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.

在牛分枝杆菌 BCG 感染早期产生的各种炎症细胞因子中，干扰素-γ 对保护性 T 细胞的发育起着关键作用。我们已经证明，IL-12 和 IL-18 均诱导产生 IFN-γ。由感染 BCG 或结核分枝杆菌 (MTB) 的巨噬细胞产生，培养物上清液中的某些分枝杆菌因子通过激活Toll 样受体 2 (TLR2) 途径由于 IL-12p40（IL-12 的一个亚基）是 IFN-γ 诱导的重要因子，因此 IL-12p40 诱导的分枝杆菌成分似乎在保护性细胞生成中发挥着关键作用。为了鉴定该因子，我们从MTB培养物中制备了培养物滤液（CF），并通过凝胶过滤色谱法分级分离CF。在含有MW 150-200k的分子的级分中发现了该级分，并且该级分能够通过TLR2途径诱导NF-κB的激活。通过在非还原条件下的SDS-PAGE分析，我们检测到级分中提取了约190kDa和200kDa的蛋白质。在还原条件下进行凝胶和 SDS-PAGE 分析，这些蛋白质的大小分别变为 22k 和 19k，表明。我们发现，培养上清液中的这些蛋白质进一步由每个小亚基组成。这些结果表明，分枝杆菌铁蛋白可能通过激活 TLR2 途径来诱导 IL-12p40。