Porphyromonas gingivalis Mfa1 fimbria putatively binds to TLR2 and induces both IL-6 and IL-8 production in human bronchial epithelial cells.

Porphyromonas gingivalis (Pg) a major periodontal pathogen involved in periodontal disease development and progression. Moreover, Pg has two fimbriae surface proteins (FimA and Mfa1) that are genetically distinct and make-up the fimbrial shaft which in-turn form crucial attachment to oral bacteria and multiple host cells. However, unlike FimA, Mfa1 attachment to non-periodontal cells has not been fully elucidated. Considering Pg-associated periodontal disease contributes to pulmonary disease development, we investigated whether Mfa1 can functionally interact with human bronchial epithelial cells and, likewise, trigger a functional response. Initially, we simulated molecular docking and performed both luciferase and neutralization assays to confirm Mfa1-related functional interaction. Subsequently, we treated BEAS-2B cells with purified Mfa1 and performed cytokine quantification through real time-PCR and ELISA to establish Mfa1-related functional response. We found that both Mfa1-TLR2 and Mfa1-TLR4 docking is possible, however, only Mfa1-TLR2 showed a functional interaction. Additionally, we observed that both IL-8 and IL-6 gene expression and protein levels were induced confirming Mfa1-related functional response. Taken together, we propose that BEAS-2B human bronchial epithelial cells are able to recognize Pg Mfa1 and induce both IL-8 and IL-6 inflammatory responses.

Epstein-Barr Virus Promotes the Production of Inflammatory Cytokines in Gingival Fibroblasts and RANKL-Induced Osteoclast Differentiation in RAW264.7 Cells.

Periodontitis is an inflammatory condition that causes the destruction of the supporting tissues of teeth and is a major public health problem affecting more than half of the adult population worldwide. Recently, members of the herpes virus family, such as the Epstein-Barr virus (EBV), have been suggested to be involved in the etiology of periodontitis because bacterial activity alone does not adequately explain the clinical characteristics of periodontitis. However, the role of EBV in the etiology of periodontitis is unknown. This study aimed to examine the effect of inactivated EBV on the expression of inflammatory cytokines in human gingival fibroblasts (HGFs) and the induction of osteoclast differentiation. We found that extremely high levels of interleukin (IL)-6 and IL-8 were induced by inactivated EBV in a copy-dependent manner in HGFs. The levels of IL-6 and IL-8 in HGFs were higher when the cells were treated with EBV than when treated with lipopolysaccharide and lipoteichoic acid. EBV induced IκBα degradation, NF-κB transcription, and RAW264.7 cell differentiation into osteoclast-like cells. These findings suggest that even without infecting the cells, EBV contributes to inflammatory cytokine production and osteoclast differentiation by contact with oral cells or macrophage lineage, resulting in periodontitis onset and progression.

SARS-CoV-2 Infection and Significance of Oral Health Management in the Era of "the New Normal with COVID-19".

More than a year ago, the coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was declared a pandemic by the World Health Organization, with the world approaching its fourth wave. During this period, vaccines were developed in a much shorter period than thought possible, with the initiation of the pertinent vaccination. However, oral cavities have come under renewed scrutiny worldwide because saliva, a mixture of salivary secretions, pharyngeal secretions, and gingival crevicular fluid, have not only been shown to contain infective viral loads, mediating the route of SARS-CoV-2 transmission via droplet, aerosol, or contagion, but also used as a sample for viral RNA testing with a usefulness comparable to the nasopharyngeal swab. The oral cavity is an important portal for ingress of SARS-CoV-2, being an entryway to the bronchi, alveoli, and rest of the lower respiratory tract, causing inflammation by viral infection. Moreover, angiotensin-converting enzyme 2, a host receptor for SARS-CoV-2, coupled with proteases responsible for viral entry have been found to be expressed on the tongue and other oral mucosae, suggesting that the oral cavity is the site of virus replication and propagation. Furthermore, there is a possibility that the aspiration of oral bacteria (such as periodontal pathogens) along with saliva into the lower respiratory tract may be a complicating factor for COVID-19 because chronic obstructive pulmonary disease and diabetes are known COVID-19 comorbidities with a greater risk of disease aggravation and higher death rate. These comorbidities have a strong connection to chronic periodontitis and periodontal pathogens, and an oral health management is an effective measure to prevent these comorbidities. In addition, oral bacteria, particularly periodontal pathogens, could be proinflammatory stimulants to respiratory epithelia upon its exposure to aspirated bacteria. Therefore, it may be expected that oral health management not only prevents comorbidities involved in aggravating COVID-19 but also has an effect against COVID-19 progression. This review discusses the significance of oral health management in SARS-CoV-2 infection in the era of "the new normal with COVID-19" and COVID-19 prevention with reference to the hypothetical mechanisms that the authors and the other researchers have proposed.

