Expression Analysis And Functional Characterization Of Lipopolysaccharide-induced TNFα Factor(LITAF) From Zebrafish Danio Rerio

When pathogens invade the body,the innate immune system will quickly release pro-inflammatory cytokines as agents to clear the pathogens.Among the proinflammatory cytokines,TNFα is a small molecule protein secreted by macrophages and a toxic factor for many malignant tumor cells.It also is one of the most powerful biologically active factors that can directly kill tumor cells,which plays roles in resisting viral,bacterial and parasitic infections and autoimmune responses.As an important inflammatory factor,TNFα plays vital roles in homoestasis,inflammation and autoimmune diseases.The expression of TNFα can be activated by a variety of transcription factors,including NF-κB,NF-AT,AP-1,Ets,cAMP and lipopolysaccharide-induced TNFα factor(LITAF).LITAF is an important transcription factor that activates the expression of TNFα and regulates apoptosis and inflammation,which initially identified in human macrophage THP-1 cells by the stimulation of lipopolysaccharide(LPS).It not only induces the expression of TNFα,but also participates in apoptosis,cellular immunity,cell proliferation,autophagy and other life activities,and regulates other cytokines,such as ILlα,IFNγ and IL10.Zebrafish(Danio rerio),as a typical model animal for the study of fish and human diseases,has been applied in many fields.So the study of its innate immune response is also of great significance.In this study,a LITAF homologue was identified in zebrafish and was shown to compare with human LITAF in amino acid sequence,genomic organization and synteny.ZF4 cells were stimulated with pathogen associated molecular patterns(PAMPs),spring viremia of carp virus(SVCV)and zebrafish recombinant type I interferon proteins in vitro,while zebrafish were intraperitoneally(i.p.)injected with LPS,poly(I:C),Edwardsiella tarda(E.tarda),Aeromonas hydrophila(A.hydrophila)and SVCV in vivo.The expression profiles of zebrafish LITAF in healthy tissues and after challenges in vitro or in vivo were analyzed by quantitative real-time PCR.In this study,the functions of the zebrafish LITAF was also detected.The cytotoxic effect of LITAF in HEK293 T cells was analyzed by quantitative real-time PCR and flow cytometry,and its subcellular localization in HEK293 T and ZF4 cells was deteced.The results were shown as follows.1.The LITAF gene in zebrafish was identified,which was composed of 163 amino acids.The sequence homology rate of zebrafish and human LITAF was 39.76%,and both of them had a conservative LITAF domain.Sequence alignment,genomic organization,synteny and phylogenetic tree analysis of human and zebrafish LITAF show that the zebrafish LITAF is conserved,suggesting that its function may be conserved as well.2.Zebrafish LITAF was constitutively expressed in tissues,with the highest expression detected in the gills.In the embryos,its expression was steadily increased during embryonic development.It could be modulated by infection with LPS,poly(I:C),E.tarda,A.hydrophila and SVCV in vivo.When ZF4 cells were stimulated in vitro,the expression of LITAF in zebrafish was significantly increased by 2.3 times and 2.8 times respectively at 6 and 12 h after LPS stimulation,but the expression was not affected by SVCV,recombinant type I interferon protein,PHA and poly(I:C)stimulation,and the expression of LITAF was down-regulated after PMA stimulation.The results suggest that the zebrafish LITAF is involved in the immune defense against bacterial and viral infections,and it may function differently in cell types such as leucocytes and fibroblast cells.3.Overexpression of zebrafish LITAF in HEK293 T cells induced the up-regulation of TNFα,resulting in increased intercellular voids,decreased density and proliferation rate.While the number of cells in the control group increased rapidly and had a bigger density.It was confirmed by flow cytometry that LITAF could induce apoptosis in zebrafish,which might be mediated by caspase3.4.The zebrafish LITAF protein aggregated on the cellular and nuclear membrane and the subcellular structure associated with nuclear membrane.After the transfection of LITAF,the cell membrane was atrophied and deformed,and the cytoplasmic density was increased.The activation of LPS led to the rapid translocation of LITAF into the nucleus and induced apoptosis.In HEK293 T cells,LITAF is mainly located in the endoplasmic reticulum,rather than in the lysosome.