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类细胞因子如干扰素-γ、白介素12(IL-12)和白介素18(IL-18)在保护性T细胞的发育中起着关键作用。人们还知道，这些细胞因子的产生是由能够产生保护性免疫的活细菌诱导的，而不是由被杀死的细菌诱导的。为了鉴定影响TH1细胞因子产生的分枝杆菌因子，我们制备了活体卡介苗一维培养滤液(VBCG)，并测定了其细胞因子诱导活性。培养滤液可诱导巨噬细胞产生IL-12p40。然而，灭活卡介苗的类似制剂不能诱导细胞因子的产生。在C3H/HeJ小鼠的巨噬细胞中检测到细胞因子诱导活性，但不受内毒素拮抗剂E5531的影响。此外，培养滤液不能诱导转染TLR4和MD-2的HEK293细胞中的NF-κB活化。然而，在vBCG来源的培养滤液刺激下，转导TLR2的细胞中转录因子被激活。细胞因子诱导活性对脱氧核糖核酸酶耐受，对蛋白酶K部分敏感，提示一些蛋白质和非蛋白质因子参与了细胞因子的产生。在活的但未被杀死的结核分枝杆菌H37Rv的培养滤液中也检测到类似的活性。这些结果表明，活体分枝杆菌早期分泌的成分通过TLR2依赖的途径激活巨噬细胞产生IL-12。这很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.