Porphyromonas gingivalis Outer Membrane Vesicles Stimulate Gingival Epithelial Cells to Induce Pro-Inflammatory Cytokines via the MAPK and STING Pathways

Porphyromonas gingivalis (Pg) is a keystone pathogen associated with chronic periodontitis and produces outer membrane vesicles (OMVs) that contain lipopolysaccharide (LPS), gingipains, and pathogen-derived DNA and RNA. Pg-OMVs are involved in the pathogenesis of periodontitis. Pg-OMV-activated pathways that induce the production of the pro-inflammatory cytokines, interleukin (IL)-6, and IL-8 in the human gingival epithelial cell line, OBA-9, were investigated. The role of mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB in levels of Pg-OMV-induced pro-inflammatory cytokines was investigated using Western blot analysis and specific pathway inhibitors. Pg-OMVs induced IL-6 and IL-8 production via the extracellular signal-regulated kinase (Erk) 1/2, c-Jun N-terminal kinase (JNK), p38 MAPK, and NF-κB signaling pathways in OBA-9 cells. In addition, the stimulator of interferon genes (STING), an essential innate immune signaling molecule, was triggered by a cytosolic pathogen DNA. Pg-OMV-induced IL-6 and IL-8 mRNA expression and production were significantly suppressed by STING-specific small interfering RNA. Taken together, these results demonstrated that Pg-OMV-activated Erk1/2, JNK, p38 MAPK, STING, and NF-κB signaling pathways resulting in increased IL-6 and IL-8 expression in human gingival epithelial cells. These results suggest that Pg-OMVs may play important roles in periodontitis exacerbation by stimulating various pathways.


Introduction
Periodontitis is a chronic inflammatory disease caused by periodontopathogenic bacteria in dental plaques [1]. Continuous stimulation and an increase in the number of periodontopathogenic bacteria cause inflammatory responses, including pro-inflammatory cytokine production that is executed by host innate immune responses in periodontal tissue, leading to bone and tissue destruction, and eventually tooth loss [1]. In addition, chronic periodontitis is associated with various systemic diseases, such as cardiovascular disease, diabetes, rheumatoid arthritis, and kidney disease [2]. Porohyromonas gingivalis (Pg) is a keystone pathogen associated with chronic periodontitis and contributes to systemic diseases [3]. Pg expresses several virulence factors, such as lipopolysaccharide (LPS), capsular polysaccharide, fimbriae, and gingipains (cysteine proteinases) [4]. Pg-LPS strongly aggravates periodontitis through stimulation of intracellular signaling events by the host immune system, such as the induction of pro-inflammatory cytokines, chemokines, and other inflammation-related molecules in periodontal tissue [4].
Both pathogenic and commensal Gram-negative bacteria can produce outer membrane vesicles (OMVs) and release them into the extracellular environment [5]. OMVs contain LPS, periplasmic and membrane-bound proteins, enzymes, toxins, DNA, RNA, and peptidoglycan [5]. OMVs have been proposed to be involved in cell-cell interactions, nutrient acquisition, host immune dysregulation and modulation, host cell interaction, and biofilm formation [5]. Several studies using proteomic tools have validated that Pg-OMVs contain various virulence factors, including FimA, HagA, mating factors, and gingipains, which are involved in periodontitis pathology [6][7][8]. Since Pg-OMVs are smaller than bacterial cells, Pg-OMVs can be transported rapidly through the blood circulation and therefore affect distant tissues and organs [9], resulting in the modulation of various responses. For instance, Pg-OMVs containing gingipains accumulate in the liver and inhibit insulinstimulated glycogen synthesis [8]. Moreover, Pg-OMVs induce apoptosis and destroy the lung epithelial cell barrier [10]. These results suggest that gingipains in Pg-OMVs play an important role in systemic diseases related to Pg infection.
Pg-OMVs penetrate epithelial cells in a fimbria-dependent manner and are endocytosed through actin-dependent lipid raft-mediated pathways [11]; further, they cause host cells to produce a variety of inflammatory factors [12]. Moreover, Pg-OMVs induce nuclear factor (NF)-κB (NF-κB) activation, promote apoptosis and glycolysis, and inhibit proliferation in epithelial cells [12]. Therefore, Pg-OMVs contribute to host cell invasion, host cell destruction, escape from the immune system, and antibiotic resistance therefore leading to inflammation induction in gingival tissue, and thereby facilitating the progression of periodontitis [12].
Stimulator of interferon genes (STING) is a critical and essential innate immune signaling adaptor molecule for controlling the transcription of numerous host defense genes, including type I interferon (IFNs) and inflammatory cytokines. STING is activated by recognizing cyclic GMP-AMP (cGAMP), which is synthesized by the cytoplasmic enzyme-cyclic GMP-AMP synthase (cGAS)-in response to pathogen-derived DNA [13][14][15]. In periodontitis, a strong STING accumulation was observed in the basal epithelium and around vessel walls in the connective tissue, however, STING was also weakly present in healthy gingiva [16]. Hence, we considered that the STING-mediated pathway may play an important role in the induction of inflammation by Pg-OMV-derived DNA in periodontitis.
Based on these findings, we hypothesized that gingipains and DNA in Pg-OMVs may contribute to the pathogenesis and the progression of periodontitis. Since the molecular mechanisms underlying the effects of Pg-OMVs on gingival epithelial cells and the establishment of local infection remain obscure, the possible mechanisms underlying Pg-OMV-mediated gingival epithelial cell events including the STING-mediated pathway were investigated.

