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

Abstract

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 AjIKKε could 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 AjIKKε were 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 AjIKKε may 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.