Periodontitis is a dental plaque-induced chronic inflammatory disease. Long-term exposure of the host to periodontal pathogens leads to a hyporesponsive state to the following stimulations, which is described as endotoxin tolerance. Neutrophils are the most abundant innate immune cells in the body. To clarify the roles of endotoxin tolerance in periodontitis, inflammatory responses in Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS)-tolerized neutrophils were explored in this study. Here, apoptosis and respiratory burst in neutrophils upon single or repeated P. gingivalis LPS stimulations were explored by flow cytometry. Cytokine production (TNF-α, IL-8, and IL-10) in tolerized neutrophils or neutrophils co-cultured with peripheral blood mononuclear cells was determined by ELISA. Phagocytosis of P. gingivalis by tolerized neutrophils was also assayed by flow cytometry. In addition, quality and quantitation of neutrophil extracellular trap (NET) formation were detected using immunofluorescence microscope and microplate reader, respectively. The protein expressions of extracellular signal-regulated kinase1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK) were examined to identify possible mechanisms for the abovementioned changes. Tolerance induced by P. gingivalis LPS significantly suppressed apoptosis, reactive oxygen species (ROS) generation, and phagocytosis in neutrophils (p < 0.05). In both neutrophils alone and co-culture system, repeated P. gingivalis LPS stimulations significantly decreased TNF-α production, but increased IL-10 secretion (p < 0.05). Moreover, in tolerized neutrophils, NET formations were strengthened and there were more released extracellular DNA (p < 0.05). In P. gingivalis LPS-tolerized neutrophils, phosphorylation of ERK1/2 was suppressed compared with that in non-tolerized cells. Taken together, immune responses in neutrophils were reprogrammed by P. gingivalis LPS-induced tolerance, which might be related with the development of inflammation in periodontal tissues. Moreover, ERK1/2 might play important roles in endotoxin tolerance triggered by P. gingivalis LPS.

中文翻译：

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牙龈卟啉单胞菌脂多糖诱导的内毒素耐受性对中性粒细胞炎性反应的影响。

牙周炎是一种由牙菌斑引起的慢性炎症性疾病。宿主长期暴露于牙周病原体导致对以下刺激的反应低下，这被描述为内毒素耐受性。中性粒细胞是体内最丰富的先天免疫细胞。为了阐明内毒素耐受性在牙周炎中的作用，本研究探讨了牙龈卟啉单胞菌（P. gingivalis）脂多糖（LPS）耐受的中性粒细胞的炎症反应。在这里，通过流式细胞术探讨了单次或重复牙龈假单胞菌LPS刺激后嗜中性粒细胞的凋亡和呼吸爆发。通过ELISA确定在耐受的嗜中性粒细胞或与外周血单核细胞共培养的嗜中性粒细胞中的细胞因子产生（TNF-α，IL-8和IL-10）。P的吞噬作用 还通过流式细胞术测定了耐受性中性粒细胞引起的牙龈炎。此外，分别使用免疫荧光显微镜和酶标仪检测中性粒细胞胞外陷阱（NET）形成的质量和定量。检查了细胞外信号调节激酶1/2（ERK1 / 2），c-Jun N-末端激酶（JNK）和p38促分裂原活化蛋白激酶（p38 MAPK）的蛋白表达，以确定上述变化的可能机制。牙龈卟啉单胞菌LPS诱导的耐受性显着抑制了中性粒细胞的凋亡，活性氧（ROS）生成和吞噬作用（p <0.05）。在单独的嗜中性粒细胞和共培养系统中，重复的牙龈卟啉单胞菌LPS刺激均显着降低了TNF-α的产生，但增加了IL-10的分泌（p <0.05）。此外，在耐受性中性粒细胞中，NET形成增强，并且释放的细胞外DNA增多（p <0.05）。在牙龈卟啉单胞菌对LPS耐受的嗜中性粒细胞中，ERK1 / 2的磷酸化被抑制。两者合计，中性粒细胞的免疫反应被牙龈卟啉单胞菌脂多糖诱导的耐受性重新编程，这可能与牙周组织炎症的发展有关。此外，ERK1 / 2可能在牙龈卟啉单胞菌LPS触发的内毒素耐受中起重要作用。牙龈LPS诱导的耐受性可能与牙周组织炎症的发展有关。此外，ERK1 / 2可能在牙龈卟啉单胞菌LPS触发的内毒素耐受中起重要作用。牙龈LPS诱导的耐受性可能与牙周组织炎症的发展有关。此外，ERK1 / 2可能在牙龈卟啉单胞菌LPS触发的内毒素耐受中起重要作用。