Molecular characterization, expression analysis and function identification of TNF? in black rockfish (Sebastes schlegelii)

TNF alpha, as a pro-inflammatory cytokine, plays an important role in inflammation and immune homeostasis maintaining. However, the knowledge about the immune functions of teleost TNF alpha against bacterial infections is still limited. In this study, the TNF alpha was characterized from black rockfish (Sebastes schlegelii). The bioinformatics analyses showed the evolutionary conservations in sequence and structure. The expression levels of Ss_TNF alpha mRNA were significantly up-regulated in the spleen and intestine after Aeromonas salmonicides and Edwardsiella tarda infections, and dramatically down-regulated in PBLs after LPS and poly I:C stimulations. Meanwhile, the extremely up-regulated expressions of other inflammatory cytokines (especially for IL-1 beta and IL17C) were observed in the intestine and spleen after bacterial infection and down-regulations were obtained in PBLs. The significant regulation with expression patterns of Ss_TNF alpha and other inflammatory cytokine mRNAs illustrated the variations of immunity in different tissues and cells of black rockfish. The regulated functions of Ss_TNF alpha in the up/downstream signaling pathways were preliminarily verified on the transcription and translation levels. Subsequently, in vitro knockdown of Ss_TNF alpha in the intestine cells of black rockfish confirmed the important immune roles of Ss_TNF alpha. Finally, the apoptotic analyses were conducted in PBLs and intestine cells of black rockfish. The rapid increases of the apoptotic rates were obtained in both PBLs and intestine cells after treatment with rSs_TNF alpha, but distinct apoptotic rates at the early and late stages of apoptosis were observed between these two types of cells. The results of apoptotic analyses suggested that Ss_TNF alpha could trigger apoptosis of different cells in different strategies in black rockfish. Overall, the findings in this study indicated the important roles of Ss_TNF alpha in the immune system of black rockfish during pathogenic infection, as well as the potential function on biomarker for monitoring the health status.

Automated summary

In this study, the TNF alpha was characterized and its roles in the immune system of black rockfish during bacterial infections were explored. The results showed that Ss_TNF alpha was significantly up-regulated in the spleen and intestine after infections, while down-regulated in PBLs. The expression patterns of other inflammatory cytokines were also observed.