Functions Of TNF-α1 And TNF-α2 In Large Yellow Croaker On Monocyte/Macrophage Activation

Tumor necrosis factor-α（TNF-α）plays crucial roles in cell development,proliferation,apoptosis,inflammation,and immune response.There are two or even three isoforms of TNF-αin some fishes,but the functional differences between different TNF-αisoforms are still uncertain and need to be further explored.A TNF-αisoform has been reported in large yellow croaker,which is named TNF-α1 here.In this study,we identified a novel TNF-αhomologue（Lc TNF-α2）from large yellow croaker（Larimichthys crocea）,which shares a low amino acid sequence identity to the previously reported large yellow croaker TNF-α（Lc TNF-α1）.The open reading frame of Lc TNF-α2 is 714 nucleotides long,encoding a peptide of 237 amino acids（aa）.The deduced Lc TNF-α2 protein contains a 23-aa transmembrane region,a tumor necrosis factor-αconverting enzyme（TACE）restriction site at residues T⁷¹/L⁷²,a TNF family signature(I¹⁰⁸?F¹³⁵),and two conserved cysteine residues(C³⁹and C¹⁷⁹),as found in other known TNF-αsequences.Both Lc TNF-α1 and Lc TNF-α2 were constitutively expressed in all examined tissues and significantly up-regulated in the spleen and head kidney by Vibrio alginolyticus.Their transcripts were also detected in primary head kidney monocytes/macrophages（MO/Mφs）,lymphocytes（PKLs）,granulocytes（PKGs）,and large yellow croaker head kidney（LYCK）cell line and significantly increased in MO/Mφs by inactivated Vibrio alginolyticus.Recombinant Lc TNF-α1 and Lc TNF-α2 proteins（r Lc TNF-α1and r Lc TNF-α2）produced in Pichia pastoris not only significantly increased the production of reactive oxygen species（ROS）,but also promoted the expression of proinflammatory cytokines（IL-1β,IL-6,IL-8,and TNF-α1）in MO/Mφs from large yellow croaker.After stimulation with r Lc TNF-α1 and r Lc TNF-α2,the production of nitrogen oxide（NO）and the expression of inducible NO synthase（i NOS）gene were significantly increased.However,only r Lc TNF-α1 remarkedly enhanced the phagocytosis of MO/Mφs and increased the expression of TNF-α2 in MO/Mφs.All these results indicated that Lc TNF-α1 and Lc TNF-α2 have the similar but differential roles in promoting activation of MO/Mφs from head kidney of large yellow croaker.Through this study,we have a further understood the relationship between TNF-αand macrophage function,and further proved that TNF-αplays an important role in innate immune resonse.