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Photobiomodulation of oral fibroblasts stimulated with periodontal pathogens.

ABSTRACT: Photobiomodulation (PBM) utilises light energy to treat oral disease, periodontitis. However, there remains inconsistency in the reporting of treatment parameters and a lack of knowledge as to how PBM elicits its molecular effects in vitro. Therefore, this study aimed to establish the potential immunomodulatory effects of blue and near infra-red light irradiation on gingival fibroblasts (GFs), a key cell involved in the pathogenesis of periodontitis. GFs were seeded in 96-well plates in media + / - Escherichia coli lipopolysaccharide (LPS 1 μg/ml), or heat-killed Fusobacterium nucleatum (F. nucleatum, 100:1MOI) or Porphyromonas gingivalis (P. gingivalis, 500:1MOI). Cultures were incubated overnight and subsequently irradiated using a bespoke radiometrically calibrated LED array (400-830 nm, irradiance: 24 mW/cm² dose: 5.76 J/cm²). Effects of PBM on mitochondrial activity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and adenosine triphosphate (ATP) assays, total reactive oxygen species production (ROS assay) and pro-inflammatory/cytokine response (interleukin-8 (IL-8) and tumour growth factor-β1 (TGFβ1)) were assessed 24 h post-irradiation. Data were analysed using one-way ANOVA followed by the Tukey test. Irradiation of untreated (no inflammatory stimulus) cultures at 400 nm induced 15%, 27% and 13% increases in MTT, ROS and IL-8 levels, respectively (p < 0.05). Exposure with 450 nm light following application of P. gingivalis, F. nucleatum or LPS induced significant decreases in TGFβ1 secretion relative to their bacterially stimulated controls (p < 0.001). Following stimulation with P. gingivalis, 400 nm irradiation induced 14% increases in MTT, respectively, relative to bacteria-stimulated controls (p < 0.05). These findings could identify important irradiation parameters to enable management of the hyper-inflammatory response characteristic of periodontitis.

SUBMITTER: Serrage HJ

PROVIDER: S-EPMC8593050 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Periodontitis is closely related to oral cancer, but the molecular mechanism of periodontal pathogens involved in the occurrence and development of oral cancer is still inconclusive. Here, we demonstrate that, in vitro, the cell proliferation ability and S phase cells of the periodontitis group (colonized by Porphyromonas gingivalis and Fusobacterium nucleatum, P+) significantly increased, but the G1 cells were obviously reduced. The animal models with an in situ oral squamous cell carcinoma (OSCC) and periodontitis-associated bacteria treatment were constructed, and micro-CT showed that the alveolar bone resorption of mice in the P+ group (75.3 ± 4.0 μm) increased by about 53% compared with that in the control group (48.8 ± 1.3 μm). The tumor mass and tumor growth rate in the P+ group were all higher than those in the blank control group. Hematoxylin-eosin (H&E) staining of isolated tumor tissues showed that large-scale flaky necrosis was found in the tumor tissue of the P+ group, with lots of damaged vascular profile and cell debris. Immunohistochemistry (IHC) of isolated tumor tissues showed that the expression of Ki67 and the positive rate of cyclin D1 were significantly higher in tumor tissues of the P+ group. The qRT-PCR results of the expression of inflammatory cytokines in oral cancer showed that periodontitis-associated bacteria significantly upregulated interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-18, apoptosis-associated speck-like protein containing a CARD (ASC) (up to six times), and caspase-1 (up to four times), but it downregulated nuclear factor (NF)-κB, NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3), and IL-1β (less than 0.5 times). In addition, the volume of spleen tissue and the number of CD4+ T cells, CD8+ T cells, and CD206+ macrophages in the P+ group increased significantly. IHC and Western blotting in tumor tissues showed that expression levels of γ-H2AX, p-ATR, RPA32, CHK1, and RAD51 were upregulated, and the phosphorylation level of CHK1 (p-chk1) was downregulated. Together, we identify that the periodontitis-related bacteria could promote tumor growth and proliferation, initiate the overexpressed NLRP3, and activate upstream signal molecules of ATR-CHK1. It is expected to develop a new molecular mechanism between periodontitis-related bacteria and OSCC.