Expression of the SARS-CoV-2 Receptor ACE2 and Proinflammatory Cytokines Induced by the Periodontopathic Bacterium Fusobacterium nucleatum in Human Respiratory Epithelial Cells.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is currently a global public health emergency. Periodontitis, the most prevalent disease that leads to tooth loss, is caused by infection by periodontopathic bacteria. Periodontitis is also a risk factor for pneumonia and the exacerbation of chronic obstructive pulmonary disease, presumably because of the aspiration of saliva contaminated with periodontopathic bacteria into the lower respiratory tract. Patients with these diseases have increased rates of COVID-19 aggravation and mortality. Because periodontopathic bacteria have been isolated from the bronchoalveolar lavage fluid of patients with COVID-19, periodontitis may be a risk factor for COVID-19 aggravation. However, the molecular links between periodontitis and COVID-19 have not been clarified. In this study, we found that the culture supernatant of the periodontopathic bacterium Fusobacterium nucleatum (CSF) upregulated the SARS-CoV-2 receptor angiotensin-converting enzyme 2 in A549 alveolar epithelial cells. In addition, CSF induced interleukin (IL)-6 and IL-8 production by both A549 and primary alveolar epithelial cells. CSF also strongly induced IL-6 and IL-8 expression by BEAS-2B bronchial epithelial cells and Detroit 562 pharyngeal epithelial cells. These results suggest that when patients with mild COVID-19 frequently aspirate periodontopathic bacteria, SARS-CoV-2 infection is promoted, and inflammation in the lower respiratory tract may become severe in the presence of viral pneumonia.

How Does Epstein-Barr Virus Contribute to Chronic Periodontitis?

Chronic periodontitis is spreading worldwide and mutually interacts with systemic diseases like diabetes mellitus. Although periodontopathic bacteria are inevitable pathogens in their onset and progression, many cases are not ascribable to the virulence of these bacteria because the effect of plaque control is limited. In contrast, Epstein-Barr virus (EBV) in the periodontium has been correlated with chronic periodontitis and has recently been considered as a promising pathogenic candidate for this disease. However, several important questions have yet to be addressed. For instance, although EBV latently infects more than 90% of individuals over the world, why do patients with chronic periodontitis exclusively harbor progeny EBV in the oral cavity? In addition, how does latently infected or reactivated EBV in the periodontium relate to the onset or progression of chronic periodontitis? Finally, is periodontitis incurable because EBV is the pathogen for chronic periodontitis? In this review, we attempt to answer these questions by reporting the current understanding of molecular relations and mechanisms between periodontopathic bacteria and EBV reactivation in the context of how this relationship may pertain to the etiology of chronic periodontitis.

The Periodontopathic Bacterium Fusobacterium nucleatum Induced Proinflammatory Cytokine Production by Human Respiratory Epithelial Cell Lines and in the Lower Respiratory Organs in Mice.

