The Role And Regulatory Mechanism Of TRIM10 In The Innate Antiviral Immunity

Innate immunity represents the first defense line against microbial infection.The activation of innate immune responses requires the recognition of pathogen-associated molecular patterns(PAMPs)by pattern recognition receptors(PRRs).There are many PRRs in cells,including TLR,RLR,NLR,CLR and DNA receptors.The recognition of PAMPs by PRRs triggers the respective signal transduction pathway,ultimately leading to the production of type ? interferons and proinflammatory factors.Among them,interferons play a major role in resisting viral infection in the body,causing apoptosis of infected cells or exerting antiviral effects to inhibit viral replication.However,abnormal interferon regulatory signals elicit autoimmune disease.Therefore,the precise regulation of the expression of interferon is of great significance for maintaining the homeostasis of the human immune system.TRIM 10(Tripartite motif-containing protein 10)is a member of the TRIM family of E3 ubiquitin ligase.In humans,the TRIM family has more than 70 members.Members of the TRIM family have similar structural features,including a RING domain at the N-terminus,one or two B-boxes domains,a coiled-coil domain and a C-terminal variable domain.TRIM10 is localized on the human chromosome 6 short arm,where several other TRIM family members have been reported to play important regulatory roles in antiviral immunity.Of note,human major histocompatibility complex(MHC)class I molecules also reside in this region,suggesting that TRIM 10 is likely to possess an immune regulatory function.Therefore,my study explores whether TRIM10 plays a regulatory role in antiviral innate immunity.My study found that TRIM 10 can positively regulate the production of type I interferon in antiviral innate immunity and TRIM10-deficient mice are more susceptible to viral infection.ObjectivesTo explore the role and molecular mechanism of TRIM10 in antiviral innate immunity.Methods1)Measures interferon's expression mediated by innate immunity signaling pathway with overexpression of TRIM10.In HEK293T cells,the TRIM10 plasmid and signaling pathway adaptors cGAS+STING,MAVS,TRIF overexpression vector were co-transfected.After transfection for 24 hours,qRT-RCR(quantitative real-time PCR)and luciferase reporter assay were performed to test the expression and activation of IFN-? induced by different signaling pathway adaptors.2)Measures interferon s expression mediated by innate immunity signaling pathway with knockdown the expression of TRIM10.The primary peritoneal macrophages of wild-type mice were transfected with negative control siRNA and TRIM 10 siRNA.After 48h,SeV stimulates Oh,8h;VSV stimulates Oh,4h,8h,12h;ISD,HSV-60 transfects Oh,2h,4h,6h and LPS stimulates Oh,3h,6h.Then total RNA was extracted and reverse transcribed into cDNA.Expression of IFN-a and IFN-? was measured through qRT-PCR.3)Measures interferon s expression mediated by innate immunity signaling pathway in TRIM10 knockout macrophages.The primary peritoneal macrophages of wild-type mice and TRIM 10 knockout mice were stimulated with SeV,5'-pppRNA,ISD,HSV-60,LPS for indicated time points.Then total RNA was extracted and reverse transcribed into cDNA.Expression of IFN-?,Cxc110 and IL-6 was measured through qRT-PCR.And supernatant was measured through ELISA.4)Measures antiviral response in TRIM10 knockout macrophages.The primary peritoneal macrophages of WT and TRIM 10 knockout mice were stimulated with VSV for indicated time points.Expression of IFN-?,Cxc110 and IL-6 was measured through qRT-PCR.And expression of VSV was measured through Western Blot.5)Identifies the signaling molecules targeted by TRIM10 in innate immunity signaling pathway.The potential target of TRIM10 in signaling pathway were filtered through dualluciferase reporter gene assay and Co-Immunoprecipitation(Co-IP).Results1)Overexpression of TRIM10 upregulates the expression of interferon in the innate immune signaling pathway.After overexpressing TRIM 10 and adaptors in HEK293T cells,the changes of interferon at mRNA levels were detected by qRT-PCR,and the changes of interferon promoter activity were detected by dual luciferase reporter gene.After TRIM 10 overexpression,IFN-? mRNA levels and promoter activity were increased compared with the control group.2)Knockdown of TRIM10 downregulates the expression of interferon in the innate immune signaling pathway.TRIM 10 siRNA was transfected into primary peritoneal macrophages from wild-type mice to knock down the expression of TRIM 10.After transfection for 48h,cells were stimulated with RLR,TLR,DNA signaling pathway activator.qRT-PCR was used to detect the changes of interferon at the mRNA level.We found that the expression of IFN-? mRNA and IFN-a mRNA was decreased after knockdown of TRIM 10.We repeated SeV stimulation in THP-1 cells and obtained similar experimental results.3)Knockout of TRIM10 downregulates the expression of multiple cytokines in the innate immune signaling pathway.The primary peritoneal macrophages of wild-type mice and TRIM 10 knockout mice were tested for different time periods after stimulation with SeV,ISD,HSV-60,LPS or 5'-pppRNA and qRT-PCR was used to detect the changes of downstream cytokines at mRNA levels.We found that type I interferon and its downstream factors Cxc110 expression decreased and inflammatory factors IL-6 decreased.4)TRIM10 deficiency impairs macrophages antiviral response.The primary peritoneal macrophages of WT and TRIM10 knockout mice were tested for different time periods after stimulation with VSV.qRT-PCR was used to detect the changes of downstream cytokines at mRNA level and WB was used to detect the change of VSVG at protein level.5)TRIM10 targets TBK1.TRIM10 overexpression plasmid and cGAS+STING,RIG-IN,MDA5,MAYS,TBK1,IRF3-5D overexpression plasmid were co-transfected into HEK293T cells.Compared with the control group,TRIM10 overexpression group enhanced the cGAS+STING,RIG-IN,MDA5,MAVS,TBK1 mediated increase of IFN-? expression and promoter activity,but had no effect on IRF3-5D-induced IFN-P expression and reporter gene activity.Co-IP results showed that TRIM10 only interacted with TBK1 instead of other signaling molecules.Conclusion1)TRIM10 plays a positive role in antiviral innate immunity.2)TRIM10 targets TBK1,but the precise regulatory mechanisms need further study.