Ctenopharyngodon Idella TBK1 Activates Innate Immune Response Via IRF7

The innate immune response protects host cells from the invasion of pathogenic microorganisms.In this signaling pathway,a large number of cytokines are involved,eventually IFN and ISGs expression activates the innate immune response.In mammals,IRF3 and IRF7 are critical in signaling pathways that induce type I IFN responses under variable pathogenic infections.Interferon regulatory factor(IRF)7 is also a central regulator of IFN-? expression response.Multiple signal transduction pathways can be activated when pathogenic sensors detect mammalian pathogen invasion.Its recognition and signal transduction are key events in host immune responses and are associated with many inflammatory diseases that trigger IRF7-mediated responses through different pathways.TANK binding kinase 1(TBK1)is a key mediator of IRF7 activation following pathogen infection.Among fish,there are many reports on TBK1,IRF3 and IRF7,especially regarding the TBK1-IRF3 signaling pathway.However,the mechanism of action of TBK1-IRF7 in the innate immune signaling pathway is unclear.In this study,we investigated how TBK1 up-regulates IFN,ISG expression,and how TBK1 initiates an innate immune response in fish through IRF7 under LPS stimulation.Lipopolysaccharide(LPS)is the main component of the cell wall of Gram-negative bacteria.After stimulation with LPS,the expression levels of IRF3 and IRF7 in grass carp are significantly up-regulated,indicating that both IRF3 and IRF7 are involved in TLR-mediated antiviral.signal path.Interestingly,grass carp IRF3 responded to LPS earlier than IRF7.Furthermore,IRF7,but not IRF3,acts as a stronger positive regulator of IFN and ISG transcription in grass carp kidney cells(CIKs).The functional differences between fish IRF3 and IRF7 after LPS infection are shown.The dual luciferase assay also showed that overexpression of grass carp IRF7 and TBK1 up-regulated the transcript levels of IFN and PKR,and fish IRF7 was able to amplify IFN and PKR promoter activity compared to IRF3.To further understand the innate immune response mechanism mediated by TBK1-IRF7 in fish,it was found that overexpression of TBK1 significantly up-regulated IRF7,PKR and PKZ protein levels.However,knocking out IRF7 inhibited ISG expression,indicating that grass carp TBK1 regulates ISG through IRF7.Transcription.Later,co-immunoprecipitation and GST pull-down experiments demonstrated that grass carp IRF7 can bind to TBK1.Fluorescence colocalization experiments have further confirmed that they can be combined directly in vivo.Our study found that binding of TBK1 to IRF7 activates IRF7.Under normal conditions,fish IRF7 and TBK1 have similar subcellular localizations,all present in the cytoplasm,but after stimulation with LPS or transfection with TBK1,laser confocal microscopy Both nuclear and nuclear extraction experiments indicated that IRF7 translocated from the cytoplasm to the nucleus,and it was also detected that the dimerization of IRF7 was also increased.In summary,our study found that when foreign substances stimulate host cells,after a series of signal transduction,TBK1 can bind to IRF7 to activate IRF7,and translocate to the nucleus to bind to IFN and ISGs promoters to activate innate immunity.reaction.First,both grass carp IRF3 and IRF7 can participate in the TLR-mediated signaling pathway,and IRF7 is found to be more responsive to IFN and ISG transcription.Overexpression of TBK1 can then up-regulate the expression of IRF7 and ISG,while knockdown of IRF7 inhibits ISG expression.The results showed that grass carp TBK1 up-regulated the expression of IFN and PKR by IRF7.Finally,co-immunoprecipitation,GST-Pulldown and colocalization experiments demonstrated that fish TBK1 interacted with IRF7.Moreover,fish TBK1 promotes dimerization and nuclear translocation of IRF7,thereby activating innate immune responses.