-like receptors signaling pathway via inhibiting DAK which is the inhibitors of MDA5. The expression of miR-122 and DAK showed negative relationship in both miiuy croaker spleen and macrophages, which imply that miR-122 may regulate DAK at the post-transcriptional level. These results will enhance our understanding about the regulation of miRNAs on immune response in fish.


Assuntos
Regiões 3' não Traduzidas/genética , Proteínas de Peixes/metabolismo , Macrófagos/imunologia , MicroRNAs/genética , Perciformes/imunologia , Animais , Células Cultivadas , Biologia Computacional , Helicase IFIH1 Induzida por Interferon/metabolismo , Camundongos , Poli I-C/imunologia , Receptores do Ácido Retinoico/metabolismo , Transdução de Sinais , Proteínas de Peixe-Zebra/genética
10.
Mol Immunol ; 85: 123-129, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28236773

RESUMO

Proinflammatory cytokines and type I IFNs were produced by TLR signaling and these responses are crucial for host defensive responses against pathogens. In order to avoid harmful and inappropriate inflammatory responses, there are multiple mechanisms to negatively regulate TLR signaling. In this paper, we have firstly studied IRF9 functions as a negative regulator involved in TRIF-mediated NF-κB pathway. Moreover, we found inhibitory effect of IRF9 primary depends on DBD domain. Interestingly, we also demonstrated that else mutants of IRF9, except for IRF9-ΔDBD, have different inhibitory effects upon TRIF-mediated NF-κB pathway. This study provides a novel evidence about the negatively regulation of innate immune signaling pathway in teleost fish. In addition, this finding provides new insights into the regulatory mechanism in mammals.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/imunologia , Imunidade Inata/imunologia , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/imunologia , NF-kappa B/imunologia , Perciformes/imunologia , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Western Blotting , Imuno-Histoquímica , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/metabolismo , NF-kappa B/metabolismo , Perciformes/metabolismo , Reação em Cadeia da Polimerase , Transdução de Sinais/imunologia
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