Fusobacterium nucleatum triggers senescence phenotype in gingival epithelial cells.

The prevalence of periodontitis increases with physiological aging. However, whether bacteria associated with periodontal diseases foster aging and the mechanisms by which they may do so are unknown. Herein, we hypothesize that Fusobacterium nucleatum, a microorganism associated with periodontitis and several other age-related disorders, triggers senescence, a chief hallmark of aging responsible to reduce tissue repair capacity. Our study analyzed the senescence response of gingival epithelial cells and their reparative capacity upon long-term exposure to F. nucleatum. Specifically, we assessed (a) cell cycle arrest by analyzing the cyclin-dependent kinase inhibitors p16INK4a and p14ARF and their downstream cascade (pRb, p53, and p21) at both gene and protein levels, (b) lysosomal mediated dysfunction by using assays targeting the expression and activity of the senescence-associated ß-galactosidase (SA-ß-Gal) enzyme, and (c) nuclear envelope breakdown by assessing the expression of Lamin-B1. The consequences of the senescence phenotype mediated by F. nucleatum were further assessed using wound healing assays. Our results revealed that prolonged exposure to F. nucleatum promotes an aging-like phenotype as evidenced by the increased expression of pro-senescence markers (p16INK4a , p21, and pRb) and SA-ß-Gal activity and reduced expression of the counter-balancing cascade (p14ARF and p53) and Lamin-B1. Furthermore, we also noted impaired wound healing capacity of gingival epithelial cells upon prolong bacterial exposure, which was consistent with the senescence-induced phenotype. Together, our findings provide a proof-of-concept evidence that F. nucleatum triggers a pro-senescence response in gingival epithelial cells. This might affect periodontal tissue homeostasis by reducing its repair capacity and, consequently, increasing susceptibility to periodontitis during aging.

Selective BET inhibitor RVX-208 ameliorates periodontal inflammation and bone loss.

AIM: To determine the effects of RVX-208, a selective bromodomain and extra-terminal domain (BET) inhibitor targeting bromodomain 2 (BD2), on periodontal inflammation and bone loss. MATERIALS AND METHODS: Macrophage-like cells (RAW264.7) and human gingival epithelial cells were challenged by Porphyromonas gingivalis (Pg) with or without RVX-208. Inflammatory gene expression and cytokine production were measured by reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. RAW264.7 cells were induced to osteoclast differentiation. After RVX-208 treatment, osteoclast differentiation was evaluated by histology, tartrate-resistant-acid-phosphatase (TRAP) activity and the expression of osteoclast-specific genes. The effect of RVX-208 on osteoclast transcriptome was studied by RNA sequencing. Periodontitis was induced in rats by ligature and local RVX-208 treatment was administered every other day. Alveolar bone loss was measured by micro-computed tomography. RESULTS: RVX-208 inhibited inflammatory gene expression and cytokine production in Pg-infected cells. Osteoclast differentiation was inhibited by RVX-208, as evidenced by reduced osteoclast number, TRAP activity and osteoclast-specific gene expression. RVX-208 displayed a more selective and less profound suppressive impact on transcriptome compared with pan-BET inhibitor, JQ1. RVX-208 administration prevented the alveolar bone loss in vivo. CONCLUSIONS: RVX-208 regulated both upstream (inflammatory cytokine production) and downstream (osteoclast differentiation) events that lead to periodontal tissue destruction, suggesting that it may be a promising 'epi-drug' for the prevention of periodontitis.

Quercetin Preserves Oral Cavity Health by Mitigating Inflammation and Microbial Dysbiosis.

Failure to attenuate inflammation coupled with consequent microbiota changes drives the development of bone-destructive periodontitis. Quercetin, a plant-derived polyphenolic flavonoid, has been linked with health benefits in both humans and animals. Using a systematic approach, we investigated the effect of orally delivered Quercetin on host inflammatory response, oral microbial composition and periodontal disease phenotype. In vivo, quercetin supplementation diminished gingival cytokine expression, inflammatory cell infiltrate and alveolar bone loss. Microbiome analyses revealed a healthier oral microbial composition in Quercetin-treated versus vehicle-treated group characterized by reduction in the number of pathogenic species including Enterococcus, Neisseria and Pseudomonas and increase in the number of non-pathogenic Streptococcus sp. and bacterial diversity. In vitro, Quercetin diminished inflammatory cytokine production through modulating NF-κB:A20 axis in human macrophages following challenge with oral bacteria and TLR agonists. Collectively, our findings reveal that Quercetin supplement instigates a balanced periodontal tissue homeostasis through limiting inflammation and fostering an oral cavity microenvironment conducive of symbiotic microbiota associated with health. This proof of concept study provides key evidence for translational studies to improve overall health.

A20 Restricts Inflammatory Response and Desensitizes Gingival Keratinocytes to Apoptosis.

The pathophysiology of periodontal disease involves a perturbed immune system to a dysbiotic microflora leading to unrestrained inflammation, collateral tissue damage, and various systemic complications. Gingival epithelial cells function as an important part of immunity to restrict microbial invasion and orchestrate the subsequent innate responses. A20 (TNFAIP3), an ubiquitin-editing enzyme, is one of the key regulators of inflammation and cell death in numerous tissues including gastrointestinal tract, skin, and lungs. Emerging evidence indicates A20 as an essential molecule in the oral mucosa as well. In this study, we characterized the role of A20 in human telomerase immortalized gingival keratinocytes (TIGKs) through loss and gain of function assays in preclinical models of periodontitis. Depletion of A20 through gene editing in TIGKs significantly increased IL-6 and IL-8 secretion in response to Porphyromonas gingivalis infection while A20 over-expression dampened the cytokine production compared to A20 competent cells through modulating NF-κB signaling pathway. In the subsequent experiments which assessed apoptosis, A20 depleted TIGKs displayed increased levels of cleaved caspase 3 and DNA fragmentation following P. gingivalis infection and TNF/CHX challenge compared to A20 competent cells. Consistently, there was reduced apoptosis in the cells overexpressing A20 compared to the control cells expressing GFP further substantiating the role of A20 in regulating gingival epithelial cell fate in response to exogenous insult. Collectively, our findings reveal first systematic evidence and demonstrate that A20 acts as a regulator of inflammatory response in gingival keratinocytes through its effect on NF-κB signaling and desensitizes cells to bacteria and cytokine induced apoptosis in the oral mucosa. As altered A20 levels can have profound effect on different cellular responses, future studies will determine whether A20-targeted therapies can be exploited to restrain periodontal inflammation and maintain oral mucosa tissue homeostasis.

