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Periodontitis is a prevalent polymicrobial disease. It damages soft tissues and alveolar bone, and causes a significant public-health burden. Development of an advanced therapeutic approach and exploration of vaccines against periodontitis hold promise as potential treatment avenues. Clinical trials for a periodontitis vaccine are lacking. Therefore, it is crucial to address the urgent need for developing strategies to implement vaccines at the primary level of prevention in public health. A deep understanding of the principles and mechanisms of action of vaccines plays a crucial role in the successful development of vaccines and their clinical translation. This review aims to provide a comprehensive summary of potential directions for the development of highly efficacious periodontitis vaccines. In addition, we address the limitations of these endeavors and explore future possibilities for the development of an efficacious vaccine against periodontitis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Background: Malondialdehyde-acetaldehyde (MAA) adducts lead to generation of anti-MAA autoantibodies and have been independently identified in inflamed periodontal and rheumatoid arthritis (RA) tissues. This study evaluates serum samples from RA cases and osteoarthritis (OA) controls to quantify associations between periodontal clinical measures, alveolar bone loss (ABL), and anti-Porphyromonas gingivalis, anti-Prevotella intermedia, and anti-Fusobacterium nucleatum antibody concentrations with anti-MAA antibody concentrations., Methods: Participants (n = 284 RA cases, n = 330 OA controls) underwent periodontal clinical assessments and ABL measurements. Serum immunoglobulin (Ig) A, IgG, and IgM anti-MAA and serum IgG antibacterial antibody concentrations were quantified by enzyme-linked immunosorbent assay (ELISA). Analyses utilized simple linear regression and multivariable adjusted models., Results: No significant associations of periodontal clinical measures with serum anti-MAA were found. Moderate (p = 0.038 and p = 0.036, respectively) and high ABL (p = 0.012 and p = 0.014, respectively) in RA cases (but not in OA) were positively associated with IgG and IgM anti-MAA. Anti-P. gingivalis and anti-P. intermedia antibody concentrations were positively associated with IgA (p = 0.001 for both), IgG (p = 0.007 and p = 0.034, respectively), and IgM anti-MAA antibody concentrations (p < 0.001 and p = 0.020, respectively), while anti-F. nucleatum was positively associated with IgG anti-MAA (p = 0.042), findings that were similar across groups., Conclusions: A positive association was demonstrated between ABL and serum IgG and IgM anti-MAA antibody concentrations that was unique to RA and not observed in OA. Serum anti-P. gingivalis, anti-P. intermedia, and anti-F. nucleatum antibody concentrations displayed significant associations with anti-MAA antibody in both groups. These findings suggest MAA may play a role in the interrelationship between the periodontium and RA., (© 2024 The Authors. Journal of Periodontology published by Wiley Periodicals LLC on behalf of American Academy of Periodontology.)

Porphyromonas gingivalis is the most pathogenic periodontal bacterium in the world. Recently, P. gingivalis has been considered responsible for dysbiosis during the development of periodontitis. This study aimed to evaluate a novel immunochromatographic device using monoclonal antibodies against P. gingivalis in subgingival plaques. A total of 72 patients with chronic periodontitis and 53 periodontally healthy volunteers underwent clinical and microbiological examinations. Subgingival plaque samples were analyzed for the presence of P. gingivalis and compared using real-time polymerase chain reaction (PCR). In the periodontitis group, a significant positive correlation was observed between the test device scores and the real-time PCR results. The specificity, positive predictive value, negative predictive value, and accuracy of the test device for P. gingivalis , as determined by real-time PCR, were 98%, 94%, 89%, and 90%, respectively. There were significant differences in bacterial counts by real-time PCR among the groups with different ranges of device scores. Additionally, there was a significant positive correlation between the device scores for P. gingivalis and periodontal parameters. These results suggest that this novel immunochromatographic device can be effectively used for rapid detection and semi-quantification of P. gingivalis in subgingival plaques.

Periodontitis is a chronic inflammatory disease that affects the periodontal tissues. Although it is associated with various systemic diseases, the impact of periodontitis on kidney transplantation (KT) outcomes, particularly allograft rejection, remains unclear. This study investigated the effect of periodontitis on transplant immunity, specifically examining Porphyromonas gingivalis-derived lipopolysaccharide (LPS-PG). In vitro experiments revealed that LPS-PG increased regulatory T cells (Tregs) in Lewis rat spleen cells. In a mixed lymphocyte reaction assay, concentrations of interferon-γ, indicative of alloreactivity, were lower than in controls when LPS-PG was added to the culture and when LPS-PG-administered Lewis rat spleen cells were used as responders. In a rat KT model, LPS-PG administration to recipients promoted mild tubulitis and low serum creatinine and blood urea nitrogen levels 5 days post-KT compared with PBS-administered controls. Furthermore, LPS-PG-administered recipients had an elevated Treg proportion in their peripheral blood and spleen cells, and increased infiltrating Tregs in kidney allografts, compared with controls. The elevated Treg proportion in peripheral blood and spleen cells had a significant negative correlation with serum creatinine, suggesting elevated Tregs modulated allograft rejection. These findings suggest that periodontitis might modulate alloimmune reactivity through LPS-PG and Tregs, offering insights to refine immunosuppressive strategies for KT recipients., (© 2024. The Author(s).)

Background: The aim of this study was to investigate the impact of Porphyromonas gingivalis (P. gingivalis) on the progression of oral squamous cell carcinoma (OSCC) through neutrophil extracellular traps (NETs) in the tumor immune microenvironment., Methods: The expression of NETs-related markers was identified through immunohistochemistry, immunofluorescence, and Western blotting in different clinical stages of OSCC samples. The relationship between NETs-related markers and clinicopathological characteristics in 180 samples was analyzed using immunohistochemistry data. Furthermore, the ability to predict the prognosis of OSCC patients was determined by ROC curve analysis and survival analysis. The effect of P. gingivalis on the release of NETs was identified through immunofluorescence and immunohistochemistry, both in vitro and in vivo. CAL27 and SCC25 cell lines were subjected to NETs stimulation to elucidate the influence of NETs on various cellular processes, including cell proliferation, migration, invasion, and metastasis in vitro. Furthermore, the impact of NETs on the growth and metastatic potential of OSCC was assessed using in vivo models involving tumor-bearing mice and tumor metastasis mouse models., Results: Immunochemistry analysis revealed a significant correlation between the NETs-related markers and clinical stage, living status as well as TN stage. P. gingivalis has demonstrated its ability to effectively induce the release of NETs both in vivo and in vitro. NETs have the potential to facilitate cell migration, invasion, and colony formation. Moreover, in vivo experiments have demonstrated that NETs play a pivotal role in promoting tumor metastasis., Conclusion: High expression of NETs-related markers demonstrates a strong correlation with the progression of OSCC. Inhibition of the NETs release process stimulated by P. gingivalis and targeted NETs could potentially open up a novel avenue in the field of immunotherapy for patients afflicted with OSCC., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Background: Periodontitis is a chronic infectious disease, characterized by an exacerbated inflammatory response and a progressive loss of the supporting tissues of the teeth. Porphyromonas gingivalis is a key etiologic agent in periodontitis. Cystatin C is an antimicrobial salivary peptide that inhibits the growth of P. gingivalis . This study aimed to evaluate the antimicrobial activity of this peptide and its effect on cytokine production, nitric oxide (NO) release, reactive oxygen species (ROS) production, and programmed cell death in human macrophages infected with P. gingivalis., Methods: Monocyte-derived macrophages generated from peripheral blood were infected with P. gingivalis (MOI 1:10) and stimulated with cystatin C (2.75 µg/ml) for 24 h. The intracellular localization of P. gingivalis and cystatin C was determined by immunofluorescence and transmission electron microscopy (TEM). The intracellular antimicrobial activity of cystatin C in macrophages was assessed by counting Colony Forming Units (CFU). ELISA assay was performed to assess inflammatory (TNFα, IL-1β) and anti-inflammatory (IL-10) cytokines. The production of nitrites and ROS was analyzed by Griess reaction and incubation with 2',7'-dichlorodihydrofluorescein diacetate (H 2 DCFDA), respectively. Programmed cell death was assessed with the TUNEL assay, Annexin-V, and caspase activity was also determined., Results: Our results showed that cystatin C inhibits the extracellular growth of P. gingivalis . In addition, this peptide is internalized in the infected macrophage, decreases the intracellular bacterial load, and reduces the production of inflammatory cytokines and NO. Interestingly, peptide treatment increased ROS production and substantially decreased bacterial-induced macrophage apoptosis., Conclusions: Cystatin C has antimicrobial and immuno-regulatory activity in macrophages infected with P. gingivalis. These findings highlight the importance of understanding the properties of cystatin C for its possible therapeutic use against oral infections such as periodontitis., Competing Interests: The authors declare there are no competing interests., (©2024 Blancas-Luciano et al.)

