Regulatory Roles Of Wnt5a And SOX2 Signaling In Apoptosis And Inflammatory Response Of Alveolar Epithelial Cells To BCG Infection

Tuberculosis(Tuberculosis,TB)is a worldwide outbreak of chronic lethal zoonosis caused by the infection of Mycobacterium tuberculosis(Mtb).A large number of studies on the pathogenesis of tuberculosis focus on the interaction between MTB,alveolar macrophages and dendritic cells.It has also been shown that alveolar epithelial cells(AEC)are also target cells and host cells of MTB,which play an important role in the regulation of anti-Mycobacterium tuberculosis infection.However,the molecular mechanism of the interaction between AEC and Mycobacterium tuberculosis has not been clarified.The newest research show that Wnt signal of alveolar epithelial cells can play an immunomodulatory role by binding to membrane receptor protein.Wnt5a is an important member of the Wnt family of secretory glycoproteins,which can activate nonclassical Wnt signaling pathway and participate in the molecular regulation of cellular inflammatory response.SOX2 has a series effect on many cell signals including Wnt signals,which also is an important regulator of autophagy,inflammation,apoptosis,development and so on.In order to reveal the immunomodulatory effect of Wnt5a signal on the anti-tuberculosis infection of alveolar epithelial cells,the key scientific problems to be solved in this study are as follows:1.Whether mycobacterium tuberculosis activates Wnt5a and SOX2signals after infection of alveolar epithelial cells?2.Whether Wnt5a signal and SOX2 signal are involved in the regulation of apoptosis of alveolar epithelial cells infected by Mycobacterium tuberculosis and the inflammatory response mediated by TLR signal?What is the molecular mechanism of the interaction between the two signals?Based on the infection of alveolar epithelial cell A549 by Mycobacterium tuberculosis vaccine strain BCG,in this study,Wnt5a/JNK signaling pathway was activated or inhibited by Wnt5a conditioned medium and JNK inhibitor SP600125 respectively.The overexpression of SOX2 by lentivirus transfected A549 cells.Combined with MTT assay,real-time fluorescence quantitative PCR,Western blotting,flow cytometry and enzyme-linked immunosorbent assay,the expression levels of Wnt5a signaling pathway related protein,TLR signaling pathway related protein,inflammatory cytokines and apoptosis related protein were detected.Meanwhile,the regulatory effect and mechanism of Wnt5a and SOX2 on alveolar epithelial cell A549 infected by MTB were explored.The results are as follows:1.The expression of the key molecules of Wnt5a,Ror2,JNK and CaMKⅡ on signaling pathway,was significantly up-regulated at mRNA level after MTB infection of A549 cells.Compared with the uninfected control group,Wnt5a,PKC,CaMKⅡ,Fzd5,Ror2 and JNK protein expression levels in A549 cells were significantly increased under the stimulation of Wnt5a or BCG alone.The expression of Wnt5a,PKC,Fzd5 and JNK in Wnt5a and BCG group was significantly higher than that in BCG group alone(P<0.05).After treatment of A549 cells treated with SP600125,BCG and Wnt5a alone or in combination,compared with the control group,the expression of Wnt5a,Ror2,JNK,p-JNK and AP-1 protein in each treatment group decreased significantly(P<0.01).The results show that Wnt5a can effectively promote the opening of Wnt5a signal pathway after BCG infection of A549 cells,and block Wnt5a/JNK signal pathway,which can inhibit Wnt5a signal activated by BCG alone or in cooperation with Wnt5a.2.After A549 cells treated with Wnt5a and BCG alone or in cooperation,the TLR signal key molecules and inflammatory cytokines were detected.The expression of TLR2,TLR6,MyD88,NF-κB,phosphorylated NF-κB p65 and IRF5 in the BCG group and the Wnt5a+BCG co-treatment group were significantly up-regulated.Compared with control group,this difference was highly significant(P<0.01),and the expression level of the Wnt5a+BCG group was significantly higher than the BCG group.After treatment of A549 cells with inhibitor SP600125,the expressions of TLR2,TLR6,MyD88,NF-κB,phosphorylated NF-κBp65 and IRF5 were significantly down-regulated in the BCG group and the Wnt5a+BCG group(P<0.05).Compared with the u control group,the levels of inflammatory factors IL-6 and TNF-α were significantly