Neutrophil extracellular traps induced by Porphyromonas gingivalis lipopolysaccharide modulate inflammatory responses via a Ca2+-dependent pathway

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 mu g/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 Ca2+ levels in neutrophils were explored by flow cytometry. Expressions of phospho-tumor progression locus 2 (p-TPL2), phospho-mitogen-activated protein kinase kinasel/2 (p-MEK1/2), phospho-extracellular signal-regulated kinasel/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 Ca2+, 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 Ca2+ by two Ca2+ 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 Ca2+ -TPL2-MEK-ERK-PAD4 signaling pathway, which contribute to the elimination of P. gingivalis.