Lipopolysaccharides of Fusobacterium nucleatum and Porphyromonas gingivalis increase RANKL-expressing neutrophils in air pouches of mice

Ae Ri Kim,Yun Kyong Lim,Eun-Jung Bak,Joong-Ki Kook,Yun-Jung Yoo

doi:10.1186/s42826-020-00080-y

Abstract

Increases of neutrophils and osteoclasts are pathological changes of periodontitis. RANKL is an osteoclast differentiation factor. The effect of periodontopathogen LPS on RANKL-expressing neutrophils has not been clarified yet. We evaluated numerical changes of RANKL-expressing neutrophils in air pouches of mice injected with LPSs of Fusobacterium nucleatum and Porphyromonas gingivalis. Mice with air pouches were assigned into saline (C)-, E. coli LPS- (Ec LPS)-, F. nucleatum LPS (Fn LPS)-, P. gingivalis LPS (Pg LPS)-, and Fn LPS and Pg LPS (Fn + Pg LPS)-injected groups. CD11b+Ly6G+ neutrophils and CD11b+Ly6G+RANKL+ neutrophils in blood and air pouch exudates were determined by flow cytometry. In blood, compared to the C group, the Fn LPS group showed increases of CD11b+Ly6G+ neutrophils and CD11b+Ly6G+RANKL+ neutrophils whereas the Pg LPS group showed no significant differences. These increases in the Fn LPS group were not different to those in the Ec LPS group. In exudates, Fn LPS and Pg LPS groups showed increases of CD11b+Ly6G+ neutrophils and CD11b+Ly6G+RANKL+ neutrophils compared to the C group. Increased levels in the Fn LPS group were not different to those in the Ec LPS group, but Pg LPS group was lower than those in the Ec LPS group. In blood and exudates, the Fn + Pg LPS group showed no difference in levels of these neutrophils compared to the Ec LPS group. LPSs of F. nucleatum and P. gingivalis increased RANKL-expressing neutrophils although the degrees of increases were different. These suggest that periodontopathogen LPS can act as a stimulant to increase RANKL-expressing neutrophils.

Highlights

Periodontitis is an inflammatory disease caused by several bacteria
To determine whether periodontopathogen LPS could act as a stimulant to increase of Receptor activator of NF-κB ligand (RANKL)-expressing neutrophils, RANKL-expressing neutrophils in Porphyromonas gingivalis (Pg) LPS and Fusobacterium nucleatum (Fn) LPS-injected air pouches of mice were estimated in the present study
Effects of F. nucleatum LPS (Fn LPS) on RANKL-expressing neutrophils in blood and exudates of air pouches In order to evaluate the effect of Fn LPS on numbers of RANKL-expressing neutrophils in blood and exudates of air pouches of mice, numbers of CD11b+Ly6G+ neutrophils and CD11b+Ly6G+RANKL+ neutrophils were measured by flow cytometry (Fig. 2a and b)

Summary

Introduction

Periodontitis is an inflammatory disease caused by several bacteria. Bacteria in subgingival biofilm was grouped into five complexes (red, orange, green, yellow, and purple complexes) by cluster analysis and community ordination technique [1]. F. nucleatum LPS (Fn LPS) is a stronger stimulator for the secretion of IL-1β and TNF-α than P. gingivalis LPS (Pg LPS) in neutrophils, suggesting that secretion levels of cytokines induced by LPSs from various periodontopathogens might play important roles in the onset and progression of periodontal disease [11]. A previous in vivo study has shown that RANKL-expressing neutrophils are increased in air pouches of mice injected with Escherichia coli LPS (Ec LPS) [19]. To determine whether periodontopathogen LPS could act as a stimulant to increase of RANKL-expressing neutrophils, RANKL-expressing neutrophils in Pg LPS and Fn LPS-injected air pouches of mice were estimated in the present study. LPS injection into air pouch Ec LPS (Sigma-Aldrich, St Louis, Mo, USA) was used as a positive control. All statistical analyses were performed using SPSS (IBM SPSS Statistics version 25, Armonk, NY, USA)

Results

Discussion

Conclusions