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

在牛分枝杆菌卡介苗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。卡介苗或结核分枝杆菌感染的巨噬细胞产生的IL-12和IL-18均可诱导产生干扰素-γ，培养上清液中的某些分枝杆菌因子通过激活Toll样受体2途径参与IL-12p40的产生。由于IL-12p40是IL-12的一个亚基，是诱导干扰素-γ的重要因素，因此，IL-12p40诱导的分枝杆菌成分在保护性免疫的产生中起着关键作用。为了确定这一因素，我们从MTB的1D培养中制备了培养滤液(CF)，并用凝胶过滤层析分离了CF。在相对分子质量为150-200K的组分中具有较强的活性，并能通过TLR2途径诱导核因子-κB的活化。通过非还原条件下的SDS-PAGE分析，检测到约190 kDa和200 kDa的蛋白质。从凝胶中提取蛋白质，在还原条件下进行SDS-PAGE分析。这些蛋白质的大小分别移动到22K和19K，表明培养上清液中的蛋白质是由每个小亚基组成的。我们进一步发现19K分子是分枝杆菌铁蛋白家族蛋白Brf2。上述结果提示，分枝杆菌铁蛋白可能通过激活TLR2途径参与IL-12p40的诱导。

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.