RESUMO
Leukocyte cell-derived chemotaxin 2 (LECT2) is a multifunctional cytokine involved in many diseases in which immune dysfunction is present. Ayu LECT2 (PaLECT2), which interacts with a C-type lectin receptor (PaCLR), was shown to activate ayu head kidney-derived monocytes/macrophages (MO/MΦ) to improve the outcomes of fish upon bacterial infections. However, it is not known if PaCLR mediates PaLECT2 effects on ayu MO/MΦ. In this study, we determined the role of PaCLR in signal transduction of PaLECT2 on ayu MO/MΦ. We expressed the PaCLR ectodomain in Escherichia coli and produced a refolded recombinant protein (rPaCLR) that was then used to produce the anti-PaCLR IgG (anti-PaCLR) for neutralization. Addition of the refolded PaLECT2 mature peptide (rPaLECT2m) to ayu MO/MΦ cultures, increased cytokine expression, induced chemotaxis, and enhanced phagocytosis and bactericidal activity of these cells were observed. When we added anti-PaCLR to block the ectodomain of PaCLR, these effects were significantly inhibited. Based on our previous works and the data presented here, we conclude that PaCLR mediates the immunomodulatory effects of PaLECT2 on ayu MO/MΦ, thus defining a mechanism by which LECT2 protects fish against pathogens.
Assuntos
Proteínas de Peixes/genética , Imunidade Inata , Peptídeos e Proteínas de Sinalização Intercelular/genética , Lectinas Tipo C/genética , Lectinas/genética , Osmeriformes/genética , Animais , Quimiotaxia , Escherichia coli/genética , Proteínas de Peixes/metabolismo , Rim Cefálico/metabolismo , Imunomodulação , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Lectinas/metabolismo , Lectinas Tipo C/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Organismos Geneticamente Modificados/genética , Osmeriformes/imunologia , Osmeriformes/metabolismoRESUMO
C-type lectin receptors (CTLRs) play vital roles in immune responses as pattern-recognition receptors (PRRs). In this study, we identified a novel C-type lectin receptor (PaCTLRC) gene from ayu, Plecoglossus altivelis. Predicted PaCTLRC is a single transmembrane receptor with a typical carbohydrate recognition domain (CRD) at its C-terminus. Sequence comparison and phylogenetic tree analysis showed that PaCTLRC was most closely related to Atlantic salmon (Salmo salar) CLRC, but was significantly different from two other ayu CTLRs, aCLR and PaCD209L. PaCTLRC transcript was detected in all tested tissues and cells, with high levels in the liver; and its expression was significantly altered upon Vibrio anguillarum infection. Refolded recombinant PaCTLRC (rPaCTLRC) agglutinated three types of Gram-positive bacteria (Listeria monocytogenes, Staphylococcus aureus and Streptococcus iniae) and four types of Gram-negative bacteria (Aeromonas hydrophila, Escherichia coli, V. anguillarum and Vibrio parahaemolyticus) in a Ca(2+)-dependent manner in vitro, and Gram-positive bacteria were shown to be biologically relevant ligands for PaCTLRC. rPaCTLRC bound to d-mannose, d-galactose, l-fucose, N-acetyl-d-glucosamine (GlcNAc), lipopolysaccharide (LPS) and peptidoglycan (PGN), exhibiting a relative binding strength to d-mannose and PGN. d-Mannose, l-fucose, GlcNAc, LPS and PGN could inhibit the agglutinating activity of rPaCTLRC, while d-galactose did not functioned. PaCTLRC neutralization using anti-PaCTLRC IgG resulted in the inhibition of phagocytosis by ayu monocytes/macrophages (MO/MΦ) of S. aureus but not of E. coli, and produced a consistently higher survival rate of S. aureus than that of E. coli. d-Mannose, LPS and PGN treatment had no significant influence on the phagocytosis of ayu MO/MΦ. These results suggest that PaCTLRC may serve as a Gram-positive bacteria-preferred PRR which is involved in pathogen recognition and signal transduction in ayu MO/MΦ.
Assuntos
Proteínas de Peixes/imunologia , Macrófagos/imunologia , Monócitos/imunologia , Osmeriformes/imunologia , Receptores Mitogênicos/imunologia , Aeromonas hydrophila/imunologia , Sequência de Aminoácidos , Animais , Escherichia coli/imunologia , Proteínas de Peixes/química , Proteínas de Peixes/genética , Regulação da Expressão Gênica , Hexoses/imunologia , Hexoses/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/metabolismo , Listeria monocytogenes/imunologia , Macrófagos/microbiologia , Dados de Sequência Molecular , Monócitos/microbiologia , Osmeriformes/classificação , Osmeriformes/genética , Fagocitose , Filogenia , Estrutura Terciária de Proteína , Receptores Mitogênicos/química , Receptores Mitogênicos/genética , Salmo salar/classificação , Salmo salar/genética , Salmo salar/imunologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Staphylococcus aureus/imunologia , Vibrio/imunologiaRESUMO
Interleukin 1ß (IL-1ß), the first interleukin to be characterized, plays a key role in regulating the immune response. In this study, we determined the cDNA and genomic DNA sequences of the IL-1ß gene from the large yellow croaker, Larimichthys crocea. Phylogenetic analysis indicated that the IL-1ß (LcIL-1ß) gene was most closely related to that of European seabass (Dicentrarchus labrax), sharing 67.8% amino acid identity. In healthy large yellow croaker, LcIL-1ß transcription was detected in all tested tissues, with the highest level found in the head kidney. Upon Vibrio alginolyticus infection, LcIL-1ß transcription in all tested tissues was significantly upregulated. Intraperitoneal injection of recombinant LcIL-1ß (rLcIL-1ß) improved the survival rate and reduced the tissue bacterial load after V. alginolyticus infection. In addition, rLcIL-1ß induced monocytes/macrophages (MO/MΦ) chemotaxis and increased phagocytosis and bactericidal activity in vitro. These results suggest that LcIL-1ß plays an important role in the large yellow croaker immune response against V. alginolyticus.