During periodontitis, the extracellular capsule of Porphyromonas gingivalis favors alveolar bone loss by inducing Th1 and Th17 patterns of lymphocyte response in the infected periodontium. Dendritic cells recognize bacterial antigens and present them to T lymphocytes, defining their activation and polarization. Thus, dendritic cells could be involved in the Th1 and Th17 response induced against the P. gingivalis capsule. Herein, monocyte-derived dendritic cells were obtained from healthy individuals and then stimulated with different encapsulated strains of P. gingivalis or two non-encapsulated isogenic mutants. Dendritic cell differentiation and maturation were analyzed by flow cytometry. The mRNA expression levels for distinct Th1-, Th17-, or T-regulatory-related cytokines and transcription factors, as well as TLR2 and TLR4, were assessed by qPCR. In addition, the production of IL-1β, IL-6, IL-23, and TNF-α was analyzed by ELISA. The encapsulated strains and non-encapsulated mutants of P. gingivalis induced dendritic cell maturation to a similar extent; however, the pattern of dendritic cell response was different. In particular, the encapsulated strains of P. gingivalis induced higher expression of IRF4 and NOTCH2 and production of IL-1β, IL-6, IL-23, and TNF-α compared with the non-encapsulated mutants, and thus, they showed an increased capacity to trigger Th1 and Th17-type responses in human dendritic cells.

The interaction between the gut microbiota and invariant Natural Killer T (iNKT) cells plays a pivotal role in colorectal cancer (CRC). The pathobiont Fusobacterium nucleatum influences the anti-tumor functions of CRC-infiltrating iNKT cells. However, the impact of other bacteria associated with CRC, like Porphyromonas gingivalis , on their activation status remains unexplored. In this study, we demonstrate that mucosa-associated P. gingivalis induces a protumour phenotype in iNKT cells, subsequently influencing the composition of mononuclear-phagocyte cells within the tumor microenvironment. Mechanistically, in vivo and in vitro experiments showed that P. gingivalis reduces the cytotoxic functions of iNKT cells, hampering the iNKT cell lytic machinery through increased expression of chitinase 3-like-1 protein (CHI3L1). Neutralization of CHI3L1 effectively restores iNKT cell cytotoxic functions suggesting a therapeutic potential to reactivate iNKT cell-mediated antitumour immunity. In conclusion, our data demonstrate how P. gingivalis accelerates CRC progression by inducing the upregulation of CHI3L1 in iNKT cells, thus impairing their cytotoxic functions and promoting host tumor immune evasion.

Background/purpose: The signaling mechanisms for Porphyromonas gingivalis lipopolysaccharide (PgLPS)-induced inflammation in human dental pulp cells are not fully clarified. This in vitro study aimed to evaluate the involvement of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway in PgLPS-induced pulpal inflammation., Methods: Human dental pulp cells (HDPCs) were challenged with PgLPS with or without pretreatment and coincubation with a PI3K/Akt inhibitor (LY294002). The gene or protein levels of PI3K, Akt, interleukin (IL)-6, IL-8, alkaline phosphatase (ALP), osteocalcin and osteonectin were analyzed by reverse transcription polymerase chain reaction (PCR), real-time PCR, western blotting, and immunofluorescent staining. In addition, an enzyme-linked immunosorbent assay was used to analyze IL-6 and IL-8 levels in culture medium., Results: In response to 5 μg/ml PgLPS, IL-6, IL-8, and PI3K, but not Akt mRNA expression of HDPCs, was upregulated. IL-6, IL-8, PI3K, and p-Akt protein levels were stimulated by 10-50 μg/ml of PgLPS in HDPCs. PgLPS also induced IL-6 and IL-8 secretion at concentrations higher than 5 μg/ml. Pretreatment and co-incubation by LY294002 attenuated PgLPS-induced IL-6 and IL-8 mRNA expression in HDPCs. The mRNA expression of ALP, but not osteocalcin and osteonectin, was inhibited by higher concentrations of PgLPS in HDPCs., Conclusion: P. gingivalis contributes to pulpal inflammation in HDPCs by dysregulating PI3K/Akt signaling pathway to stimulate IL-6 and IL-8 mRNA/protein expression and secretion. These results are useful for understanding the pulpal inflammation and possible biomarkers of inflamed pulp diagnosis and treatment., Competing Interests: Declaration of competing interest The authors have no conflicts of interest relevant to this article., (Copyright © 2022 Formosan Medical Association. Published by Elsevier B.V. All rights reserved.)

Objectives: There is accumulating evidence that periodontal disease is associated with atrial fibrillation (AF) or stroke, but it is unclear which causative species of periodontal disease are present in stroke patients with AF. We aimed to investigate the associations between AF and specific periodontal pathogens using serum titers of IgG antibodies of bacteria in acute stroke patients., Materials and Methods: Acute stroke patients were registered at two hospitals. Serum samples were evaluated for titers of antibodies against 9 periodontal pathogens (16 genotypes) using ELISAs. We identified AF in patients according to the following criteria: (1) a history of sustained or paroxysmal AF or (2) AF detection upon arrival or during admission. We carried out propensity score matching to categorize the patients as those with AF and those without., Results: Of the 664 acute stroke patients, 123 (18.5%) had AF. After propensity score matching, 234 patients were selected. Patients with AF had a higher prevalence of positive serum titers of antibodies against Porphyromonas gingivalis (FimA type III) and Porphyromonas gingivalis (FimA type V) than those without AF (59.0% vs. 39.3%, p=0.004 and 58.2% vs. 40.2%, p=0.009, respectively)., Conclusions: Porphyromonas gingivalis, especially FimA type III and type V, might be associated with AF in stroke patients., Competing Interests: Declaration of Competing Interest Dr. Maruyama received Grants-in-Aid for Scientific Research (20K07887) and research support from Eisai, Pfizer, Takeda Pharmaceutical, Otsuka Pharmaceutical, Nihon Pharmaceutical, Shionogi, Teijin Pharma, Fuji Film, Sumitomo Dainippon Pharma, Nihon Medi-Physics, Bayer, MSD, Daiichi Sankyo, Kyowa Hakko Kirin, Sanofi, Novartis, Kowa Pharmaceutical, Astellas Pharma, Tsumura, and Japan Blood Products Organization, which are unrelated to the submitted work., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Porphyromonas gingivalis is a Gram-negative pathogenic bacterium associated with chronic periodontitis. The development of a chimeric peptide-based vaccine targeting this pathogen could be highly beneficial in preventing oral bone loss as well as other severe gum diseases. We applied a computational framework to design a multi-epitope-based vaccine candidate against P. gingivalis . The vaccine comprises epitopes from subunit proteins prioritized from the P. gingivalis reference strain ( P. gingivalis ATCC 33277) using several reported vaccine properties. Protein-based subunit vaccines were prioritized through genomics techniques. Epitope prediction was performed using immunoinformatic servers and tools. Molecular modeling approaches were used to build a putative three-dimensional structure of the vaccine to understand its interactions with host immune cells through biophysical techniques such as molecular docking simulation studies and binding free energy methods. Genome subtraction identified 18 vaccine targets: six outer-membrane, nine cytoplasmic membrane-, one periplasmic, and two extracellular proteins. These proteins passed different vaccine checks required for the successful development of a vaccine candidate. The shortlisted proteins were subjected to immunoinformatic analysis to map B-cell derived T-cell epitopes, and antigenic, water-soluble, non-toxic, and good binders of DRB1*0101 were selected. The epitopes were then modeled into a multi-epitope peptide vaccine construct (linked epitopes plus adjuvant) to enhance immunogenicity and effectively engage both innate and adaptive immunity. Further, the molecular docking approach was used to determine the binding conformation of the vaccine to TLR2 innate immune receptor. Molecular dynamics simulations and binding free energy calculations of the vaccine-TLR2 complex were performed to highlight key intermolecular binding energies. Findings of this study will be useful for vaccine developers to design an effective vaccine for chronic periodontitis pathogens, specifically P. gingivalis ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Shaker, Ahmad, Shen, Kim and Na.)

Periodontitis is characterized by bacterially induced inflammatory destruction of periodontal tissue. This also affects fibroblasts of the human periodontal ligaments (HPdLF), which play a coordinating role in force-induced tissue and alveolar bone remodeling. Excessive inflammation in the oral tissues has been observed with simultaneous stimulation by pathogens and mechanical forces. Recently, elevated levels of growth differentiation factor 15 (GDF15), an immuno-modulatory member of the transforming growth factor (TGFB) superfamily, were detected under periodontitis-like conditions and in force-stressed PdL cells. In view of the pleiotropic effects of GDF15 in various tissues, this study aims to investigate the role of GDF15 in P. gingivalis -related inflammation of HPdLF and its effect on the excessive inflammatory response to concurrent compressive stress. To this end, the expression and secretion of cytokines (IL6, IL8, COX2/PGE2, TNFα) and the activation of THP1 monocytic cells were analyzed in GDF15 siRNA-treated HPdLF stimulated with P. gingivalis lipopolysaccharides alone and in combination with compressive force. GDF15 knockdown significantly reduced cytokine levels and THP1 activation in LPS-stimulated HPdLF, which was less pronounced with additional compressive stress. Overall, our data suggest a pro-inflammatory role for GDF15 in periodontal disease and demonstrate that GDF15 partially modulates the force-induced excessive inflammatory response of PdLF under these conditions.

