Porphyromonas gingivalisリポ多糖はToll-like receptor 2および4を介して好中球細胞外トラップを発現する

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.

要旨

歯周病患者の歯周ポケットにおいて，好中球細胞外トラップ（NETs）が観察されるが，歯周病原細菌によりNETsが発現するメカニズムは解明されていない。そこで本研究は，Porphyromonas gingivalisのリポ多糖（PG-LPS）によるNETs発現とToll-like receptor（TLR）の関係を解明することを目的とした。末梢血好中球を，PMA，PG-LPS，Escherichia coli LPS（EC-LPS）で刺激した。また，好中球に発現するTLRを阻害した後，同様に上記物質で刺激した。SEM，蛍光免疫染色により観察し，細胞外DNA量，好中球エラスターゼ（NE）量を測定した。その結果，SEM像において，好中球から糸状の構造物の放出を確認した。蛍光免疫染色像において，糸状の構造物がDNA，ヒストン，NEにより染色されていることが確認され，NETsであることを確認した。細胞外DNA量とNE量も，コントロールと比較して有意な増加が認められた。また，TLR2およびTLR4を阻害した場合はPG-LPS刺激においてNETs発現の低下が観察された。本研究の結果から，PG-LPSが，TLR2およびTLR4を介してNETs発現を誘導することが示唆された。

Introduction

Periodontal disease involves chronic inflammation affecting the periodontal tissue and is caused by the host immune response to periodontopathic bacteria as foreign antigens¹⁾. In general, the immune response in nature exhibits a pathway from innate immunity to acquired immunity, and the response to periodontopathic bacteria is considered to be a similar pathway²⁾.

Polymorphonuclear leukocytes (PMNs) are the main cells that function in innate immunity³⁾. PMNs are phagocytic cells similar to macrophages that are thought to migrate to periodontopathic bacteria and phagocytose them³⁾. Subsequently, acquired immunity is induced through the presentation of antigenic information by macrophages⁴⁾. In other words, the response of the innate immune system to periodontopathic bacterial infection plays an important role in subsequent development of the immune response. In particular, PMNs, which account for approximately 90% of the cellular components in gingival crevicular fluid (GCF), play a central role in the initial defense response against periodontopathic bacterial invasion in the gingival crevice^5,6) and are closely related to the progression of periodontal disease.

Brinkmann et al. proposed the concept of neutrophil extracellular traps (NETs) as a new function of PMNs, which play an important role in innate immunity⁷⁾. NETs are reportedly formed by active release by PMNs of a mesh-like structure of DNA, histone, and intracellular antimicrobial proteins such as neutrophil elastase (NE). The function of NETs is the physical capture of pathogens via the mesh-like structure and subsequent destruction by the bactericidal action of the antimicrobial proteins⁷⁾. The process of NET expression is initiated by stimulation with foreign antigens, such as bacteria, via recognition by Toll-like receptors (TLRs) and Fc receptors of PMNs, thereby inducing nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and peptidylarginine deiminase (PAD) 4 and leading to the deconstruction of chromatin within PMNs. Subsequently, the nuclear membrane is disrupted via nuclear translocation of NE and myeloperoxidase (MPO) and chromatin unfolding. The release of chromatin into the cytoplasm modifies granular and cytoplasmic proteins, and disruption of the plasma membrane releases NETs into the extracellular space^8-10). The main functions of these NETs are the physical capture of pathogens via reticular structures and their destruction by antimicrobial proteins⁷⁾.

Periodontal studies have reported the presence of NETs expressed by PMNs in GCF of patients with chronic periodontitis^11,12). PMNs in the saliva of patients with chronic periodontitis show higher NET expression than salivary PMNs from healthy subjects¹³⁾. In addition, stimulation of PMNs by periodontopathic bacteria induces NET expression, suggesting that periodontopathic bacteria are involved in NET expression^14,15). In other words, the role of NETs in the development and progression of periodontal disease is thought to be protective against periodontopathic bacteria, which are foreign antigens, and the involvement of periodontopathic bacteria in the expression of NETs is strongly inferred.

