Porphyromonas gingivalis Lipopolysaccharide Expresses Neutrophil Extracellular Traps via Toll-like receptors 2 and 4

Abstract

Neutrophil extracellular traps (NETs) occur in periodontal pockets of patients with periodontal disease, but the mechanism by which NETs are expressed by periodontopathic bacteria has not been clarified. Therefore, the aim of this study was to elucidate the relationship between NET expression and Toll-like receptors (TLRs) induced by Porphyromonas gingivalis lipopolysaccharide (PG-LPS).

Neutrophils were isolated from peripheral blood and stimulated with 1, 10, 25, 50, or 75 nM phorbol myristate acetate (PMA), 1, 10, or 100 μg/ml PG-LPS, or 1, 10, or 100 μg/ml Escherichia coli LPS (EC-LPS). Polymorphonuclear leukocytes were also stimulated after TLRs were blocked using specific antibodies. NETs were observed using scanning electron microscopy (SEM), fluorescence immunostaining of NET component DNA, histone, and neutrophil elastase (NE), and the amounts of extracellular DNA and NE were measured by NET quantification assay and ELISA, respectively.

SEM images showed that 50 nM PMA, 100 μg/ml PG-LPS, and 100 μg/ml EC-LPS stimulated the release of filamentous structures from neutrophils. Fluorescence immunostaining images showed that the filamentous structures stained positive for DNA, histone, and NE, confirming that the structures were NETs. The amounts of extracellular DNA and NE were also significantly increased by treatment with PMA, PG-LPS, and EC-LPS compared to control. A decrease in NET expression was observed in the PG-LPS group when TLR2 and TLR4 were inhibited.

The results of this study suggest that PG-LPS induces NET expression via TLR2 and TLR4.