Periodontal diseases are common inflammatory diseases that are induced by infection with periodontal bacteria such as Porphyromonas gingivalis (Pg). The association between periodontal diseases and many types of systemic diseases has been demonstrated; the term "periodontal medicine" is used to describe how periodontal infection/inflammation may impact extraoral health. However, the molecular mechanisms by which the factors produced in the oral cavity reach multiple distant organs and impact general health have not been elucidated. Extracellular vesicles (EVs) are nano-sized spherical structures secreted by various types of cells into the tissue microenvironment, and influence pathophysiological conditions by delivering their cargo. However, a detailed understanding of the effect of EVs on periodontal medicine is lacking. In this study, we investigated whether EVs derived from Pg-infected macrophages reach distant organs in mice and influence the pathophysiological status. EVs were isolated from human macrophages, THP-1 cells, infected with Pg. We observed that EVs from Pg-infected THP-1 cells (Pg-inf EVs) contained abundant core histone proteins such as histone H3 and translocated to the lungs, liver, and kidneys of mice. Pg-inf EVs also induced pulmonary injury, including edema, vascular congestion, inflammation, and collagen deposition causing alveoli destruction. The Pg-inf EVs or the recombinant histone H3 activated the NF-κB pathway, leading to increase in the levels of pro-inflammatory cytokines in human lung epithelial A549 cells. Our results suggest a possible mechanism by which EVs produced in periodontal diseases contribute to the progression of periodontal medicine., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Periodontitis involves chronic inflammation of the tissues around the teeth caused by plaque and the corresponding immune response. Growth arrest-specific protein 6 (GAS6) and AXL receptor tyrosine kinase (AXL) are known to be involved in inflammatory diseases, while signal transducer and activator of transcription-1 (STAT1) and suppressor of cytokine signaling (SOCS) are related to inflammatory processes. Moreover, miRNA34a directly targets AXL to regulate the AXL expression. However, the specific roles of GAS6 and AXL in periodontitis remain unclear. This study was designed to explore the effect and mechanism of AXL on the expression of inflammatory cytokines induced by Porphyromonas gingivalis lipopolysaccharide ( P. gingivalis LPS) in human periodontal ligament cells (hPDLCs). The effects of different concentrations of P. gingivalis LPS on the expression of GAS6/AXL in hPDLCs were observed. Additionally, the effect of LPS on AXL was investigated by transfection of the miRNA34a inhibitor. AXL was knocked down or overexpressed to observe the release of inflammatory cytokines interleukin- (IL-) 8 and IL-6. The results showed that the expression levels of GAS6 and AXL decreased after P. gingivalis LPS infection. Transfection of a miR-34a inhibitor to hPDLCs demonstrated a role of miR-34a in the downregulation of AXL expression induced by LPS. Moreover, AXL knockdown or overexpression influencing the expression of IL-8 and IL-6 was investigated under LPS stimulation. AXL knockdown decreased the expression of STAT1 and SOCS1/3. Overall, these results demonstrate that AXL inhibits the expression of LPS-induced inflammatory cytokines in hPDLCs and that STAT1 and SOCS1/3 are involved in the regulation of inflammation by GAS6/AXL., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2021 Shengnan Zhang et al.)

Periodontitis induced by bacteria especially Porphyromonas gingivalis (P. gingivalis) is the most prevalent microbial disease worldwide and is a significant risk factor for systemic diseases such as rheumatoid arthritis (RA). RA and periodontitis share similar clinical and pathologic features. Moreover, the prevalence of RA is much higher in patients with periodontitis than in those without periodontitis. To explore the immunologic mechanism of periodontitis involved in RA, we established a mouse model of periodontitis and then induced RA. According to the results of paw thickness, arthritis clinical score, arthritis incidence, microscopic lesion using H&E staining, and micro-CT analysis, periodontitis induced by P. gingivalis promoted the occurrence and development of collagen-induced arthritis (CIA) in mice. Furthermore, periodontitis enhanced the frequency of CD19 + B cells, Th17, Treg, gMDSCs, and mMDSCs, whereas down-regulated IL-10 producing regulatory B cells (B10) in CIA mice preinduced for periodontitis with P. gingivalis. In vitro stimulation with splenic cells revealed that P. gingivalis directly enhanced differentiation of Th17, Treg, and mMDSCs but inhibited the process of B cell differentiation into B10 cells. Considering that adoptive transfer of B10 cells prevent RA development, our study, although preliminary, suggests that down-regulation of B10 cells may be the key mechanism that periodontitis promotes RA as the other main immune suppressive cells such as Treg and MDSCs are up-regulated other than down-regulated in group of P. gingivalis plus CIA., (©2021 Society for Leukocyte Biology.)

Aims: Periodontitis is an independent risk factor for cardiovascular disease, but the mechanistic link is not fully understood. In atherosclerotic cardiovascular disease, monocytes can adopt a persistent hyperresponsive phenotype, termed trained immunity. We hypothesized that periodontitis-associated bacteria can induce trained immunity in monocytes, which subsequently accelerate atherosclerosis development., Materials and Methods: We combined in vitro experiments on human primary monocytes and in vivo techniques in patients with periodontitis to test this hypothesis. Adherent peripheral blood mononuclear cells (PBMCs) were transiently exposed in vitro to Porphyromonas gingivalis for 24 hours, and restimulated with lipopolysaccharide (LPS) or Pam3CysK4 (P3C) six days later, to measure interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) production. In an exploratory observational study, patients with severe periodontitis (63 ± 6 years, n=14) and control subjects with no-to-mild periodontitis (54 ± 10 years, n=14) underwent venipuncture and 2'-deoxy-2'-[ 18 F]fluoro-D-glucose positron-emission-tomography ([ 18 F]FDG PET/CT) scanning., Results: When adherent peripheral blood mononuclear cells (PBMCs) were transiently exposed in vitro to Porphyromonas gingivalis for 24 hours, and restimulated with LPS or P3C six days later, IL-6 and TNFα production was significantly increased (TNFα/P3C, p<0.01). Circulating leukocytes, IL-6 and interleukin-1 receptor antagonist (IL-1Ra) concentrations were generally higher in patients compared to controls (leukocytes: p<0.01; IL-6: p=0.08; IL-1Ra: p=0.10). Cytokine production capacity in PBMCs after 24h stimulation revealed no differences between groups. [ 18 F]FDG PET/CT imaging showed a trend for increased [ 18 F]FDG-uptake in the periodontium [mean standard uptake value (SUV mean ), p=0.11] and in femur bone marrow (SUV mean , p=0.06), but no differences were observed for vascular inflammation. Positive correlations between severity of periodontitis, measured by The Dutch Periodontal Screening Index and pocket depth, with circulating inflammatory markers and tissue inflammation were found., Conclusions: P. gingivalis induces long-term activation of human monocytes in vitro (trained immunity). Patients with severe periodontitis did have signs of increased systemic inflammation and hematopoietic tissue activation. However, their circulating monocytes did not show a hyperresponsive phenotype. Together we suggest that trained immunity might contribute to local periodontal inflammation which warrants further investigation., Competing Interests: LJ and MGN are scientific founders of TTxD. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Noz, Plachokova, Smeets, Aarntzen, Bekkering, Vart, Joosten, Netea and Riksen.)

Titanium is often used in the medical field and in dental implants due to its biocompatibility, but it has a high rate of leading to peri-implantitis, which progresses faster than periodontitis. Therefore, in the present study, the expression of cytokines from gingival epithelial cells by nanotitania was investigated, which is derived from titanium in the oral cavity, and the additional effect of Porphyromonas gingivalis (periodontopathic bacteria) lipopolysaccharide (PgLPS) was investigated. Ca9-22 cells were used as a gingival epithelial cell model and were cultured with nanotitania alone or with PgLPS. Cytokine expression was examined by reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. In addition, cellular uptake of nanotitania was observed in scanning electron microscopy images. The expression of interleukin (IL)-6 and IL-8 significantly increased in Ca9-22 cells by nanotitania treatment alone, and the expression was further increased by the presence of PgLPS. Nanotitania was observed to phagocytose Ca9-22 cells in a dose- and time-dependent manner. Furthermore, when the expression of IL-11, related to bone resorption, was investigated, a significant increase was confirmed by stimulation with nanotitania alone. Therefore, nanotitania could be associated with the onset and exacerbation of peri-implantitis, and the presence of periodontal pathogens may worsen the condition. Further clinical reports are needed to confirm these preliminary results.

Periodontal disease (PD) is an inflammatory disease of the supporting tissues of the teeth that develops in response to formation of a dysbiotic biofilm on the subgingival tooth surface. Although exacerbated inflammation leads to alveolar bone destruction and may cause tooth loss, the molecular basis of PD initiation and progression remains elusive. Control over the inflammatory reaction and return to homeostasis can be efficiently restored by negative regulators of Toll-like receptor (TLR) signaling pathways such as monocyte chemoattractant protein-induced protein 1 (MCPIP-1), which is constitutively expressed in gingival keratinocytes and prevents hyperresponsiveness in the gingiva. Here, we found that inflammophilic periodontal species influence the stability of MCPIP-1, leading to an aggravated response of the epithelium to proinflammatory stimulation. Among enzymes secreted by periodontal species, gingipains-cysteine proteases from Porphyromonas gingivalis-are considered major contributors to the pathogenic potential of bacteria, strongly influencing the components of the innate and adaptive immune system. Gingipain proteolytic activity leads to a rapid degradation of MCPIP-1, exacerbating the inflammatory response induced by endotoxin. Collectively, these results establish a novel mechanism of corruption of inflammatory signaling by periodontal pathogens, indicating new possibilities for treatment of this chronic disease. IMPORTANCE Periodontitis is a highly prevalent disease caused by accumulation of a bacterial biofilm. Periodontal pathogens use a number of virulence strategies that are under intensive study to find optimal therapeutic approaches against bone loss. In our work, we present a novel mechanism utilized by the key periodontal pathogen Porphyromonas gingivalis, based on the selective degradation of the negative regulator of inflammation, MCPIP-1. We found that the diminished levels of MCPIP-1 in gingival keratinocytes-cells at the forefront of the fight against bacteria-cause sensitization to endotoxins produced by other oral species. This results in an enhanced inflammatory response, which promotes the growth of inflammophilic pathobionts and damage of tooth-supporting tissues. Our observation is relevant to understanding the molecular basis of periodontitis and the development of new methods for treatment.