We focused our research on the effect of lipopolysaccharide (LPS) of Porphyromonas gingivalis (P. gingivalis) ¹⁶⁾, a representative periodontopathic bacterium expressing virulence factors such as gingipains, proteases, and lineage hairs, on the expression of NETs. Previous reports on NETs and P. gingivalis have been diverse and suggested that P. gingivalis induces the expression of NETs^17-20). Although P. gingivalis LPS is an agonist of TLR2 and TLR4²¹⁾, and NET expression is induced by stimulation with TLR2 and TLR4^8,22), the relationship between P. gingivalis LPS and TLRs in the expression of NETs is unclear.

In this study, scanning electron microscopy (SEM) and fluorescence immunostaining were used to investigate the formation of NET-like structures based on morphological changes in PMNs in response to P. gingivalis LPS stimulation. In addition, histological and biochemical evaluations of NET-like structures were carried out to confirm that they were NETs. Subsequently, the extent of NET expression induced by P. gingivalis LPS was used to investigate the mechanism of NET formation. The overall aim of the present study, therefore, was to elucidate the relationship between NET expression and TLRs induced by P. gingivalis LPS.

Materials and Methods

1  Selection of subjects

Six male healthy volunteers (mean age 32.5 years) who had given informed consent, had no systemic disease or notable history, non-smokers and had not received any medication in the past 3 months were selected as subjects. This study was conducted with the approval of the Ethics Review Committee of the Nippon Dental University School of Life and Dental Sciences (NDU-T2021-67). The study was also conducted in accordance with the Declaration of Helsinki of 1975, as revised in 2013.

2  Collection of peripheral blood PMNs

A total of 10 ml of peripheral blood was collected from the forearm vein using a heparinized tube (TERUMO, Tokyo, Japan). A sample tube (Corning, Lowell, MA, USA) was filled with 5 ml of Polymorphprep blood cell separation solution (MP Biomedicals, Bernburg, Germany), onto which 5 ml of blood was gently layered. The blood was then centrifuged at 500 g for 30 min at 20 °C, and the buffy coat containing PMNs was removed and transferred to another sample tube (AGC Techno Glass, Shizuoka, Japan) and washed with phosphate-buffered saline (PBS; pH 7.4, Takara Bio, Shiga, Japan). PMNs were adjusted to 1.0 × 10⁶ cells/ml using PMN culture medium RPMI 1640 (Nacalai Tesque, Kyoto, Japan). PMNs were confirmed to be highly pure (>95%) by May-Grünwald Giemsa staining (Sysmex, Hyogo, Japan), with the presence of segmental lobe nuclei in the leukemic cells and granules in the cytoplasm. High viability (>95%) was confirmed by Trypan blue staining (Life Technologies, Grand Island, NY, USA).

3  Analysis of NET expression

3.1  SEM

Morphological analysis of PMNs was carried out by SEM when PMNs were stimulated as described below. Peripheral blood PMNs were preincubated with 200 μl of 1.0×10⁶ PMNs/ml in each well of a 24-well plate (AGC Techno) containing 12-mm glass slides (Matsunami, Osaka, Japan) coated with 0.01% poly-L-lysine (Sigma, St. Louis, MO, USA) for 30 min. The wells were then stimulated with RPMI 1640 as a negative control or 50 nM phorbol myristate acetate (PMA) (Sigma) as a positive control, 100 μg/ml P. gingivalis LPS (PG-LPS) (InvivoGen, San Diego, CA, USA), and 100 μg/ml Escherichia coli LPS (EC-LPS) (Sigma) and incubated at 37 °C for 4 h in a 5% CO₂ environment. After 4 h, the cells were fixed, dehydrated, dried and deposited, and then photographed and observed by SEM on a JSM-IT200 instrument (JEOL, Tokyo, Japan).

