The Effects Of Salmonella Plasmid On DC Maturation, Autophagy And The Development Of T Cell Immunity

Typhoid, also known as enteric fever, is an ancient acute diarrheal diseases causedby Salmonella typhi(S.typhi). The epidemic of multi-drug resistant S.typhi affected asmany as13provinces and cities in China in the mid-and late1980’s. In a survey ofantimicrobial susceptibility,591strains of S.typhi isolated from clinical samples werestudied. Plasmid electrophoresis, conjugation and elimination tests indicated that thedrug resistance was mediated by a large and conjugative resistant plasmid (R plasmid)of98.6Md(159Kb), which belongs to IncC group. This98.6Md R plasmid of S.typhiwas designated as pRST98. Further research indicated that pRST98is a hybrid resistance-virulence plasmid carrying both antibiotic resistance and virulence genes. Salmonellaare facultative intracellular pathogen and able to invade both phagocytic and non-phagocytic cells. Dendritic cell (DC) play a major role in the activation of adaptiveimmunity by capturing, processing, and presenting antigen to na ve T cells in lymphoidorgans. DC are important for protective immunity to the Gram-negative, intracellularbacterial pathogen, S.typhi.In an effort to gain more precise and further insight into the role of pRST98in theinteraction between S.typhi and DC, we examined cell maturation, autophagy and thedevelopment of T cell immunity in the present study. This study helps to further revealthe virulence phenotype of pRST98.Part1The effects of Salmonella plasmid on DC maturation andautophagyIn this study, S.typhi strain carrying a virulence plasmid pRST98, pRST98-deletionstrain S.typhi-Δ-pRST98and pRST98-complemented strain S.typhi-c-pRST98constructed byconjugative transfer were used as experimental strains. Three strains of bacteria wereco-cultured with mouse bone marrow derived DC (DC) in vitro respectively at amultiplicity of infection (MOI) of50:1. After incubated at37℃for1h (0-h time point), then RPMI1640containing amikacin (100μg/ml) was added to kill remainingextracellular bacteria. After2h of further incubation at37℃, medium in the plates wasreplaced with RPMI1640containing amikacin (10μg/ml) to inhibit the propagation ofpossible extracellular bacteria in the medium. Infected cells were collected and detectedat2h,4h,12h and24h time points of infection. The expression of co-stimulatorymolecules and endocytosis rate were determined by flow cytometry (FCM). Th1-typecytokines such as interferon-γ (IFN-γ) as well as interleukin-12(IL-12) secretion ofinfected cells and Th2-type cytokines such as interleukin-10(IL-10) of infected cellswere examined by enzyme-linked immunosorbent assay (ELISA). The expression ofautophagic protein LC3-II and Beclin-1were detected by Western Blot (WB) assay. Thegold standard of autophagy phenomenon such as double-or multilayer membrane ofautophagosome in the cytoplasma was observed by transmission electron microscopy(TEM).In this part, we demonstrated that the expressions of CD40, CD80and CD86onDC surface stimulated with either strain were increased; while the mean fluorescenceintensity (MFI) in S.typhi-WT-or S.typhi-c-pRST98-treated DC was significant lowerthan in S.typhi-ΔpRST98-treated DC. The endocytosis activity of DC in each group wasdeclining after infection. S.typhi-WT and S.typhi-c-pRST98group were both higher thanS.typhi-Δ-pRST98group. All groups’ cytokines levels were significantly increased, andIFN-γ、IL-12secretion of DC infected by S.typhi-Δ-pRST98was significantly higher thanboth S.typhi-WT and S.typhi-c-pRST98group, while IL-10secretion of DC infected byS.typhi-Δ-pRST98was significantly lower than S.typhi-WT group. LC3-II and Beclin-1protein expression in DC infected by S.typhi-WT and S.typhi-c-pRST98containing pRST98was weaker than in DC infected by S.typhi-Δ-pRST98at2h. Ultrastructure of infectedcells under TEM showed, DC infected by S.typhi-WT and S.typhi-c-pRST98did notappear typical autophagic vacuoles in the cytoplasm, however, S.typhi-Δ-pRST98infected-DC displayed typical autophagic vacuoles with double or multilayer membranein the cytoplasm at2h and4h. Then the autophagic vacuoles fused with lysosomes toform phagolysosome and bacteria were degradation. Part2The effect of Salmonella plasmid on immune responseS.typhi is highly adapted to humans and no ideal animal model is suitable forS.typhi. For further investigation of pRST98function in vivo, Salmonella typhimurium, aclose relative to S.typhi, was employed to set up the mouse infection model. Virulenceplasmid-deletion S.typhimurium strain χ3337and S.typhimurium3337/pRST98were usedin this section. Two kinds of bacteria were co-cultured with mouse DC in vitrorespectively. Then we collected the infected cells and detected the entry of Salmonellato murine DC and DC viability. Mice were infected through intravenous injection.Spleen cell suspension was collected after mice were sacrificed at different timepoints(1d,3d,5d and10d). The proportion, absolute number and the phenotype ofDC were determined by FCM. The cytokines secretion of DC was identified by FCMthrough intracellular cytokine staining. The activation of T cells from S.typhimurium-infected mice was also detected by FCM. Organ loads of bacteria from S.typhimurium-infected mice were quantified by plate count method.We observed that among cells infected by S.typhimurium, the number ofintracellular bacteria had no significant difference between χ3337/pRST98-andχ3337-treated-DC. Among cells infected by conjugant χ3337/pRST98containing pRST98,the apoptotic rate was significant higher than χ3337-treated-DC. Total cell number andabsolute number of DC from mice spleen were increased significantly after infection.The absolute DC number and the proportion of DC after infection with S.typhimuriumχ3337were significant higher than conjugant χ3337/pRST98group. The CD40, CD80,CD86and MHCⅡ expression on DC surface infected with either strain were increased.The proportions of CD80, CD86, MHCⅡ positive DC in χ3337/pRST98group weresignificant lower than χ3337group. The proportion of CD40positive DC had nosignificant difference between two groups. Expression of Th1type cytokine such asIFN-γ and IL-12from splenic DC in χ3337infected mice were higher than cells fromχ3337/pRST98infected mice. Both Th-and Tc-cell percentages of spleen had nosignificant difference between two groups. But splenic CD44highand CD62LlowT cellproportion were increased at d10after infection, and the percentages of CD44highandCD62LlowT cell from χ3337group were higher than those of χ3337/pRST98infectedmice. Conclusions:1. Salmonella plasmid pRST98can not influence bacterial invasion ability, but caninhibit cellular autophagy and enhance apoptosis of infected DC and consequentlylessen the injury of host cells.2. Salmonella plasmid pRST98can regulate Th1/Th2shift away through increasingTh2type cytokines IL-10secretion and decreasing Th1type cytokine IFN-γ and IL-12secretion. However, pRST98has the ability of convert different type of immune response.3. Salmonella strains carrying pRST98can decrease the antigen presenting functionthrough inhibiting maturation of DC, and consequently suppress DC and T cellsactivation and result in the more serious damage of host.