BACKGROUND/AIMS: The most prevalent infectious disease, chronic periodontitis which leads to alveolar bone destruction and subsequent tooth loss, develops due to proinflammatory cytokine production induced by periodontopathic bacteria. Chronic obstructive pulmonary disease (COPD), a non-infectious disease, is the third leading cause of death globally. This condition exacerbates frequently, and which is attributable to proinflammatory cytokine production induced by infection by respiratory microorganisms such as Streptococcus pneumoniae. Although a positive association has recently been revealed between chronic periodontitis and COPD, how periodontitis contributes to the pathogenesis of COPD remains unclear. Therefore, we hypothesized that some periodontopathic bacteria are involved in the exacerbation of COPD through the induction of proinflammatory cytokine production by respiratory epithelial cells. In this connection, COPD develops in the airways; however, because most periodontopathic bacteria are anaerobic, they are unlikely to exhibit stable virulence in the lower respiratory organs in humans. Hence, we aimed to elucidate whether exposure to heat-inactivated periodontopathic bacteria induces proinflammatory cytokine production by several human respiratory epithelial cell lines and in the lower respiratory organs and serum in mice. METHODS: Real-time polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) were used to investigate in vitro induction by heat-inactivated periodontopathic bacteria and S. pneumoniae for mRNA expression and protein production of interleukin (IL)-8 and IL-6 by human respiratory epithelial cell lines. ELISA was also used to determine in vivo induction of cytokine production in the lower respiratory organs and serum of intratracheally heat-inactivated Fusobacterium nucleatum-inoculated mice. RESULTS: Some, but not all, periodontopathic bacteria, especially F. nucleatum, strongly induced IL-8 and IL-6 production by BEAS-2B bronchial epithelial cells. In addition, F. nucleatum induced IL-8 production by A549 alveolar epithelial cells as well as IL-8 and IL-6 production by Detroit 562 pharyngeal epithelial cells. Furthermore, F. nucleatum induced considerably higher cytokine production than S. pneumoniae. This was also observed in the entire lower respiratory organs and serum in mice. CONCLUSION: Exposure to increased number of F. nucleatum potentially induces proinflammatory cytokine production by human bronchial and pharyngeal epithelial cells, which may trigger exacerbation of COPD.

Bactericidal effect of hydroxyl radicals generated by the sonolysis and photolysis of hydrogen peroxide for endodontic applications.

The aim of endodontic root canal treatment is the elimination of bacteria and their products from an infected tooth root canal. To effectively disinfect a root canal, an ultrasonic irrigation system, in which hydroxyl radicals (HO·) generated artificially by sonolysis of H2O2, was developed previously for endodontic applications and was demonstrated to have bactericidal efficacy against Enterococcus faecalis. To improve this system, we examined the in vitro bactericidal effects of HO· generated from H2O2, activated by simultaneous irradiation with ultrasound for sonolysis and dental LED light for photolysis with a peak wavelength of 405 nm. Regarding the LED irradiation, two methods were used: (i) 'ideal' experimental conditions (irradiation close to the glass tube), and (ii) simulated endodontic conditions (more distant irradiation of a masked glass tube). In these conditions, HO· generation from H2O2 was detected by electron spin resonance (ESR) spectroscopy, and bactericidal efficacy against E. faecalis was assessed by measuring the colony forming units (CFU)/mL. The results indicated that HO· generation by ESR measurements and the bactericidal effect on E. faecalis by viable count using CFU/mL were enhanced significantly in a time-dependent manner in both conditions. In a comparison of these conditions, bactericidal activity under 'ideal' experimental conditions was similar to that under simulated endodontic conditions. Moreover, the irradiation time for effective killing of E. faecalis through the sonolysis and photolysis of H2O2 under simulated endodontic conditions was shorter than that with sonolysis alone. These results demonstrate that H2O2 activated by ultrasound and LED light may be a safe and effective disinfection technique for endodontic root canal treatment.

Prevalence of Epstein-Barr virus DNA and Porphyromonas gingivalis in Japanese peri-implantitis patients.

BACKGROUND: Peri-implantitis (PI) is an inflammatory reaction associated with functional deterioration of supporting bones around the dental implant. Recent studies suggested Epstein-Barr virus (EBV) is involved in the pathogenesis of periodontitis. We investigated the association between EBV and Porphyromonas gingivalis in Japanese PI patients. METHODS: Fifteen periodontally healthy individuals, 15 healthy implant patients and 15 PI patients were recruited. Forty five subgingival plaque samples were collected from the deepest probing pocket depth (PPD) site from each patient. Real-time PCR was used to detect EBV DNA and P. gingivalis. RESULTS: EBV and P. gingivalis were detected in 7 and 3 PPD sites of the healthy controls, in 9 and 4 PPD sites of the healthy implants, and in 13 and 14 PPD sites of the PI patients. P. gingivalis and coexistence of EBV and P. gingivalis were detected significantly higher in the PI patients than healthy controls and healthy implant patients. EBV was detected significantly higher in the PI patients than healthy controls. CONCLUSIONS: Higher levels of EBV and P. gingivalis were detected in PPD sites of PI patients. These results suggest that coexistence of EBV and P. gingivalis may serve pathogenic factors cause for PI in Japanese dental patients.

Prevalence and quantitative analysis of Epstein-Barr virus DNA and Porphyromonas gingivalis associated with Japanese chronic periodontitis patients.