Porphyromonas gingivalis ( P. gingivalis ) is a major bacterial pathogen that causes periodontitis, a chronic inflammatory disease of tissues around the teeth. Periodontitis is known to be related to other diseases, such as oral cancer, Alzheimer's disease, and rheumatism. Thus, a precise and sensitive test to detect P. gingivalis is necessary for the early diagnosis of periodontitis. The objective of this study was to optimize a rapid visual detection system for P. gingivalis . First, we performed a visual membrane immunoassay using 3,3',5,5'-tetramethylbenzidine (TMB; blue) and coating and detection antibodies that could bind to the host laboratory strain, ATCC 33277. Antibodies against the P. gingivalis surface adhesion molecules RgpB (arginine proteinase) and Kgp (lysine proteinase) were determined to be the most specific coating and detection antibodies, respectively. Using these two selected antibodies, the streptavidin-horseradish peroxidase (HRP) reaction was performed using a nitrocellulose membrane and visualized with a detection range of 10 3 -10 5 bacterial cells/ml following incubation for 15 min. These selected conditions were applied to test other oral bacteria, and the results showed that P. gingivalis could be detected without crossreactivity to other bacteria, including Streptococcus mutans and Escherichia fergusonii . Furthermore, three clinical strains of P. gingivalis , KCOM 2880, KCOM 2803, and KCOM 3190, were also recognized using this optimized enzyme immunoassay (EIA) system. To conclude, we established optimized conditions for P. gingivalis detection with specificity, accuracy, and sensitivity. These results could be utilized to manufacture economical and rapid detection kits for P. gingivalis .

Our previous studies have shown that inoculation of the oral cavity of "humanized" B6.DR1/4 mice with the periodontal pathogen Porphyromonas gingivalis results in an increase in the percentage of circulating Th17 cells, loss of bone and an exacerbation of experimental autoimmune arthritis. The aim of this study was to assess the role played by the human HLA-DRβ molecule containing the shared epitope supplied as a transgene to I-A˚ (murine class II null) C57BL/6 (B6) mice in driving these findings. We compared various immune response parameters as well as alveolar and peri-articular bone loss between humanized B6.DR1 (or B6.DR4) mice and their WT (B6) counterparts. We found that the presence of the shared epitope in the context of inoculation with P. gingivalis enhanced the percentage of Th17 cells generated, dramatically enhanced bone loss and importantly allowed for the generation of CCP2⁺ ACPAs that are not found in C57BL/6 or DBA/1 arthritic mouse serum. Due to the exceedingly complex nature of environmental factors impacting on genetic elements, it has been difficult to unravel mechanisms that drive autoimmune arthritis in susceptible individuals. The findings in this study may provide one small piece of this puzzle that can help us to better understand part of this complexity., Competing Interests: The authors have declared that no competing interests exist.

Objective: Both periodontal disease and cardiovascular disease (CVD) are overrepresented in rheumatoid arthritis (RA). This study was undertaken to investigate the contribution of periodontal pathogens to CVD in RA., Methods: RA patients underwent assessments of coronary artery calcification (CAC), carotid intima-media thickness and plaque, and ankle-brachial index via computed tomography, ultrasound, and Doppler ultrasound, respectively. Sera were assayed for antibodies targeting Porphyromonas gingivalis (Pg), Aggregatibacter actinomycetemcomitans serotype B (Aa), and Aa-derived leukotoxin A (LtxA). Associations of antibodies against these periodontal pathogens with measures of atherosclerosis were explored using generalized linear models., Results: Among 197 RA patients, anti-Pg was detected in 72 patients (37%), anti-Aa in 41 patients (21%), and anti-LtxA in 84 patients (43%). Adjusting for relevant confounders and reported tooth loss, the mean CAC score was 90% higher in those with anti-Aa and/or anti-LtxA compared with those without either antibody (19 units versus 10 units; P = 0.033). The adjusted odds of CAC ≥100 units were 2.23-fold higher in those with anti-Aa and/or anti-LtxA compared with those without either antibody (P = 0.040). Anti-Aa and/or anti-LtxA seropositivity was significantly associated with all other assessed measures of atherosclerosis except carotid plaque. Anti-Pg was not associated with any measure of atherosclerosis. Higher swollen joint count was associated with CAC exclusively in the group with anti-Aa and/or anti-LtxA., Conclusion: Immunoreactivity against Aa and/or its major virulence factor LtxA was associated with atherosclerosis in multiple vascular beds of RA patients and amplified the effect of swollen joints on coronary atherosclerosis, suggesting a role for treatment/prevention of periodontal disease in the prevention of CVD in RA., (© 2021, American College of Rheumatology.)

Sphingolipids (SLs) are essential structural components of mammalian cell membranes. Our group recently determined that the oral anaerobe Porphyromonas gingivalis delivers its SLs to host cells and that the ability of P. gingivalis to synthesize SLs limits the elicited host inflammatory response during cellular infection. As P. gingivalis robustly produces outer membrane vesicles (OMVs), we hypothesized that OMVs serve as a delivery vehicle for SLs, that the SL status of the OMVs may impact cargo loading to OMVs, and that SL-containing OMVs limit elicited host inflammation similar to that observed by direct bacterial challenge. Transwell cell culture experiments determined that in comparison to the parent strain W83, the SL-null mutant elicited a hyperinflammatory immune response from THP-1 macrophage-like cells with elevated tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), and IL-6. Targeted assessment of Toll-like receptors (TLRs) identified elevated expression of TLR2, unchanged TLR4, and elevated expression of the adaptor molecules MyD88 and TRIF (Toll/IL-1 receptor domain-containing adaptor-inducing beta interferon) by SL-null P. gingivalis No significant differences in gingipain activity were observed in our infection models, and both strains produced OMVs of similar sizes. Using comparative two-dimensional gel electrophoresis, we identified differences in the protein cargo of the OMVs between parent and SL-null strain. Importantly, use of purified OMVs recapitulated the cellular inflammatory response observed in the transwell system with whole bacteria. These findings provide new insights into the role of SLs in P. gingivalis OMV cargo assembly and expand our understanding of SL-OMVs as bacterial structures that modulate the host inflammatory response., (Copyright © 2021 Rocha et al.)

Our previous studies showed that a combination of a DNA plasmid encoding Flt3 ligand (pFL) and CpG oligodeoxynucleotides 1826 (CpG ODN) (FL/CpG) as a nasal adjuvant provoked antigen-specific immune responses. In this study, we investigated the efficacy of a nasal vaccine consisting of FimA as the structural subunit of Porphyromonas gingivalis ( P. gingivalis ) fimbriae and FL/CpG for the induction of FimA-specific antibody (Ab) responses and their protective roles against nasal and lung infection by P. gingivalis , a keystone pathogen in the etiology of periodontal disease. C57BL/6 mice were nasally immunized with recombinant FimA ( r FimA) plus FL/CpG three times at weekly intervals. As a control, mice were given nasal r FimA alone. Nasal washes (NWs) and bronchoalveolar lavage fluid (BALF) of mice given nasal r FimA plus FL/CpG resulted in increased levels of r FimA-specific secretory IgA (SIgA) and IgG Ab responses when compared with those in controls. Significantly increased numbers of CD8- or CD11b-expressing mature-type dendritic cells (DCs) were detected in the respiratory inductive and effector tissues of mice given r FimA plus FL/CpG. Additionally, significantly upregulated Th1/Th2-type cytokine responses by r FimA-stimulated CD4 + T cells were noted in the respiratory effector tissues. When mice were challenged with live P. gingivalis via the nasal route, mice immunized nasally with r FimA plus FL/CpG inhibited P. gingivalis colonization in the nasal cavities and lungs. In contrast, controls failed to show protection. Of interest, when IgA-deficient mice given nasal r FimA plus FL/CpG were challenged with nasal P. gingivalis , the inhibition of bacterial colonization in the respiratory tracts was not seen. Taken together, these results show that nasal FL/CpG effectively enhanced DCs and provided balanced Th1- and Th2-type cytokine response-mediated r FimA-specific IgA protective immunity in the respiratory tract against P. gingivalis. A nasal administration with r FimA and FL/CpG could be a candidate for potent mucosal vaccines for the elimination of inhaled P. gingivalis in periodontal patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial and financial relationships that could be construed as a potential conflict of interest. The reviewer SM declared a shared affiliation with one of the authors, KF, to the handling editor at the time of review. The reviewer ZM declared a shared affiliation with one of the authors, KF, to the handling editor at the time of review., (Copyright © 2021 Kataoka, Kawabata, Koyanagi, Hashimoto, Miyake and Fujihashi.)

