Ding Pei-Hui, Darveau Richard P, Wang Cun-Yu, Jin Lijian
Department of Periodontology, The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China.
Discipline of Periodontology, Faculty of Dentistry, The University of Hong Kong, Hong Kong SAR, China.
PLoS One. 2017 Apr 6;12(4):e0173223. doi: 10.1371/journal.pone.0173223. eCollection 2017.
Lipopolysaccharide (LPS)-binding protein (LBP) as an acute-phase protein plays a crucial role in innate host response to bacterial challenge. Our previous study shows that LBP expression in human gingiva is associated with periodontal status. Porphyromonas gingivalis is a keystone periodontopathogen, and its LPS with lipid A structural heterogeneity critically accounts for periodontal pathogenesis. This study investigated the effects of LBP and its interactions with two featured isoforms of P. gingivalis LPS (tetra-acylated LPS1435/1449 and penta-acylated LPS1690) on the expression of pro-inflammatory cytokines in human oral keratinocytes (HOKs), and the involvement of Toll-like receptor (TLR) signaling. HOKs were pre-incubated with recombinant human LBP (rhLBP) at 10ng/ml, 100ng/ml and 1μg/ml for 1 h, followed by the treatment of P. gingivalis LPS1690 or LPS1435/1449 for 3h or 24h respectively. The expression of IL-6 and IL-8, and involvements of TLR2 and TLR4 were analyzed. The genes associated with TLR signaling were assessed by PCR array. Interestingly, rhLBP per se significantly up-regulated the expression of IL-6 and IL-8 in HOKs (p<0.05), which was blocked by TLR2 antibody (p<0.001). LPS1435/1449 down-regulated more significantly rhLBP-induced IL-6 and IL-8 mRNAs with reference to P. gingivalis LPS1690 (approximately 80% vs. 40%, p<0.05; and 90% vs. 36%, p<0.001, respectively). Moreover, rhLBP markedly down-regulated the gene expression of TLRs and their adaptors such as CD180 (-2.44 folds) and MD-1 (-9.62 folds), while the interaction of P. gingivalis LPS1435/1449 with rhLBP greatly up-regulated both transcripts (7.11 and 4.05 folds, respectively). Notably, P. gingivalis LPS1690-rhLBP interaction dramatically up-regulated CD180 transcript (20.86 folds) and significantly down-regulated MD-1 transcript (-6.93 folds). This pioneering study shows that rhLBP enables to enhance the expression of pro-inflammatory cytokines in HOKs through TLR2 signaling pathway. P. gingivalis LPS with different lipid A structures down-regulates to different extents rhLBP-induced cytokine expression, possibly through fine-tuning of the CD180-MD1 complex and relevant TLRs.
脂多糖（LPS）结合蛋白（LBP）作为一种急性期蛋白，在宿主对细菌攻击的固有反应中起关键作用。我们之前的研究表明，人牙龈中LBP的表达与牙周状况相关。牙龈卟啉单胞菌是一种关键的牙周病原体，其具有脂质A结构异质性的LPS是牙周发病机制的关键因素。本研究调查了LBP及其与牙龈卟啉单胞菌LPS的两种特征性异构体（四酰化LPS1435/1449和五酰化LPS1690）的相互作用对人口腔角质形成细胞（HOKs）中促炎细胞因子表达的影响，以及Toll样受体（TLR）信号通路的参与情况。将HOKs分别与10ng/ml、100ng/ml和1μg/ml的重组人LBP（rhLBP）预孵育1小时，然后分别用牙龈卟啉单胞菌LPS1690或LPS1435/1449处理3小时或24小时。分析了IL-6和IL-8的表达以及TLR2和TLR4的参与情况。通过PCR阵列评估与TLR信号通路相关的基因。有趣的是，rhLBP本身显著上调了HOKs中IL-6和IL-8的表达（p<0.05），这被TLR2抗体阻断（p<0.001）。与牙龈卟啉单胞菌LPS1690相比，LPS1435/1449更显著地下调了rhLBP诱导的IL-6和IL-8 mRNA（分别约为80%对40%，p<0.05；90%对36%，p<0.001）。此外，rhLBP显著下调了TLRs及其衔接蛋白如CD180（-2.44倍）和MD-1（-9.62倍）的基因表达，而牙龈卟啉单胞菌LPS1435/1449与rhLBP的相互作用则显著上调了这两种转录本（分别为7.11倍和4.05倍）。值得注意的是，牙龈卟啉单胞菌LPS1690-rhLBP相互作用显著上调了CD180转录本（20.86倍）并显著下调了MD-1转录本（-6.93倍）。这项开创性研究表明，rhLBP能够通过TLR2信号通路增强HOKs中促炎细胞因子的表达。具有不同脂质A结构的牙龈卟啉单胞菌LPS可能通过对CD180-MD1复合物和相关TLRs的微调，不同程度地下调rhLBP诱导的细胞因子表达。
