Molecular characterization, expression and immune functional analysis of cystatin 10 in turbot.

BACKGROUND: Cystatin is a protease inhibitor that also regulates genes expression linked to inflammation and plays a role in defense and regulation. METHODS AND RESULTS: Cystatin 10 (Smcys10) was cloned from Scophthalmus maximus and encodes a 145 amino acid polypeptide. The results of qRT-PCR showed that Smcys10 exhibited tissue-specific expression patterns, and its expression was significantly higher in the skin than in other tissues. The expression level of Smcys10 was significantly different in the skin, gill, head kidney, spleen and macrophages after Vibrio anguillarum infection, indicating that Smcys10 may play an important role in resistance to V. anguillarum infection. The recombinant Smcys10 protein showed binding and agglutinating activity in a Ca2+-dependent manner against bacteria. rSmcys10 treatment upregulated the expression of IL-10, TNF-α and TGF-ß in macrophages of turbot and hindered the release of lactate dehydrogenase (LDH) from macrophages after V. anguillarum infection, which confirmed that rSmcys10 reduced the damage to macrophages by V. anguillarum. The NF-κB pathway was suppressed by Smcys10, as demonstrated by dual-luciferase analysis. CONCLUSIONS: These results indicated that Smcys10 is involved in the host antibacterial immune response.

C-type lectin 12B/4E of black rockfish (Sebastes schlegelii) macrophages as pattern recognition receptors in the antibacterial mechanism of exploration.

As lower vertebrates, fish have both innate and adaptive immune systems, but the role of the adaptive immune system is limited, and the innate immune system plays an important role in the resistance to pathogen infection. C-type lectins (CLRs) are one of the major pattern recognition receptors (PRRs) of the innate immune system. CLRs can combine with pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) to trigger NF-κB signaling pathway and exert immune efficacy. In this study, Ssclec12b and Ssclec4e of the C-type lectins, were found to be significantly up-regulated in the transcripts of Sebastes schlegelii macrophages stimulated by bacteria. The identification, expression and function of these lectins were studied. In addition, the recombinant proteins of the above two CLRs were obtained by prokaryotic expression. We found that rSsCLEC12B and rSsCLEC4E could bind to a variety of bacteria in a Ca2+-dependent manner, and promoted the agglutination of bacteria and blood cells. rSsCLEC12B and rSsCLEC4E assisted macrophages to recognize PAMPs and activate the NF-κB signaling pathway, thereby promoting the expression of inflammatory factors (TNF-α, IL-1ß, IL-6, IL-8) and regulating the early immune inflammation of macrophages. These results suggested that SsCLEC12B and SsCLEC4E could serve as PRRs in S. schlegelii macrophages to recognize pathogens and participate in the host antimicrobial immune process, and provided a valuable reference for the study of CLRs involved in fish innate immunity.

The TRAF gene family in turbot (Scophthalmus maximus): Identification, characterization, molecular evolution and expression patterns analysis.

Tumor necrosis factor receptor-associated factor (TRAF) is an important structural protein, which can bind to TNF receptors and participate in the regulation of TNF signaling pathway. Nonetheless, few studies have been conducted to investigate the systematic identification of TRAF gene family in teleost and role in innate immunity of turbot (Scophthalmus maximus). In this study, eight TRAF genes, namely SmTRAF2aa, SmTRAF2ab, SmTRAF2b, SmTRAF3, SmTRAF4a, SmTRAF5, SmTRAF6 and SmTRAF7, were identified and annotated in turbot by using bioinformatics methods. Analysis of the phylogenetic, syntenic and molecular evolution demonstrated that all SmTRAF members were evolutionarily conserved in teleost. Domain analysis showed all SmTRAF proteins contained a typical conserved N-terminal RING finger domain. Most SmTRAF proteins contained a MATH domain at the C-terminal, while SmTRAF7 contains seven duplicate WD40 domains. In addition, quantitative real-time PCR was performed to detect the expression patterns of SmTRAFs in tissues from healthy and Vibrio anguillarum infected turbots. The results indicated SmTRAFs had diverse tissue expression patterns and the expression of TRAF gene changed significantly after V. anguillarum infection. This study provided a basis for understanding the roles of TRAFs in the innate immune response of turbot.