Gu Jian-Yu, Liu Yu-Jie, Zhu Xiang-Qing, Qiu Jia-Ying, Sun Ying
Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University, Nanjing, China.
Department of Periodontology, Affiliated Hospital of Stomatology, Nanjing Medical University, Nanjing, China.
Inflammation. 2020 Oct;43(5):1692-1706. doi: 10.1007/s10753-020-01243-8.
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.
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中会导致对后续刺激产生低反应状态，这被称为内毒素耐受。中性粒细胞是体内最丰富的天然免疫细胞。为了阐明内毒素耐受在牙周炎中的作用，本研究探讨了牙龈卟啉单胞菌（P. gingivalis）脂多糖（LPS）耐受的中性粒细胞中的炎症反应。在此，通过流式细胞术探讨了单次或重复牙龈卟啉单胞菌LPS刺激后中性粒细胞的凋亡和呼吸爆发。通过酶联免疫吸附测定法（ELISA）测定耐受的中性粒细胞或与外周血单核细胞共培养的中性粒细胞中细胞因子的产生（肿瘤坏死因子-α、白细胞介素-8和白细胞介素-10）。还通过流式细胞术检测了耐受的中性粒细胞对牙龈卟啉单胞菌的吞噬作用。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诱捕网（NET）形成的质量和定量。检测细胞外信号调节激酶1/2（ERK1/2）、c-Jun氨基末端激酶（JNK）和p38丝裂原活化蛋白激酶（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诱导的耐受显著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刺激均显著降低肿瘤坏死因子-α的产生，但增加白细胞介素-10的分泌（p < 0.05）。此外，在耐受的中性粒细胞中，NET形成增强，释放的细胞外DNA更多（p < 0.05）。与未耐受的细胞相比，牙龈卟啉单胞菌LPS耐受的中性粒细胞中ERK1/2的磷酸化受到抑制。综上所述，牙龈卟啉单胞菌LPS诱导的耐受对中性粒细胞的免疫反应进行了重新编程，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引发的内毒素耐受中发挥重要作用。
