TNF-α敲除对创伤弧菌血流感染小鼠肝脏和脾脏中性粒细胞的影响

Effects of TNF-αknockout on liver and spleen neutrophil responses to Vibrio vulnificus bloodstream infection in a mouse model

目的在创伤弧菌血流感染小鼠模型中探讨TNF-α敲除对宿主肝脏和脾脏中性粒细胞的影响。方法(1)选取6~8周龄C57BL/6J品系的TNF-α敲除(TNF-α-/-)小鼠和野生型(WT)小鼠,随机分成WT未感染组、WT感染组、TNF-α-/-未感染组和TNF-α-/-感染组,每组各6只。小鼠腹腔注射2×108 CFU/200μl的创伤弧菌CGMCC1.1758构建血流感染模型,未感染组小鼠腹腔注射等量的无菌PBS。(2)感染4 h后,分别分离肝脏免疫细胞和脾细胞,流式细胞术检测中性粒细胞百分比、数量和存活率,以及活性氧簇(ROS)和细胞吞噬功能变化,分析创伤弧菌血流感染对小鼠肝脏和脾脏中性粒细胞mTOR信号通路的影响。结果(1)与WT未感染组相比,WT感染组肝脏与脾脏中性粒细胞百分比和数量均显著增加。与WT感染组相比,TNF-α-/-感染组中仅肝脏中性粒细胞百分比和数量增加明显,而脾脏中性粒细胞差异无统计学意义。(2)与WT感染组比,TNF-α-/-感染组肝脏中性粒细胞吞噬功能增强,而脾脏中性粒细胞吞噬功能并无明显改变。(3)与WT未感染组相比,WT感染组肝脏与脾脏中性粒细胞存活率均降低,ROS水平明显上升;与WT感染组相比,TNF-α-/-感染组肝脏与脾脏中性粒细胞存活率均增加,但细胞ROS水平明显下降。(4)WT感染组肝脏与脾脏中性粒细胞中p-AKT(S473)水平均低于WT未感染组,TNF-α-/-感染组肝脏中性粒细胞中p-AKT(S473)水平低于WT感染组,脾脏中性粒细胞中p-AKT(S473)水平高于WT感染组。与WT感染组相比,WT未感染组p-4E-BP1(T37/46)水平差异无统计学意义,TNF-α-/-感染组肝脏中性粒细胞中p-4E-BP1(T37/46)水平降低而脾脏中性粒细胞中p-4E-BP1(T37/46)水平差异无统计学意义。结论TNF-α对创伤弧菌血流感染后小鼠肝脏和脾脏中性粒细胞有不同影响,主要对肝脏中性粒细胞的募集、吞噬功能改变及mTOR信号强度有重要影响。

Objective To investigate the effects of TNF-αknockout on liver and spleen neutrophil responses to Vibrio vulnificus bloodstream infection in a mouse model.Methods(1)TNF-α-knockout(TNF-α-/-)and wild-type(WT)C57BL/6J mice aged 6-8 weeks were randomly divided into four groups with six in each group:uninfected WT group,infected WT group,uninfected TNF-α-/-group and infected TNF-α-/-group.The mouse model of bloodstream infection was constructed by intraperitoneal injection of Vibrio vulnificus CGMCC1.1758(2×108 CFU/200μl),while the mice in the uninfected groups were injected intraperitoneally with equal amount of PBS.(2)Liver immune cells and splenocytes were isolated 4 h after infection and subjected to analyze the percentages and numbers of neutrophils,and the changes in cell viability,cellular reactive oxygen species(ROS)level and phagocytosis by flow cytometry.In addition,effects of Vibrio vulnificus bloodstream infection on mTOR signaling pathway in murine neutrophils were evaluated in vivo.Results(1)Compared with the uninfected WT group,the percentages and numbers of neutrophils in liver and spleen tissues of the infected WT group increased significantly.The percentage and number of liver neutrophils were significantly higher in the infected TNF-α-/-group than in the infected WT group,but no significant difference in spleen neutrophils was detected between the two groups.(2)Compared with the infected WT group,the phagocytosis of liver neutrophils rather than that of spleen neutrophils was enhanced in the infected TNF-α-/-group.(3)The survival rates of neutrophils in both liver and spleen were decreased,while the cellular ROS level was significantly increased in the infected WT group compared with those of the uninfected WT group.Compared with the infected WT group,the infected TNF-α-/-group had increased survival rates of both liver and spleen neutrophils,but decreased level of ROS.(4)The levels of p-AKT(S473)in liver and spleen neutrophils of the infected WT group were lower than those of the uninfected WT group.Compared with the infected WT group,the infected TNF-α-/-group had lower level of p-AKT(S473)in liver neutrophils,but higher p-AKT(S473)level in spleen neutrophils.There were no significant differences in p-4E-BP1(T37/46)levels between the uninfected WT group and the infected WT group.The p-4E-BP1(T37/46)level in liver neutrophils was lower in the infected TNF-α-/-group than in the infected WT group,but no significant difference in p-4E-BP1(T37/46)levels in spleen neutrophils was observed between the two groups.Conclusions TNF-αhad different effects on the neutrophils in spleen and liver tissues of mice with Vibrio vulnificus bloodstream infection.It played a critical role in regulating the recruitment,phagocytic function and mTOR signaling of liver neutrophils after Vibrio vulnificus infection in vivo.