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1.
Fish Shellfish Immunol ; 119: 587-601, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34743023

RESUMO

Toll-like receptors (TLRs) are evolutionarily conserved proteins of pattern recognition receptors (PRRs) and play a crucial role in innate immune systems recognition of conserved pathogen-related molecular samples (PAMPs). We identified and characterized TLR18 from Nile tilapia (Oreochromis niloticus), OnTLR18, to elucidate its role in tissue expression patterns, modulation of gene expression after microbial challenge and TLR ligands, subcellular localization in fish and human cells, and the possible effectors TLR18 induces in a melanomacrophage-like cell line (tilapia head kidney (THK) cells). OnTLR18 expression was detected in all tissues examined, with the highest levels in the intestine and the lowest in the liver. OnTLR18 transcript was up-regulated in immune-related organs after bacterial and polyinosinic-polycytidylic acid (poly I:C) challenges and in the THK cells after lipopolysaccharide (LPS) stimulation. In transfected THK and human embryonic kidney (HEK) 293 cells, OnTLR18 localizes in the intracellular compartment. OnMyD88 and OnTRIF, but not OnTIRAP, were co-immunoprecipitated with OnTLR18, suggesting that the former two molecules are recruited by OnTLR18 as adaptors. The constitutively active form of OnTLR18 induced the production of pro-inflammatory cytokines, type I interferon (IFN), and antimicrobial peptides such as tumor necrosis factor α, interferon (IFN) d2.13, tilapia piscidin (TP)2, TP3, TP4, and hepcidin in THK cells. Our results suggest that OnTLR18 plays an important role in innate immunity through initiating nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and IFN signaling pathways via OnMyD88 and OnTRIF and induces the production of various effectors in melanomacrophages.


Assuntos
Ciclídeos , Doenças dos Peixes , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Animais , Peptídeos Antimicrobianos , Ciclídeos/genética , Ciclídeos/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Células HEK293 , Humanos , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Poli I-C/farmacologia
2.
Dev Comp Immunol ; 117: 103991, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33387560

RESUMO

Toll/interleukin 1 receptor domain-containing adaptor protein (TIRAP) and toll/interleukin 1 receptor-domain-containing adapter-inducing interferon-ß (TRIF) are crucial adaptors of signal transduction for the signaling pathways of toll-like receptors (TLRs). TIRAP and TRIF perform an essential function in an antimicrobial immune response; however, their function in Nile tilapia remains unknown. Herein, TIRAP and TRIF from Nile tilapia were identified and functionally characterized. Phylogenetic analysis showed that OnTIRAP and OnTRIF clustered with corresponding homologs from other fish species, with comparable gene structures to those of select vertebrate TIRAP and TRIF genes, respectively. The expression profiles of OnTIRAP and OnTRIF were broadly distributed in the ten tissues investigated, with high transcript levels noticed in immune organs. The transcription levels of OnTIRAP and OnTRIF were upregulated in response to bacterial and poly (I:C) challenges. GFP signals were only detected in the cytoplasmic region of fish cells transfected with OnTIRAP-GFP and OnTRIF-GFP expression plasmids. Moreover, overexpression of OnTIRAP and OnTRIF activated interferon-ß (IFN-ß) and activator protein 1 (AP1) reporters in HEK 293 cells. Activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) reporter was only observed in OnTRIF-overexpressing HEK 293 cells. Furthermore, the results of the co-immunoprecipitation analysis showed that OnTRIF, but not OnTIRAP, was recruited as an adaptor protein by OnTLR25. This study provides the first evidence on the functions of OnTIRAP and OnTRIF in the immune system of Nile tilapia against pathogens and may serve as the basis for further investigations on TLR signaling in fish.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Adaptadoras de Transporte Vesicular/imunologia , Ciclídeos/imunologia , Proteínas de Peixes/imunologia , Expressão Gênica/imunologia , Transdução de Sinais/imunologia , Proteínas Adaptadoras de Transdução de Sinal/classificação , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transporte Vesicular/classificação , Proteínas Adaptadoras de Transporte Vesicular/genética , Sequência de Aminoácidos , Animais , Bactérias/imunologia , Bactérias/patogenicidade , Linhagem Celular , Ciclídeos/genética , Ciclídeos/microbiologia , Doenças dos Peixes/genética , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Perfilação da Expressão Gênica/métodos , Células HEK293 , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata/genética , Imunidade Inata/imunologia , Filogenia , Homologia de Sequência de Aminoácidos , Transdução de Sinais/genética , Virulência/imunologia
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