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

Abstract

ObjectiveThe 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). DesignNETs 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 Ca2+ 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. ResultsP. 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). ConclusionP. gingivalis LPS induced the formation of NETs via a Ca2+-TPL2-MEK-ERK-PAD4 signaling pathway, which contribute to the elimination of P. gingivalis.