Endotoxin Activity Assay
The endotoxin activity level of OMV was determined via using the colorimetric LAL assay (Limulus Color KY Test; Wako Pure Chemical Industries, Osaka, Japan), according to the manufacturer's instructions. As a positive control, LPS from Pg (Pg-LPS, InvivoGen, San Diego, CA, USA) was incubated under the same conditions. In the present study, Pg-OMVs containing the same level of endotoxin activity as Pg-LPS were added to cells to compare the induction of cytokines, using Pg-LPS as a positive control. Fn-OMVs was matched to the same level of endotoxin activity as Pg-LPS for comparison.

DNA Quantification
DNA concentration in purified Pg-OMVs were measured using a Qubit 4 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions.

Cell Culture
The Simian virus-40 antigen-immortalized human gingival epithelial cell line OBA-9 was kindly provided by Prof. Shinya Murakami (Osaka University Graduate School of Dentistry, Osaka, Japan). OBA-9 cells were cultured as described previously [19]. Before the addition of OMVs or Pg-LPS, OBA-9 cells were incubated in a medium without bovine pituitary extract and epidermal growth factor for 2 h.

Enzyme-Linked Immunosorbent Assay (ELISA)
Supernatants from OBA-9 cells were collected after 6 h of stimulation with OMVs or Pg-LPS (100 ng/mL). IL-6 and IL-8 protein levels were measured using a sandwich ELISA kit from R&D Systems (Minneapolis, MN, USA), according to the manufacturer's instructions.

Inhibition of MAPK and NF-κB Signaling Pathways
To investigate potential pathways contributing to OMV-induced IL-6 and IL-8 expression, OBA-9 cells were either untreated or pre-treated for 1 h with the following inhibitors used at the given concentrations: U0126 (mitogen-activated protein kinase [MEK] inhibitor, 10 µM); SB203580 (p38 MAPK inhibitor, 10 µM); SP600125 (JNK inhibitor, 20 µM); and BAY11-7082 (NF-κB inhibitor, 10 µM) (all from Wako). After inhibitor pre-treatment, OBA-9 cells were cultured with OMVs or Pg-LPS (100 ng/mL) for a further 6 h. SiRNA targeting STING, as well as a negative control siRNA (no significant homology to any known mouse, rat, or human gene sequences) were both purchased from Sigma-Aldrich (St. Louis, MO, USA). OBA-9 cells were cultured as described previously [19]. Cells were transfected with siRNA complexes using Lipofectamine ® RNAiMAX Reagent (Thermo Fisher Scientific), according to the manufacturer's instructions. The final concentration of the siRNA transfected into cells was 50 nM. After 24 h, the total RNA and protein were extracted in order to confirm STING knockdown. OBA-9 cells were cultured with Pg-OMVs for 3 and 6 h to examine the expression of IL-6 and IL-8 mRNA levels and to measure the levels of these proteins by an ELISA program, respectively. Furthermore, OBA-9 cells were cultured with Pg-OMVs or Fn-OMVs for 30 min to analyze via Western blotting for the phosphorylation of NF-κB p65. Data were analyzed using the negative control siRNA as a baseline for mRNA knockdown efficiency.

Statistical Analysis
All experiments were performed at least three times to confirm the reproducibility of the results. The results are presented as the mean ± standard deviation (SD) of three independent experiments. The mean values of the groups were compared using a one-way analysis of variance in conjunction with Holm-Sidak's multiple comparisons test. Statistical significance was set at p < 0.05. Further, GraphPad Prism 6.00 software (GraphPad Software, Inc., La Jolla, CA, USA) was used for data analysis.

