IKKεpositively regulates NF-κB,MAPK,and IRF3-mediated type Ⅰ IFN signaling pathways in Japanese eel(Anguilla japonica)

IKKε is an IκB kinase participating in the control of NF-κB and type Ⅰ IFN signal pathways in mammals.However,the function of IKKε in regulating immune response is largely unknown in teleost.Herein,an IKKε homologue named AjIKKε was characterized in Japanese eel(Anguilla japonica).AjIKKε has an N-terminal kinase domain,a ubiquitin-like domain,and a coiled coil-containing domain(CC),which is conserved and similar to its counterpart in mammals.Expression analysis showed that AjIKKεcould be up-regulated in kidney,spleen,and particularly in liver under the stimulation of poly Ⅰ:C,LPS,and Aeromonas hydrophila infection.In vitro,the mRNA levels of AjIKKεwere significantly provoked in eel liver cells stimulated by LPS and poly I:C,or the different concentrations of A.hydrophila.The overexpression of AjIKKε could not only induce a significantly higher level of promoter activity of human NF-κB,AP-1,and IFN-β in a dose-dependent manner but also up-regulate the activation of promoters of Japanese eel cRel,AP1,IL6,IFN4,IRF3,and IRF7 in HEK293 cells.RNAi studies showed that after AjIKKε was knocked down,the expression levels of IL1,IL6,TNFα,c-Jun,IFN2,IFN3,MX1,MX2,and IRF3 genes were significantly down-regulated in liver,spleen,and kidney of Japanese eels.In addition,the mutants of AjIKKε-K39A,AjIKKε-S174A,and AjIKKε-ΔCC failed to activate Japanese eel IFN4,IRF3 and human IFN-β promoters in HEK293 cells.Collectively,these results suggest that AjIKKεmay function as a positive regulator of NF-κB,MAPK,and IRF3-mediated type Ⅰ IFN signaling pathways related to immune response evoked by bacterial and viral infection.