细粒棘球绦虫微囊对小鼠腹腔巨噬细胞活化及炎症因子水平的影响

Effects of Echinococcus granulosus microcysts on the activation of peritoneal macrophages and the level of inflammatory factors in mice

目的探索细粒棘球绦虫微囊感染对小鼠腹腔不同表型巨噬细胞活化及腹腔灌洗液中炎症因子水平的影响。方法体外培养细粒棘球绦虫原头蚴形成微囊,通过腹腔注射微囊和原头蚴,分别建立BALB/c小鼠细粒棘球蚴继发感染模型,对照组小鼠腹腔注射等体积的磷酸盐缓冲液(PBS)。于感染后的第8、30、60、90、180、240 d,收集各组小鼠的腹腔灌洗液和腹腔细胞,流式细胞术检测F4/80+CD16/32+(M1型)与F4/80+CD206+(M2型)巨噬细胞比例变化,流式细胞微球阵列法(Cytometric Bead Array,CBA)检测6种炎症因子IL-12p70、TNF-α、IFN-γ、MCP-1、IL-10、IL-6水平。结果感染后240 d,微囊感染组(微囊组)存活囊泡和囊团数分别为(237.67±4.5)、(10.33±1.21)个,与原头蚴感染组(原头蚴组)的囊泡和囊团数(70.33±3.5)、(7.17±1.17)个比较差异有统计学意义(均P<0.01)。微囊组和原头蚴组小鼠腹腔M1型巨噬细胞比例均在感染早期开始升高,8~30 d逐渐升高达峰值,后逐渐下降,在240 d有一定程度回升。感染后30、60、90、180 d,微囊组M1型巨噬细胞比例分别为(78.6±7.52)、(72.95±3.07)、(63.33±3.89)、(28.7±4.84)%,与原头蚴组(66.30±3.47)、(64.03±2.67)、(47.42±2.46)、(18.00±6.77)%比较差异有统计学意义(均P<0.05)。而微囊组和原头蚴组的M2型巨噬细胞细胞比例均在感染早期第8 d处于较高水平,8 d后下降但在感染晚期90~240 d持续升高。感染后8、30、180和240 d,微囊组M2型巨噬细胞比例分别为(60.24±1.35)、(78.6±7.52)、(28.7±4.84)、(51.93±9.49)%,与原头蚴组(56.70±4.25)、(66.30±3.47)、(18.00±6.77)、(43.92±2.66)%比较差异有统计学意义(均P<0.05)。CBA显示,微囊组和原头蚴组腹腔灌洗液中促炎因子IL-12p70和TNF的水平均在8~90 d升高,90 d后下降,其中8、30、60和90 d,微囊组IL-12p70和TNF水平均与原头蚴组比较差异有统计学意义(均P<0.01)。微囊组和原头蚴组促炎因子MCP-1、IL-6均在8~180 d升高,180 d后降低,其中8、30、60、90和180 d,微囊组MCP-1和IL-6水平与原头蚴组比较差异均有统计学意义(均P<0.01)。微囊组IL-6水平在感染晚期240 d下降为(503.89±97.62)pg/ml,但仍与原头蚴组比较差异有统计学意义(179.34±30.31)pg/ml(P<0.01)。微囊组IFN-γ水平在感染早期在8、30 d分别为(25.34±1.94)、(25.90±1.71)pg/mL,与原头蚴组(21.32±3.15)、(21.22±2.46)pg/ml比较差异有统计学意义(均P<0.05),但在30 d后下降,而原头蚴组IFN-γ水平升高至90 d出现峰值为(38.16±3.13)pg/ml,与微囊组(20.22±2.08)pg/ml比较差异有统计学意义(P<0.01)。抑炎因子IL-10水平变化缓慢,增长至90 d达峰值后下降,其中8、30、60、90、240 d时两感染组比较差异无统计学意义(均P>0.05)。结论微囊感染在棘球蚴继发感染寄生虫免疫逃避及宿主早期腹膜炎症反应中发挥重要作用,导致M1型巨噬细胞比例及促炎细胞因子水平表达较原头蚴感染更显著。

