牙龈卟啉单胞菌脂多糖诱导的中性粒细胞胞外诱捕网通过 Ca 依赖性途径调节炎症反应。
Neutrophil extracellular traps induced by Porphyromonas gingivalis lipopolysaccharide modulate inflammatory responses via a Ca-dependent pathway.
机构信息
Department of Periodontology, The Affiliated Stomatological Hospital of Nanjing Medical University, Nanjing, China,; Jiangsu Province Key Laboratory of Oral Diseases, Nanjing, China,; Jiangsu Province Engineering Research Center of Stomatological Translational Medicine, Nanjing, China.
Department of Periodontology, The Affiliated Stomatological Hospital of Nanjing Medical University, Nanjing, China,; Jiangsu Province Key Laboratory of Oral Diseases, Nanjing, China,; Jiangsu Province Engineering Research Center of Stomatological Translational Medicine, Nanjing, China.
出版信息
Arch Oral Biol. 2022 Sep;141:105467. doi: 10.1016/j.archoralbio.2022.105467. Epub 2022 Jun 2.
OBJECTIVE
The aim of this study was to explore the effects and underlying mechanisms of Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) on the formation of neutrophil extracellular traps (NETs).
DESIGN
NETs induced by 1 μg/ml P. gingivalis LPS were observed by a fluorescence microscope and quantified by a microplate reader. Quantities of extra- and intracellular P. gingivalis in neutrophils were determined to assay the bactericidal efficiency of NETs. Intracellular Ca levels in neutrophils were explored by flow cytometry. Expressions of phospho-tumor progression locus 2 (p-TPL2), phospho-mitogen-activated protein kinase kinase1/2 (p-MEK1/2), phospho-extracellular signal-regulated kinase1/2 (p-ERK1/2), ORAI1, ORAI2 and peptidylarginine deiminase 4 (PAD4) were detected by Western blot. In addition, neutrophil elastase activities in NETs incubated with macrophages were assayed to evaluate their clearance.
RESULTS
P. gingivalis LPS contributed to the formation of NETs and the increased levels of extracellular DNA (p < 0.05), which enhanced bactericidal activity of neutrophils (p < 0.05). Levels of intracellular Ca, p-TPL2, p-MEK1/2, p-ERK1/2, ORAI1, ORAI2 and PAD4 were increased in P. gingivalis LPS-treated neutrophils compared with control group (p < 0.05). In addition, inhibition of intracellular Ca by two Ca chelators, and PAD4 knockdown resulted in decreased levels of extracellular DNA (p < 0.05). After co-culture of NETs with macrophages, neutrophil elastase activities were decreased (p < 0.05).
CONCLUSION
P. gingivalis LPS induced the formation of NETs via a Ca-TPL2-MEK-ERK-PAD4 signaling pathway, which contribute to the elimination of P. gingivalis.
目的
本研究旨在探讨牙龈卟啉单胞菌（P. gingivalis）脂多糖（LPS）对中性粒细胞胞外诱捕网（NETs）形成的影响及其作用机制。
设计
荧光显微镜观察并通过微孔板读数器定量检测 1μg/ml P. gingivalis LPS 诱导的 NETs。通过检测中性粒细胞内外的牙周卟啉单胞菌数量来测定 NETs 的杀菌效率。通过流式细胞术检测中性粒细胞内 Ca 水平。通过 Western blot 检测磷酸化肿瘤进展位点 2（p-TPL2）、磷酸化丝裂原活化蛋白激酶激酶 1/2（p-MEK1/2）、磷酸化细胞外信号调节激酶 1/2（p-ERK1/2）、ORAI1、ORAI2 和肽基精氨酸脱亚氨酶 4（PAD4）的表达。此外，测定与巨噬细胞共培养的 NETs 中的中性粒细胞弹性蛋白酶活性，以评估其清除能力。
结果
牙龈卟啉单胞菌 LPS 促进 NETs 的形成和细胞外 DNA 水平的增加（p<0.05），从而增强中性粒细胞的杀菌活性（p<0.05）。与对照组相比，牙龈卟啉单胞菌 LPS 处理的中性粒细胞中细胞内 Ca、p-TPL2、p-MEK1/2、p-ERK1/2、ORAI1、ORAI2 和 PAD4 的水平升高（p<0.05）。此外，两种 Ca 螯合剂抑制细胞内 Ca 和 PAD4 敲低导致细胞外 DNA 水平降低（p<0.05）。NETs 与巨噬细胞共培养后，中性粒细胞弹性蛋白酶活性降低（p<0.05）。
结论
牙龈卟啉单胞菌 LPS 通过 Ca-TPL2-MEK-ERK-PAD4 信号通路诱导 NETs 的形成，有助于清除牙周卟啉单胞菌。
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