Simultaneous blocking of human toll-like receptors 2 and 4 suppresses myeloid dendritic cell activation induced by Mycobacterium bovis bacillus Calmette-Guerin peptidoglycan
Simultaneous blocking of human toll-like receptors 2 and 4 suppresses myeloid dendritic cell activation induced by Mycobacterium bovis bacillus Calmette-Guerin peptidoglycan
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DOI：
10.1128/iai.71.8.4238-4249.2003
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发表时间：
2003-08-01
影响因子：
3.1
通讯作者：
Seya, T
中科院分区：
文献类型：
--
作者：
Uehori, J;Matsumoto, M;Seya, T
牛分枝杆菌卡介苗（BCG）细胞壁骨架（CWS）由分枝菌酸、阿拉伯半乳聚糖和肽聚糖（PGN）组成，并激活Toll样受体2（TLR 2）和TLR 4。在这里，我们通过使用以下标准研究了高度纯化的BCG CWS的必需部分支持TLR激动剂功能的能力：骨髓树突状细胞（DC）成熟，即，肿瘤坏死因子α（TNF-α）产生和CD 83/CD 86上调。纯化的PGN区域足以激活小鼠DC和巨噬细胞中的TLR 2和TLR 4;在TLR 2和TLR 4双敲除细胞中，BCG PGN介导的TNF-α产生能力完全受损。同样，用BCG CWS刺激表达人TLR 2或TLR 4、MD-2和CD 14的HEK 293细胞导致NF-κ B活化，如通过报告基因测定所确定的。特别是特异性阻滞剂;细胞外人TLR 2（单克隆抗体TLR2.45和TH2.1的原始混合物）和TLR 4（E5531）抑制BCG CWS介导的NF-κ B活化80%。使用这种人TLR阻断系统，我们测试了人髓样DC成熟是否是TLR 2和TLR 4依赖性的。BCG PGN介导的DC成熟通过抑制TLR 2和TLR 4被阻断70%，通过抑制这些TLRs中的任一个被阻断30 - 40%。观察到BCG CWS介导的DC成熟的类似但不太深刻的抑制。因此，BCG PGN的存在是在人DC中激活TLR 2和TLR 4两者的最低要求，不像革兰氏阳性细菌的PGN的存在，其仅激活TLR 2。然而，出乎意料的是，与BCG CWS不同，BCG PGN在共表达TLR 2加TLR 1、TLR 2加TLR 4、TLR 2加TLR 6或TLR 2加TLR 10的HEK 293细胞中几乎不激活NF-κ B，这表明存在于人DC上而不存在于HEK 293细胞上的除TLR 2和TLR 4之外的PGN受体参与DC激活的TLR信号传导。
The Mycobacterium bovis bacillus Calmette-Guerin (BCG) cell wall skeleton (CWS) consists of mycolic acids, arabinogalactan, and peptidoglycan (PGN) and activates Toll-like receptor 2 (TLR2) and TLR4. Here we investigated the ability of the essential portion of highly purified BCG CWS to support the TLR agonist function by using the following criteria: myeloid dendritic cell (DC) maturation, i.e., tumor necrosis factor alpha (TNF-alpha) production and CD83/CD86 up-regulation. The purified PGN region was sufficient to activate TLR2 and TLR4 in mouse DCs and macrophages; in TLR2 and TLR4 double-knockout cells the BCG PGN-mediated TNF-alpha production ability was completely impaired. Likewise, stimulation with BCG CWS of HEK293 cells expressing either human TLR2 or TLR4, MD-2, and CD14 resulted in NF-kappaB activation as determined by a reporter assay. Notably, specific blockers; of extracellular human TLR2 (an original cocktail of monoclonal antibodies TLR2.45 and TH2.1) and TLR4 (E5531) inhibited BCG CWS-mediated NF-kappaB activation by 80%. Using this human TLR blocking system, we tested whether human myeloid DC maturation was TLR2 and TLR4 dependent. BCG PGN-mediated DC maturation was blocked by 70% by suppression of both TLR2 and TLR4 and by 30 to 40% by suppression of either of these TLRs. Similar but less profound suppression of BCG CWS-mediated DC maturation was observed. Hence, the presence of BCG PGN is a minimal requirement for activation of both TLR2 and TLR4 in human DCs, unlike the presence of PGNs of gram-positive bacteria, which activate only TLR2. Unexpectedly, however, BCG PGN, unlike BCG CWS, barely activated NF-kappaB in HEK293 cells coexpressing TLR2 plus TLR1, TLR2 plus TLR4, TLR2 plus TLR6, or TLR2 plus TLR10, suggesting that PGN receptors other than TLR2 and TLR4 present on human DCs but not on HEK293 cells are involved in TLR signaling for DC activation.