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Background: Porphyromonas gingivalis exacerbates tissue hypoxia and worsens periodontal inflammation. This study investigated the effect of a therapeutic oxygen carrier (M101), derived from Arenicola marina, on hypoxia and associated inflammation in the context of periodontitis., Methods: The effect of M101 on GLUT-1, GLUT-3, HIF-1α, and MMP-9 expression, hypoxia, and antioxidant status in oral epithelial cells (EC) exposed to CoCl 2 (1000 μM), P. gingivalis (MOI 100), and CoCl 2 + P. gingivalis was evaluated through hypoxia detection fluorescence assay, antioxidant concentration colorimetric assay, and RTqPCR. Evaluation of M101 on EC proliferation was evaluated in an in vitro wound assay. In experimental periodontitis, periodontal wound healing and osteoclastic activity were compared among natural wound healing, placebo, and gels containing M101 (1  and 2 g/L) groups through histomorphometry and TRAP (tartrate-resistant acid phosphatase activity assay) assay respectively. The expression of HIF-1α, MMP-9, and NFκB in periodontal tissues was also evaluated through immunofluorescence studies., Results: M101 downregulated GLUT-1, GLUT-3, HIF-1α, and MMP-9 levels in EC exposed to CoCl 2 , P. gingivalis, and CoCl 2 + P. gingivalis (p < 0.05). Fluorescence and colorimetric analyses confirmed hypoxia reduction and antioxidant capacity improvement in such EC upon M101 treatment. Moreover, M101 improved significantly the in vitro wound closure. In vivo, the attachment level was significantly improved, and osteoclastic activity was reduced in mice treated with M101 gels compared to placebo and natural wound healing groups (p < 0.05). HIF-1α, MMP-9, and NFκB expression in periodontal tissues was reduced in M101 gels treated mice compared to the controls., Conclusion: M101 showed promise in resolving hypoxia and associated inflammation-mediated tissue degradation. Its potential in the clinical management of periodontitis must be further investigated., (© 2022 American Academy of Periodontology.)

The aim of this study was to evaluate the potential anti-inflammatory and anti-resorptive effects of lenabasum in the context of Porphyromonas gingivalis (Pg)-induced inflammation. Lenabasum or ajulemic acid (1',1'-dimethylheptyl-THC-11-oic-acid), a synthetic analog of THC-11-oic acid, has already demonstrated anti-inflammatory properties for the treatment of several inflammatory diseases. In vitro, the cytocompatibility of lenabasum was evaluated in human oral epithelial cells (EC), oral fibroblasts and osteoblasts by metabolic activity assay. The effect of lenabasum (5 µM) treatment of Pg-LPS- and P. gingivalis-infected EC on the pro- and anti-inflammatory markers was studied through RTqPCR. In vivo, lenabasum was injected subcutaneously in a P. gingivalis-induced calvarial abscess mouse model to assess its pro-healing effect. Concentrations of lenabasum up to 5 µM were cytocompatible in all cell types. Treatment of Pg-LPS and Pg-infected EC with lenabasum (5 µM; 6 h) reduced the gene expression of TNF-α, COX-2, NF-κB, and RANKL, whereas it increased the expression of IL-10 and resolvin E1 receptor respectively (p < 0.05). In vivo, the Pg-elicited inflammatory lesions' clinical size was significantly reduced by lenabasum injection (30 µM) vs untreated controls (45%) (p < 0.05). Histomorphometric analysis exhibited improved quantity and quality of bone (with reduced lacunae) and significantly reduced calvarial soft tissue inflammatory score in mice treated with lenabasum (p < 0.05). Tartrate-resistant acid phosphatase activity assay (TRAP) also demonstrated decreased osteoclastic activity in the treatment group compared to that in the controls. Lenabasum showed promising anti-inflammatory and pro-resolutive properties in the management of Pg-elicited inflammation, and thus, its potential as adjuvant periodontal treatment should be further investigated., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Periodontitis is induced by periodontal dysbiosis characterized by the predominance of anaerobic species. TLRs constitute the classical pathway for cell activation by infection. Interestingly, the Toll/IL-1 receptor homology domain adapters initiate signaling events, leading to the activation of the expression of the genes involved in the host immune response. The aim of this study was to evaluate the effects of Porphyromonas gingivalis on the expression and protein-protein interactions among five TIR adapters (MAL, MyD88, TRIF, TRAM and SARM) in gingival epithelial cells and endothelial cells. It was observed that P. gingivalis is able to modulate the signaling cascades activated through its recognition by TLR4/2 in gingival epithelial cells and endothelial cells. Indeed, MAL-MyD88 protein-protein interactions associated with TLR4 was the main pathway activated by P. gingivalis infection. When transient siRNA inhibition was performed, cell viability, inflammation, and cell death induced by infection decreased and such deleterious effects were almost absent when MAL or TRAM were targeted. This study emphasizes the role of such TIR adapter proteins in P. gingivalis elicited inflammation and the precise evaluation of TIR adapter protein interactions may pave the way for future therapeutics in both periodontitis and systemic disease with a P. gingivalis involvement, such as atherothrombosis.