Objectives: An increased prevalence of periodontitis and perturbation of the oral microbiome has been identified in patients with rheumatoid arthritis (RA). The periodontal pathogen Porphyromonas gingivalis may cause local citrullination of proteins, potentially triggering anti-citrullinated protein antibody production. However, it is not known if oral dysbiosis precedes the onset of clinical arthritis. This study comprehensively characterised the oral microbiome in anti-cyclic citrullinated peptide (anti-CCP) positive at-risk individuals without clinical synovitis (CCP+at risk)., Methods: Subgingival plaque was collected from periodontally healthy and diseased sites in 48 CCP+at risk, 26 early RA and 32 asymptomatic healthy control (HC) individuals. DNA libraries were sequenced on the Illumina HiSeq 3000 platform. Taxonomic profile and functional capability of the subgingival microbiome were compared between groups., Results: At periodontally healthy sites, CCP+at risk individuals had significantly lower microbial richness compared with HC and early RA groups (p=0.004 and 0.021). Microbial community alterations were found at phylum, genus and species levels. A large proportion of the community differed significantly in membership (523 species; 35.6%) and structure (575 species; 39.1%) comparing CCP+at risk and HC groups. Certain core species, including P. gingivalis , had higher relative abundance in the CCP+at risk group. Seventeen clusters of orthologous gene functional units were significantly over-represented in the CCP+at risk group compared with HC (adjusted p value <0.05)., Conclusion: Anti-CCP positive at-risk individuals have dysbiotic subgingival microbiomes and increased abundance of P. gingivalis compared with controls. This supports the hypothesis that the oral microbiome and specifically P. gingivalis are important in RA initiation., Competing Interests: Competing interests: ZC reports scholarship from the China Scholarship Council during the conduct of the study. Dr TD and JM reports grants from Colgate Palmolive, outside the submitted work. KM reports grants from the National Institute for Health Research (NIHR) Leeds Biomedical Research Unit and grants from Leeds Biomedical Research Centre during the conduct of the study. DD reports grants from NIHR, grants from Wellcome Trust, during the conduct of the study; grants from Colgate Palmolive, outside the submitted work. PE reports grants and personal fees from Pfizer, Merck Sharp & Dohme, AbbVie, Bristol-Myers Squibb, Roche, Samsung, Sandoz, and Eli Lilly and Company; and personal fees from Novartis and UCB outside the submitted work., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Skeletal muscles have a high metabolic capacity, which play key roles in glucose metabolism. Although periodontal disease increases the risk of metabolic syndrome, the relationship between periodontal bacterial infection and skeletal muscle metabolic dysfunction is unclear. We found that anti-Porphyromonas gingivalis (Pg) antibody titers positively correlated with intramuscular adipose tissue content (IMAC), fasting blood glucose, and HOMA-IR in metabolic syndrome patients. In C57BL/6J mice fed a high-fat diet, recipients of oral Pg (HFPg) had impaired glucose tolerance, insulin resistance, and higher IMAC compared to recipients of saline (HFco). The soleus muscle in HFPg mice exhibited fat infiltration and lower glucose uptake with higher Tnfa expression and lower insulin signaling than in HFco mice. Gene set enrichment analysis showed that TNFα signaling via NFκB gene set was enriched in the soleus muscle of HFPg mice. Moreover, TNF-α also decreased glucose uptake in C2C12 myoblast cells in vitro. Based on 16S rRNA sequencing, Pg administration altered the gut microbiome, particularly by decreasing the abundance of genus Turicibacter. Microbial network of the gut microbiome was dramatically changed by Pg administration. Our findings suggest that infection with Pg is a risk factor for metabolic syndrome and skeletal muscle metabolic dysfunction via gut microbiome alteration., (© 2020 Federation of American Societies for Experimental Biology.)

Porphyromonas gingivalis (Pg) is a periodontopathic pathogen that may affect MUC5AC-related mucus hypersecretion along airway epithelial cells. Here, we attempted to establish whether Pg virulence factors (lipopolysaccharide, FimA fimbriae, gingipains) affect MUC5AC in immortalized and primary bronchial cells. We report that MUC5AC gene expression and protein levels are affected by Pg culture supernatant, but not by lipopolysaccharide or FimA fimbriae. Cells treated with either Pg single (Kgp or Rgp) or double (Kgp/Rgp) mutants had altered levels of MUC5AC gene expression and protein levels, and MUC5AC staining of double mutant-treated mouse lung cells showed that MUC5AC protein levels were unaffected. Taken together, we propose that Pg gingipains may be the primary virulence factor that influences both MUC5AC gene expression and protein levels., (© 2020 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The majority of Gram-negative bacteria elicit a potent immune response via recognition of lipid A expressed on the outer bacterial membrane by the host immune receptor Toll-like receptor 4 (TLR4). However, some Gram-negative bacteria evade detection by TLR4 or alter the outcome of TLR4 signaling by modification of lipid A species. Although the role of lipid A modifications on host innate immunity has been examined in some detail, it is currently unclear how lipid A remodeling influences host adaptive immunity. One prototypic Gram-negative bacterium that modifies its lipid A structure is Porphyromonas gingivalis , an anaerobic pathobiont that colonizes the human periodontium and induces chronic low-grade inflammation that is associated with periodontal disease as well as a number of systemic inflammatory disorders. P. gingivalis produces dephosphorylated and deacylated lipid A structures displaying altered activities at TLR4. Here, we explored the functional role of P. gingivalis lipid A modifications on TLR4-dependent innate and adaptive immune responses in mouse bone marrow-derived dendritic cells (BMDCs). We discovered that lipid A 4'-phosphate removal is required for P. gingivalis to evade BMDC-dependent proinflammatory cytokine responses and markedly limits the bacterium's capacity to induce beta interferon (IFN-β) production. In addition, lipid A 4'-phosphatase activity prevents canonical bacterium-induced delay in antigen degradation, which leads to inefficient antigen cross-presentation and a failure to cross-prime CD8 T cells specific for a P. gingivalis -associated antigen. We propose that lipid A modifications produced by this bacterium alter host TLR4-dependent adaptive immunity to establish chronic infections associated with a number of systemic inflammatory disorders., (Copyright © 2021 American Society for Microbiology.)

Our previous study demonstrated that IL-10 secreting B (B10) cells alleviate inflammation and bone loss in experimental periodontitis. The purpose of this study is to determine whether antigen-specificity is required for the local infiltration of B10 cells. Experimental periodontitis was induced in the recipient mice by placement of silk ligature with or without the presence of live Porphyromonas gingivalis (P. gingivalis). Donor mice were pre-immunized by intraperitoneal (IP) injection of formalin-fixed P. gingivalis, or PBS as non-immunized control. Spleen B cells were purified and treated with LPS and CpG for 48 h to expand the B10 population in vitro. Fluorescence-labelled B10 cells were transferred into the recipient mice by tail vein injection and were tracked on day 0, 3, 5 and 10 using IVIS Spectrum in vivo imaging system. The number of B10 cells and P. gingivalis-binding B cells were significantly increased after in vitro treatment of LPS and CpG. On day 5, the fluorescence intensity in gingival tissues was the highest in mice transferred with B10 cells from pre-immunized donor mice. Gingival expression of IL-6, TNF-α, RANKL/OPG ratio and periodontal bone loss in recipient mice were significantly reduced, and the expression of IL-10 and the number of CD19+ B cells were significantly increased after pre-immunized B10 cell transfer in the presence of antigen, compared to those with non-immunized B10 cell transfer or no antigen presence. This study suggests that antigen specificity dictate the local infiltration of B10 cells into periodontal tissue and these antigen-specific B10 cells promote anti-inflammatory responses., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Bacteria release spherical nanobodies, known as membrane vesicles (MVs), during various growth phases. MVs have been gaining recognition as structurally stable vehicles in the last two decades because they deliver a wide range of antigens, virulence factors, and immunomodulators to the host. These functions suggest not only the possible contribution of MVs to pathogenicity but also the potential applicability of low-dose MVs for use as vaccines. Here, we describe a series of methods for isolating MVs of Porphyromonas gingivalis, which is an important species among periodontopathic bacteria. The present chapter also introduces a mouse model of intranasal immunization using MVs from P. gingivalis.

Periodontitis is a chronic inflammatory disease with alveolar bone resorption and subsequent tooth loss as its ultimate outcomes. Gastrin-releasing peptide (GRP) is a neuropeptide with growth-stimulatory and tumorigenic properties, and neuropeptides have previously been suggested to play a role in the complex cascade of chemical activity associated with periodontal inflammation. In this study, GRP treatment enhanced the differentiation of bone marrow-derived macrophages (BMMs) into osteoclasts, and gastrin-releasing peptide receptor (GRPR) antagonists suppressed the pro-osteoclastogenic effect of GRP. Grpr -siRNA knockdown resulted in a significantly lower number of osteoclasts formed as compared with the control. Interestingly, gene expression analysis indicated downregulation of Grp and Grpr expressions in BMMs during osteoclastogenesis. Moreover, ligature-induced periodontitis model in mice and gingival samples from patients with periodontitis displayed increased immunostaining of GRP in the oral epithelium. Subsequently, stimulation of mouse primary epithelial cells (ECs) and HaCaT cells, human epidermal keratinocytes, with lipopolysaccharides (LPS) of Porphyromonas gingivalis or live P. gingivalis upregulated Grp and Grpr expressions. Finally, coculture of P. gingivalis -stimulated ECs and BMMs using Transwell system revealed that the differentiation of BMMs was induced when subjected to paracrine activation by LPS- as well as live- P. gingivalis stimulated ECs. Taken together, our results demonstrate that the pro-osteoclastogenic properties of BMMs may be modulated by GRP produced by ECs in the periodontal microenvironment.