3.2  Fluorescence immunostaining

Morphological analysis of DNA, histone, and NE, the primary structures of NETs, was performed by fluorescence immunostaining²³⁾ using 24-well plates containing 12-mm glass slides coated with 0.01% poly-L-lysine and preincubated for 30 min with 200 μl of peripheral blood PMNs adjusted to 1.0 × 10⁶ cells/ml in each well. The cells were stimulated with RPMI 1640 as a negative control or 1, 10, 25, 50, or 75 nM PMA, 1, 10, or 100 μg/ml PG-LPS, and 1, 10, or 100 μg/ml EC-LPS as a positive control and incubated at 37 °C in 5% CO₂ for 0, 1, 2, 3, or 4 h. NETs were confirmed using the DNA-degrading enzyme 100 U/ml deoxyribonuclease (DNase; Takara Bio). PMNs were fixed by adding 16% paraformaldehyde solution (Nacalai Tesque) to a final concentration of 4% and incubating for 15 min at room temperature. After treatment with 0.5% Triton X-100 (Nacalai Tesque), cells were incubated with anti-DNA/Histone H1 antibody (1:500, Sigma) or anti-neutrophil elastase rabbit pAb (1:300, Sigma) as primary antibodies. The isotype controls were mouse IgG2a isotype control from murine myeloma (1:300, Sigma) and IgG from rabbit serum (1:588, Sigma). Secondary antibodies used included goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, DyLight™ 488 (1:500, Thermo Fisher Scientific, Waltham, MA, USA) and goat anti-rabbit IgG cross-adsorbed secondary antibody, Alexa Fluor™ 633 (1:1000, Thermo Fisher Scientific). DNA was then stained using Hoechst 33342 (1:1000, Thermo Fisher Scientific) and encapsulated using ProLong™ Gold (Thermo Fisher Scientific). A laser scanning confocal microscope (LSM 700, Zeiss, Jena, Germany) and LSM software (ZEN 2014, Zeiss) were used for imaging and observation.

3.3  NET quantification assay

Extracellular DNA was quantified using a NET quantification assay²⁴⁾. Peripheral blood PMNs were adjusted to 1.0 × 10⁶ cells/ml, and 100 μl was placed in each well of a 96-well plate (AGC Techno). The cells were pre-incubated for 30 min and then stimulated with RPMI, PMA, or each LPS for 4 h. The cells were then incubated with RPMI, PMA, or each LPS for 10 min. Micrococcal nuclease (Takara Bio) was added to each well, and after 10 min of incubation, Sytox Green nucleic acid fluorescence stain (Thermo Fisher Scientific) was added. Fluorescence intensity (arbitrary fluorescence units, AFU) was measured in triplicate using a fluorescent plate reader (SH-9000, Corona Electric, Ibaraki, Japan).

3.4  ELISA

A Neutrophil Elastase Activity ELISA kit (Cayman Chemical, Ann Arbor, MI, USA) was used to quantify NE. Peripheral blood PMNs were adjusted to 1.0 × 10⁶ cells/ml, and 100 μl was placed in each well of a 96-well plate. The cells were then pre-incubated for 30 min and stimulated with RPMI, PMA, or the respective LPS for 4 h. Fluorescence intensity (AFU) was measured in triplicate using a fluorescent plate reader (Corona Electric).

4  Analysis of TLR2 and TLR4 of PMNs during NET expression induced by PG-LPS

TLR analysis was performed according to the method of Zhang et al.²⁵⁾. Peripheral blood PMNs were adjusted to 1.0 × 10⁶ cells/ml and incubated with 5 μg/ml anti-TLR2 (Novus Biologicals, LLC, Littleton, CO, USA) or 5 μg/ml anti-TLR4 antibodies (Novus Biologicals) for 1 h at 37 °C. The cells were then stimulated with RPMI, 50 nM PMA, 100 μg/ml PG-LPS, and 100 μg/ml EC-LPS and incubated for 4 h at 37 °C in a 5% CO₂ environment. NET expression was assessed using fluorescence immunostaining, ELISA, and the NET quantification assay.

