Stannous fluoride protects gingival keratinocytes against infection and oxidative stress by Porphyromonas gingivalis outer membrane vesicles

Abstract

Objective: To determine whether outer membrane vesicles (OMVs) of the periodontal pathogen Porphyromonas gingivalis (P. gingivalis) can infect gingival keratinocytes and stimulate reactive oxygen species (ROS) production, and to assess whether stannous fluoride (SnF₂), stannous chloride (SnCl₂) or 0.454% SnF₂ toothpaste diluents can inhibit OMV infection.

Methods: OMVs were isolated from P. gingivalis culture and their morphology was characterized using scanning electron microscopy and transmission electron microscopy. OMVs were harvested, separated from parent bacteria, labeled with fluorescent probes, and added to proliferating gingival keratinocytes. Infection was monitored by measuring uptake of fluorescence. Free radicals and ROS were quantified by adding a separate CellROX fluorescent probe following 24 h incubation with OMVs, and automated fluorescence imaging was used to assess ROS generation rates. A dose response range of SnF₂ and SnCl₂ concentrations as well as 0.454% SnF₂ toothpaste dilutions were added to OMVs to examine their potential to neutralize OMV infectivity and protect gingival keratinocytes from development of oxidative stress. The mechanism of SnF₂ inhibition of OMV infection was studied by binding SnF₂ with purified lipopolysaccharides (LPS) from the bacterial culture and examining the binding of stannous to LPS using mass spectrometry.

Results: Large numbers of OMVs were formed in P. gingivalis culture medium. They were purified along with isolating soluble LPS. Fluorescence imaging revealed that OMVs infected gingival keratinocytes and promoted oxidative stress in a dose-dependent manner. SnF₂, SnCl₂, and SnF₂ toothpaste inhibited OMV infectivity (p < 0.05) and likewise protected gingival keratinocytes from oxidative stress (p < 0.05). Stannous precipitated LPS and OMVs from solution, forming insoluble aggregates easily isolated by centrifugation. Mass spectroscopic analysis revealed that stannous was bound to LPS in a one-to-one molecular equivalent ratio.

Conclusion: SnF₂ not only kills bacteria, but also inhibits bacterial virulence factors, such as LPS and OMVs. SnF₂, SnCl₂ and stannous-containing toothpastes can precipitate OMVs and LPS to in principle protect gingival keratinocyte cells from infection leading to inflammation and oxidative stress.

Keywords: Porphyromonas gingivalis; antibacterial agents; keratinocyte infection; periodontal diseases; reactive oxygen species; scanning electron microscopy; stannous fluoride; transmission electron microscopy.

Figure 1

Isolation and characterization of P. gingivalis OMVs. (A) SEM of P. gingivalis bacteria with small outgrowths on surface. (B) Several OMVs (yellow arrow) adjacent to a P. gingivalis bacterium (blue arrow). (C) P. gingivalis OMVs in the culture medium. (D) A visible band of OMVs in OptiPrep gradient gel. (E,F) Purified OMVs of P. gingivalis.

Figure 2

SnF₂ and SnCl₂ inhibited OMV entry into gingival keratinocytes. (A) Images of fluorescent-labeled OMVs (green) entry into gingival keratinocytes. (B) Entry of OMVs into gingival keratinocytes in a dose curve. Each time point represented the mean and SE of three separate experiments. (C) SnF₂ and (D) SnCl₂ inhibited entry of OMVs into gingival keratinocytes at 42 h. Each bar represented the mean and SE of three separate experiments. The plots were generated using ggpubr package in RStudio. The results were analyzed using one-way ANOVA and t-test conducted to compare the difference with OMV alone (stannous is 0 μM) as the reference group in stat_compare_means of Rstudio packages.

Figure 3

Effects of P. gingivalis OMVs on oxidation of CellROX dyes in gingival keratinocytes. (A) Images of OMV (red) infection and oxidation of CellROX (green). (B) Entry of OMVs into gingival keratinocytes. (C) Oxidation of CellROX dyes in gingival keratinocytes. (D) Cell confluence evaluated by phase contract images. Each time point represented the mean and SE of five separate experiments.

Figure 4

Effects of SnF₂ and SnCl₂ on the intake of fluorescence-labeled OMV (Red) and oxidized CellROX Dye (green): (A) Images of OMV intake and oxidized CellROX green in cells infected with 0.65 μg/ml OMVs without or with SnF₂ or with SnCl₂. (B) SnF₂ and (C) SnCl₂ inhibited cellular oxidation of CellROX. Each point represents the mean and SE from four separate experiments at 42 h. CellROX fluorescence was expressed as relative fluorescence. P-values listed here were from pairwise comparisons with the stannous 0 µM (OMV at 0.65 µg/ml alone) as the reference group.

Figure 5

Effects of stannous-based toothpastes on OMV entry and oxidative stress. (A) Images showing OMV (red) entry into gingival keratinocytes and oxidized CellROX Green stains (green). (B) Entry of fluorescence-labeled OMVs into gingival keratinocytes. (C) Quantification of oxidized CellROX dyes inside the cells. CellROX fluorescence and OMV fluorescence are presented as relative fluorescence. Results from two SnF₂-containing toothpastes were pooled for analysis. Each bar represents the mean and SE of four separate experiments.

Figure 6

OMVs of P. gingivalis and SnF₂ formed precipitates. (A) SnF₂ binds OMVs and forms precipitates. (B) Time course of stannous binding to OMVs. Each time point represents the mean and SE of 24 independent measurements. 0 mM SnF₂ was not represented in B since the OD600 was 0. Relative OD600 values were obtained by dividing OD600 by the average OD600 of the 2 µg/ml OMV + 1 mM SnF₂ samples at the 60 min timepoint. Mixed effect models were fitted to the relative OD600, and all comparisons were significant at 0.01 after Bonferroni adjustment. These comparisons include SnF₂ + OMV vs. SnF₂, OMV + 0.5 mM SnF₂ vs. OMV, and OMV + 1 mM SnF₂ vs. OMV.

Figure 7

Mass spectrum analysis of stannous and LPS binding. P. gingivalis LPS is highly heterogeneous in size and structure. Each peak represents one molecule of LPS. There were two groups of LPS peaks in the LPS alone panel as indicated by the red circles. With SnF₂, two more groups of peaks were generated as represented by the blue circles. The peaks in the blue circles were shifted by 116, 118 or 120 da to the right as indicated by the green arrows.

Figure 8

SnF₂ interacts with OMVs to form aggregates. (1). OMVs are highly effective in entering gingival keratinocytes. (2). LPS on the surface of OMVs can interact with stannous, which is bivalent. Stannous can bind two OMV structures or bind one OMV and another stannous. (3). As more OMVs are linked together, they form a large aggregate. (4). Large aggregates of OMVs reduce the effective concentration of OMVs in the solution, and retard entry into gingival keratinocytes.

Figure 9

Proposed model for relationship between OMV infection and systemic exposure. (A) P. gingivalis within plaque in the gingival sulcus produces OMVs. (B) OMVs adhere to gingival keratinocytes, leading to invasion of keratinocytes. (C) OMVs can migrate through the disrupted epithelial barrier and into the underlying capillary beds creating a potential pathway to introduce OMVs into the circulatory system where they could be carried to distant tissues and organs, including the brain, liver and heart.