A link between periodontitis and atherothrombosis has been highlighted. The aim of this study was to determine the influence of Porphyromonas gingivalis on endothelial microvesicles (EMV Pg ) shedding and their contribution to endothelial inflammation. Endothelial cells (EC) were infected with P. gingivalis (MOI = 100) for 24 h. EMV Pg were isolated and their concentration was evaluated by prothrombinase assay. EMV Pg were significantly increased in comparison with EMV Ctrl shedded by unstimulated cells. While EMV Ctrl from untreated EC had no effect, whereas, the proportion of apoptotic EC was increased by 30 nM EMV Pg and viability was decreased down to 25%, a value elicited by P. gingivalis alone. Moreover, high concentration of EMV Pg (30 nM) induced a pro-inflammatory and pro-oxidative cell response including up-regulation of TNF-α, IL-6 and IL-8 as well as an altered expression of iNOS and eNOS at both mRNA and protein level. An increase of VCAM-1 and ICAM-1 mRNA expression (4.5 folds and 3 folds respectively (p < 0.05 vs untreated) was also observed after EMV Pg (30 nM) stimulation whereas P. gingivalis infection was less effective, suggesting a specific triggering by EMV Pg . Kinasome analysis demonstrated the specific effect induced by EMV Pg on main pro-inflammatory pathways including JNK/AKT and STAT. EMV Pg are effective pro-inflammatory effectors that may have detrimental effect on vascular homeostasis and should be considered as potential autocrine and paracrine effectors involved in the link between periodontitis and atherothrombosis.

Porphyromonas gingivalis-induced inflammatory effects are mostly investigated in monolayer cultured cells. The aim of this study was to develop a 3D spheroid model of gingiva to take into account epithelio-fibroblastic interactions. Human gingival epithelial cells (ECs) and human oral fibroblasts (FBs) were cultured by hanging drop method to generate 3D microtissue (MT) whose structure was analyzed on histological sections and the cell-to-cell interactions were observed by scanning and transmission electron microscopy (SEM and TEM). MTs were infected by P. gingivalis and the impact on cell death (Apaf-1, caspase-3), inflammatory markers (TNF-α, IL-6, IL-8) and extracellular matrix components (Col-IV, E-cadherin, integrin β1) was evaluated by immunohistochemistry and RT-qPCR. Results were compared to those observed in situ in experimental periodontitis and in human gingival biopsies. MTs exhibited a well-defined spatial organization where ECs were organized in an external cellular multilayer, while, FBs constituted the core. The infection of MT demonstrated the ability of P. gingivalis to bypass the epithelial barrier in order to reach the fibroblastic core and induce disorganization of the spheroid structure. An increased cell death was observed in fibroblastic core. The development of such 3D model may be useful to define the role of EC-FB interactions on periodontal host-immune response and to assess the efficacy of new therapeutics.

Porphyromonas gingivalis is able to invade and modulate host-immune response to promote its survival. This bacterium modulates the cell cycle and programed cell death, contributing to periodontal lesion worsening. Several molecular pathways have been identified as key triggers of apoptosis, including apoptosome apoptotic peptidase activating factor 1 (APAF-1). Apaf-1 and X-linked inhibitor of apoptosis protein (Xiap) mRNA were differentially expressed between gingival samples harvested from human healthy and chronic periodontitis tissues (Apaf-1, 19.2-fold; caspase-9, 14.5-fold; caspase-3, 6.8-fold; Xiap: 2.5-fold in chronic periodontitis) (P < 0.05), highlighting their potential role in periodontitis. An increased proteic expression of APAF-1 was also observed in a murine experimental periodontitis model induced by P. gingivalis-soaked ligatures. In vitro, it was observed that P. gingivalis targets APAF-1, XIAP, caspase-3, and caspase-9, to inhibit epithelial cell death at both mRNA and protein levels. Opposite effect was observed in fibroblasts in which P. gingivalis increased cell death and apoptosis. To assess if the observed effects were associated to APAF-1, epithelial cells and fibroblasts were transfected with siRNA targeting Apaf-1. Herein, we confirmed that APAF-1 is targeted by P. gingivalis in both cell types. This study identified APAF-1 apoptosome and XIAP as intracellular targets of P. gingivalis, contributing to the deterioration of periodontal lesion through an increased persistence of the bacteria within tissues and the subversion of host-immune response., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Periodontitis, an inflammatory disease initiated by Gram-negative bacteria such as Porphyromonas gingivalis ( Pg), is considered as a risk factor for rheumatoid arthritis (RA). Our study aimed to determine the effect of Pg and its LPS on the expression of peptidyl arginine deiminase isotypes (PADs) in human primary chondrocytes (HC). HCs were infected with Pg and activated by its LPS (LPS- Pg). The mRNA expression levels of human PADs (1, 2, 3, 4 and 6) and bacterial enzyme (PADPg) were quantified by RT-qPCR. Cellular extracts served to measure the enzymatic activities of PADs and PADPg and to visualize the profiles of citrullinated proteins/peptides by Western blotting. Our data showed significant inhibitions of mRNA expressions of human PAD-2, PAD-3 and PAD-4 during infection of HC with live Pg. Activation of HC by LPS- Pg increased mRNA expressions of human PAD-2 and PAD-3. The PADPg enzymatic activity was significantly increased in only infected HC. Analysis of citrullinated proteins/peptides profiles revealed the occurrence of low molecular bands only in cellular extracts from HC infected with Pg. Our data showed that Pg and its LPS differentially regulate the expression of PADs in human chondrocytes and that Pg favors the apparition of new citrullinated proteins/peptides.