Molecular characterization, expression analysis and function identification of TNFα in black rockfish (Sebastes...

TNFα, as a pro-inflammatory cytokine, plays an important role in inflammation and immune homeostasis maintaining. However, the knowledge about the immune functions of teleost TNFα against bacterial infections is still limited. In this study, the TNFα was characterized from black rockfish (Sebastes schlegelii). The bioinformatics analyses showed the evolutionary conservations in sequence and structure. The expression levels of Ss_TNFα 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-1β 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_TNFα and other inflammatory cytokine mRNAs illustrated the variations of immunity in different tissues and cells of black rockfish. The regulated functions of Ss_TNFα in the up/downstream signaling pathways were preliminarily verified on the transcription and translation levels. Subsequently, in vitro knockdown of Ss_TNFα in the intestine cells of black rockfish confirmed the important immune roles of Ss_TNFα. 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_TNFα, 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_TNFα could trigger apoptosis of different cells in different strategies in black rockfish. Overall, the findings in this study indicated the important roles of Ss_TNFα in the immune system of black rockfish during pathogenic infection, as well as the potential function on biomarker for monitoring the health status.