Microbiota, cirrhosis, and the emerging oral-gut-liver axis.

Cirrhosis is a prevalent cause of morbidity and mortality, especially for those at an advanced decompensated stage. Cirrhosis development and progression involves several important interorgan communications, and recently, the gut microbiome has been implicated in pathophysiology of the disease. Dysbiosis, defined as a pathological change in the microbiome, has a variable effect on the compensated versus decompensated stage of cirrhosis. Adverse microbial changes, both in composition and function, can act at several levels within the gut (stool and mucosal) and have also been described in the blood and oral cavity. While dysbiosis in the oral cavity could be a source of systemic inflammation, current cirrhosis treatment modalities are targeted toward the gut-liver axis and do not address the oral microbiome. As interventions designed to modulate oral dysbiosis may delay progression of cirrhosis, a better understanding of this process is of the utmost importance. The concept of oral microbiota dysbiosis in cirrhosis is relatively new; therefore, this review will highlight the emerging role of the oral-gut-liver axis and introduce perspectives for future research.

Chemokine function in periodontal disease and oral cavity cancer.

The chemotactic cytokines, or chemokines, comprise a superfamily of polypeptides with a wide range of activities that include recruitment of immune cells to sites of infection and inflammation, as well as stimulation of cell proliferation. As such, they function as antimicrobial molecules and play a central role in host defenses against pathogen challenge. However, their ability to recruit leukocytes and potentiate or prolong the inflammatory response may have profound implications for the progression of oral diseases such as chronic periodontitis, where tissue destruction may be widespread. Moreover, it is increasingly recognized that chronic inflammation is a key component of tumor progression. Interaction between cancer cells and their microenvironment is mediated in large part by secreted factors such as chemokines, and serves to enhance the malignant phenotype in oral and other cancers. In this article, we will outline the biological and biochemical mechanisms of chemokine action in host-microbiome interactions in periodontal disease and in oral cancer, and how these may overlap and contribute to pathogenesis.

Oral bacterial DNA differ in their ability to induce inflammatory responses in human monocytic cell lines.

BACKGROUND: Deoxyribonucleic acids (DNA) of periodontal pathogens, Porphyromonas gingivalis (Pg) and Tannerella forsythia, stimulate cytokine production in human monocytic cells (THP-1) through Toll-like receptor 9 (TLR-9) and nuclear factor-κB signaling. Fusobacterium nucleatum (Fn) is one of the most frequently isolated bacteria in periodontally diseased tissues and is reported to synergize with Pg, enhancing the pathogenicity. We investigate inflammatory mediator production in THP-1 cells challenged with Fn and Streptococcus sanguinis (Ss) DNA, a non-pathogenic oral bacteria, and further assess whether cytokines triggered by whole pathogens or Pg lipopolysaccharide (LPS) are affected by TLR-9 signaling inhibitors (chloroquine). METHODS: THP-1 cells were stimulated with Pg-DNA (100 ng/µL), Fn-DNA (100 ng/µL), Ss-DNA (100 ng/µL), Pg-LPS (10 ng/µL), and heat-killed whole bacteria (multiplicity of infection, 1:100) for 16 hours with or without chloroquine pretreatment (10 µg/mL). Interleukin (IL)-1ß, IL-6, IL-8, and tumor necrosis factor-α levels were determined using enzyme-linked immunosorbent assay. Statistical analyses included analysis of variance with multiple comparisons using Dunnett or Tukey methods and paired t test. A value of P <0.05 was significant. RESULTS: Inflammatory mediator levels were increased in response to all the stimuli with the exception of Ss-DNA (P <0.05). Chloroquine pretreatment significantly decreased cytokine production from THP-1 cells with the exception of IL-6 production triggered by whole Fn and Ss (P <0.05). CONCLUSIONS: Differences exist among oral bacterial DNA in inducing immune responses. By altering the conditions in cytosolic compartments, we can interfere with cellular responses triggered by extracellular receptor activation. Thus, alternative treatment approaches targeted to intracellular receptors might be of benefit in controlling periodontal inflammation.

Esthetic management of peripheral giant cell granuloma.

BACKGROUND: Peripheral giant cell granuloma (PGCG) is a relatively rare hyperplastic lesion of the oral cavity. The lesion occurs in females more frequently than males and more often in the mandible than the maxilla. Although the precise etiology of PGCG is unknown, it might represent a local reaction to trauma or irritation. METHODS: In general, treatment requires a wide excision of the lesion due to its possible recurrence. RESULTS: This report describes the clinical and histopathological findings of PGCG diagnosed in the maxilla of a young male, as well as the successful treatment of a gingival defect that occurred following excision of the lesion, by placement of a subepithelial connective tissue graft concurrently with the biopsy procedure. CONCLUSIONS: This report emphasizes the importance of having histopathological data to confirm the clinical diagnosis, and the importance of an adequate excision to prevent recurrence even in less extensive cases.