Ctenopharyngodon idella TBK1 activates innate immune response via IRF7

Abstract

In mammals, IFN regulatory factor (IRF) 7 is a central regulator of IFN-α expression in response to variable pathogenic infections. There are several pathogenic sensors involved in monitoring pathogen intrusion in mammals. These sensors trigger IRF7-mediated responses through different pathways. TANK-binding kinase 1 (TBK1) is a critical mediator of IRF7 activation upon pathogen infection. In fish, there are many reports on TBK1, IRF3 and IRF7, especially on TBK1-IRF3 signaling pathway. However, it is not very clear how TBK1-IRF7 works in innate immune signaling pathway. In this study, we explored how TBK1 up-regulates IFN, ISG expression, and how TBK1 initiates innate immune response through IRF7 in fish under lipopolysaccharides (LPS) stimulation. After stimulation with LPS, grass carp IRF3 and IRF7 transcriptions were up-regulated, indicating they participate in TLR-mediated antiviral signaling pathway. It is interesting that the response time of grass carp IRF3 to LPS was earlier than that of IRF7. In addition, IRF7 rather than IRF3 acted as a stronger positive regulator of IFN and ISG transcription in Ctenopharyngodon idella kidney cells (CIKs). It is suggested the potential function differentiation between IRF3 and IRF7 upon LPS infection in fish. Dual luciferase assays also showed that overexpression of grass carp IRF7 and TBK1 up-regulated the transcription level of IFN and PKR. However, knockdown of IRF7 inhibits ISG expression, suggesting that grass carp TBK1 regulates the transcription via IRF7. Co-immunoprecipitation and GST pull-down assays proved the binding of grass carp IRF7 to TBK1. Furthermore, grass carp TBK1 can promote the nuclear translocation of IRF7. The results indicated that grass carp TBK1 can bind directly to and activate IRF7.