Blocking of TLR2 and TLR4 was assessed by measuring IL-8 in the supernatant of PMNs stimulated with the TLR2 agonist Pam2CSK4 (Selleck, Houston, TX, USA) and the TLR4 agonist EC-LPS, respectively²⁵⁾. IL-8 was measured using an ELISA (Human CXCL8/IL-8 Quantikine ELISA Kit, R&D Systems, Minneapolis, MN, USA).

5  Statistical analysis

The normality of the distribution of measured data was confirmed using the Shapiro-Wilk test. The Levene test was used to test for equal variance in the data. Data that showed a normal distribution were then subjected to one-way analysis of variance, followed by group comparisons using the Games-Howell test. Data that did not show a normal distribution were subjected to the Kruskal-Wallis test, followed by group comparisons using the Dunn test. Statistical evaluations were carried out using IBM SPSS software (version 28.0; IBM Japan, Tokyo, Japan) and Ky Plot (version 6.0; KyensLab, Tokyo, Japan). Analysis results are presented as the mean±standard deviation, with significance levels of <5%.

Results

1  SEM observations after each stimulus

The results of SEM analyses after each stimulus are shown in Figure 1. Control PMNs were approximately 10 μm in diameter, with a circular morphology and rough cell surface. In the positive control stimulated with PMA, PMNs adhered to the basal surface, and reticulate structures (i.e., NET-like structures) were observed on the cell surface together with granules. Furthermore, NET-like structures were also observed with PG-LPS and EC-LPS stimulation, as was the case with PMA stimulation.

Figure 1

SEM analysis of PMNs after stimulation with 50 nM PMA, 100 μg/ml PG-LPS, or 100 μg/ml EC-LPS for 4 h. Left figures: magnification ×2000, scale bars＝10 μm. Right figures: magnification ×6000, scale bars＝2 μm. Three similar experiments were performed, and representative results are shown.

2  Expression of NETs in each group

NET expression by PMNs stimulated under various conditions was assessed using fluorescence immunostaining and determination of extracellular DNA and NE levels. Stimulated PMNs were examined using fluorescence immunostaining (Figure 2A). PMNs stimulated with PMA, the positive control, showed protrusions of reticular structures composed of DNA, histone, and NE (the primary components of NETs) from the plasma membrane and swelling of the cells, which precedes NET expression. Similar findings were observed upon PG-LPS and EC-LPS stimulation. It should be noted that no staining was observed in the isotype control (Figure 3). Furthermore, fluorescence immunostaining images acquired at different durations of stimulation (0-4 h) showed no changes in any group up to 2 h, but NET expression was observed beginning at 3 h and became more pronounced at 4 h (Figure 4). Fluorescence immunostaining images of the PMA group showed that NET expression was induced at concentrations >10 nM, and NET expression increased in a concentration-dependent manner (Figure 5A). The amount of extracellular DNA as determined using the NET quantification assay was significantly higher (p<0.05) in the 50 and 75 nM PMA, 100 μg/ml PG-LPS, and 100 μg/ml EC-LPS groups than in the control (Figure 5B). NE levels were significantly higher in the 25 and 75 nM PMA, 100 μg/ml PG-LPS, and 100 μg/ml EC-LPS groups (p<0.05) (Figure 5C). No significant difference was observed in the 50 nM PMA group, but the values were comparable. The 50 nM PMA did not increase significantly, presumably because of the large variation in values. Fluorescence immunostaining images following stimulation with the NET-degrading enzyme DNase showed no NET expression in the PMA, PG-LPS, and EC-LPS groups, which showed NET structures, confirming that the structures seen in this study were NETs (Figure 2B).

Figure 2

(A) Evaluation of stimulated PMNs using fluorescence immunostaining. Localization of DNA (blue), histone (green), and NE (red) in PMNs stimulated with 50 nM PMA, 100 μg/ml PG-LPS, or 100 μg/ml EC-LPS for 4 h. Magnification ×200; scale bars＝100 μm (magnification ×630, scale bars＝50 μm). Four similar experiments were performed, and representative results are shown.

