Viperin Negatively Regulates Inflammatory Cytokine And NO Through IRAK1-TRAF6-TAK1 To Facilitate Mycobacterium Tuberculosis Infection

BackgroundTuberculosis(TB),which is caused by Mycobacterium tuberculosis(Mtb)infection,represents the most emerging infectious diseases in the world.At the same time,multidrug-resistant tuberculosis(MDR-TB)is becoming increasingly widespread,with a cure rate of only 55%.At present,the most commonly used drugs for the effective treatment of TB are rifampicin and isoniazid.However,first-line drugs mainly target Mtb to exert bactericidal effects and are prone to drug resistance problems.In recent years,interferons(IFNs)have been used clinically in combination with first-line drugs in the treatment of TB,especially MDR-TB.Interferons have diverse effects on regulating host immune response in infectious diseases.IFNs activate the JAK-STAT signaling pathway and promote the production of hundreds of interferon-stimulated genes(ISGs).Different ISGs have different effects on infectious diseases.Some important ISGs such as ISG15,IFITMs,IRF1,and MxA have been reported to regulate Mtb infection,and the effects and mechanisms of most ISGs remain unknown.As one of ISGs,Viperin has a broad-spectrum antiviral effect,and plays an important role in the natural immune defense of macrophages and dendritic cells and the maintenance of host homeostasis.However,whether Viperin is involved in the regulation of macrophage function and the inmune response against Mtb infection has not yet been studied.Therefore,in-depth study of the interaction between Viperin and Mtb infection is of great significance for regulating Mtb infection and development of anti-Mtb drugs.Objective1.Determine the expression of Viperin in TB patients' PBMC/hMDMs;2.Explore the effect of Viperin on macrophage killing Mycobacterium tuberculosis;3.Investigate the pathways that Viperin participates in promoting Mtb infection in macrophage(inflammatory cytokines,ROS,RNS,M1/M2 polarization);4.Investigate whether Viperin binds IRAK1 and TAK1 to competitively interrupt the IRAK1-TRAF6-TAK1 interaction,thus reducing IRAK1/TRAF6 ubiquitination and inhibiting NF-?B/MAPKs pathway signal axis;5.To explore the ability of Rsad2-/-C57BL/6J mice to resistant Mtb infection.Method1.RT-PCR and WB were used to detect the expression of Viperin in PBMCs/hMDMs of TB patients and healthy individuals;the expression of Viperin in hMDMs,THP-1M?,and BMDM infected by Mtb H37Rv.2.The effect of Viperin on the killing of Mycobacterium tuberculosis by macrophages was measured by H37Rv plate colony counting test(CFU).3.The expression of inflammatory factors(TNF-?,IL-6,IL-10,TGF-? and IFN-?/?/?)involved in killing Mycobacterium tuberculosis in hMDMs/BMDM were detected by RT-PCR,ELISA and Luminex.4.H37Rv infection Rsad2-/-BMDM:Nitric oxide synthase(iNOS)was detected by RT-PCR and WB.Nitric Oxide(NO)secreted by macrophages was detected by Griess-Reagent-System.Phosphorylation levels of TAK1-IKK?/?-NF-?B/MAPKs pathway kinases were detected by WB.Reactive oxygen species(ROS)in macrophages was detected by ROS-Activity-Assay-Kit fluorescent probes.Pretreatment of macrophages with NO inhibitor(L-NAME HC1),NF-?B inhibitor(JSH-23),and JNK inhibitor(SP600125).H37Rv CFU was used to detect the ability of each inhibitor to restore Viperin's ability to promote Mycobacterium tuberculosis infection in macrophages.5.H37Rv infection Rsad2-/-BMDM:Co-Immunoprecipitation(CO-IP)was used to detect whether IRAK1,TRAF6,TAK1 and Myd88 bound to Viperin.293T cells overexpress the corresponding plasmid.CO-IP was used to detect the Viperin functional domain bound to IRAK1,TRAF6 and TAK1.6.Viperin expression was detected by immunohistochemistry(IHC)in lung and lymph nodes of TB patients;7.Rsad2-/-mice were infected with Mtb in vivo for 1 and 4 weeks.The ability of Rsad2-/-mice to resist Mtb infection was verified by the CFU of the mouse spleen and lung;Results1.Viperin is highly expressed in the lungs and lymph nodes of TB patients.Mtb infection promotes high expression of Viperin in TB patients' PBMCs/hMDMs.Viperin promotes Mtb infection of macrophages;2.Viperin inhibits the production of proinflammatory cytokines in Mtb-infected macrophages;3.Viperin negatively regulates NO production through NF-?B/MAPKs signaling pathway during Mtb infection;4.Viperin binds to IRAK1 and TAK1,interrupting the interaction of IRAK1-TRAF6-TAK1 complex,thus reducing IRAK1/TRAF6 ubiquitination and inhibiting TAK1-IKK?/? signaling;5.Rsad2-/-mice are more resistant to Mtb infection in vivo.ConclusionsViperin was significantly induced in PBMC and monocytes of pulmonary TB patients in vivo and in Mtb-infected macrophages in vitro.In addition,this research found that Viperin inhibits the production of pro-inflammatory cytokines and NO,and promotes Mtb infection of macrophages,indicating that Viperin participates in activation of innate immunity upon Mtb infection.Rsad2-/-mice experiments in vitro demonstrated that Viperin binds to IRAK1 and TAK1,interrupting the interaction of IRAK1-TRAF6-TAK1 complex,reducing IRAK1/TRAF6 ubiquitination.Thus,it inhibits TAK1-IKK?/?-NF-?B/MAPKs signaling pathway,reduces the production of pro-inflammatory cytokines and NO,and promotes Mtb infection of macrophages.Given the importance of Viperin on regulation of innate immune responses,Viperin may represent a promising target for HDT in TB therapy.