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Characterization of a novel BCG-derived TLR2 ligand capable of preferentially inducing Th1 cytokine production and investigation of the cytokine-inducing mechanism

能够优先诱导 Th1 细胞因子产生的新型 BCG 衍生 TLR2 配体的表征以及细胞因子诱导机制的研究

基本信息

批准号：
15590385
负责人：
KAWAMURA Ikuo
金额：
$ 2.3万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

Among various inflammatory cytokines produced at the early stage of infection with Mycobacterium bovis BCG, Interferon-γ plays a pivotal role for development of protective T cells. We have shown that IFN-γ production is induced by both IL-12 and IL-18 that are produced from macrophages infected with BCG or M.tuberculosis(MTB), and that some mycobacterial factor in the culture supernatant contributes to IL-12p40 production via activation of Toll-like Receptor 2(TLR2) pathway. Because IL-12p40, a subunit of IL-12, is an important factor for IFN-γ induction, it seems that IL-12p40-inducing mycobacterial component plays a critical role in generation of protective immunity. To identify the factor, we prepared a culture filtrate(CF) from 1d-culture of MTB and fractionated CF by gel filtration chromatography. The strong activity was found in the fractions containing molecules of MW 150-200k and the fractions were able to induce activation of NF-κB via TLR2 pathway. By SDS-PAGE analysis under nonreducing condition, we detected about 190kDa and 200kDa proteins in the fractions. The proteins were extracted from the gel and SDS-PAGE analysis was performed under reducing condition. The size of those proteins was shifted to 22k and 19k, respectively, indicating that those proteins in the culture supernatant are composed of each small subunit. We further found that 19k molecule was a mycobacterial ferritin familiy protein, Brf2. Those results suggest that the mycobacterial ferritin protein probably contribute to IL-12p40 induction via activation of TLR2 pathway.

在BCG感染早期产生的多种炎性细胞因子中，干扰素-γ在保护性T细胞的发育中起着关键作用。我们已经表明，IFN-γ的产生是由BCG或结核分枝杆菌（MTB）感染的巨噬细胞产生的IL-12和IL-18诱导的，并且培养上清液中的一些分枝杆菌因子通过激活Toll样受体2（TLR 2）途径促进IL-12 p40的产生。由于IL-12亚基IL-12 p40是IFN-γ诱导的重要因子，因此IL-12 p40诱导的分枝杆菌组分似乎在保护性免疫的产生中起关键作用。为了鉴定该因子，我们从MTB的1d培养物和通过凝胶过滤色谱法分级的CF制备培养物滤液（CF）。在分子量为150- 200 k的组分中发现了较强的活性，并且这些组分能够通过TLR 2途径诱导NF-κB的活化。在非还原条件下进行SDS-PAGE分析，我们在组分中检测到约190 kDa和200 kDa的蛋白质。从凝胶中提取蛋白质，并在还原条件下进行SDS-PAGE分析。这些蛋白质的大小分别变为22 k和19 k，表明培养上清液中的这些蛋白质由每个小亚基组成。我们进一步发现19 k分子是分枝杆菌铁蛋白家族蛋白Brf 2。这些结果表明，分枝杆菌铁蛋白可能通过激活TLR 2途径参与IL-12 p40的诱导。