| Salmonella-carrying food is one of the major source of salmonellosis in human.After infection,it can escape the killing of host immune system through regulating cell function and then survive in host cells,such as macrophages.In the early stage of infection,the innate immunity can be activated to clear the bacteria,such as secreting inflammatory cytokines.Therefore,it is significant to analyse the relationship between them.In this study,the mouse cytometric bead array(CBA)kit was optimized.Then the relationship between the secretion of inflammatory cytokines and the colonization of Salmonella in host was analysed in the early stage of infection.1 Optimization of mouse inflammation cytometric bead array kit by flow cytometryCytometric bead array(CBA)is a good technique to analyze the contents of cytokines,while its cost is expensive.In this experiment,the mouse inflammation CBA kit was used as reference,and the mean fluorescence intensity by flow cytometry was compared through changing reagent dose and incubation time,respectively.The results showed that similar tendency could be detected when decreasing reagent dose from 50 uL to 20 uL and shortening incubation time from 2 hours to 30 minutes,respectively.These data will be beneficial for the application of CBA assay.2 Responses of mouse against Salmonella infection analyzed by CBAAfter infection with Salmonella,the immune system will respond to Salmonella and secret inflammatory cytokines.In this experiment,the mouse responses to Salmonella typhimurium were analyzed by CBA technique after intraperitoneal or oral infection,respectively.The results showed that Salmonella infection could accelerate blood coagulation after peritoneal infection with 1×104 CFU per mouse or oral infection with 2×107 CFU per mouse,when compared with that for uninfected mouse.Additionally,the contents of inflammatory cytokines(IFN-γ,IL-6 and TNF)in mouse serum increased in the early stage of infection and then decreased later.This change tendency is in accordance with mouse spleen weight and bacterial colonization in mouse spleens and livers.Same changes were found between peritoneal and oral infection.These results indicate that there are strong and positive correlations between the secretion of inflammatory cytokines in mice and bacterial colonization.3 In vitro analysis of macrophages RAW264.7 after infection with the recombinant Salmonella expressing fusion protein SspH2-EscIThe previous study has shown that the Salmonella-induced activation of intracellular inflammasome pathway in macrophages can be enhanced after infection with the recombinant Salmonella expressing fusion protein SspH2-EscI.In this experiment,the recombinant Salmonella X4550(pYA3334)and X4550(pYA3334-P-SspH2)infection were used as control,and the responses of macrophage cell line RAW264.7 against Salmonella were analyzed after infection with the recombinant Salmonella,X4550(pYA3334-P-SspH2-EscI),expressing fusion protein SspH2-EscI.The results showed that no significant difference was found between.X4550(pYA3334)-and X4550(pYA3334-P-SspH2)-infection.After infection with X4550(pYA3334-P-SspH2-EscI)for five hours(MOI=100),the damage of cell membrane,the activation of intracellar caspase-1 and the release of lactate dehydrogenase increased,compared with that for X4550(pYA3334)-and X4550(pYA3334-P-SspH2).These results indicate that the intracellular inflammasome pathway are activated.Furthermore,we found that the contents of inflammasome cytokines in the supernatants decreased as infection time extended and it was different from those infected with X4550(pYA3334)or X4550(pYA3334-P-SspH2).With regard to cell function,no significant difference in mitochondrial membrane potential,reactive oxygen species,nitric oxide production and intracellular Ca2+ concentration was found among different infection group.For intracellular pH value,it increased after infection with X4550(pYA3334-P-SspH2-EscI)for 5 hours,which was significantly different from those infected with X4550(pYA3334)or X4550(pYA3334-P-SspH2).These results indicate that the activation of intracellular inflammasome pathway influences the secretion of inflammatory cytokines and the change of intracellular pH value.4 In vivo analysis of mouse after infection with the recombinant Salmonella expressing fusion protein SspH2-EscIIt has been reported that the activation of intracellular inflammasome pathway is beneficial for the clearance of Salmonella.In this experiment,the recombinant Salmonella X4550(pYA3334)and X4550(pYA3334-P-SspH2)infection were used as control,and the colonization of bacteria in mice and the secretion of mouse inflammatory cytokines were analyzed after intraperitoneal or oral infection with the recombinant Salmonella expressing fusion protein SspH2-EscI.The results showed that there was no significant difference between X4550(pYA3334)-and X4550(pYA3334-P-SspH2)-infection,and both could accelerate the coagulation of blood,the secretion of inflammatory cytokines in serum,the increase of spleen weight and the colonization of bacteria in mouse spleens.However,those for X4550(pYA3334-P-SspH2-EscI)were significantly different from those for X4550(pYA3334)-or X4550(pYA3334-P-SspH2).The same tendency was found between intraperitoneal and oral infection.These results indicate that the expression of Ssph2-Escl can decrease the secretion of inflammatory cytokines and the colonization of Salmonella in mice... |
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