(B) Fluorescence immunostaining analysis of NET expression after DNase treatment. Localization of DNA (blue), histone (green), and NE (red) in PMNs stimulated with 50 nM PMA, 100 μg/ml PG-LPS, or 100 μg/ml EC-LPS for 4 h after 1-h treatment with 100 U/ml DNase. Magnification ×400. Scale bars＝100 μm. Three similar experiments were performed, and representative results are shown.

Figure 3

Fluorescence immunostaining images when using isotype controls. Localization of DNA (blue), mouse IgG2a isotype control from murine myeloma cells (green), and IgG from rabbit serum (red) against PMNs stimulated with 50 nM PMA, 100 μg/ml PG-LPS, or 100 μg/ml EC-LPS for 4 h. Magnification ×200; scale bars＝100 μm. Three similar experiments were performed, and representative results are shown.

Figure 4

Fluorescence immunostaining images of NET expression over time. Localization of DNA (blue), histone (green), and NE (red) in PMNs stimulated with 50 nM PMA, 100 μg/ml PG-LPS, or 100 μg/ml EC-LPS for 0, 1, 2, 3, and 4 h. Magnification ×200; scale bars＝100 μm. Four similar experiments were performed, and representative results are shown.

Figure 5

(A) Fluorescence immunostaining images of NET expression at different stimulant concentrations. Localization of DNA (blue), histone (green), and NE (red) in PMNs stimulated with 1, 10, 25, 50, or 75 nM PMA, 1, 10, or 100 μg/ml PG-LPS, and 1, 10, or 100 μg/ml EC-LPS for 4 h. Magnification ×200; scale bars＝100 μm. Four similar experiments were performed, and representative results are shown.

(B) Assessment of extracellular DNA using NET quantification assay. Extracellular DNA in cultures of PMNs stimulated with 1, 10, 25, 50, or 75 nM PMA, 1, 10, or 100 μg/ml PG-LPS, or 1, 10, or 100 μg/ml EC-LPS for 4 h. ^＊p<0.05, ^＊＊p<0.01, n＝6.

(C) Assessment of NE by ELISA. Measurement of NE secreted by PMNs stimulated with 1, 10, 25, 50, or 75 nM PMA, 1, 10, or 100 μg/ml PG-LPS, and 1, 10, or 100 μg/ml EC-LPS for 4 h. ^＊p<0.05, ^＊＊p<0.01, n＝5.

3  NET expression following TLR blockade

TLR blockade was confirmed by ELISA (Figure 6A), and NET expression by PMNs after stimulation was assessed using fluorescence immunostaining and determination of extracellular DNA and NE content. Fluorescence immunostaining images showed that NET expression was suppressed when cells were stimulated with PG-LPS after treatment with anti-TLR2 and anti-TLR4 antibodies, and also when stimulated with EC-LPS after anti-TLR4 antibody blockade (Figure 6B). A significant difference in extracellular DNA levels was observed after anti-TLR2 and anti-TLR4 antibody treatment and stimulation with PG-LPS compared to the group without TLR blockade (p<0.05). A significant difference was observed when anti-TLR4 antibodies were applied followed by stimulation with EC-LPS compared to the group without TLR blockade (Figure 6C) (p<0.05). No significant difference was observed with PMA stimulation following TLR blockade. A significant difference in the NE level was observed when cells were treated with anti-TLR2 and anti-TLR4 antibodies and then stimulated with PG-LPS compared to the group without TLR blockade (p<0.05). No significant difference was observed with EC-LPS or PMA stimulation following TLR blockade (Figure 6D).

Figure 6

(A) ELISA confirmation of blockade of TLR2 and TLR4 in PMNs. Measurement of IL-8 in PMNs stimulated with Pam2CSK4 or EC-LPS for 1 h after blocking TLR2 or TLR4. ^＊p<0.05, n＝5.

