梅毒螺旋体诱导巨噬细胞分泌的外泌体对巨噬细胞极化的影响

Treponema pallidum induces the polarization of macrophages via macrophage-derived exosomes

目的探讨梅毒螺旋体(Tp)诱导巨噬细胞产生的外泌体(EXOTp)对巨噬细胞极化的影响。方法将人单核巨噬细胞(THP-1)诱导为巨噬细胞,分别行Tp刺激和不用Tp刺激,作用24 h后,更换培养基继续培养48 h,收集其巨噬细胞外泌体悬液,用差速离心法分离外泌体,运用Western blot、透射电镜鉴定外泌体,纳米颗粒跟踪分析技术定量分析外泌体,在激光共聚焦显微镜下观察巨噬细胞对两组外泌体的内化作用。实验组将分离的EXOTp加入M0型巨噬细胞中共培养24 h,阴性对照组加入未经Tp感染的巨噬细胞自身产生的外泌体(EXO),空白对照组不加入外泌体。3组以6、12、24 h为时间点,通过qRT-PCR检测INOS、IL-12、Arg-1、IL-10等mRNA的表达,通过流式细胞术对巨噬细胞表型MHC-Ⅱ、CD86、CD206、CD209进行鉴定。结果qRT-PCR结果显示,实验组INOS、IL-12 mRNA表达量均明显上调(P<0.05),且随共培养时间的延长,上调更明显,而Arg-1、IL-10表达无明显变化;阴性对照组INOS、IL-12、Arg-1、IL-10 mRNA表达均无明显变化(P>0.05)。流式细胞术结果显示MHC-Ⅱ阳性(P<0.05),而CD86、CD206、CD209变化无统计学意义(P>0.05)。结论THP-1来源的巨噬细胞分泌的外泌体不能诱导M0巨噬细胞向M1型巨噬细胞极化,而经Tp刺激后,能诱导M0巨噬细胞向M1型巨噬细胞极化。

Objective To explore the effect of Treponema pallidum(Tp)-induced exosomes(EXOTp)by macrophages on macrophage polarization.Methods Human monocyte-macrophages(THP-1)were induced into macrophages,followed by stimulation with or without Tp for 24 h.After changing medium,cells were cultured for additional 48 h.The macrophage exosome suspension was collected,and exosomes were isolated by differential speed centrifugation.Isolated exosomes were identified by both Western blotting and transmission electron microscopy,and were quantitated with nanoparticle tracking technology.The internalization of the macrophages in the two groups was observed under a laser confocal microscope.In the experimental group,the isolated EXOTp was incubated with M0 macrophages for 24 h.Incubation of M0 macrophages with exosomes produced by non-Tp-treated macrophages served as negative controls,while M0 macrophages incubated without exosomes served as blank controls.At 6,12,and 24 h,the expression levels of INOS,IL-12,Arg-1,IL-10 and other mRNAs in all three groups were detected by qRT-PCR.Flow cytometry was used to identify macrophage phenotypes using MHC-Ⅱ,CD86,CD206,and CD209 markers.Results qRT-PCR showed that the expression levels of INOS and IL-12 mRNA in experimental group were time-dependently up-regulated(P<0.05),while expression levels of Arg-1 and IL-10 were not significantly changed.Moreover,expression levels of INOS,IL-12,Arg-1,and IL-10 mRNA were not significantly altered in the negative controls(P>0.05).Flow cytometry results showed that MHC-Ⅱ was positive(P<0.05),but the changes in CD86,CD206,and CD209 were not significant(P>0.05).Conclusions The exosomes secreted by THP-1-derived macrophages cannot induce the polarization of M0 macrophages to M1 macrophages.Upon the stimulation with Tp,the exosomes secreted by THP-1-derived macrophages can induce polarization of M0 macrophages to M1 macrophages.