MxA Suppresses TAK1-IKK?/?-NF-?B Signaling Pathway To Facilitate Mycobacterium Tuberculosis Infection

BackgroundTuberculosis(TB)is a fatal chronic infectious disease caused by Mycobacterium tuberculosis(MTB)infection.It has existed for thousands of years and remains a major threat of global human health as it is one of the top ten causes of death worldwide.Interferons(IFNs)have become a highly effective drug for TB treatment,especially against multiple drug resistance(MDR)-TB.IFNs regulate the host immune response to control the outcomes of infectious diseases by inducing a series of downstream IFN-stimulated genes(ISGs)via the JAK-STAT pathway after binding to different types of IFN receptors.Several ISGs(such as ISG15,IFITM3,and IRF1)have been reported to play important roles in anti-TB responses,however,it is unknown whether other ISGs participate in host immune responses to MTB infection.Myxovirus resistance protein 1(Myxovirus resistance protein 1,MxA)is one of the classic ISGs,which has a broad-spectrum antiviral function.It has been reported that MxA is highly expressed in TB patients and the positive tuberculin skin reactions,however,how MxA regulate MTB infection is unknown.Objective1.Screen the ISGs that affect MTB infection in macrophages(M?s).2.Determine the expression of MxA in MTB-infected M?s and the effect of MxA on the survival of MTB in M?s.3.Explore the molecular mechanism of MxA acting on MTB infection.Methods1.mRNA expression detection:real-time quantitative PCR(qRT-PCR).2.Protein expression and activation detection:cellular immunofluorescence,Western blot.3.Cytokine expression detection:Multiplex Luminex,ELISA.4.Intracellular MTB Survival Detection:Colony forming unit(CFU).Results1.The mRNA expression of 8 ISGs in M?s after MTB infection are significantly up-regulated.2.Silence of MxA and Ddx58 can limit the survival of MTB in Mcps.3.The expression of MxA at mRNA and protein level is significantly up-regulated in THP-1-M?s and hMDMs after MTB-infecttion.4.Silencing MxA in THP-1-M?s and hMDMs limits MTB survival.5.Silencing MxA limits the survival of MTB in M?s by promoting the secretion of key antibacterial factors such as proinflammatory cytokines IL-1?,IL-6 and TNF-?,without affecting the expression of NO or chemokines,autophagy,apoptosis and pyroptosis.6.MxA promotes MTB infection independent of JAK-STAT1-IFNs signaling.7.MxA silencing controls the intracellular MTB by activating the NF-?B signaling pathway.8.Silencing MxA activates the NF-?B pathway by increasing IKK?/?phosphorylation.9.Silencing MxA activates the IKK?/?-NF-?B pathway by increasing TAK1 phophorylation.10.Low concentration of INH combined with MxA silencing facilitate enhanced antibacterial effect through activating TAK1-IKK?/?-NF-?B signaling pathway.ConclusionsIn this study,we examined eight important ISGs,which were significantly induced during MTB infection.We profiled their potential effects on MTB infection in human macrophages(M?s)by RNA interference and identified Myxovirus resistance protein 1(MxA)and DExD/H-Box helicase 58(Ddx58,also termed RIG-1)as the key ISGs to promote intracellular bacterial growth.Furthermore,we demonstrated that mycobacterial infection potently induced MxA expression in human peripheral blood mononuclear cell-derived macrophages(hMDMs)and THP-1 cell line-derived macrophages(THP-1-M(ps)at both mRNA and protein levels and MxA silencing effectively inhibited intracellular survival of MTB.Mechanistically,we found that MxA silencing was surprisingly able to induce NF-?B p65 phosphorylation and NF-?B p65 translocation into the nucleus.In addition,TAK1 and IKK?/? phosphorylation and activation were found important for the activation of NF-?B p65 after silencing MxA in M?s.Subsequently,we treated cells with MxA silencing combined with low concentration of isoniazid(INH).The results showed that the combination could promote the activation of TAK1-IKK?/?-NF-?B signaling pathway and independently fight MTB infection.Our results reveal a novel role of MxA in regulating TAK1-IKK?/?-NF-?B signal activation and production of antibacterial inflammatory cytokines,which provides a potential target for clinical treatment.