The current paradigm of onset and progression of periodontitis includes oral dysbiosis directed by inflammophilic bacteria, leading to altered resolution of inflammation and lack of regulation of the inflammatory responses. In the construction of explanatory models of the etiopathogenesis of periodontal disease, autoimmune mechanisms were among the first to be explored and historically, for more than five decades, they have been described in an isolated manner as part of the tissue damage process observed in periodontitis, however direct participation of these mechanisms in the tissue damage is still controversial. Autoimmunity is affected by genetic and environmental factors, leading to an imbalance between the effector and regulatory responses, mostly associated with failed resolution mechanisms. However, dysbiosis/infection and chronic inflammation could trigger autoimmunity by several mechanisms including bystander activation, dysregulation of toll-like receptors, amplification of autoimmunity by cytokines, epitope spreading, autoantigens complementarity, autoantigens overproduction, microbial translocation, molecular mimicry, superantigens, and activation or inhibition of receptors related to autoimmunity by microorganisms. Even though autoreactivity in periodontitis is biologically plausible, the associated mechanisms could be related to non-pathologic responses which could even explain non-recognized physiological functions. In this review we shall discuss from a descriptive point of view, the autoimmune mechanisms related to periodontitis physio-pathogenesis and the participation of oral dysbiosis on local periodontal autoimmune responses as well as on different systemic inflammatory diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Suárez, Garzón, Arboleda and Rodríguez.)

Background: The R1441G mutation in the leucine-rich repeat kinase 2 (LRRK2) gene results in late-onset Parkinson's disease (PD). Peripheral inflammation and gut microbiota are closely associated with the pathogenesis of PD. Chronic periodontitis is a common type of peripheral inflammation, which is associated with PD. Porphyromonas gingivalis (Pg), the most common bacterium causing chronic periodontitis, can cause alteration of gut microbiota. It is not known whether Pg-induced dysbiosis plays a role in the pathophysiology of PD., Methods: In this study, live Pg were orally administrated to animals, three times a week for 1 month. Pg-derived lipopolysaccharide (LPS) was used to stimulate mononuclear cells in vitro. The effects of oral Pg administration on the gut and brain were evaluated through behaviors, morphology, and cytokine expression., Results: Dopaminergic neurons in the substantia nigra were reduced, and activated microglial cells were increased in R1441G mice given oral Pg. In addition, an increase in mRNA expression of tumor necrosis factor (TNF-α) and interleukin-1β (IL-1β) as well as protein level of α-synuclein together with a decrease in zonula occludens-1 (Zo-1) was detected in the colon in Pg-treated R1441G mice. Furthermore, serum interleukin-17A (IL-17A) and brain IL-17 receptor A (IL-17RA) were increased in Pg-treated R1441G mice., Conclusions: These findings suggest that oral Pg-induced inflammation may play an important role in the pathophysiology of LRRK2-associated PD.

Bone destruction in inflammatory osteolytic diseases including periodontitis is related to excessive activity of osteoclasts (OC), which originate from precursor cells of the myeloid lineage, termed osteoclast precursors (OCP). In contrast to ample knowledge that we currently have on mature OC, little is known about OCP and their regulation during bacterial infection. Therefore, this study aimed to identify and characterize OCP following chronic infection with a periodontal bacteria Porphyromonas gingivalis ( Pg ). We used a micro-osmotic pump to continually release Pg subcutaneously in a murine model. Two weeks after Pg infection, the frequency of CD11b + c-fms + Ly6C hi population is significantly elevated within the bone marrow, spleen and peripheral blood. In vitro and in vivo studies identified these cells as the OCP-containing population and Pg infection significantly enhanced the osteoclastogenic activity of these cells. Furthermore, mRNA sequencing analysis indicated a unique gene and pathway profile in CD11b + c-fms + Ly6C hi population following Pg infection, with changes in genes and pathways related to OC differentiation, cell proliferation and apoptosis, inflammatory response, phagocytosis and immunity, as well as antigen processing and presentation. Moreover, using IL-6 knockout mice, we found that IL-6 is important for Pg -induced accumulation of CD11b + c-fms + Ly6C hi population from the bone marrow and periphery. Our results provide new insights into the characterization and regulation of OCP following a chronic bacterial infection. This knowledge is relevant to the understanding of the pathogenesis of bacteria-induced bone loss, and to the identification of potential therapeutic targets of bone loss diseases., Competing Interests: DISCLOSURE The authors declare no conflict of interest.

Bone destruction in inflammatory osteolytic diseases including periodontitis is related to excessive activity of osteoclasts (OC), which originate from precursor cells of the myeloid lineage, termed osteoclast precursors (OCP). In contrast to ample knowledge that we currently have on mature OC, little is known about OCP and their regulation during bacterial infection. Therefore, this study aimed to identify and characterize OCP following chronic infection with a periodontal bacteria Porphyromonas gingivalis (Pg). We used a microosmotic pump to continually release Pg subcutaneously in a murine model. Two weeks after Pg infection, the frequency of CD11b + c-fms + Ly6C hi population is significantly elevated within the bone marrow, spleen, and peripheral blood. In vitro and in vivo studies identified these cells as the OCP-containing population and Pg infection significantly enhanced the osteoclastogenic activity of these cells. Furthermore, mRNA sequencing analysis indicated a unique gene and pathway profile in CD11b + c-fms + Ly6C hi population following Pg infection, with changes in genes and pathways related to OC differentiation, cell proliferation and apoptosis, inflammatory response, phagocytosis, and immunity, as well as antigen processing and presentation. Moreover, using IL-6 knockout mice, we found that IL-6 is important for Pg-induced accumulation of CD11b + c-fms + Ly6C hi population from the bone marrow and periphery. Our results provide new insight into the characterization and regulation of OCP following a chronic bacterial infection. This knowledge is relevant to the understanding of the pathogenesis of bacteria-induced bone loss, and to the identification of potential therapeutic targets of bone loss diseases., (©2020 Society for Leukocyte Biology.)

Objectives: Porphyromonas gingivalis ( P.g .) is discussed to be involved in triggering self-reactive immune responses. The aim of this study was to investigate the autocitrullinated prokaryotic peptidylarginine deiminase (PPAD) from P.g . CH2007 (RA CH2007 -PPAD) from a rheumatoid arthritis (RA) patient and a synthetic citrullinated PPAD peptide (CPP) containing the main autocitrullination site as potential targets for antibody reactivity in RA and to analyse the possibility of citrullinating native human proteins by PPAD in the context of RA., Methods: Recombinant RA CH2007 -PPAD was cloned and expressed in Escherichia coli . Purified RA CH2007 -PPAD and its enzymatic activity was analysed using two-dimensional electrophoresis, mass spectrometry, immunoblot and ELISA. Autoantibody response to different modified proteins and peptides was recorded and bioinformatically evaluated., Results: RA CH2007 -PPAD was capable to citrullinate major RA autoantigens, such as fibrinogen, vimentin, hnRNP-A2/B1, histone H1 and multiple peptides, which identify a common RG/RGG consensus motif. 33% of RA patients (n=30) revealed increased reactivity for α-cit-RA CH2007 -PPAD before RA onset. 77% of RA patients (n=99) presented α-cit-specific signals to CPP amino acids 57-71 which were positively correlated to α-CCP2 antibody levels. Interestingly, 48% of the α-CPP-positives were rheumatoidfactor IgM/anti-citrullinated peptide/protein antibodies (ACPA)-negative. Anti-CPP and α-RA CH2007 -PPAD antibody levels increase with age. Protein macroarrays that were citrullinated by RA CH2007 -PPAD and screened with RA patient sera (n=6) and controls (n=4) uncovered 16 RA CH2007 -PPAD citrullinated RA autoantigens and 9 autoantigens associated with lung diseases. We showed that the α-CPP response could be an important determinant in parenchymal changes in the lung at the time of RA diagnosis (n=106; p=0.018)., Conclusions: RA CH2007 -PPAD induced internal citrullination of major RA autoantigens. Anti-RA CH2007 -PPAD correlates with ACPA levels and interstitial lung disease autoantigen reactivity, supporting an infection-based concept for induction of ACPAs via enzymatic mimicry., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

The host-microbe relationship is pivotal for oral health as well as for peri-implant diseases. Peri-implant mucosa and commensal biofilm play important roles in the maintenance of host-microbe homeostasis, but little is known about how they interact. We have therefore investigated the early host-microbe interaction between commensal multispecies biofilm (Streptococcus oralis, Actinomyces naeslundii, Veillonella dispar, Porphyromonas gingivalis) and organotypic peri-implant mucosa using our three-dimensional model. After 24 hr, biofilms induced weak inflammatory reaction in the peri-implant mucosa by upregulation of five genes related to immune response and increased secretion of IL-6 and CCL20. Biofilm volume was reduced which might be explained by secretion of β-Defensins-1, -2, and CCL20. The specific tissue reaction without intrinsic overreaction might contribute to intact mucosa. Thus, a relationship similar to homeostasis and oral health was established within the first 24 hr. In contrast, the mucosa was damaged and the bacterial distribution was altered after 48 hr. These were accompanied by an enhanced immune response with upregulation of additional inflammatory-related genes and increased cytokine secretion. Thus, the homeostasis-like relationship was disrupted. Such profound knowledge of the host-microbe interaction at the peri-implant site may provide the basis to improve strategies for prevention and therapy of peri-implant diseases., (© 2020 The Authors. Cellular Microbiology published by John Wiley & Sons Ltd.)

