Study On The Mechanisms Of IFIT5 Negatively Regulates Type ? Interferon Signaling Pathway

Innate immunity,also known as natural immunity or non-specific immunity,refers to the immune defense ability gradually developed by the organism in genetic evolution to prevent the invasion of foreign microorganisms or pathogens.Innate immunity constitutes the first line of defense of the organism against pathogenic microorganisms.Innate immunity is mainly completed by the synergistic effect of innate immune cell-and molecule-mediated signaling pathways.The type I interferon signaling pathway is a critical component of the innate immune signaling pathway,which can participate in the immune defense and clearance of various pathogenic microorganisms.When microorganisms or pathogens invade the organism,the cascade reaction of signaling pathways mediated by pattern recognition receptors induces the production of interferons,activates the Jak-STAT signaling pathway,and produces a series of interferon stimulating genes.Interferons directly or indirectly mediate the clearance of pathogens,regulate the immune responses,and maintain the immune homeostasis of the organism.Interferon-inducible protein with tetratricopeptide(IFITs)family repeats is a typical and conservative ISGs,which plays an essential role in regulating pathogen-mediated immune response and maintaining immune homeostasis.The protein family mainly includes four members: IFIT1,IFIT2,IFIT3,and IFIT5.Comparing with IFIT1,IFIT2,and IFIT3,the role and detailed mechanism of IFIT5 in regulating innate immune response,especially IFN signaling pathway response,remain to be further studied.This study explores the role of IFIT5 in IFN-I signaling pathway response and its potential regulatory mechanism.To explore the role of IFIT5 in the virus-induced IFN-I signaling pathway,the dual-fluorescence reporter gene system was used in this study to detect the effects of IFIT5 on the promoter activity of IFN-?,ISRE,IRF3,and NF-?B induced by Se V and poly(I:C)in HEK293 T cells.Also,the m RNA levels of IFN-? and ISGs were detected by real-time PCR.The results showed that IFIT5 overexpression significantly inhibited the promoter activity of IFN-?,ISRE,IRF3,and NF-?B induced by Se V and poly(I:C).The m RNA levels of IFN-? and ISGs were upregulated upon IFIT5 overexpression.Meanwhile,IFIT5 inhibited the secretion of IFN-? in the cell culture supernatant,which is conducive to replicating the Se V genome.On the contrary,IFIT5 knockdown or knockout facilitates IFN-?,ISRE,IRF3,and NF-?B promoter activation,as well as IFN-?,ISGs production.Therefore,IFIT5 negatively regulates the Se V-induced IFN-I signaling pathway.To determine the target molecule of IFIT5 in the IFN-I signaling pathway,the recombinant eukaryotic plasmids expressing each molecule in the IFN-I signaling pathway were constructed.These plasmids were co-transfection with IFIT5 expression plasmids and dual-luciferase reporter gene plasmids into HEK293 T cells,and the effect of IFIT5 on the activity of IFN-? promoter activated by each molecule in the signaling pathway was evaluated by the dual-luciferase reporter system.The results showed that IFIT5 inhibited the IFN-? promoter activity induced by IRF3 and its upstream molecules but had no significant effect on the IFN-? promoter activity caused by the IRF3-5D activation mutant.Further investigations demonstrated that IFIT5 interacted with IRF3 and competitively bounnd to the IRF3 IAD domain,thereby inhibited IRF3 phosphorylation,dimerization,and nuclear entry.The above findings indicate that IFIT5 inhibits the IFN-I signaling pathway by targeting IRF3,inhibiting IRF3 activation,and damaging the TBK1-IKK?-IRF3 signaling complex.To further elucidate the regulatory mechanism of IFIT5 on the IFN-I pathway,the interaction of IFIT5 with TBK1 and IKK? and its effects on TBK1-IRF3 and IKK?-IRF3 complexes were examined.The results showed that IFIT5 interacted with TBK1 and IKK?,recruited IKK? and degraded IKK? and IRF3 through autophagy-lysosome pathway,thereby inhibiting the production of interferon.In summary,this study revealed that IFIT5 inhibits Se V-induced IFN-I innate immune response by targeting IRF3,recruiting and degrading IKK?,thus disrupting the formation of TBK1-IKK?-IRF3 signaling complexes.The detailed molecular mechanism of the negative regulatory function of IIFT5 on the natural immune signaling pathway was elaborated for the first time,providing a new horizon and perspective for further exploring the innate immune signaling pathway.