Project description:BackgroundPeriodontal disease is associated with systemic conditions such as diabetes, arthritis, and cardiovascular disease, all diseases with large inflammatory components. Some, but not all, reports show periopathogens Porphyromonas gingivialis and Tannerella forsythia at higher levels orally in people with one of these chronic diseases and in people with more severe cases. These oral pathogens are thought to be positively associated with systemic inflammatory diseases through induction of oral inflammation that works to distort systemic inflammation or by directly inducing inflammation at distal sites in the body. This study aimed to determine if, among patients with severe periodontal disease, those with multi-morbidity (or many chronic diseases) showed higher levels of periodontal pathogens.MethodsA total of 201 adult subjects, including 84 with severe periodontal disease were recruited between 1/2017 and 6/2019 at a city dental clinic. Electronic charts supplied self-reported diseases and conditions which informed a morbidity index based on the number of chronic diseases and conditions present. Salivary composition was determined by 16S rRNA gene sequencing.ResultsAs expected, patients with severe periodontal disease showed higher levels of periodontal pathogens in their saliva. Also, those with severe periodontal disease showed higher levels of multiple chronic diseases (multimorbidity). An examination of the 84 patients with severe periodontal disease revealed some subjects despite being of advanced age were free or nearly free of systemic disease. Surprisingly, the salivary microbiota of the least healthy of these 84 subjects, defined here as those with maximal multimorbidity, showed significantly lower relative numbers of periodontal pathogens, including Porphyromonas gingivalis and Tannerella Forsythia, after controlling for active caries, tobacco usage, age, and gender. Analysis of a control group with none to moderate periodontal disease revealed no association of multimorbidity or numbers of medications used and specific oral bacteria, indicating the importance of severe periodontal disease as a variable of interest.ConclusionThe hypothesis that periodontal disease patients with higher levels of multimorbidity would have higher levels of oral periodontal pathogens is false. Multimorbidity is associated with a reduced relative number of periodontal pathogens Porphyromonas gingivalis and Tannerella forsythia.

Project description:Acylhomoserine lactones (AHLs), the quorum-sensing (QS) signals produced by a range of Gram-negative bacteria, are involved in biofilm formation in many pathogenic and environmental bacteria. Nevertheless, the current paradigm excludes a role of AHLs in dental plaque formation, while other QS signals, such as AI-2 and autoinducer peptides, have been demonstrated to play an important role in biofilm formation and virulence-related gene expression in oral pathogens. In the present work, we have explored the effect of externally added AHLs on in vitro oral biofilm models for commensal, cariogenic, and periodontal dental plaque. While little effect on bacterial growth was observed, some AHLs specifically affected the lactic acid production and protease activity of the biofilms. Most importantly, the analysis of bacterial diversity in the biofilms showed that the addition of C6-homoserine lactone (C6-HSL) results in a shift toward a periodontal bacterial composition profile by increasing the relative presence of the orange-complex bacteria Peptostreptococcus and Prevotella These results point to a relevant role of AHL-mediated QS in dental plaque formation and might be involved in the development of dysbiosis, the mechanism of which should be further investigated. This finding potentially opens new opportunities for the prevention or treatment of the periodontal disease.IMPORTANCE Dental plaque is omnipresent in healthy oral cavities and part of our commensal microbial colonization. At the same time, dental plaque is the cause of the most common human diseases, caries and gum disease. Dental plaque consists of billions of microbes attached to the surface of your teeth. Communication among these microbes is pivotal for development of these complex communities yet poorly studied in dental plaque. In the present study, we show that a specific communication molecule induces changes within the community related to the development of gum disease. This finding suggests that interfering with microbial communication may represent an interesting novel strategy to prevent gum disease that should be further investigated.

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Photobiomodulation of oral fibroblasts stimulated with periodontal pathogens.

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