Maintaining barrier function of infected gingival epithelial cells by inhibition of DNA methylation

Abstract

Background: Infection and inflammation induce epigenetic changes that alter gene expression. In periodontal disease, inflammation, and microbial dysbiosis occur, which can lead to compromised barrier function of the gingival epithelia. Here, we tested the hypotheses that infection of cultured human gingival epithelial (HGEp) cells with Porphyromonas gingivalis disrupts barrier function by inducing epigenetic alterations and that these effects can be blocked by inhibitors of DNA methylation. Methods: Primary HGEp cells were infected with P. gingivalis either in the presence or absence of the non-nucleoside DNA methyltransferase (DNMT) inhibitors RG108, (-) epigallocatechin-3-gallate (EGCG), or curcumin. Barrier function was assessed as transepithelial electrical resistance (TEER). DNA methylation and mRNA abundance were quantified for genes encoding components of three cell-cell junction complexes, CDH1, PKP2, and TJP1. Cell morphology and the abundance of cell-cell junction proteins were evaluated by confocal microscopy. Results: Compared to non-infected cells, P. gingivalis infection decreased TEER (P < 0.0001) of HGEp cells; increased methylation of the CDH1, PKP2, and TJP1 (P < 0.0001); and reduced their expression (mRNA abundance) (P < 0.005). Pretreatment with DNMT inhibitors prevented these infection-induced changes in HGEp cells, as well as the altered morphology associated with infection. Conclusion: Pathogenic infection induced changes in DNA methylation and impaired the barrier function of cultured primary gingival epithelial cells, which suggests a mechanism for systemic consequences of periodontal disease. Inhibition of these events by non-nucleoside DNMT inhibitors represents a potential strategy to treat periodontal disease.

The effects of mouthwashes in human gingiva epithelial progenitor (HGEPp) cells

“…For apoptosis results, the mean of the reference substances tested (61.2% ± 4.44) is below the value of commercially available mouthwashes (108.7% ± 9.01). In the case of Gum Paroex, which has a strong apoptotic effect (148% ± 8.99), the proapoptotic nature of vitamin E analogs seems to be responsible among its ingredients (see Table 1 and Table 4) [51]. Nevertheless, the weak viability-reducing effect of Gum Paroex seems to balance its apoptotic character on HGEPp cells, especially when compared to the other reference substances.…”

“…The principal aim of the present study was to investigate the effect of a few very commonly used mouthwashes (8) and their main active components (4) on eukaryotic gingival epithelial progenitor cells (HGEPp). The oral epithelium being a squamous epithelium (providing most of the protecting barrier function), recent literature [51] shows that the barrier function still develops in HGEPp cultures, which made these cells ideal models for our experiments. A complex panel of three cell physiology assays (real-time impedimetry, computer-based morphometry, and apoptosis test) proved to be suitable to characterize the cytotoxic or viability modulator behavior of each investigated compound.…”

Epigenetic regulation of inflammation in periodontitis: cellular mechanisms and therapeutic potential

“…These findings indicate that, at least in this model, the amelioration of disease severity by HDACi was unrelated to inflammation. In light of the well-documented ability of [53] TSA, butyrate Upregulation of hBD2, IL-8 and CCL20 in GECs infected with P. gingivalis or F. nucleatum [35] butyrate Suppression of LPS-induced TNF and IL-1β expression and ROS production in PDL cells [56] TSA, apicidin Suppression of T. denticola-induced MMP2 activation in PDL cells [29] TSA, butyrate Upregulation of osteoblast markers and induction of osteogenic differentiation of PDL cells [56,57] 1179.4b Reduction of alveolar bone destruction in experimental periodontitis in mice [69] TSA Reduction of inflammation and increased alveolar bone volume in experimental periodontitis in rats [68] BET proteins I-BET151, JQ1 Suppression of inflammatory mediator production by GECs and gingival fibroblasts [65] JQ1 Amelioration of inflammation and alveolar bone resorption in experimental periodontitis in mice [70] DNMTs AZA Induction of differentiation of gingival fibroblasts into osteoblasts and induction of ectopic bone formation in mice [120] Suppression of T. denticola-induced MMP2 activation in PDL cells [29] AZA, decitabine Modulation of inflammatory cytokine production by GECs [121] RG108 Prevention of P. gingivalis-mediated impairment of GEC barrier function [114] HDACi to suppress osteoclastogenesis [71], and recent evidence that these compounds induce osteogenic differentiation of PDL cells [56,57], the protective effect of 1179.4b may be attributed to the influence of HDACi on alveolar bone regeneration. Indeed, the bone-protective effects of TSA in ligature-induced periodontitis in rats were associated with the ability of HDACi to induce osteogenic differentiation of mesenchymal stem cells [68], though this effect has not been formally demonstrated in vivo.…”

“…2). P. gingivalis infection of GECs also leads to increased methylation and reduced expression of genes coding for the components of the cell-cell junction complexes (CDH1, PKP2, and TJP1), which results in functional impairment of the epithelial barrier [114]. Chronic stimulation of PDL cells with P. gingivalis LPS promotes hypermethylation of several genes encoding ECM components, including FANK1, COL4A1-A2, COL12A1, COL15A1, LAMA5, LAMB1, MMP25, POMT1, and EMILIN3, that is associated with reduced expression levels of these genes [115].…”

“…However, in these studies, GECs were exposed to DNMT1 inhibitors for a short period before infection (4 h) [35,121], which may be insufficient to induce DNA hypomethylation. Finally, a recent study demonstrated that pretreatment of GECs with non-nucleoside DNMT inhibitors (RG-108, EGCG, or curcumin) could reverse the effects of P. gingivalis on methylation and expression of a cluster of genes encoding components of cell-cell junction complexes, as well as functional impairment of the gingival epithelial barrier [114].…”