(B) Analysis of NET expression by fluorescence immunostaining after blockade of TLR2 and TLR4. Localization of DNA (blue) and NE (red) in PMNs stimulated with 50 nM PMA, 100 μg/ml PG-LPS, or 100 μg/ml EC-LPS for 4 h after blockade of each TLR. Magnification ×400. Scale bars＝100 μm. Four similar experiments were performed, and representative results are shown.

(C) Analysis of extracellular DNA using an NET quantification assay after blockade of TLR2 and TLR4. Measurement of extracellular DNA in PMNs stimulated with 50 nM PMA, 100 μg/ml PG-LPS, or 100 μg/ml EC-LPS for 4 h after blockade of each TLR. ^＊p<0.05, n＝6.

(D) Measurement of NE by ELISA following blockade of TLR2 and TLR4. Measurement of NE in PMNs stimulated with 50 nM PMA, 100 μg/ml PG-LPS, or 100 μg/ml EC-LPS for 4 h after blockade of each TLR. ^＊p<0.05, n＝5.

Discussion

In innate immunity, PMNs exert bactericidal action. The bactericidal action pathway involves chemotactic migration, adhesion, and phagocytosis. NETs, as a new function of PMNs, are attracting increasing attention in the field of natural sciences, both in medicine and dentistry.

Brinkmann et al. reported NET expression as a new function of PMNs⁷⁾, but the process by which they are expressed and the factors that induce their expression remain unclear. Early reports relating to NETs in periodontal tissue confirmed their presence in periodontal pockets of patients with chronic periodontal disease, and reports have accumulated indicating that periodontopathic bacteria such as P. gingivalis, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans are involved in the expression of NETs^{14,15,17,20,26)}. However, there is currently no unified view regarding the process by which periodontopathic bacteria release NETs. Therefore, the present study focused on LPS, a virulence factor of P. gingivalis, a typical periodontopathic bacterium, and investigated its histochemical and biochemical effects on NET expression.

The results of the present study showed that PG-LPS stimulation of PMNs was confirmed by SEM with images of thread-like structures released outside the cells, as reported previously⁷⁾. Fluorescent immunostaining of DNA, histone, and NE, the major NET structures, was also performed to confirm that the released substances were NETs. However, reports of the expression of NETs following PG-LPS stimulation have been inconsistent, with some reports supporting the expression of NETs^18,19) and others reporting no NET expression²⁷⁾. In contrast, Jayaprakash et al. reported phagocytosis when PMNs were stimulated for 3 h with P. gingivalis ATCC33277, which is of the same origin as the PG-LPS used in the present study²⁰⁾, whereas Zhang et al. reported NETosis when PMNs were stimulated with the same organism¹⁵⁾. These results were thought to be caused by procedural differences in duration of exposure and concentration, as well as differences in protocols for collecting PMNs and the composition of the medium used. For example, many studies have reported the use of erythrocyte lysis buffer when isolating PMNs. Phillips et al. investigated the effect of ammonium chloride, which plays a major role in this erythrocyte-lysing effect, on PMNs and showed that the use of ammonium chloride increased the iodination activity of PMNs²⁸⁾. In addition, there is no unified protocol for assays of NET expression, but the most common approaches combine fluorescence immunostaining, measurement of extracellular DNA, measurement of NE and myeloperoxidase by ELISA, measurement of reactive oxygen species, and detection of NETs by flow cytometry. The particular combination used in previous studies varies widely, and it is thought that there is no consensus of opinion on the matter. Furthermore, there are no specific markers for NETs, so caution is needed in the interpretation of results. However, the present study was based on staining evidence suggesting NET expression and clearly indicated that the key factor for NET expression is LPS, a major virulence factor.

