The Relationship Between Salmonella Plasmid Virulence Gene And Dendritic Cells Autophagy, Apoptosis And Immune Regulation

Objective: To study the influence of Salmonella plasmid virulence genes (spv) on autophagy, apoptosis and immune function of dendritic cells (DCs), and explore the molecular mechanisms by cell and animal model, respectively. Then search for a new strategy for the control of infection process by regulating autophagy and apoptosis levels to enhance the immune function, and provide theoretical and experimental basis for discovering new drug target sites.Methods:1. The in vitro experiment using a cell model. Salmonella typhi ST8 carrying spv gene is the object of this study. Salmonella typhimurium strain SR-11 containing spv gene was used as a positive control, and Salmonella typhi ST10 as a negative one. Then the 3 strains of Salmonella were co-cultured with mouse bone marrow derived DCs (BMDCs). Another group of BMDCs were treated with an autophagy agonist rapamycin (RAPA) overnight before co-cultured with bacteria, and the uninfected BMDCs were used as normal control group. BMDCs in each group were harvested at different time points, and the autophagy protein Beclin-1 was detected by Western blot; Caspase-3 activity was assayed by colorimetry; the apoptosis rate, endocytosis rate and the expression of co-stimulatory molecules CD40, CD80 and CD86 were determined by flow cytometry (FCM); the proliferation of CD4+ and CD8+ naive T cells stimulated with BMDCs were detected by mixed lymphocyte reaction (MLR); IL-12, IFN-γ, IL-10 and IL-4 secretions were assayed by ELISA; the survival rate of BMDCs was determined by trypan blue stain and the viable bacterial quantities inside BMDCs were detected by plate count method.2. The in vivo experiment using a mouse model. Salmonella typhi only strictly infects human beings and has no animal model. Salmonella typhimurium, a close relative to Salmonella tyhpi, was employed to setup the mouse infection model. Standard Salmonella typhimurium virulence strain SR-11 carrying spv gene were used in the intraperitoneal injection of mouse, and the attenuated strain BRD509 without spv gene was a negative control. Another group of mice were injected with RAPA after bacterial infection and the uninfected mice were used as normal control group. At different time points, the common condition of mouse was obsereved, then the pathological change of livers and spleens were compared after sacrifice of mice; Beclin-1 and Caspase-3 expression in livers and spleens were detected by immunohistochemisty; the apoptosis rate, endocytosis rate and CD40, CD80 and CD86 expression of splenic DCs were determined by FCM; the proliferation of naive T cells stimulated with splenic DCs were detected by MLR; IL-12, IFN-γ, IL-10 and IL-4 secretions in serum were assayed by ELISA, and the viable bacterial amounts in livers and spleens were detected by plate count method.Results:1. Before RAPA intervention, Beclin-1 expression of ST10 group was higher than SR-11 and ST8 group at 2 h post co-culture, and this difference could be observed since 0 h for those Salmonella-infected BMDCs pretreated with RAPA. The in vivo results showed that at the early stage of infection, Beclin-1 expression in organs of BRD509 group was higher than that in SR-11 group. RAPA intervention increased Beclin-1 expression in all groups.2. Before RAPA intervention, the apoptosis rate and Caspase-3 activity of each group increased gradually. The in vitro results showed that SR-11 and ST8 group were higher than ST10 group, and the in vivo results displayed that SR-11 group was higher than BRD509 group. After RAPA treatment, SR-11 and ST8 group were decreased, while ST10 and BRD509 group were increased.3. Before RAPA intervention, the endocytosis activity of DCs in each group was declining with the infection time. ST10 group was lower than SR-11 and ST8 group; BRD509 group was lower than SR-11 group. After intervention with RAPA, BMDCs endocytosis rate of each group were all decreased significantly.4. Before RAPA intervention, CD40, CD80 and CD86 expression on DCs surface and the proliferation of naive T cells stimulated with DCs in all groups were gradually increased; the capability of ST10 group was significantly stronger than SR-11 and ST8 groups; the in vivo BRD509 group was significantly higher than that of SR-11 group. After RAPA intervention, both indexes in each group were increased.5. Before RAPA intervention, all groups'IL-12 and IFN-γlevels were significantly increased, and ST10 group was significantly higher than both SR-11 and ST8 group.The in vivo results showed that BRD509 group was significantly higher than SR -11 group. After RAPA intervention, IL-12 and IFN-γof each group was increased.6. Before RAPA intervention, IL-10 in each group could not detected during the early stage of infection. In the advanced infection stage, IL-10 secretion of BMDCs in SR-11 and ST8 group was detected significantly higher than ST10 group, and IL-10 of SR-11 group in vivo was detected, while BRD509 group still could not be detected. IL-10 expression of each group was decreased after intervented with RAPA. The expression of IL-4 was not detected in any group.7. Before RAPA intervention, the BMDCs'survival rate of SR-11 and ST8 group were lower than ST10 group, but the numbers of intracellular viable bacteria in BMDCs of SR-11 and ST8 group were more than ST10 group. At the advanced stage of infection, mice of SR-11 group showed a worse common condition and more severe pathological changes in organs than BRD509 group, and the viable numbers of bacteria in organs was also higher. After RAPA intervention, the survival rate of BMDCs in SR-11 and ST8 group were increased, while ST10 group reduced, and the viable bacterial numbers in organs of SR-11 and ST8 group were decreased but ST10 group increased. The viable bacteria in organs of SR-11 group were decreased while BRD509 group increased, and the organ injury of SR-11 group was reduced but BRD509 group became more serious.Conclusions:1. Salmonella strains carrying spv gene can promote apoptosis to aggravate the infection by inhibit autophagy. RAPA intervention can protect cells and body by moderate activation of autophagy. Salmonella strains without spv gene can activate autophagy to reduce the bacterial damage to cells and body to protect bacteria themselves. RAPA intervention can lead to host injury when autophagy was hyper-enhanced.2. Salmonella strains carrying spv gene can inhibit DCs maturation to decrease the proliferation of naive T cells. RAPA can promote DCs maturation and enhance the capability of antigen presentation.3. Salmonella infection can stimulate murine DCs to induce a Th1 type immune response. Appropriate immune response can eradicate the infection of attenuated strains. However, spv containing Salmonella strains can resist and escape from immune defense. RAPA can promote the secretion of Th1 type cytokines and suppress Th2 type immune response by enhancing autophagy to strengthen the resistance to Salmonella infection.