Mechanism Of BAG2-Mediated Immunoregulation In Macrophage Resistance To Mycobacterium Tuberculosis Infection

Tuberculosis(TB)is a chronic infectious disease.As a zoonosis,it seriously threats human health and development of animal husbandry.Mycobacterium tuberculosis(Mtb)is a pathogenic bacterium of tuberculosis and successfully parasitizes the macrophages in host.On the one hand,the host initiates the immune response to suppress the proliferation and spread of the pathogen;on the other hand,Mtb evolves various strategies to evade the elimination of the host immune system.In order to solve this problem,it is essential to fully understand the molecular mechanism of host-pathogen interaction.During infection,apoptosis,autophagy and inflammatory reactions all affect cell fate and survival of Mtb in host.Previous studies have shown that BAG2 is related to the fate of cells under different pathological conditions.However,the role of BAG2 in Mtb infection remains unclear.In this study,we focused on the effect of BAG2 in infected macrophages and molecular mechanisms of function.The main research contents and results were as follows:1.To study the expression of BAG2 and the mechanism of inhibition of BAG2 expression after Mtb infection.First,the influence of infection on ER stress and BAG2 expression were investigated by qPCR and western blot.The results confirmed that the expression of ER stress marker protein HSPA5 and CHOP were increased,and Mtb infection induces ER stress.Meanwhile,the study results showed that BAG2 expression was down-regulated in a time-dependent manner in Mtb H37 Rv or H37Ra-infected macrophages.Then,the Bag2 promoter activity and the effect of ERN1-XBP1 signaling pathways on Bag2 transcription were assessed by dual-luciferase reporter assay and ChIP.Silence of the Ern1 or Xbp1 restored BAG2 expression.Moreover,XBP1 binds to the Bag2 promoter and transcriptionally inhibits Bag2 expression during Mtb H37Ra-induced ER stress.2.Studies of the function of BAG2 in Mtb-infected macrophages.First,we analyzed the effect of BAG2 on the survival of cells and pathogens.The results showed that knockdown of Bag2 increased cell apoptosis,inhibited cell viability,and decreases the survival of mycobacteria.Then,by gain of function and loss of function experiments,the effects of BAG2 on autophagy and whether it affects cell survival through autophagy were studied.BAG2 enhanced the expression of autophagic marker protein LC3-II and autophagosome formation,and promoted autophagy flow,and activated ER-phagy.Besides,autophagy inhibitors treatments rendered macrophages more sensitive to killing by H37 Ra.Inhibition of autophagy through Baf A1 pretreatment abolished BAG2's protective capability in cell survival.3.Analysis of the mechanism by which BAG2 activates autophagy and ER-phagy.The effect of interfering Bag2 on the interaction between BCL2 and BECN1 was studied by Co-IP and western blot.The results showed that knockdown of Bag2 inhibited the phosphorylation of ERK1/2 and BCL2,and disrupted BCL2–BECN1 disassociation induced by H37 Ra.Similarly,the addition of ERK inhibitor U0126 blocks the effect of BAG2 on autophagy,indicating that BAG2 promotes BECN1 dissociation from BCL2 through ERK signaling pathway and activates autophagy.Then,BAG2 and SQSTM1 interaction were analyzed.Interaction between BAG2 and SQSTM1 depends on PB1 and UBA domains.In addition,overexpression of BAG2 significantly increased the localization of SQSTM1 in ER and promoted ER-phagy.4.To investigate the influence of miR-27 b /BAG2 on inflammatory response.Through qPCR and dual-luciferase reporter assay,the transcription of miR-27 b and its regulatory mechanism were studied during infection.Studies confirmed that miR-27 b is gradually upregulated after infection.Further investigation demonstrated that upregulation of miR-27 b relies on the TLR2/MyD88/NF-?B signaling pathway.In addition,we predicted and verified the target gene of miR-27 b through dual-luciferase reporter assay and western blot combined with bioinformatics analysis.It turns out that miR-27 b directly targets Bag2 3'UTR and inhibits endogenous Bag2 expression.This study further explored the effect and mechanism of miR-27b/BAG2 on inflammatory factors.The results showed that miR-27 b inhibited the production of inflammatory factors via restraining the activity of NF-?B.Functional rescue experiment demonstrated that BAG2 weakened the inhibitory effect of miR-27 b on inflammatory factors,indicating that miR-27b/BAG2 affected the expression of inflammatory factors by regulating the activity of NF-?B.Based on the above research,we revealed the function and molecular mechanism of BAG2 on Mtb-infected macrophages.On the one hand,BAG2 plays an important role on regulating intracellular homeostasis and bacterial survival through cell apoptosis and autophagy;on the other hand,miR-27b/BAG2 and the NF-?B pathway form a negative feedback loop to avoid an excessive inflammatory response,thereby balancing the infection and tissue damage.