Montenegro Raudales Jorge Luis, Yoshimura Atsutoshi, Sm Ziauddin, Kaneko Takashi, Ozaki Yukio, Ukai Takashi, Miyazaki Toshihiro, Latz Eicke, Hara Yoshitaka
Department of Periodontology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.
Center for Oral Diseases, Fukuoka Dental College, Fukuoka, Japan.
PLoS One. 2016 Sep 15;11(9):e0162865. doi: 10.1371/journal.pone.0162865. eCollection 2016.
Dental calculus is a mineralized deposit associated with periodontitis. The bacterial components contained in dental calculus can be recognized by host immune sensors, such as Toll-like receptors (TLRs), and induce transcription of proinflammatory cytokines, such as IL-1β. Studies have shown that cellular uptake of crystalline particles may trigger NLRP3 inflammasome activation, leading to the cleavage of the IL-1β precursor to its mature form. Phagocytosis of dental calculus in the periodontal pocket may therefore lead to the secretion of IL-1β, promoting inflammatory responses in periodontal tissues. However, the capacity of dental calculus to induce IL-1β secretion in human phagocytes has not been explored. To study this, we stimulated human polymorphonuclear leukocytes (PMNs) and peripheral blood mononuclear cells (PBMCs) with dental calculus collected from periodontitis patients, and measured IL-1β secretion by ELISA. We found that calculus induced IL-1β secretion in both human PMNs and PBMCs. Calculus also induced IL-1β in macrophages from wild-type mice, but not in macrophages from NLRP3- and ASC-deficient mice, indicating the involvement of NLRP3 and ASC. IL-1β induction was inhibited by polymyxin B, suggesting that LPS is one of the components of calculus that induces pro-IL-1β transcription. To analyze the effect of the inorganic structure, we baked calculus at 250°C for 1 h. This baked calculus failed to induce pro-IL-1β transcription. However, it did induce IL-1β secretion in lipid A-primed cells, indicating that the crystalline structure of calculus induces inflammasome activation. Furthermore, hydroxyapatite crystals, a component of dental calculus, induced IL-1β in mouse macrophages, and baked calculus induced IL-1β in lipid A-primed human PMNs and PBMCs. These results indicate that dental calculus stimulates IL-1β secretion via NLRP3 inflammasome in human and mouse phagocytes, and that the crystalline structure has a partial role in the activation of NLRP3 inflammasome.
牙石是一种与牙周炎相关的矿化沉积物。牙石中含有的细菌成分可被宿主免疫传感器（如Toll样受体（TLRs））识别，并诱导促炎细胞因子（如IL-1β）的转录。研究表明，细胞摄取结晶颗粒可能触发NLRP3炎性小体激活，导致IL-1β前体裂解为成熟形式。因此，牙周袋中牙石的吞噬作用可能导致IL-1β的分泌，促进牙周组织的炎症反应。然而，牙石在人吞噬细胞中诱导IL-1β分泌的能力尚未得到研究。为了研究这一点，我们用从牙周炎患者收集的牙石刺激人多形核白细胞（PMNs）和外周血单核细胞（PBMCs），并通过ELISA测量IL-1β的分泌。我们发现牙石可诱导人PMNs和PBMCs分泌IL-1β。牙石还可诱导野生型小鼠巨噬细胞分泌IL-1β，但不能诱导NLRP3和ASC缺陷小鼠的巨噬细胞分泌IL-1β，表明NLRP3和ASC参与其中。多粘菌素B可抑制IL-1β的诱导，表明LPS是牙石中诱导前IL-1β转录的成分之一。为了分析无机结构的作用，我们将牙石在250°C下烘烤1小时。这种烘烤后的牙石未能诱导前IL-1β转录。然而，它确实能在脂质A预处理的细胞中诱导IL-1β分泌，表明牙石的晶体结构可诱导炎性小体激活。此外，牙石的成分羟基磷灰石晶体可在小鼠巨噬细胞中诱导IL-1β，烘烤后的牙石可在脂质A预处理的人PMNs和PBMCs中诱导IL-1β。这些结果表明，牙石通过NLRP3炎性小体刺激人和小鼠吞噬细胞分泌IL-1β，并且晶体结构在NLRP3炎性小体的激活中起部分作用。
