TOLL-LIKE RECEPTOR MEDIATES INFLAMMATORY RESPONSES TO BORRELIA BURGDORFERI

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 我们研究了导致人类单核细胞在体外暴露于活的伯氏疏螺旋体（Bb）螺旋体时产生促炎介质的机制。 我们首先关注髓样分化初级反应蛋白88（MyD 88），这是一种在Toll样受体（TLR）通路中必不可少的衔接分子。实时PCR，流式细胞术和共聚焦显微镜实验表明，MyD 88最大限度地表达在THP-1细胞24小时后，这些细胞与活Bb刺激。使用小干扰RNA沉默MyD 88基因导致用活Bb刺激的THP-1细胞中TNF-γ、IL-8和IL-6的产生分别下调24%、35%和84%。通过RNA干扰对TLR 1、TLR 2或TLR 5基因的特异性沉默进一步揭示，单独或组合的TLR 1和TLR 2基因的沉默，而不是TLR 5基因的沉默，导致在活的Bb刺激的THP-1细胞中IL-6、IL-8和TNF-γ的下调。总的来说，用纯化的脂蛋白刺激的THP-1细胞获得了类似的结果。我们的研究结果表明TLR通路至少部分介导了用肝B刺激的人单核细胞中炎症介质的释放。burgdorferi螺旋体，并进一步表明这些细胞和活螺旋体之间的TLR依赖性相互作用是由螺旋体脂蛋白介导的，而不是鞭毛蛋白。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We investigated the mechanisms that lead to the production of pro-inflammatory mediators by human monocytes when these cells are exposed in vitro to live Borrelia burgdorferi (Bb) spirochetes. We first focused on myeloid differentiation primary response protein 88 (MyD88), an adapter molecule that is essential in the Toll-like receptor (TLR) pathway. Real-time PCR, flow cytometry and confocal microscopy experiments revealed that MyD88 was maximally expressed in THP-1 cells after 24-h stimulation of these cells with live Bb. Silencing of the MyD88 gene using small interfering RNA resulted in 24%, 35% and 84% down-modulation of the production of TNF-¿, IL-8 and IL-6, respectively in THP-1 cells stimulated with live Bb. Specific silencing of the TLR1, TLR2 or TLR5 genes by RNA interference further revealed that silencing of the TLR1 and TLR2 genes alone or combined, but not the TLR5 gene caused a down-regulation of IL-6, IL-8 and TNF-¿ in live Bb-stimulated THP-1 cells. Overall, similar results were obtained for THP-1 cells stimulated with purified lipoproteins. Our results indicate that the TLR pathway mediates at least in part the release of inflammatory mediators in human monocytes stimulated with live B. burgdorferi spirochetes, and further suggest that the TLR-dependent interaction between these cells and live spirochetes is mediated by spirochetal lipoproteins but not flagellin.