| Mycobacterium bovis(M.bovis)is the main cause of tuberculosis(TB)in cattle,deer and other mammals and it also can infect people by air and drinking or eating contaminated,unpasteurized dairy products.Like mycobacterium tuberculosis(Mtb),M.bovis is highly adapted to macrophages and has developed multiple mechanisms to resist intracellular assaults.However,host macrophages induce an apoptotic signal to control bacterial infection.Besides exogenous death receptor pathway and endogenous mitochondrial damage pathway,endoplasmic reticulum(ER)stress this new apoptotic pathway is becoming more and more important.Here,we discovered whether M.bovis could induce macrophages apoptosis and confirmed the apoptosis is mediated by ER stress.We also investigated whether ER stress-induced apoptosis is mediated by STING-TBK1-IRF3 pathway.The results could provide a new experimental evidence for the mechanism of ER stress-regulated cell death and innate immunity in response to bacterial escape.1.We first detected the percentage of apoptosis and caspase expression.The results indicated M.bovis could induce macrophages apoptosis.We also detected the morphology of ER and ER stress-related factors expression after M.bovis infection.The results indicated M.bovis could induce ER stress.Next,we investigated whether the apoptosis is induced by ER stress.Chemical chaperone 4-phenyl butyric acid(4-PBA),an ER stress inhibitor was used to treat cells prior to M.bovis infection.The results were shown that 4-PBA inhibited macrophages apoptosis and caspase expression,which confirmed M.bovis-induced apoptosis is mediated by ER stress.We detected intracellular survival of M.bovis by colony-forming unit assay(CFU)when Tm(an ER stress-agonist)and 4-PBA were used.The results were shown that ER stress could induce apoptosis,which effectively control bacteria.2.We studied the mechanism of the activated ER stress induced apoptosis.We first investigated the translocation of STING and subsequently the activation of the downstream molecule TBK1 in the cytoplasm.We detected co-localization of STING and TBK1 and the phosphorylation of TBKI1.The results were indicated that M.bovis could induce the translocation of STING and the phosphorylation of TBK1.Next,4-PBA was used to treat cells prior to M.bovis infection.We found out 4-PBA inhibited co-localization of STING and TBK1 and the phosphorylation of TBK1,which confirmed the activation of STING-TBK1 pathway is connected with ER stress.To verify whether the phosphorylation of TBK1 induces downstream IRF3 activation,we used BX-795,a specific inhibitor of TBK1 to treat cells prior to M.bovis infection.The results were shown that BX-795 inhibited the phosphorylation of IRF3.Finally,we confirmed the activation of IRF3 by nuclear translocation of IRF3 after M.bovis infection by immunofluorescence.3.We studied the mechanism of the activated STING-TBK1-IRF3 pathway-induced apoptosis.We detected the mitochondrial transmembrane potential and the translocation of cytochrome c and Bax.The results were shown that the mitochondrial transmembrane potential decreased and Bax was detected in isolated mitochondria and was reduced in the cytoplasm.In contrast,cytochrome c was released from mitochondria to the cytoplasm after M.bovis infection.We further confirmed the activation of IRF3 is related to mitochondrial dysfunction by BX-795.Then we detected the interaction between IRF3 and Bax by co-immunoprecipitation.The result was shown that the phosphorylated IRF3 bound to Bax and together moved to the mitochondria during M bovis infection,which finally induced the apoptosis.We also found Caspase-8 and Caspase-3 were decreased in a dose-dependent manner in response to BX-795,which means the extrinsic cell death pathway may also be involved in IRF3-mediated apoptosis.We detected intracellular survival of M.bovis by CFU when BX-795 was used.The result was shown that ER stress-activated STING-TBK1-IRF3 pathway could induce apoptosis,which effectively control bacteria. |
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