Excessive neutrophil extracellular trap formation induced by Porphyromonas gingivalis lipopolysaccharide exacerbates inflammatory responses in high glucose microenvironment

Yue Tong; Yue Xin; Lanqing Fu; Jia Shi; Ying Sun

doi:10.3389/fcimb.2023.1108228

Excessive neutrophil extracellular trap formation induced by Porphyromonas gingivalis lipopolysaccharide exacerbates inflammatory responses in high glucose microenvironment

Front Cell Infect Microbiol. 2023 Jan 20:13:1108228. doi: 10.3389/fcimb.2023.1108228. eCollection 2023.

Authors

Yue Tong^{1

2

3}, Yue Xin^{1

2

3}, Lanqing Fu^{1

2

3}, Jia Shi^{1

2

3}, Ying Sun^{1

2

3}

Affiliations

¹ Department of Periodontology, The Affiliated Stomatological Hospital of Nanjing Medical University, Nanjing, China.
² Jiangsu Province Key Laboratory of Oral Diseases, Nanjing, China.
³ Jiangsu Province Engineering Research Center of Stomatological Translational Medicine, Nanjing, China.

Abstract

Introduction: Neutrophil extracellular trap (NET) is a novel defense strategy of neutrophils and found to be induced by Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) or high glucose. The aim of this study was to investigate the roles and mechanisms of NET formation in high glucose inflammatory microenvironment.

Methods: NETs induced by 1 μg/ml P. gingivalis LPS and/or 25 mM glucose were visualized using a fluorescence microscopy and the levels of extracellular DNA were determined by a microplate reader. The bactericidal efficiency of NETs was assessed by quantifying the survival P. gingivalis in neutrophils. The levels of NLRP3 and IL-1β in THP-1 derived-macrophages, and the expressions of p-PKC βII, p-MEK1/2, p-ERK1/2, ORAI1 and ORAI2 in neutrophils were detected by Western blot. Moreover, levels of intracellular Ca2+ and reactive oxygen species (ROS) in neutrophils were explored by flow cytometry.

Results: P. gingivalis LPS enhanced the formation of NETs and increased the levels of extracellular DNA in high glucose microenvironment (p < 0.05). Compared with normal glucose inflammatory microenvironment, quantities of extra- and intracellular viable P. gingivalis in neutrophils exposed to NETs induced in high glucose inflammatory one were increased (p < 0.05) and the expressions of NLRP3 and IL-1β were dramatically increased in macrophages co-cultured with NETs from high glucose inflammatory microenvironment (p < 0.05). In addition, levels of ROS, intracellular Ca²⁺, p-PKC βII, p-MEK1/2, p-ERK1/2, ORAI1 and ORAI2 were increased in neutrophils stimulated with both high glucose and P. gingivalis LPS compared with the single stimulus groups (p < 0.05).

Discussion: In high glucose inflammatory microenvironment, formation of NETs was enhanced via oxidative stress, which failed to reverse the decreased bactericidal capacity in high glucose microenvironment, and instead aggravated the subsequent inflammatory responses.

Keywords: Porphyromonas gingivalis; inflammatory responses; lipopolysaccharide; neutrophil extracellular traps; reactive oxygen species.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Antibodies
DNA / metabolism
Extracellular Traps* / metabolism
Humans
Lipopolysaccharides / metabolism
NLR Family, Pyrin Domain-Containing 3 Protein / metabolism
Neutrophils / metabolism
Porphyromonas gingivalis* / metabolism
Reactive Oxygen Species / metabolism
THP-1 Cells

Substances

Antibodies
DNA
Lipopolysaccharides
NLR Family, Pyrin Domain-Containing 3 Protein
Reactive Oxygen Species

Grants and funding

This research was funded by National Natural Science Foundation of China, grant number 81771075 and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, grant number 2018-87.