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Abstract

Several studies have demonstrated the remarkable properties of microbiota and their metabolites in the pathogenesis of several inflammatory diseases. 10-Hydroxy-cis-12-octadecenoic acid (HYA), a bioactive metabolite generated by probiotic microorganisms during the process of fatty acid metabolism, has been studied for its protective effects against epithelial barrier impairment in the intestines. Herein, we examined the effect of HYA on gingival epithelial barrier function and its possible application for the prevention and treatment of periodontal disease. We found that GPR40, a fatty acid receptor, was expressed on gingival epithelial cells; activation of GPR40 by HYA significantly inhibited barrier impairment induced by Porphyromonas gingivalis, a representative periodontopathic bacterium. The degradation of E-cadherin and beta-catenin, basic components of the epithelial barrier, was prevented in a GPR40-dependent manner in vitro. Oral inoculation of HYA in a mouse experimental periodontitis model suppressed the bacteria-induced degradation of E-cadherin and subsequent inflammatory cytokine production in the gingival tissue. Collectively, these results suggest that HYA exerts a protective function, through GPR40 signaling, against periodontopathic bacteria-induced gingival epithelial barrier impairment and contributes to the suppression of inflammatory responses in periodontal diseases.

Protective effect of HYA-induced GPR40 signaling on epithelial barrier impairment

To examine a possible role of bioactive metabolites in gingival epithelial barrier function, we performed an in vitro permeability assay using fluorescein isothiocyanate (FITC) -conjugated dextran after the optimization of HYA concentration (Supplementary Fig. ). The epithelial barrier impairment induced by P. gingivalis was significantly inhibited by pretreatment with HYA but not HYB (Fig. ). Pretreatment with the selective GPR40 antagonist GW1100 partially diminished the protective property of HYA, indicating a GPR40-dependent mechanism (Fig. ). Taken together, these results demonstrate that HYA prevents P. gingivalis-induced barrier impairment, via its activation of GPR40 signaling.

HYA reduces local inflammatory cytokine production in gingival tissue in vivo

In order to examine the clinical relevance of HYA in periodontitis, an in vivo study was carried out using a mouse experimental periodontal disease model. After validation of epithelial GPR40 expression in the gingiva, periodontitis was induced by applying a ligature on the molars with repeated oral inoculation of P. gingivalis (Supplementary Figs ,). The mRNA expression levels of inflammatory cytokines such as IL-1β, TNF-α and IL-6 in gingival tissues of the ligated HYA-treated group were significantly decreased in comparison with those of the ligated sham-treated group, with a tendency of suppression of alveolar bone destruction (Fig. ). The ligated HYA-treated group showed a higher immunofluorescence intensity for E-cadherin in the epithelium when compared to that of the ligated sham-treated group (Fig. ). No differences of E-cadherin intensity were observed between the unligated groups (Supplementary Fig. ). In consistent with in vitro study, no alteration of E-cadherin mRNA expression was observed for all groups (Supplementary Fig. ). Collectively, these results indicate that HYA reinforces gingival epithelial barrier function by inhibiting E-cadherin degradation, resulting in the suppression of periodontal inflammatory responses and subsequent alveolar bone destruction in vivo.

Reagents and antibodies

10-hydroxy-cis-12-octadecenoic acid (HYA) and 10-hydroxyoctadecanoic acid (HYB) were synthesized according to methods published previously. Anti-GPR40 antibody was purchased from GeneTex Inc. (San Antonio, TX, USA). Anti-E-cadherin, anti-Beta-catenin, anti-ERK and anti-phosphorylated ERK antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Rabbit anti-mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Cell Signaling Technology) and peroxidase-labeled anti-rabbit IgG antibody (Cell Signaling Technology) were used for Western blotting experiments. A selective GPR40 antagonist, GW1100, was purchased from Cayman Chemical (Ann Arbor, MI, USA).

Abstract

Background: Gut microbiota plays a pivotal role in regulating host metabolism that affects the systemic health. To date, several studies have confirmed the fact that microbiota interacts with host, modulating immunity, controlling the homeostasis environment, and maintaining systemic condition. Recent studies have focused on the protective function of poly unsaturated fatty acids, 10-oxo-trans-11-oxadecenoic acid (KetoC) and 10-hydroxy-cis-12-octadecenoic acid (HYA), generated by gut microbiota on periodontal disease. Nevertheless, the mechanism remains unclear as investigations are limited to in vivo and in vitro studies. In this present review, we found that the administration of metabolites, KetoC and HYA, by a probiotic gut microbiota Lactobacillus plantarum from linoleic acid is found to inhibit the oxidation process, possess an antimicrobial function, and prevent the inflammation. These findings suggest the promising use of functional lipids for human health. Conclusion: Protective modalities of bioactive metabolites may support periodontal therapy by suppressing bacterial dysbiosis and regulating periodontal homeostasis in the clinical setting.

2.2.1. Anti-Inflammatory Function

Regarding the signaling pathway regulating the inflammation, HYA is found to decrease TNFR2 expression in Dextran sulfate sodium (DSS)-induced colitis mice []. In respect to this, the upregulation of TNFR2 subsequently reduced the ratio of phospho-IκBα/IκBα and NFκB p65, thereby decreasing proinflammatory cytokine production. HYA decreases local inflammatory cytokine mRNA level (IL-1β, TNFα, and IL-6) in gingival tissue in vivo with a tendency of alveolar bone loss suppression []. HYA induces ERK phosphorylation; on the other hand, the blockade of the MEK-ERK signaling deteriorates the HYA-GPR40 anti-inflammatory action [].

2.2.3. Effect on Epithelial Barrier Function

HYA treatment suppressed this degradation by promoting the proteolytic resistance of E-cadherin/β-catenin toward P. gingivalis in the gingival epithelial cells [] and colorectal epithelial adenocarcinoma cells (Caco-2) [], thereby reducing the inflammation and injury. An investigation reported that tight junction permeability (TER) and protein molecules (occluding, ZO-1, ZO-2, and claudin-3) were impaired by INF-γ and TNFα challenge in Caco-2 cells through the upregulation of IL-8, and then recovered by the pretreatment of HYA in a dose-dependent manner []. Moreover, the action of HYA depends on the expression of GPR40 in gingival epithelial cells [].