Effect of Pg-OMVs on IL-6 and IL-8 mRNA Levels in OBA-9 Cells
The effect of Pg-OMVs, or Pg-LPS, on the expression of IL-6 and IL-8 mRNA levels was compared in OBA-9 cells using qRT-PCR. The amount of Pg-OMVs added to cells was determined by using the same level of endotoxin activity at each concentration (10, 100, or 1000 ng/mL) of Pg-LPS. As shown in Figure 1A, Pg-OMVs significantly induced IL-6 and IL-8 mRNA expression at the same levels as endotoxin activity at 10 and 100 ng/mL Pg-LPS, whereas Pg-LPS significantly promoted the induction of IL-6 and IL-8 mRNA expression in a dose-dependent manner compared to that of the unstimulated controls. Pg-OMVs at the same level of endotoxin activity as 100 ng/mL Pg-LPS had significantly increased IL-6 and IL-8 mRNA levels compared to that of the unstimulated control (by 6.7-fold and 12.6-fold, respectively) and when also compared to that of 100 ng/mL Pg-LPS. Pg-OMVs at the same level of endotoxin activity as 1000 ng/mL Pg-LPS did not change the expression of IL-6 and IL-8 mRNA when compared to that of the unstimulated control. In fact, many floating dead cells were observed in cells that were cultured with high amounts of Pg-OMVs, but this was not observed in cells cultured with PgKDP-OMVs (data not shown). Thus, OBA-9 cells were treated with Pg-OMVs at the same level of endotoxin activity as 100 ng/mL Pg-LPS in the subsequent experiments. As shown in Figure 1B, Pg-OMVs significantly increased IL-6 and IL-8 production in OBA-9 cells. The IL-6 concentration in the supernatant of cells treated with Pg-OMVs was significantly higher than that of Pg-LPS, whereas the IL-8 concentration levels were similar between Pg-OMVs and Pg-LPS.

Involvement of NF-κB p65 in IL-6 and IL-8 Induction by Pg-OMVs
To investigate the involvement of NF-κB in OBA-9 cells treated with Pg-OMVs, PgKDP-OMVs, or Pg-LPS, the phosphorylation level of NF-κB was determined using Western blotting. An increase in NF-κB phosphorylation was observed within 5 min after the stimulation with Pg-OMVs or PgKDP-OMVs compared to that of the unstimulated control; however, Pg-LPS slightly increased the phosphorylation level in OBA-9 cells at 5 min after the stimulation ( Figure 4A). Moreover, the NF-κB inhibitor BAY11-7082 strongly attenuated Pg-OMVor PgKDP-OMV-induced IL-6 and IL-8 production, and Pg-LPS-induced IL-6 production ( Figure 4B). These results suggested that Pg-OMVs activated the MAPK and NF-κB pathways to produce IL-6 and IL-8 by other factors, in addition to gingipains. were pre-treated with BAY11-7082 (10 µM) for 1 h before the addition of Pg-OMVs, PgKDP-OMVs, or 100 ng/mL of Pg-LPS, for 6 h. IL-6 and IL-8 concentrations in the cell culture supernatants were determined using an ELISA program. Data are the mean ± SD of three independent experiments. * p < 0.05 and ** p < 0.01, compared to the unstimulated control. ## p < 0.01 compared to the control without inhibitor.

Effect of STING Knockdown on IL-6 and IL-8 Expression
Next, we examined whether the nucleic acids in Pg-OMVs can induce the expression of IL-6 and IL-8 via the STING pathway. In this study, DNA concentrations contained in Pg-OMVs ranged from approximately 300-1500 µg/mL. Transfection with STING-specific siRNA significantly inhibited STING mRNA expression ( Figure 5A, left panel) and protein levels ( Figure 5A, right panel), by more than 65%. STING downregulation via STINGspecific siRNA partially suppressed IL-6 and IL-8 mRNA expression as well as protein expression induced by Pg-OMVs ( Figure 5B,C). These results suggest that the nucleic acid-activating STING pathway induced by Pg-OMVs is important for the induction of pro-inflammatory cytokines from human gingival epithelial cells. Data are the mean ± SD of three independent experiments. * p < 0.05 and ** p < 0.01, compared to the unstimulated control. # p < 0.05 and ## p < 0.01, compared to the negative control siRNA.

