The Influence Of Mycobacterium Tuberculosis Protein CFP10 And ESAT6 On The Inflammatory Cytokine Response In A549 Cells

Background:Tuberculosis(TB)is a zoonotic infectious disease caused by Mycobacterium tuberculosis(Mtb).The alveolar epithelial cells are one of the Mtb-infected target cells.Both CFP10 and ESAT6 protein are immune-protective antigens that are absent in Bacille Calmette-Guerin(BCG).The TLR signaling pathway of the host plays an important regulatory role in the inflammatory response caused by Mtb infection.Purpose and methods:In this study,prokaryotic expression systems of E.coli were used to express recombiant proteins of CFP10 and ESAT6.After purification,the human alveolar epithelial cells A549 were treated with BCG and recombinant proteins of CFP10 and ESAT6,respectively,and the expression levels of some key molecules of TLR signaling pathway were accessed by western blotting assay.Meanwhile,cytokines production was assessed by determining the concentrations of IL-6 and TNF-a using cell culture supernatants after CFP10,ESAT6 proteins treatment.Finally,the effects of CFP10 and ESEAT6 on cell survival and death of A549 was also evaluated using the MTT assay.The following results were obtained through our experiments:Results:(1)After optimization of the expression and purification conditions,the two high-purity proteins CFP10 and ESAT6 were successfully abtained.(2)The results showed that the expression leves of TLR2,TLR4,MyD88,IRAK1,TRAF6 and NF-?B p65 were up-regulated and the expression levels of TIC AMI,TRAF3 and TBK1 were down-regulated after the cells were treated with CFP10 or CFP10+ESAT6 as compared to the control groups.In addition,the expression of some of the inflammatory cytokines such as IL-6 and TNF-a were significantly elevated;After treatment with ESAT6,the expression levels of TLR2,TLR4,MyD88,IRAK1,TRAF6,NF-?B p65,TICAM1,TRAF3,TBK1 and IRF3 were significantly down-regulated,and the expression levels of cytokines of IL-6 and TNF-a were also down-regulated.These results indicated that both CFP10 and ESAT6 could be recognized by Toll-like receptors.(3)BCG activates TLR signaling pathway signaling in A549 cells and promotes the secretion of inflammatory factors.We detected the expression levels of several key components of the TLR signaling after A549 cells were co-treated with BCG and the purified proteins.The results showed that the expression of TLR2,TLR4,MyD88,IRAK1,TRAF6,TAB1,NF-?B p65,TICAM1,TRAF3,TBK1 and IRF3 as well as IL-6 and TNF-a cytokines levels were up-regulated in BCG+CFP10 group as compared to the BCG group.However,in the BCG+ESAT6 group and BCG+CFP10+ESAT6 group,the expression of TLR2,MyD88,IRAK1,TRAF6,TAB1,NF-?B p65,TICAM1,TRAF3,TBK1,IRF3 and the expression of inflammatory factors such as IL-6 and TNF-? were significantly down-regulated as compared to the BCG group.The results indicated that CFP10 enhanced the TLR signaling pathway-mediated inflammatory response in BCG-infected A549 cells,whereas ESAT6 or co-treatment of both proteins inhibited the inflammatory response of A549 cells induced by BCG infection.(4)CFP10 and ESAT6 could induce A549 death,and the cell survival rate decreased with increasing protein treatment concentration and treatment time.And the survival rate of A549 cells co-treated with the two proteins was higher than that of CFP10 or ESAT6 treatment at the same concentration and time,which may be related to the interaction between CFP10 and ESAT6.(5)CFP10 and ESAT6 proteins have different effects on TLR signaling pathway and inflammatory response in A549 cells.The results suggested that CFP10 and ESAT6 proteins have important regulatory effects on Toll-like receptor p-athway and inflammatory response.When A549 cells were co-treated with CFP10 and ESAT6 proteins,they can exert different effects as compare to the groups treated with CFP10 or ESAT6 protein alone.Conclusion:In summary,we have succesfully expressed and purifiedthe recombiant proteins of CFP10 and ESAT6.After treatment A549 cells with the recombinant proteins,we found that the expression of several key components of the TLR signaling changed significantly,suggesting that both MyD88-dependent and indepent TLR signaling pathway may play key roles in the the airway epithelial cell upon Mtb infection.CFP10 and ESAT6 have different physiological effects and are capable of interacting with each other.This study further lays a theoretical foundation for deepening the understanding of the pathogenesis of Mtb and the mechanism of anti-tuberculosis immunity.