Mechanism Of STAT1-Mediated Macrophage Signaling Pathway And Immunoregulation During Mycobacterium Tuberculosis Infection

Tuberculosis(TB)is an anthropozoonosis caused by mycobacterium tuberculosis(Mtb).Pathogenic bacteria can be spread to people by sick animals.The Mycobacterium bovis shows a world-wide bacterial disease of poultry,and the spread of the epidemic not only influence the development of animal husbandry,but also a serious threat to human health.So far,the loss caused by bovine tuberculosis is far more than the total number of the loss caused by other diseases.Due to the lack of effective drugs and drug resistant strains appear constantly,TB can not get effective control,and becomes the first killer of adults' infectious diseases causing great damage to public health in thosebovine tuberculosis-endemic countries.In order to solve this problem fundamentally,an in-depth research is crucial to illuminate the mechanism of transmission.Tuberculosis bacili is cell parasitic bacteria thatcaused tuberculosis.After infection of the Mtb,it causedthe phagocytosis bymacrophages.The Mtb is either eliminated by the macrophages or long-term survival in macrophages.Mtb activated macrophagesby contacting toll-like receptors on the surface of the macrophages.The activated macrophage induced multiple signaling pathways in cells,which lead to apoptosis of macrophage so as to achieve the purpose of clearing pathogenic bacteria.However,as the coevolution of pathogen and the host immune system,the tuberculosis bacili is developed to reduce macrophage apoptosis rate so as to obtain the chance for immune escape for survival in the macrophages.This study mainly focused onthe JAK/STAT pathway mediatedimmune regulation in macrophages RAW264.7 cells during the infection of Mtb H37 Ra strain.The concrete research content of this study are as follows:1.During the early infection of Mtb(1 hour),exposure of macrophages to H37 Ra induces rapid phosphorylation STAT1(PSTAT1)formation following with production of Tnf-? and caspase-3 activation enhance macrophages sensitivity to apoptosis induction.Pretreatment with JAK inhibitor AG-490,correlatedwith impairment of Tnf-? and caspase-3activation,and rescued the cells from Mtbinduced apoptosis.2.During the late stage of infection byMtb(48 hour),the concentration of PSTAT1 decreased over the next several hours.As PSTAT1 decreased,there was a reciprocal increase in the concentration of unphosphorylation STAT1(USTAT1).At each time points after infection,the viability of H37 Ra was determined by CFU assay and the intracellular bacterial burden shows similar trend with the level of USTAT1 expression in a time dependent manner during the late infection.To investigate the role of H37Ra-induced USTAT1,we transduced mouse macrophage cell line RAW264.7 lentiviruses encoding USTAT1.Our results indicted the U-STAT1 induced by Mtb moves into nuclei,where it can function as a novel transcription factor to increase the expression of immune regulatory genes.Additionally,the inhibition activation of Caspase-3,Cytochrome c translocation and increase of Mcl-1 in USTAT1 overexpressed macrophages,lead to a severely obstruction in H37 Ra infection-induce apoptosis.The represses expression of Jak1 regulated by USTAT1 may lead to the inhibition of STAT1 phosphorylation,which terminated the PSTAT1 signaling pathway.3.To map the USTAT1 interactome in macrophages,a one-step pull-down procedure with biotinylated tag by streptavidin was taken in RAW264.7 cells.Weconstructed RAW264.7 cells stably expressing biotinylated USTAT1.Subsequently,the streptavidin-mediated affinitypurification was used to isolated USTAT1 protein complexesand sequenced them by using mass-spectrometer.A total of 149 USTAT1 interactingproteins were identified by our mass-spectrometric analysis.KEGG pathway analysis revealed that USTAT1-interacting proteins were enriched in antigen processing and immunoregulation.4.The binding cooperation between USTAT1/STAT3 complex and key element on the promoter of CD95 results in downregulation of CD95 expression,which leads to the reduction of susceptibility in macrophages to CD95 L induced apoptosis.Additionally,a competitive binding reactions between USTAT1 and IFIT1 to eEF1 A was verified,and such reactions may inhibit the apoptosis processes mediated by eEF1A/IFIT1 proapoptoticcomplex.Together,we propose that STAT1 may play a double-edged sword role during H37 Ra infection in macrophages.Our data firstly show the USTAT1 differs from the PSTAT1,and represses apoptosis in macrophages to promote immune evasion during Mtb infection.