TLRs are representative LPS receptors that are expressed on various immune cells, such as PMNs, and they are reportedly involved in the stimulation of cytokine responses via TLR2 and TLR4 during P. gingivalis infection²¹⁾. Therefore, in the present study, the NET expression pathway was analyzed with a focus on TLRs. There was a significant decrease in NET expression upon stimulation with PG-LPS in PMNs in which TLR2 and TLR4 were blocked. This indicates that TLR2 and TLR4 are involved in PG-LPS-induced NET expression and that the TLR signaling pathway is strongly involved in NET expression. The pathogenicity of PG-LPS is also determined by the molecular weight of the lipid A constituent^29,30). In particular, lipid A species with molecular weights of 1,435 and 1,450 reportedly activate TLR2 and TLR4³⁰⁾. Therefore, it is likely that these lipid A species activated TLRs 2 and 4 in the present study, thus suggesting that they are involved in NET expression.

The phagocytosis rate for PMNs is reportedly reduced in the presence of LPS³¹⁾. In the present study, PG-LPS was found to induce NET expression. Therefore, in addition to their phagocytosis-driven bactericidal activity, PMNs also contribute to host defense by expressing NETs. This supports a report indicating that, with regard to periodontal disease progression and NETs, patients with Papillon-Lefèvre syndrome are at higher risk for the development and progression of periodontitis due to their inability to produce NETs³²⁾. Other reports suggest that extracellular histone, which constitutes a major component of NETs, aggravates periodontitis by up-regulating the IL-17/Th17 response and causing alveolar bone destruction³³⁾. In other words, until now, whether NETs play a protective role in periodontal tissue or cause periodontal tissue destruction has been a matter of debate, and there is still a lack of uniformity of opinion.

The present study has several limitations. First, the LPS concentration used was high. LPS concentrations in periodontal pockets reportedly range from 0 to 2.05 μg/ml³⁴⁾. However, the concentration used in the present study was as high as 100 μg/ml. Care must be taken in interpreting the results, since this concentration is considered high even taking into account the difference between the actual intraoral and in vitro cell culture environments. Furthermore, previous studies have used peripheral blood PMNs to examine NET expression. However, Moonen et al. reported that salivary PMNs exhibit higher NET expression than peripheral blood PMNs³⁵⁾. In the future, the use of PMNs derived from GCF, which is closer to actual periodontal tissue, should provide a more clinically relevant and detailed understanding of the NET expression pathway. Various techniques exist for TLR blocking, one of which is knockdown of TLRs using siRNA or shRNA. Knockdown using siRNA or shRNA is a molecular biological technique that can block the expression of the receptor in differentiated cells, such as HL-60 cells, and can provide more reliable results. As a next step, our group is planning a study using GCF PMNs wherein we will compare them with peripheral blood PMNs. The knockdown method cannot be used with GCF PMNs. Therefore, the current method was used instead of the knockdown method.

Further studies are needed to elucidate the role of NETs in the onset and progression of periodontal disease and its pathogenesis, including the establishment of in vitro models for each cell type in periodontal tissue, the effects of NETs on each species of periodontopathic bacteria in in vivo models, and interactions with other receptors.

Conclusions

In order to elucidate the details of NET expression, which represents a newly identified function of PMNs in the pathogenesis of periodontal disease, the effects of stimulation with P. gingivalis LPS were examined morphologically, immunohistologically, and biochemically. The results suggest that P. gingivalis LPS stimulates the expression of NETs and that this expression is mediated by TLR2 and TLR4.

Ethical statement

This study was conducted with the approval of the Ethics Review Committee of the Nippon Dental University School of Life and Dental Sciences (NDU-T2021-67).

Funding

This study was supported by Grants-in-Aid from the Ministry of Education and Science Research Funds [JSPS (C) JP20K09981, JP23K09189].

Author contributions

Tomohisa Sakayori: Formal analysis; Biochemical analysis; Patient selection; Methodological verification; Writing review & editing; Writing original draft.

Hiroshi Ito: Funding acquisition; Blood collection; Writing review & editing; Writing original draft.

Yukihiro Numabe: Funding acquisition; Writing review & editing; Writing original draft.

Conflict of Interest: None.

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