Objective To explore the effect of Echinococcus granulosus microcyst infection on the activation of peritoneal macrophages and the level of inflammatory factors in the peritoneal lavage fluid of mice.Methods Microcysts of E.granulosus were formed by protoscoleces via in vitro culture.BALB/c murine models of secondary hydatid infection were respectively established via intraperitoneal injection of microcysts and protoscoleces.Mice in the control group were intraperitoneally injected with the same volume of phosphate-buffered saline(PBS).Peritoneal lavage fluid and cells from each group were collected 8,30,60,90,180,and 240 d after infection.Flow cytometry was used to detect the proportion of F4/80+CD16/32+(M1 type)and F4/80+CD206+(M2 type)macrophages.A flow cytometric bead array(CBA)was used to determine levels of six inflammatory cytokines:IL-12 p70,TNF,IFN-γ,MCP-1,IL-10,and IL-6.Results Two hundred and forty d after infection,there were 237.67±4.5 surviving cysts and 10.33±1.21 cystic masses in the group infected with microcysts(the microcyst group).The number of surviving cysts and cystic masses differed significantly from the number of surviving cysts and cystic masses(70.33±3.5 and 7.17±1.17)in the group infected with protoscoleces(the protoscolex group)(P<0.01 for all).In the microcyst and protoscolex groups,the proportion of M1 macrophages in the abdominal cavity was relatively high in the early stage of infection,it increased starting on day 8 and peaked on day 30,it then decreased gradually but rose to an extent on day 240.On days 30,60,90,and 180,the proportion of M1 macrophages in the microcyst group was 78.6±7.52,72.95±3.07,63.33±3.89,and 28.7±4.84%,which differed significantly from the proportion in the protoscolex group(66.30±3.47,64.03±2.67,47.42±2.46,and 18.00±6.77%)(P<0.05 for all).The proportion of M2 macrophages in both the microcyst group and the protoscolex group was high during early infection on day 8 and then decreased,but it continued to increase during the late period of infection on days 90-240.On days 8,30,180,and 240 after infection,the proportion of M2 macrophages in the microcyst group were 60.24±1.35,78.6±7.52,28.7±4.84,and 51.93±9.49%,which differed significantly from the proportion in the protoscolex group(56.70±4.25,66.30±3.47,18.00±6.77,and 43.92±2.66%)(P<0.05 for all).CBA results indicated that the levels of the pro-inflammatory cytokines IL-12 p70 and TNF in both the microcyst group and the protoscolex group increased on days 8-90 and then decreased.On days 8,30,60,and 90,the levels of IL-12 p70 and TNF in the microcyst group differed significantly from those in the protoscolex group(P<0.01 for all).On days 8-180,the levels of MCP-1 and IL-6 in the microcyst group and the protoscolex group both increased and then decreased.On days 8,30,60,90,and 180,the levels of MCP-1 and IL-6 in the microcyst group differed significantly from those in the protoscolex group(P<0.01 for all).The IL-6 level in the microcyst group was 503.89±97.62 pg/ml.The level decreased during the late stage of infection on day 240 d,and it differed significantly from that in the protoscolex group(179.34±30.31 pg/mL)(P<0.01 for all).On days 8 and 30,the IFN-γlevel in the microcyst group was 25.34±1.94 and 25.90±1.71 pg/mL,which differed significantly from that in the protoscolex group(21.32±3.15 and 21.22±2.46 pg/ml)(P<0.01).The level of IFN-γpeaked at 38.16±3.13 pg/ml on day 90 and differed significantly from that in the microcyst group(20.22±2.08 pg/mL)(P<0.01).The level of the anti-inflammatory factor IL-10 changed slowly in the microcyst group and the protoscolex group.The level peaked on day 90 d and then decreased.There were no significant differences between the two infection groups on days 8,30,60,90,and 240(P>0.05 for all).Conclusion Microcyst infection plays an important role in immune evasion by an infection secondary to echinococcosis and in the host’s peritoneal inflammatory response during the early stage of infection,leading to a markedly higher proportion of M1 macrophages and levels of pro-inflammatory cytokines than in a protoscolex infection.