| Objective:As facultative intracellular pathogens,the fate of Salmonellae in macrophage depends on the interaction between killing ability of cells and virulence of bacteria.Neutrophils are the most abundant white blood cells in mos t mammals,with the effects of chemotaxis,phagocytosis and killing.During the early stage of infection,neutrophils promptly migrate to the site of infection exerting stronger killing mechanism than macrophages.Acting as scavenger,neutrophils can effec tively eliminate apoptotic macrophages that fail to control infection,thus playing an essential role in defense against Salmonella infection as well.Autophagy is a process of degradation by lysosome,through which macromolecular substances,organelles and invading pathogens were eliminated.Type ? interferon response is the first defense line against virus,its role in antibacterial innate immune response varies from different bacteria,which can promote the secretion of cytokines and chemokines affecting the host inflammatory responses.Our previous studies showed that Salmonella plasmid virulence genes spv can inhibit autophagy and affect function of neutrophil.In order to further explore the influence of spv locus on escaping from the innate immune responses and related mechanism,the first part of this study investigated the effect of spv operon on autophagy and Type ? interferon response,using wild strains of Salmonella enterica serovar typhimurium(S.typhimurium,STM)STM-WT and spv locus deletion-mutant strain STM-?spv to infect macrophages.In the second part,we utilize the zebrafish larvae with only innate immunity to further study the inhibition of autophagy and Type ? interferon response by spv locus and their influence on the chemotaxis of neutrophils.This study aims to provide theoretical and experimental basis for clarifying the pathogenic mechanism of spv operon and the effects on innate immune function.Methods:One.The influence of spv locus on autophagy and type ? interferon response in macrophage1.The effect of spv locus on autophagyMouse peritoneal macrophages RAW264.7 were infected with STM-WT and STM-?spv at an MOI of 10.Macrophages were collected at 1,2,4 and 6 hours post infection(hpi).The expression of autophagy related protein LC3 and p62 were detected by western blot.Compare the differences between the two groups and determine when the autophagy is activated.2.The influence of spv operon on type ? interferon responseRAW264.7 were infected with STM-WT and STM-?spv.Collect cells at 2,4 and 6 hpi,respectively.The m RN A transcription levels of type ? interferon response related genes TRIF,TRAF3 and IFN-? were determined by real-time quantitative PCR(RT-q PCR).3.The effect of spv locus on autophagy and type ? interferon responseRAW264.7 were pre-treated with 5 n M autophagic agonist Torin 1 for 3 hours and then infected with STM-WT and STM-?spv.At 2 hpi,the expression of LC3 was determined by western blot.At 4 hpi,the m RNA transcription levels of TRIF,TRAF3 and IFN-? were detected by RT-q PCR.Two.The influence of spv locus on autophagy and type ? interferon response thus the chemotaxis of neutrophils in zebrafish larvae1.The effect of spv locus on autophagy and type ? interferon response in zebrafish larvaeZebrafish larvae at 5 days post fertilization(dpf)were used to be infected with 109 CFU/ml STM-WT and STM-?spv by immersion,respectively.The larvae were collected at 6,8 and 10 hpi,the expression of LC3 and p62 were determined by western blot and the m RNA transcription levels of type ? interferon response related genes IRF3,IRF7 and IFN-? in zebrafish were detected by RT-q PCR.2.The influence of spv locus on the chemotaxis of neutrophilsZebrafish larvae were infected with STM-WT and STM-?spv.At 6,8 and 10 hpi,the m RNA transcription levels of cytokine IFN-?,chemokine CXCL8-L1 and CXCL8-L2 were measured by RT-q PCR.Transgenic zebrafish larvae Tg(lyz:GFP)were immersed in the water with STM-WT and STM-?spv carrying red fluorescent protein(RFP)markers,the dissemination of bacteria and their interaction with neutrophils were observed by laser scanning confocal microscope.3.The effect of spv locus on autophagy,type ? interferon response and the chemotaxis of neutrophilsZebrafish larvae were pre-treated with Torin 1 for 48 hours,then they were infected with STM-WT and STM-?spv.At 8 hpi,the expression of LC3 was determined by western blot.The m RNA transcription level of IRF3,IRF7,IFN-?,IFN-?,CXCL8-L1 