Mechanism Study On The Role Of Salmonella Enterica Serovar Typhi Vi Capsular Polysaccharide In Macrophage Apoptosis And TRAIL Pathway

Objective:Macrophages are the main host cells during Salmonella enterica serovar Typhi(S.Typhi)infection.In this study,S.Typhi was used to infect THP-1 cells induced by PMA to explore the effects of S.Typhi and its virulence factor Vi capsular polysaccharide on macrophage apoptosis and tumor necrosis factor-related apoptosisinducing ligand(TRAIL)signaling pathway.Methods:1.Transcriptome sequencing: THP-1 cells were infected by S.Typhi wild-type strain for 0 h,4 h and 26 h,total RNAs was extracted from THP-1 cells for transcriptome sequencing(RNA-seq).2.Classification and analysis of RNA-seq results: Sorting out the genes related to apoptosis of RNA-seq results.Quantitative PCR(q PCR)was used to detect the transcriptional levels of apoptosis-related genes in the early and late stages of S.Typhi infection.3.Detection of apoptosis after THP-1 cells were infected by WT: THP-1 cells were infected by S.Typhi for 0 h,6 h and 24 h.Flow cytometry was used to determine the apoptotic rate of THP-1 cells,and western blot was used to detect the activation levels of THP-1 apoptosis-related proteins Caspase-3,Caspase-8and Caspase-9.4.Detection of TRAIL signaling pathway after THP-1 cells were infected by WT: THP-1 cells were co-cultured with S.Typhi for 0 h,6 h and 24 h.Western blot was used to detect the protein levels of ligand TRAIL and death receptor DR5 in the TRAIL signaling pathway.After neutralizing TRAIL antibody was added to the medium at 6 h,the changes of activation level of Caspase-3 and the apoptotic rate of THP-1 cells were detected by western blot and flow cytometry in the early stage of S.Typhi infection.5.Detection of Vi capsular polysaccharide expression level after THP-1 cells were infected by WT: THP-1 cells were co-cultured with S.Typhi 2 h and 24 h,RNA was extracted and q PCR was used to detect the expression of Vi-related genes tvi A and tvi B.6.Detection of apoptosis after THP-1 cells were infected by Vi mutant stains:Vi mutant strains(?tvi A??vex E??tvi B-vex E)and WT infected THP-1 cells for6 h and 24 h,the activation levels of apoptosis-related proteins Caspase-3,Caspase-8,Caspase-9 were detected by western blot and the apoptotic rate of THP-1 cells was determined by flow cytometry.7.Detection of TRAIL signaling pathway after THP-1 cells were infected by Vi mutant stains: Vi mutant strains(?tvi A??vex E??tvi B-vex E)and WT infected THP-1 cells for 6 h and 24 h,western blot was used to detect the protein expression changes of ligand TRAIL and death receptor DR5 in the TRAIL signaling pathway.The cells were infected with bacteria for 1 h,2 h and 24 h,and the expression levels of Tnfsf10(encoding TRAIL protein)and Tnfrsf10b(encoding DR5 protein)genes were analyzed by q PCR.After neutralizing TRAIL antibody was added to the medium at 6 h,western blot was used to detect the activation level of Caspase-3 and flow cytometry was used to detect the changes of THP-1 cells apoptotic rate in the early stage of ?vex E infection.Results:1.Twelve apoptosis-related genes,such as Caspase-3?Caspase-8?Caspase-9,were sorted out from RNA-seq results and analyzed.The q PCR analysis results were basically consistent with those of RNA-seq.2.After THP-1 cells were infected by WT,the apoptotic rate of THP-1 and the activation of Caspase-3,Caspase-8 and Caspase-9,which are apoptosis-related proteins,were significantly increased(p<0.05).The protein shear bands were significantly stronger than uninfected cells.The macrophage apoptosis in the late stage of infection was higher than that of early stage and showed dynamic changes(p<0.05).3.After WT infected THP-1 cells,the protein expressions of ligand TRAIL and death receptor DR5 were stronger than uninfected cells,and significantly increased with the infection time gone on.The activation of Caspase-3 reduced after TRAIL antibody was added.The shear band was weakened and the number of apoptotic macrophages was significantly reduced(p<0.05).4.In the early stage of infection,the transcriptional expression of Vi-related genes tvi A and tvi B at a high level.The expression level of Vi decreased in the late stage of infection(p<0.05).5.After Vi mutant strains(?tvi A??vex E??tvi B-vex E)infected THP-1 cells for 6 h and 24 h,the activations of apoptosis-related proteins Caspase-3,Caspase-8,and Caspase-9 were significantly increased compared with WT.At the same time,the shear band was obviously strengthened and the apoptosis rate was increased(p<0.05).6.Vi mutant strains(?tvi A??vex E??tvi B-vex E)infected THP-1 cells for 6 h and 24 h,TRAIL showed a significant up-regulation,while DR5 did not have a significant expression changes.7.Vi mutant strains(?tvi A ? ?vex E ? ?tvi B-vex E)infected THP-1 cells for 1 h,Tnfsf10 was up-regulated compared with WT(p<0.05).Tnfsf10 expression was up-regulated by ?tvi A infection with co-cultured 2 h(p<0.05),but was not affected by ?vex E and ?tvi B-vex E.Three mutant strains infection had no effect on Tnfrsf10 b.With co-cultured 24 h,three mutant strains could reduce the transcriptional levels of Tnfsf10(p<0.05),but have no effect on Tnfrsf10 b.8.When ?vex E infected THP-1 cells for 6 h,the shear band was significantly enhanced,the activation of Caspase-3 and the rate of macrophage apoptosis were significantly increased with treatment of TRAIL antibody(p<0.05).Conclusions:S.Typhi induces macrophage apoptosis and presents dynamic changes,partly by activating TRAIL signaling pathway.Both endogenous and exogenous pathways were activated to induce apoptosis.Vi capsular polysaccharide inhibits the apoptosis of macrophages induced by S.Typhi to a certain extent.TRAIL signaling pathway plays a role in the macrophage apoptosis induced by Vi mutant strains.