The Role Of GEF-H1 In Anti-mycobacterial Immune Response

BackgroundMycobacterium tuberculosis(MTB)is the causative agent of tuberculosis(TB).In 2016,about 10.4 million people acquired TB,and 1.5 million people died from this disease.Approximately 30%of exposed individuals will become tuberculin-positive patients,although only 5-10%of these infected individuals will develop clinical manifestations of active TB.In contrast,the majority of infected individuals developed latent TB,in which the invading MTB can survive asymptomatically within host macrophages for a long period.Upon recognition of the invading MTB by pattern recognition receptors(PRRs),macrophages first activate intracellular signaling cascades,including the pathways of mitogen-activated protein(MAP)kinase(MAPK)and TANK binding kinase-1(TBK1),and subsequently induce the production of cytokines,such as interleukin(IL)-1β,IL-6 and IFN-β.In addition,macrophages use myriad defense mechanisms to fight against the invading MTB,including nitric oxide(NO)production,protease activation,and autophagy induction.Rho GTPases act as molecular switches to control many basic cellular activities and are critical for several specialized cellular functions,such as actin cytoskeleton organization,cell migration and adhesion,reactive oxygen species formation,and apoptosis.GEF-H1 is also known to mediate the crosstalk between MTs and the actin cytoskeleton,which may contribute to the phagocytosis of bacteria by macrophages.Accordingly,in the present study,we evaluated the role of GEF-H1 in the macrophage-mediated antimycobacteria immune response.Our results provide important insights into the function of GEF-H1 in regulating the inflammatory cytokine response and mycobacterial elimination in macrophages during mycobacterial infection.Objective1.To determine the expression GEF-H1 during mycobacterial infection.2.To demonstrate the underlying mechanisms of GEF-H1-mediated anti-mycobacterial effect.Method1.The expression levels of GEF-H1 were detected by Real-time PCR and Western Blot after mycobacterial infection.2.Pharmacological inhibitors were used to ascertain the signalling pathways involved in MTB-induced GEF-H1 expression.3.Western blot,Real-time PCR and ELISA was used to measure the protein level of microtubule-associated protein light chain 3(LC3),a marker of autophagy and so on.Results1.Both mRNA and protein expression levels of GEF-H1 were significantly upregulated in macrophage during mycobacterial infection.2.Silencing GEF-H1 with specific siRNAs reduced the phosphorylation of p38 MAPK and TBK1,and the expression of interleukin-1β(IL-1β),IL-6 and interferon-β(IFN-β).3.Silencing GEF-H1 with specific siRNAs left the nitric oxide(NO)production and autophagy unaffected.4.GEF-H1 depletion attenuated macrophages-mediated mycobacterial phagocytosis and elimination.ConclusionsIn the present study,we demonstrated GEF-H1 functioned as a key regulator of macrophage-mediated anti-mycobacterial response.We found that both mRNA and protein expression levels of GEF-H1 were significantly upregulated in macrophage during mycobacterial infection.Moreover,silencing GEF-H1 with specific siRNAs reduced the phosphorylation of p38 MAPK and TBK1,and the expression of interleukin-1β(IL-1β),IL-6 and interferon-β(IFN-p),while leaving the nitric oxide(NO)production and autophagy unaffected.Importantly,GEF-H1 depletion attenuated macrophages-mediated mycobacterial phagocytosis and elimination.Together,our data support that GEF-H1 is a novel regulator of inflammatory cytokine production and mycobacterial elimination,and may serve as a novel potential target for clinical treatment of tuberculosis.