Comparison of Pg-OMVs and Fn-OMVs in IL-6 and IL-8 Production by STING Knockdown
Finally, to determine whether OMVs produced by other periodontopathogenic bacteria also activate IL-6 and IL-8 production via the STING-mediated pathway, we compared Pg-OMVs and Fn-OMVs in OBA-9 cells. The DNA concentration in Fn-OMVs was similar to that in Pg-OMVs. The amount of DNA in the OMVs was roughly one-tenth of the total protein content. Fn-OMVs induced IL-6 and IL-8 production in OBA-9 cells significantly more strongly than Pg-OMVs ( Figure 6A). Downregulation of STING by STING-specific siRNA significantly suppressed IL-6 production induced by Pg-OMVs by 54% and Fn-OMVs by 48%; further, IL-8 production was induced by Pg-OMVs by 58% and by Fn-OMVs by 64% ( Figure 6B). These results suggest that the STING pathways activated by nucleic acids is not only specific to Pg-OMVs, but also activated by Fn-OMVs. Moreover, transfection of OBA-9 cells with STING-specific siRNA resulted in a slight decrease in phosphorylation of NF-κB after stimulation with Pg-OMVs or Fn-OMVs compared to siRNA controls ( Figure 6C). These results suggest that IL-6 and IL-8 production induced by Pg-OMVs or Fn-OMVs might be partially dependent on the STING pathway by vesicle-associated DNA, thereby resulting in the activation of NF-κB signaling. cell supernatants were determined using an ELISA program. Data are the mean ± SD of three independent experiments. * p < 0.05 and ** p < 0.01, compared to the unstimulated control. # p < 0.05 and ## p < 0.01, compared to the negative control siRNA. (C) NF-κB p65 protein levels were determined by Western blotting; representative blots are shown. The ratio of p-NF-κB and NF-κB is shown after correction with β-actin by densitometric analysis. That same ratio for Si control was set to 1.0.

