【作者】 周莉丽； 李宇红； 樊明文；

【Author】 Zhou Lili;Li Yuhong;Fan Mingwen;Hubei-MOST & Key Laboratory of Oral Biomedicine of Ministry of Education (KLOBM),School & Hospital of Stomatology,Wuhan University;

【机构】 武汉大学口腔医学院；

【摘要】 目的:模式识别受体(PRRs)在机体识别细菌产物的过程中起着重要的作用。最近的研究表明,固有免疫和获得性免疫必须通过多种模式识别受体间的协同作用才能实现。本研究对于NOD1,NOD2与Toll样受体之间在树突状细胞激活过程中的协同作用进行分析。方法:采用TLR2,TLR3,TLR4,TLR5,TLR6,TLR7/8,NOD1和NOD2单个或两两组合刺激DC2.4细胞。刺激6小时后,收集DC2.4细胞的RNA,实时荧光定量PCR(Real-Time PCR)测定细胞因子(IL-4,IL-6,IL-10,IL-12p35,IL-12p40,IL-17,IFN-γ和TGF-β)的mRNA水平。刺激24小时后,经酶联吸附试验(ELISA)测定以上细胞因子在细胞上清中的分泌水平。同时,借助于流式细胞技术(flow cytometry)观察细胞表面分子CD83,CD40,CD80和CD86的表达水平。结果:TLR2,TLR3,TLR4,TLR5,TLR6,TLR7/8,NOD1和NOD2单个刺激均能明显增强IL-6 mRNA,双刺激时,NOD1和NOD2配体抑制Toll样受体(包括TLR2,TLR3,TLR4,TLR5,TLR6和TLR7/8)配体引起的IL-6 mRNA水平。相反的是,NOD样受体配体增强Toll样受体配体的IL-12p35 mRNA水平。二者间具有协同作用,且NOD1受体的配体iE-DAP比NOD2受体的配体MDP的协同刺激效应更强。DC细胞表面分子CD83和共刺激分子CD40,CD80,CD86表达上调,但与对照组比较没有显著性差异。结论:NOD1和NOD2受体的配体在联合Toll样受体(包括TLR2,TLR3,TLR4,TLR5,TLR6和TLR7/8)配体后能产生协同作用,明显刺激IL-12p70的表达,从而可能促进机体产生Th1类的免疫反应。相反的是,NOD1和NOD2受体的配体能有效拮抗Toll样受体(包括TLR2,TLR3,TLR4,TLR5,TLR6和TLR7/8)配体引起的IL-6的表达,可能抑制其在诱导Th17类免疫反应中的作用。更多还原

【Abstract】 Objective:Pattern recognition receptors(PRRs) like Toll-like receptors(TLRs) and NODlike receptors(NLRs) are important sensors of microbial products.Simultaneous activation of dendritic cells(DCs) via different classes of PRRs provides a powerful tool for inducing strong immune responses.Recent reports suggest that for the activation of innate immune responses and initiation of adaptive immunity,synergistic effects between two or more PRRs are necessary.No systematic analysis of the interaction between the TLRs and NLRs pathways were performed to date.In this study,a systematical analysis of the interactions between the NLRs(including NOD1 and NOD2) and their crosstalk with TLR signaling in terms of DCactivation was performed.Methods:DC2.4 cells were stimulated with either a single PRR ligand or a combination of NLR and TLR ligand.Known ligands for the major PRR families were used:TLR2,TLR3,TLR4,TLR5,TLR6,TLR7/8,NOD1 and NOD2.After 6 h of incubation,mRNA level of interleukin-4(IL-4),IL-6,IL-10,IL-12p35,IL-12p40,IL-17,interferon-gamma(IFN-γ)and tumor growth factor-beta(TGF-β)was measured by Real-time PCR.After 24 h of incubation,the secretion amount of IL-4,IL-6,IL-10,IL-12p70,IL-17,IFN-γ and TGF-β was measured in cells supernatant by enzyme-linked immunosorbent assay(ELISA).Meanwhile,the expression of CD83 and costimulatory molecules such as CD40,CD80,and CD86 was monitored by flow cytometry.Results:IL-6 mRNA increased notably after incubation with all kinds of PRR ligands.The NOD(including NOD1 and NOD2) ligands had noticeable synergistic effects with TLR(including TLR2,TLR3,TLR4,TLR5,TLR6 and TLR7/8) ligands on the mRNA expression of IL-12p35,while antagonistic effects on IL-6 mRNA.Surprisingly,the NOD1 ligand(iE-DAP) seemed more competent than N0D2 ligand(MDP) in changing TLRs-induced cytokines production.The secretion amount of IL-6 and IL-12p70 also testified the mentioned data.For the rest of cytokines,the PRR ligands showed no significant effect on the mRNA level and secretion amount.In contrast,for the expression of CD83 and costimulatory molecules such as CD40,CD80 and CD86,no significant effects were observed upon stimulation with Nod plus TLR ligands.Conclusion:These findings suggest that NOD1 and NOD2 ligands in combination with TLR2,TLR3,TLR4,TLR5,TLR6 and TLR7/8 ligands synergistically induce IL-12p70 in DC2.4 that is beneficial to Th1-lineage immune responses.On the contrary,NOD1 and NOD2 ligands in combination with these TLR ligands inhibit IL-6 in DC2.4 which plays important role in Th17-lineage immune responses.更多还原