Possible involvement of M. bovis BCG-producing TLR2 ligand to generate protective immunity by inducing TH1 cytokine productions

在感染早期产生的各种炎症介质中，TH1细胞因子如干扰素-γ、白细胞介素12 （IL-12）和IL-18在保护性T细胞的发育中起关键作用。我们进一步知道，这些细胞因子的产生是由活的而不是被杀死的细菌诱导的，这些细菌能够产生保护性免疫。为了确定分枝杆菌因子对TH1细胞因子产生的贡献，我们从活卡介苗（vBCG）的1d培养中制备了培养滤液，并测量了细胞因子诱导活性。培养滤液诱导腹腔巨噬细胞产生IL-12p40。然而，由灭活卡介苗制备的类似制剂没有诱导细胞因子的产生。在C3H/HeJ小鼠巨噬细胞中检测到细胞因子诱导活性，且不受LPS拮抗剂E5531的影响。此外，在转染TLR4和MD-2的HEK293细胞中，培养滤液未诱导NF-κB活化。然而，在转染了TLR2的细胞中，通过vbcg来源的培养滤液刺激可以诱导转录因子的激活。细胞因子诱导活性对DNase有抗性，对蛋白酶K有部分敏感性，表明一些蛋白和非蛋白因子参与了细胞因子的产生。在活的但未杀死的结核分枝杆菌H37Rv培养滤液中也检测到类似的活性。这些结果表明，活菌早期分泌的成分通过TLR2依赖途径激活巨噬细胞产生IL-12。这可能是细菌和巨噬细胞之间产生保护性免疫的关键相互作用。

Among various inflammatory mediators produced at the early stage of infection, TH1 cytokines such as interferon-γ, interleukin 12 (IL-12) and IL-18 play a pivotal role for development of protective T cells. It is further known that the production of these cytokines is induced by viable, but not killed bacteria, which are capable of generating protective immunity. To identify the mycobacterial factor contributing to the TH1 cytokine productions, we prepared a culture filtrate from 1d-culture of viable BCG (vBCG) and measured the cytokine-inducing activity. The culture filtrate elicited IL-12p40 production from peritoneal macrophages. However, the similar preparation derived from killed BCG did not induce the cytokine production. The cytokine-inducing activity was detected in macrophages of C3H/HeJ mice and was not affected by E5531, LPS antagonist. Furthermore, the culture filtrate did not induce NF-κB activation in HEK293 cells transfected with TLR4 and MD-2. However, activation of the transcription factor was induced in cells transfected with TLR2 by stimulation with vBCG-derived culture filtrate. The cytokine-inducing activity was resistant to treatment with DNase and partially sensitive to treatment with proteinase K, suggesting that some proteinaceous and non-proteinaceous factors are involved in the cytokine production. The similar activity was also detected in the culture filtrate obtained from viable, but not killed M. tuberculosis H37Rv. These results suggest that the early secreted components from viable mycobacteria activate macrophages to produce IL-12 via TLR2 dependent pathway. It is likely that this is a critical interaction between bacteria and macrophages for the generation of protective immunity.