CpG-c41对TLR9信号通路的干扰作用及其机制探究

The interference effect of Cp G-c41 on TLR9 signaling pathway and its mechanism

目的探讨Cp G-c41分子对TLR9信号通路的干扰作用及其机制。方法获取小鼠骨髓巨噬细胞,M-CSF刺激使骨髓细胞向单核巨噬细胞分化,流式细胞术(flow cytometry,FCM)检测诱导成功的巨噬细胞的比例;培养Raw264.7细胞,ELISA法检测Cp G-c41分子对TLR9激动剂Cp G-1826刺激小鼠来源骨髓巨噬细胞和Raw264.7细胞诱发的炎症因子TNF-α和IL-6的影响;Western blot检测Cp G-c41对TLR9-My D88依赖型信号通路中磷酸化NF-κB蛋白表达的影响;ELISA法检测非Cp G-ODN分子对Cp G-1826刺激Raw264.7细胞诱发的炎症因子TNF-α和IL-6的影响;免疫荧光检测Cp G-c41与TLR9的共定位情况以及Cp G-c41对Cp G-1826与TLR9结合的影响。结果 Cp G-c41抑制Cp G-1826诱导的炎症因子TNF-α和IL-6的释放(P<0.01);Cp G-c41抑制TLR9信号通路中NF-κB的磷酸化表达;非Cp G-ODN不影响Cp G-1826诱导TLR9释放TNF-α和IL-6;Cp G-c41通过与TLR9竞争性结合抑制Cp G-1826与TLR9识别。结论 Cp G-c41通过竞争性结合TLR9干扰TLR9-My D88信号通路的活化。

Toll-like receptor 9（TLR9） recognizes microbial Cp G-DNA or its analog syntheticoligonucleotides containing a Cp G motif（Cp G-ODN）, and plays an essential role in activating innate immunity.However, the excessive TLR9 activation could lead to overwhelming release of pro-inflammatory cytokines, causinginfection and autoimmune diseases. Significantly, we had found a non-stimulatory Cp G-DNA named Cp G-c41 fromthe conventional microbes using bioinformatics tool. In this study, to explore the interference effect of Cp G-c41 onTLR9 pathway, bone marrow macrophages were isolated from mouse, and then M-CSF was added to induce thedifferentiation of bone marrow cells to monocyte-macrophages. The proportion of the successfully inducedmacrophages was assayed by flow cytometry（FCM）. Besides, bone marrow macrophages and Raw264.7 cells weretreated by TLR9 agonist Cp G-1826 and Cp G-c41 alone or in combination, then the levels of TNF-α and IL-6 weremeasured by ELISA. The influence of Cp G-c41 on phosphorylation expression of NF-κB mediated byTLR9-My D88-dependent pathway was assayed by Western blotting. ELISA also used to analyze the effect of nonCp G-ODN on the release of TNF-α and IL-6 in Raw264.7 cells stimulated by Cp G-1826. The co-localization ofCp G-c41 with TLR9 and the inhibition of Cp G-c41 on the binding of Cp G-1826 to TLR9 were observed byimmunofluorescence. Data showed that the levels of TNF-α and IL- 6 induced by Cp G-1826 were all significantlyinhibited by Cp G-c41（P〈0.01） and the phosphorylation expression of NF-κB was also suppressed. However, nonCp G-ODN had no influence on the Cp G-1826-induced release of TNF-α and IL-6. On the whole, Cp G-c41 hasinterference effect on the TLR9-My D88-dependent pathway by competitively binding with TLR9.