increased in the Wnt5a+BCG group,furthermore,the expressions were the highest in the Wnt5a+BCG co-treatment group(P<0.01).The results showed that Wnt5a could effectively promote the activation of TLR signaling pathway after BCG infection of A549 cells,thus promoting the inflammatory response induced by BCG,while blocking the Wnt/JNK signaling pathway,meanwhile,inhibiting the activation of TLR signaling by BCG alone or in collaboration with Wnt5a.3.After A549 cells treated with Wnt5a and BCG alone or in cooperation,the expression levels of SOX2 protein in A549 cells in Wnt5a group,BCG group and Wnt5a+BCG group were significantly greater than the control group,the difference was highly significant(P<0.01).After blocking the JNK signal with inhibitors,the expression levels of SOX2 in the Wnt5a and BCG groups were significantly down-regulated,indicating that BCG and Wnt5a could activate the expression of SOX2 in A549 cells through the Wnt5a/JNK signaling pathway.4.The apoptosis rate and the expression of apoptosis related protein of A549 cells treated with Wnt5a and BCG were detected,the results show that:The number of apoptotic bodies,apoptotic rate,expression level of Bax,decrease range of mitochondrial membrane potential,ROS level,Caspase-3,activated caspase-3(C-caspase-3)and cyto-C expression level in A549 cells showed Wnt5a+BCG CO treatment group>BCG infection group>control group.While,the expression level of bcl XL was opposite.Compared with the uninfected control group,the release of ROS in A549 cells of BCG,Wnt5a treated group and co treated group increased significantly,and the level of ROS in NAC pretreated group decreased compared with untreated control group.The above results indicated that Wnt5a/JNK signal promoted BCG induced apoptosis of A549 cells by enhancing ROS production in A549 cells through the mitochondrial apoptosis pathway.5.In the over-expressed of SOX2 A549 cells,the Wnt5a,JNK,p-JNK,CamKⅡ and NAFT protein expression was lower;The expressions of Wnt5a,JNK,p-JNK and NAFT proteins were up-regulated after BCG infection in A549 cells,while over-expression of SOX2 inhibited the up-regulation of key proteins of Wnt5a in BCG infected A549 cells.In the expression of SOX2 A549 cells,called TLR2 and TLR4,TBK-1,and TRAF3,IRF3,AP-1 the NF-kappa B p65 protein expression was raised,BCG infection A549 cells have also performed for TLRs key protein expression level,and BCG+express SOX2 group TLR2,IRF-5,the NF-kappa B p65,TRAF3,TBK,AP-1 and IRF-3 and inflammatory cytokines IL-6 protein,the expression of TNF alpha level higher than uninfected control group but lower than that of BCG infection group,That is,over-expression of SOX2 inhibited up-regulation of key TLRs signaling proteins and inflammatory cytokines in BCG infected A549 cells.Compared with the control group without infection,the expressions of Bax/Bcl-2,C-cas3,C-cas8 and C-cas9 in BCG+over-expressed SOX2 group and BCG infected group were all up-regulated after A549,while the expression level of BCG+over-expressed SOX2 group was lower than that of BCG infected group.The results showed that the negative feedback of SOX2 over-expression inhibited the apoptosis of BCG infected A549 cells and the inflammatory response mediated by TLR signal.In summary,the regulatory mechanism of Wnt5a and SOX2 on the alveolar epithelial cells infected by mycobacterium tuberculosis was as follows:after mycobacterium tuberculosis infected the A549 cells,the expression of Wnt5a was up-regulated,and the non-canonical signaling pathway of Wnt5a/JNK was activated,which promoted the cascade amplification of TLR signals and induced cell apoptosis to clear the pathogen infection.At the same time,activation of the Wnt5a signaling pathway further up-regulated the expression of SOX2.When SOX2 is over-expressed,negative feedback can inhibit Wnt5a signal and TLR signal mediated inflammatory response and cell apoptosis of alveolar epithelial cells infected by mycobacterium tuberculosis,thus maintaining the homeostasis of epithelial cells.These findings will provide new research ideas for further studying the interaction mechanism between mycobacterium tuberculosis and host A549 cells and elucidate the immune response regulation mechanism of host epithelial cells against mycobacterium tuberculosis infection.