The relationship of Porphyromonas gingivalis and oral squamous cell carcinoma (OSCC) has been studied for several years. Previous studies have focused on the direct effect of P. gingivalis on the activities of primary epithelial cells and OSCC cells. However, the immune system is responsible for mediating cancer development, whether P. gingivalis can affect oral cancer immunity has seldom been explored to date. In this study, we investigated the role of P. gingivalis in the immunoevasion of OSCC. We evaluated the effect of P. gingivalis on the phagocytosis of Cal-27 cells (OSCC cell line) by bone marrow-derived macrophages in vitro and studied the effect of P. gingivalis on the growth of OSCC and the polarization of tumor-associated macrophages in vivo. We found that P. gingivalis was able to inhibit the phagocytosis of Cal-27 cells by macrophages, and membrane-component molecules of P. gingivalis , such as proteins, were speculated to be the effector components. In addition, sustained infection with antibiotics-inactivated P. gingivalis promoted OSCC growth in mice and induced the polarization of macrophages into M2 tumor-associated macrophages, which mainly display protumor properties. Transcriptome analysis and quantitative RT-PCR revealed that P. gingivalis infection upregulated the expression of genes encoding protumor molecules in Cal-27 cells ( suprabasin , IL-1R2 , and CD47 ) and in macrophages ( IL-1α , CCL-3 , and CCL-5 ). Our in vitro and in vivo data suggest that P. gingivalis can promote immunoevasion of oral cancer by protecting cancer from macrophage attack. To our knowledge, the present study reveals a novel mechanism by which P. gingivalis promotes OSCC development., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Correlation between periodontitis and atherosclerosis is well established, and the inherent mechanisms responsible for this relationship remain unclear. The biological function of growth arrest-specific 6 (gas6) has been discovered in both atherosclerosis and inflammation. Inhibitory effects of gas6 on the expression of inflammatory factors in human umbilical vein endothelial cells (HUVECs) stimulated by Porphyromonas gingivalis lipopolysaccharide (P. gingivalis-LPS) were reported in our previous research. Herein, the effects of gas6 on monocytes-endothelial cells interactions in vitro and their probable mechanisms were further investigated. Gas6 protein in HUVECs was knocked down with siRNA or overexpressed with plasmids. Transwell inserts and co-culturing system were introduced to observe chemotaxis and adhering affinity between monocytes and endothelial cells in vitro. Expression of gas6 was decreased in inflammatory periodontal tissues and HUVECs challenged with P. gingivalis-LPS. The inhibitory effect of gas6 on chemotaxis and adhesion affinity between monocytes and endothelial cells was observed, and gas6 promoted Akt phosphorylation and inhibited NF-κB phosphorylation. To our best knowledge, we are first to report that gas6 inhibit monocytes-endothelial cells interactions in vitro induced by P. gingivalis-LPS via Akt/NF-κB pathway. Additionally, inflammation-mediated inhibition of gas6 expression is through LncRNA GAS6-AS2, rather than GAS6-AS1, which is also newly reported., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Amyloid-β (Aβ) accumulation is one of the main pathological hallmarks of Alzheimer's disease (AD). Porphyromonas gingivalis ( P. gingivalis ), the pathogen of chronic periodontitis, could cause Aβ accumulation and was identified in the brain of AD patients. Salvianolic Acid B (SalB) has been proven to have the neuroprotective effect. Whether SalB could protect against P. gingivalis -induced cognitive impairment is still unknown. In this study, a P. gingivalis -infected mouse model was employed to study the neuroprotective role of SalB. The results showed that SalB (20 and 40 mg/kg) treatment for 4 weeks could shorten the escape latency and improve the percentage of spontaneous alternation in the P. gingivalis -infected mice. SalB inhibited the levels of reactive oxygen species and malondialdehyde, while increased the levels of antioxidative enzymes (superoxide dismutase and glutathione peroxidase). SalB decreased the levels of IL-1β and IL-6, increased the mRNA levels of bdnf and ngf in the brain of P. gingivalis -infected mice. In addition, SalB obviously decreased the level of Aβ. SalB elevated the protein expression of ADAM10, while downregulated BACE1 and PS1. SalB increased the protein expression of LRP1, while decreased RAGE. In conclusion, SalB could improve cognitive impairment by inhibiting neuroinflammation and decreasing Aβ level in P. gingivalis -infected mice.

Chronic periodontitis is an inflammatory disease caused by the formation of oral microbial biofilms. Periodontitis is associated with general health and not only oral diseases. Porphyromonas gingivalis is a well-known keystone pathogen for periodontitis and is associated with several systemic diseases, such as diabetes mellitus and Alzheimer's disease. We previously developed a system for screening periodontitis using P. gingivalis -specific serum immunoglobulin G (IgG) in an enzyme-linked immunosorbent assay with a sensitivity of 0.774 and a specificity of 0.586 and an area under the receiver operating characteristic curve of 0.708. However, the antigens elicited non-specific responses, since they were obtained from whole extracts of sonicated cultured bacteria. The purpose of this study was to identify antigens ideal for a sensitive and specific serum test. We identified the specific antigens using immunoaffinity columns immobilized with IgG antibodies from periodontitis patients. Liquid chromatography-tandem mass spectrometry identified 29 antigens from the elutes. Recombinant proteins for these candidates were synthesized using the wheat germ cell-free translation system and screened by dot blot analysis with serum from the columns. Three of the 16 candidates that reacted showed strongest affinities upon dot blot analysis; they included outer membrane protein 28, cysteine proteases, lysine gingipain Kgp, and arginine gingipain RgpA. Outer membrane protein 28 was not suitable for screening P. gingivalis infection because of its high false-negative rates. Kgp and RgpA were unstable antigens since they underwent self-digestion. They were made stable by substituting the active cysteine residues in Kgp and RgpA with alanine using site-directed mutagenesis. Using the modified antigens, we demonstrated that the patient serum IgG level against RgpA was the highest among all the antigens expressed in P. gingivalis . Moreover, the N-terminus of recombinant RgpA was excellent in differentiating between diseased and non-diseased states (with sensitivity of 0.85, specificity of 0.9, and area under the curve of 0.915). Although dot blot analysis was the only experiment used, the N-terminus of RgpA is an excellent antigen to immunologically test for P. gingivalis infection, especially for estimating the risks for periodontitis-associated systemic diseases. In conclusion, we have developed a P. gingivalis antigen for screening periodontitis., (Copyright © 2020 Hirai, Yamaguchi-Tomikawa, Eguchi, Maeda and Takashiba.)

The serine-glycine dipeptide lipid classes, including lipid 430 and lipid 654, are produced by the periodontal pathogen Porphyromonas gingivalis and can be detected in lipid extracts of diseased periodontal tissues and teeth of humans. Both serine-glycine lipid classes were previously shown to engage human and mouse Toll-like receptor 2 (TLR2) and to inhibit mouse osteoblast differentiation and function through engagement of TLR2. It is not clear if other lipids related to serine-glycine lipids are also produced by P. gingivalis The goal of this investigation was to determine whether P. gingivalis produces additional lipid classes similar to the serine-glycine lipids that possess biological properties. P. gingivalis (ATCC 33277) was grown in broth culture, and lipids were extracted and fractionated by high-performance liquid chromatography (HPLC). Lipids were separated using semipreparative HPLC, and specific lipid classes were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-multiple reaction monitoring (LC-MRM) mass spectrometric approaches. Two glycine lipid classes were identified, termed lipid 567 and lipid 342, and these lipid classes are structurally related to the serine-glycine dipeptide lipids. Both glycine lipid classes were shown to promote TLR2-dependent tumor necrosis factor alpha (TNF-α) release from bone marrow macrophages, and both were shown to activate human embryonic kidney (HEK) cells through TLR2 and TLR6 but not TLR1. These results demonstrate that P. gingivalis synthesizes glycine lipids and that these lipids engage TLR2 similarly to the previously reported serine-glycine dipeptide lipids., (Copyright © 2020 American Society for Microbiology.)

Background: We report the first protocol for a multicenter, randomized comparison study to compare the efficacies of periodontal scaling and root-planing treatment against that of tooth-brushing treatment for nonalcoholic fatty liver disease (NAFLD) (PERION: PERIOdontal treatment for NAFLD). Nonalcoholic steatohepatitis (NASH) is an advanced form of NAFLD, which can progress to cirrhosis and hepatocellular carcinoma. Increased endotoxemia is associated with the progression of NAFLD. Periodontal bacteria possess endotoxins; Porphyromonas gingivalis is well-known as a major pathogenic bacterium in periodontitis, and serum antibody levels for P. gingivalis are high in patients with periodontitis. Several reports have indicated that P. gingivalis is related to NAFLD. This study aims to investigate the effect of periodontal treatment for liver damage, P. gingivalis infection, and endotoxemia on patients with NAFLD., Methods: We will include adult patients (20-85 years old) with NAFLD, alanine aminotransferase (ALT) ≥ 40 IU/L, and equivalent steatosis grade ≥ 1 (target sample size, n = 40 patients; planned number of patients with outcome data, n = 32). Participants will be randomly assigned to one of two groups: a scaling and root-planing group or tooth-brushing as the usual group. The primary outcome will be the change in ALT levels from baseline to 12 weeks; the key secondary outcome will be the change in the serum immunoglobulin G (IgG) antibody titer for P. gingivalis at 12 weeks., Discussion: This study should determine whether periodontal treatment decreases liver damage, P. gingivalis infection, and endotoxemia in patients with NAFLD., Trial Registration: University Hospital Medical Information Network (UMIN) Clinical Trials Registry, ID: UMIN000022079.