and CXCL8-L2 were measured by RT-q PCR.Results:One.The influence of spv locus on autophagy and type ? interferon response in macrophage1.The effect of spv locus on autophagyThe results showed that LC3-II level of STM-WT infection group was significantly lower than that of STM-?spv infected group at 1 hpi and 2 hpi.At 4 hpi and 6 hpi,LC3-II level of the two infection group had no significant difference.p62 level of the two infection groups had no significant difference at 1 hpi and 2 hpi.At 4 hpi,p62 level of STM-WT infection group was significantly higher than that of STM-?spv infected group.At 6 hpi,p62 level of both infection group was significantly higher than the control group,and there was no significant difference between the infection groups.2.The influence of spv locus on type ? interferon responseAt 2 hpi,4 hpi and 6 hpi,the m RNA transcription levels of TRIF,TRAF3 and IFN-? in STM-WT infection group were significantly lower than that in STM-?spv group.3.The effect of spv locus on autophagy and type ? interferon responseResults showed that under normal infection without Torin1,LC3-II levels of STM-?spv infection group were significantly higher than STM-WT infected group at 2 hpi.While the LC3-II levels of STM-WT infection group were evidently improved by the intervention of Torin1,and had no significant difference in comparison with STM-?spv infection group.Similarly,the m RN A transcription levels of TRIF,TRAF3 and IFN-? in STM-?spv infection group were significantly higher than in STM-WT infected group at 4 hpi.While the m RNA transcription levels of TRIF,TRAF3 and IFN-? in STM-WT infection group were evidently improved by the intervention of Torin1,and had no significant difference in comparison with STM-?spv infection group.Two.The influence of spv locus on autophagy and type ? interferon response thus the chemotaxis of neutrophils in zebrafish larvae1.The effect of spv locus on autophagy and type ? interferon response in zebrafish larvaeThe results demonstrated that the LC3-II levels of STM-?spv infection group is significantly higher than that of STM-WT group at 6 hpi and 8 hpi.The p62 levels of STM-?spv infection group is significantly lower than that of STM-WT group at 8 hpi.Both the LC3-II and p62 levels of the two infection groups had no significant difference at 10 hpi.At 6 hpi and 8 hpi,the m RN A transcription levels of type ? interferon response related genes IRF3,IRF7 and IFN-? in STM-WT infection group were significantly lower than that in STM-?spv group.At 10 hpi,the m RNA transcription levels of IRF3,IRF7 and IFN-? had no significant difference between the infection groups.2.The influence of spv locus on the chemotaxis of neutrophilsThe m RNA transcription levels of IFN-?,CXCL8-L1 and CXCL8-L2 in STM-?spv infection group were significantly higher than that in STM-WT group at 6 hpi and 8 hpi.According to the results of laser scanning confocal microscope observation,the dissemination of RFP-STM-WT is much more than that of RFP-STM-?spv.In addition,fewer neutrophils were observed in infection loci in RFP-STM-WT infection group than that in RFP-STM-?spv group.3.The effect of spv locus on autophagy,type ? interferon response and the chemotaxis of neutrophilsThe LC3-II levels of STM-WT infection group is significantly lower than that of STM-?spv group at 8 hpi.However,the LC3-II levels of STM-WT infection group were improved evidently with Torin1 pre-treated,and had no significant difference in comparison with STM-?spv infection group.The m RNA transcription levels of IRF3,IRF7,IFN-?,IFN-?,CXCL8-L1 and CXCL8-L2 in STM-WT infection group is significant lower that in STM-?spv infection group under normal infection without Torin1.The m RN A transcription levels of IRF3 and IRF7 in both infection groups were evidently increased and had no significant difference between the groups by the intervention of Torin1.The m RN A transcription levels of IFN-?,IFN-?,CXCL8-L1 and CXCL8-L2 in STM-WT infection group were evidently improved by the intervention of Torin1,and had no significant difference in comparison with STM-?spv infection group.Conclusions:1.spv locus inhibits Type ? interferon response by suppressing autophagy in macrophages2.spv locus inhibits Type ? interferon response by restraining autophagy in zebrafish larvae3.spv locus affects the chemotaxis of neutrophils by suppressing Type ? interferon response... |
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