Viperin Promotes Mycobacteria Tuberculosis Infection By Inhibiting IFN-γ Production

Tuberculosis(TB),caused by the bacterium Mycobacterium tuberculosis(Mtb),has been a serious challenge to the global public health system,resulting in significant morbidity and mortality worldwide.As the main habitat of Mtb,macrophages play an unshakable role in antituberculosis infection,in which IFN-γ signal is crucial for macrophages to activate and clear Mtb.A number of clinical studies have demonstrated the effectiveness of IFN-γ against tuberculosis(including multidrug-resistant tuberculosis),improving macrophage activity and accelerating recovery in patients who do not respond well to first-line antituberculosis therapy.Interferon-induced interferon-stimulated genes(ISGs)have been shown to have antifungal and bacterial effects in addition to a wide range of antiviral effects.As one of the most easily induced ISGs,Viperin can inhibit host immune response and promote Mtb infection in macrophages,but the relationship between Viperin and interferon as well as tuberculosis infection and its mechanism remain to be studied.Method1.The expression of Viperin in BMDM of Rsad2+/+ and Rsad2-/-mice was detected by qRTPCR and Western blot analysis;2.Effect of Viperin on Mtb infection in BMDM which detected by CFU analysis;3.qRT-PCR and ELISA analysis were used to detect the effects of Viperin on the levels of IFN-α,IFN-β and IFN-γ expression in Mtb infected BMDM at different time and multiple infections;4.Effect of Viperin on IFN-γ expression in lung and spleen macrophages were determined by IHC after Mtb infection in mice;5.Effect of Viperin on Mtb infection in BMDM after IFN-γ antibody pretreatment which detected by CFU analysis;6.Western Blot analysis was used to detect the changes of TBK1/IKK-ε,IRF3,JAK1 and STAT1 signaling pathways in Mtb infected BMDM for a short time;7.qRT-PCR and ELISA analysis were used to detect the level of IFN-γ expression in Mtb infected BMDM after pretreatment with corresponding inhibitors(BX-795,BAY-985,Abrocitinib,Fludarabine);8.Effect of Viperin on Mtb infection in BMDM after pretreatment with corresponding inhibitors(BX-795,BAY-985,Abrocitinib,Fludarabine)of related pathways which detected by CFU analysis;9.The phosphorylated protein expression level of JAK1 and STAT1 in Mtb infected BMDM after IFN-y antibody pretreattment which detected by by Western Blot analysis;10.Effect of Viperin on STAT1 nuclear localization in Mtb infected BMDM which detected by by confocal analysis;11.Combined with INH in BMDM of Rsad2-/-mice to detect its antibacterial effect on Mtb.Results1.Mtb infection promoted Viperin mRNA and protein expression in BMDM;2.Viperin promoted intracellular Mtb infection in BMDM;3.Viperin did not affect the production of IFN-α and IFN-β,but promotes bacterial by inhibiting the production of IFN-γ;4.Viperin promoted Mtb intracellular infection by inhibiting the expression of IFN-γ in BMDM through regulating the TBK1/IKK-ε signaling pathway;5.Viperin promoted Mtb intracellular infection by inhibiting the expression of INF-γ in BMDM through regulating the IRF3 signaling pathway;6.Viperin promoted Mtb intracellular infection by inhibiting the expression of IFN-γ in BMDM through regulating the JAK1-STAT1 signaling pathway;7.Viperin inhibited the nuclear translocation of p-STAT1;8.Viperin regulated JAK1-STAT1 signaling pathway through direct or indirect ways;9.INH enhanced the antibacterial effect of BMDM in Rsad2-/-mice.ConclusionsThis study demonstrated that Mtb-induced Viperin in BMDN promoted intracellular Mtb infection through inhibiting IFN-γ expression through TBK1/KK-ε-IRF3 and JAK1-STAT1 signaling pathways,and had better effects when combined with INH.This study provided a new approach and target for the clinical treatment of multidrug-resistant TB.