Discussion
Pg, which is the most important pathogenic bacterium for periodontitis, produces OMVs [12]. Pg sustains a chronic inflammatory state in the host by circulating Pg-OMVs into the blood and releasing virulence factors into deep tissues [9]. Moreover, OMVs are stable because they are not affected by host-derived proteases [12]. Therefore, Pg-OMVs are thought to affect the pathogenesis and progression of periodontitis and to play roles in systemic diseases related to Pg infection [12,20]. Pg-OMVs contain several virulence factors, including FimA, LPS, and gingipains, which are key factors associated with chronic periodontitis [7]. However, little is known about the molecular mechanisms underlying the relationship between Pg-OMVs and host epithelial cells.
IL-6 and IL-8 are essential pro-inflammatory cytokines involved in the pathogenesis of periodontitis. IL-6 and IL-8 levels in gingival crevicular fluid are significantly higher in subjects diagnosed with chronic periodontitis than in healthy controls [21]. Therefore, in this study, we investigated the effect of Pg-OMVs on the production of IL-6 and IL-8 in gingival epithelial cells and compared this with Pg-LPS, which is a key factor in periodontitis development [4]. In our previous study, we showed that the levels of IL-6 and IL-8 expression in OBA-9 cells was increased by Pg-LPS [19]. Here, Pg-OMVs increased IL-6 and IL-8 mRNA expression and also protein production to be greater than those induced by Pg-LPS in OBA-9 cells (Figure 1). These results indicated that Pg-OMVs contained both Pg-LPS and other virulence factors. Gingipains, a group of proteinases produced by Pg, are a significant cause of periodontal tissue destruction [22]. Previous studies revealed a three-to five-fold enrichment of gingipains in OMVs compared to those found within their parent bacteria [23]. Here, Pg-OMVs at the same levels as endotoxin activity of 1000 ng/mL Pg-LPS causes cell detachment and changes in the morphology of OBA-9 cells due to the presence of gingipains in Pg-OMVs, which is consistent with the results of a previous study [6]. However, changes in the morphology of OBA-9 cells were not observed in cells cultured with HTPg-OMVs or PgKDP-OMVs at the same levels as endotoxin activity of 1000 ng/mL Pg-LPS. Therefore, we examined whether HTPg-OMVs or PgKDP-OMVs affected the induction of IL-6 and IL-8 mRNA expression and protein levels in OBA-9 cells. Pg-OMVs had a stronger effect on the induction of IL-6 and IL-8 than HTPg-OMVs or PgKDP-OMVs ( Figure 2). These results indicated that Pg-OMV-associated gingipains were one of the virulence factors that induce IL-6 and IL-8 expression in OBA-9 cells, as well as other remaining factors.
Little is known about the intracellular fate of pathogen OMVs in host epithelial cells. Although previous studies have demonstrated that Pg-OMVs penetrate into human oral epithelial cells [11,24], the signaling mechanisms of gingival epithelial cells stimulated with Pg-OMVs are not fully understood. OMVs cause the rapid phosphorylation of kinase signaling proteins, NF-κB transactivation, and induction of cytokines [5]. To clarify the possible mechanisms underlying the induction of IL-6 and IL-8 by Pg-OMVs in OBA-9 cells, intracellular signaling pathways were explored. Increased phosphorylation of all three major MAPK signaling molecules (Erk1/2, JNK, and p38) in OBA-9 cells was observed within 30 min after either Pg-OMV or PgKDP-OMV stimulation. Moreover, pre-treatment with the MEK, JNK, and p38 MAPK inhibitors strongly reduced the production of IL-6 and IL-8 by Pg-OMVs or PgKDP-OMVs. These results suggested that Erk1/2, JNK, and p38 participated in the induction of IL-6 and IL-8 expression in OBA-9 cells. In addition, while the NF-κB pathway is a key signaling pathway for the expression of many cytokines and chemokines, NF-κB is also activated by Pg-OMVs [25]. In agreement with previous studies, stimulation of Pg-OMVs or PgKDP-OMVs increased NF-κB phosphorylation within 15 min after treatment ( Figure 4A). The NF-κB inhibitor significantly attenuated the increased IL-6 and IL-8 production by Pg-OMVs or PgKDP-OMVs in OBA-9 cells ( Figure 4B). These results indicated that Pg-OMVs activated Erk1/2, JNK, p38 MAPK, and NF-κB signaling pathways, resulting in increased activation of IL-6 and IL-8 in human gingival epithelial cells.
Inflammatory responses in host cells require the contribution of diverse signaling pathways by various microbial products. To further determine other possible mechanisms underlying the induction of IL-6 and IL-8 by Pg-OMVs, STING-which functions as an essential innate immune signaling adaptor molecule that recognizes cytoplasmic cellular or microbial DNA [22]-was examined; this was conducted as Pg-OMVs contain DNA. Inflammatory cytokines that induce the cGAS-cGAMP-STING signaling pathway, and are triggered by pathogen-derived DNA, are important in order to protect the host from infection. However, these cytokines can cause tissue damage and immune pathology when produced in excess and sustained. In fact, activation of cGAS-STING has been implicated in the promotion of inflammatory diseases, such as acute pancreatitis [26], acute kidney injury [27], and lung injury [28]. However, activation of the STING pathway in periodontal tissues has not yet been investigated. In this study, STING-specific siRNA suppressed Pg-OMV-induced IL-6 and IL-8 mRNA expression ( Figure 5B) and markedly reduced concentrations of IL-6 and IL-8 in the cell culture supernatants ( Figure 5C). However, Pg-OMVs had no effects on STING gene and protein expression in OBA-9 cells (data not shown). Moreover, we found that induction of IL-6 and IL-8 production via the STING pathway was not Pg-OMV-specific but also occurred in Fn-OMVs ( Figure 6B). A previous study has demonstrated that activated STING induces the NF-κB signaling pathway, leading to the induction of pro-inflammatory cytokines [29]. In this study, NF-κB phosphorylation was slightly reduced when stimulated with Pg-OMV or Fn-OMV in OBA-9 cells with knockdown of STING expression, compared to siRNA controls ( Figure 6C). These results suggest that NF-κB-dependent IL-6 and IL-8 expression could be a part of the STING signaling pathway in OBA-9 cells. The cGAS-STING signaling pathway mediates interferon regulatory factor 3 (IRF3)-dependent transcription of type I IFNs and NF-κBdependent transcription of pro-inflammatory cytokines. It has been suggested that whether STING activates IRF3 or NF-κB differs between species, such as humans and mice, and even between cell types. Furthermore, how STING affects the balance and relative levels of type I IFN expression and NF-κB-dependent inflammatory cytokine production remains to be elucidated [30]. Moreover, whether OMVs predominantly activate the MAPK or STING signaling pathway also remains to be examined. The present study is significant in demonstrating the involvement of the STING pathway in pro-inflammatory cytokine production in periodontal tissues.

Conclusions
In conclusion, this study demonstrated that Pg-OMVs contain a large number of virulence factors and activate complex signaling pathways, including Erk1/2, p38, JNK, STING, and NF-κB, which therefore result in the induction of pro-inflammatory cytokine expression in human gingival epithelial cells (Figure 7). These results suggest that Pg-OMVs may play important roles in the pathogenesis and progression of periodontitis by stimulating various pathways.