Candida albicans is a pathogenic fungus capable of switching its morphology between yeast-like cells and filamentous hyphae and can associate with bacteria to form mixed biofilms resistant to antibiotics. In these structures, the fungal milieu can play a protective function for bacteria as has recently been reported for C. albicans and a periodontal pathogen- Porphyromonas gingivalis . Our current study aimed to determine how this type of mutual microbe protection within the mixed biofilm affects the contacting host cells. To analyze C. albicans and P. gingivalis persistence and host infection, several models for host-biofilm interactions were developed, including microbial exposure to a representative monocyte cell line (THP1) and gingival fibroblasts isolated from periodontitis patients. For in vivo experiments, a mouse subcutaneous chamber model was utilized. The persistence of P. gingivalis cells was observed within mixed biofilm with C. albicans . This microbial co-existence influenced host immunity by attenuating macrophage and fibroblast responses. Cytokine and chemokine production decreased compared to pure bacterial infection. The fibroblasts isolated from patients with severe periodontitis were less susceptible to fungal colonization, indicating a modulation of the host environment by the dominating bacterial infection. The results obtained for the mouse model in which a sequential infection was initiated by the fungus showed that this host colonization induced a milder inflammation, leading to a significant reduction in mouse mortality. Moreover, high bacterial counts in animal organisms were noted on a longer time scale in the presence of C. albicans , suggesting the chronic nature of the dual-species infection.

Porphyromonas gingivalis is an anaerobic bacterium commonly found in the oral cavity and associated with the development of periodontal disease. P. gingivalis has also been linked to several systemic vascular and inflammatory diseases including poor pregnancy outcomes. Little is known about the changes in the oral flora during pregnancy in connection to P. gingivalis infection. This pilot study aims to explore changes in the oral microbiome due to P. gingivalis inoculation and pregnancy in an in vivo rat model of periodontal disease. A metagenomic sequencing analysis targeting seven of the 16S rRNA gene variable regions was performed for oral samples collected at the following time points: baseline control (week 0), P. gingivalis inoculated (week 11), P. gingivalis inoculated and pregnant rat at necropsy (week 16). A second set of animals were also sampled to generate a sham-inoculated (week 11) control group. We found that the rat oral microbiome profiles were more similar to that of the human oral cavity compared to previous reports targeting one or two 16S variable regions. Overall, there appears to be a relatively stable core microbiome in the oral cavity. As expected, P. gingivalis induced periodontal disease resulted in oral microbiome dysbiosis. During pregnancy, some aspects of the oral microbiome shifted toward a more baseline-like profile. However, population analyses in terms of dissimilarity measures and especially metagenomic based predictions of select characteristics such as cell morphology, oxygen requirement, and major metabolite synthesis showed that pregnancy did not restore the composition of the oral microbiome. Rather, a uniquely altered oral microbiome composition was observed in pregnant rats with pre-established periodontal disease., (Copyright © 2020 Walkenhorst, Reyes, Perez, Progulske-Fox, Brown and Phillips.)

Objective: We investigated lipid concentrations, particle sizes and antibodies binding to periodontal bacteria Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis and to malondialdehyde-acetaldehyde (MAA) modified low-density lipoprotein in immunoglobulin (Ig) class A, G and M among patients with newly diagnosed rheumatoid arthritis (RA) in a population-based cohort. Methods: Concentrations and sizes of lipoprotein particles analysed by proton nuclear magnetic resonance spectroscopy and antibody levels to MAA modified low-density lipoprotein were studied at baseline and after one-year of follow-up. Serum Ig A and G class antibodies to periodontal bacteria were determined at baseline. Results: Sixty-three patients were divided into tertiles according to disease activity by disease activity score with 28 joint count and erythrocyte sedimentation rate (ESR) (<3.9, 3.9-4.7, >4.7). Small low-density lipoprotein concentration was lowest in the tertile with the highest disease activity. In high-density lipoprotein, the concentrations of total, medium and small particles decreased with disease activity. The particle size in low-density lipoprotein associated with disease activity and the presence of antibodies to P. gingivalis . Ig G and M antibodies to MAA modified low-density lipoprotein correlated with disease activity. Inflammation associated changes faded by one year. Conclusions: Drug naive RA patients had proatherogenic changes in lipid profiles, but they were reversible, when inflammation diminished.Key messagesPatients with drug naive rheumatoid arthritis showed proatherogenic lipid profiles.Reversible changes in lipid profiles can be achieved as response to inflammation suppression.Active therapy aimed at remission is essential in all patients with rheumatoid arthritis.

Porphyromonas gingivalis is a keystone pathogen in periodontitis. Analysis of the immunogenicity of its virulence factors may provide insight into the host response to this infection. The Kgp12 (IEDB Epitope ID 763561), an epitope of Lys-gingipain (Kgp) virulence factor from P. gingivalis ATCC 33277, elicits an immunoglobulin G (IgG) immunoreactivity with low cross-reactivity and, therefore, more specificity. The aim of the present study was to determine in silico the localization of Kgp12 within the protein and to evaluate the IgG host response to this novel Kgp peptide through its capacity to differentiate individuals with different periodontal status. Sera of 71 volunteers were tested by indirect ELISA to detect the IgG immunoreactivity specific to Kgp12, as well as to the protein HmuY and to the sonicated total extract of P. gingivalis ATCC33277, both used as gold standard. The participants had no systemic disease and were classified according to periodontal clinical parameters to comparison, firstly, into periodontitis (P) and without periodontitis (WP) groups and, secondly, into periodontitis (P), gingivitis (G) and clinically health (CH) ones. All the antigens tested, Kgp12 (p = 0.02), HmuY (p = 0.00) and P. gingivalis extract (p = 0.03), could differentiate P from WP groups considering IgG serum levels. P group also had higher IgG levels specific to Kgp12 (p = 0.03), HmuY (p < 0.01) and P. gingivalis extract (p = 0.01) when compared to G group. We conclude that the Kgp12 synthetic peptide was useful to detect the IgG-mediated host response signaling that it is a promising epitope to analyze the immunogenicity of P. gingivalis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Aging humans display an increased prevalence and severity of periodontitis, although the mechanisms underlying these findings remain poorly understood. This report examined antigenic diversity of P. gingivalis related to disease presence and patient demographics. Serum IgG antibody to P. gingivalis strains ATCC33277, FDC381, W50 (ATCC53978), W83, A7A1-28 (ATCC53977) and A7436 was measured in 426 participants [periodontally healthy (n = 61), gingivitis (N = 66) or various levels of periodontitis (N = 299)]. We hypothesized that antigenic diversity in P. gingivalis could contribute to a lack of "immunity" in the chronic infections of periodontal disease. Across the strains, the antibody levels in the oldest age group were lower than in the youngest groups, and severe periodontitis patients did not show higher antibody with aging. While 80 % of the periodontitis patients in any age group showed an elevated response to at least one of the P. gingivalis strains, the patterns of individual responses in the older group were also substantially different than the other age groups. Significantly greater numbers of older patients showed strain-specific antibody profiles to only 1 strain. The findings support that P. gingivalis may demonstrate antigenic diversity/drift within patients and could be one factor to help explain the inefficiency/ineffectiveness of the adaptive immune response in managing the infection., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Periodontitis is an infection-driven inflammatory disease, which is characterized by gingival inflammation and bone loss. Periodontitis is associated with various systemic diseases, including cardiovascular, respiratory, musculoskeletal, and reproductive system related abnormalities. Recent theory attributes the pathogenesis of periodontitis to oral microbial dysbiosis, in which Porphyromonas gingivalis acts as a critical agent by disrupting host immune homeostasis. Lipopolysaccharide, proteases, fimbriae, and some other virulence factors are among the strategies exploited by P. gingivalis to promote the bacterial colonization and facilitate the outgrowth of the surrounding microbial community. Virulence factors promote the coaggregation of P. gingivalis with other bacteria and the formation of dental biofilm. These virulence factors also modulate a variety of host immune components and subvert the immune response to evade bacterial clearance or induce an inflammatory environment. In this chapter, our focus is to discuss the virulence factors of periodontal pathogens, especially P. gingivalis, and their roles in regulating immune responses during periodontitis progression., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Periodontitis is an inflammatory disease caused by host immune response, resulting in a loss of periodontium and alveolar bone. Immune cells, such as T cells and macrophages, play a critical role in the periodontitis onset. Halofuginone, a natural quinazolinone alkaloid, has been shown to possess anti-fibrosis, anti-cancer, and immunomodulatory properties. However, the effect of halofuginone on periodontitis has never been reported. In this study, a ligature-induced mice model of periodontitis was applied to investigate the potential beneficial effect of halofuginone on periodontitis. We demonstrated that the administration of halofuginone significantly reduced the expression levels of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) in vivo, and markedly suppressed immune cell infiltration into the infected sites. Furthermore, we also observed that halofuginone treatment blocked the T-helper 17 (Th17) cell differentiation in vivo and in vitro. We demonstrated for the first time that halofuginone alleviated the onset of periodontitis through reducing immune responses.