OBJECTIVE: A number of studies have recently suggested Epstein-Barr virus (EBV) involvement in the pathogenesis of periodontitis. In this study, we investigated the association between major periodontopathic bacteria Porphyromonas gingivalis (P. gingivalis) and EBV in Japanese chronic periodontitis (CP) patients. MATERIALS AND METHODS: A group of 25 patients with CP participated in the study along with 13 individuals without periodontitis. Subgingival samples were obtained with paper points. Quantitative real-time polymerase chain reaction (PCR) was used to detect EBV DNA and P. gingivalis. RESULTS: In the CP patients, EBV DNA and P. gingivalis were detected in both 80 % of sites with probing pocket depths (PPD) of ≥5 mm and in 40 and 36 % of sites with PPD ≤3 mm, respectively. EBV DNA and P. gingivalis were detected in 50 and 27 % of the sites in periodontally healthy individuals. Coexistence of EBV DNA and P. gingivalis was significantly higher in the deeper PPD sites of CP patients (68 %) than in the PPD sites of the healthy controls (15 %) and shallow PPD sites of CP patients (12 %). PCR-positive deeper PPD sites of CP patients for EBV DNA and P. gingivalis range between 3.74 × 10(3)∼2.83 × 10(9) and 2.73 × 10(5)∼6.65 × 10(9) (copies/ml), respectively. CONCLUSION: These results suggest an association between EBV DNA, P. gingivalis, and CP in Japanese individuals. Further studies are required to clarify this association; however, we believe that our enhanced understanding of the pathogenesis of periodontal diseases involving viral infections will lead to new treatments.

Gut Bacteria-derived Membrane Vesicles Induce Colonic Dysplasia by Inducing DNA Damage in Colon Epithelial Cells.

BACKGROUND & AIMS: Colorectal cancer (CRC) is the third most common cancer in the world. Gut microbiota has recently been implicated in the development of CRC. Actinomyces odontolyticus is one of the most abundant bacteria in the gut of patients with very early stages of CRC. A odontolyticus is an anaerobic bacterium existing principally in the oral cavity, similar to Fusobacterium nucleatum, which is known as a colon carcinogenic bacterium. Here we newly determined the biological functions of A odontolyticus on colonic oncogenesis. METHODS: We examined the induction of intracellular signaling by A odontolyticus in human colonic epithelial cells (CECs). DNA damage levels in CECs were confirmed using the human induced pluripotent stem cell-derived gut organoid model and mouse colon tissues in vivo. RESULTS: A odontolyticus secretes membrane vesicles (MVs), which induce nuclear factor kappa B signaling and also produce excessive reactive oxygen species (ROS) in colon epithelial cells. We found that A odontolyticus secretes lipoteichoic acid-rich MVs, promoting inflammatory signaling via TLR2. Simultaneously, those MVs are internalized into the colon epithelial cells, co-localize with the mitochondria, and cause mitochondrial dysfunction, resulting in excessive ROS production and DNA damage. Induction of excessive DNA damage in colonic cells by A odontolyticus-derived MVs was confirmed in the gut organoid model and also in mouse colon tissues. CONCLUSIONS: A odontolyticus secretes MVs, which cause chronic inflammation and ROS production in colonic epithelial cells, leading to the initiation of CRC.

Are Viral Infections Key Inducers of Autoimmune Diseases? Focus on Epstein-Barr Virus.

It is generally accepted that certain viral infections can trigger the development of autoimmune diseases. However, the exact mechanisms by which these viruses induce autoimmunity are still not understood. In this review, we first describe hypothetical mechanisms by which viruses induce some representative autoimmune diseases. Then, we focus on Epstein-Barr virus (EBV) and discuss its role in the pathogenesis of rheumatoid arthritis (RA). The discussion is mainly based on our own previous findings that (A) EBV DNA and its products EBV-encoded small RNA (EBER) and latent membrane protein 1 (LMP1) are present in the synovial lesions of RA, (B) mRNA expression of the signaling lymphocytic activation molecule-associated protein (SAP)/SH2D1A gene that plays a critical role in cellular immune responses to EBV is reduced in the peripheral T cells of patients with RA, and (C) EBV infection of mice reconstituted with human immune system components (humanized mice) induced erosive arthritis that is pathologically similar to RA. Additionally, environmental factors may contribute to EBV reactivation as follows: Porphyromonas gingivalis peptidylarginine deiminase (PAD), an enzyme required for citrullination, engenders antigens leading to the production of citrullinated peptides both in the gingiva and synovium. Anti-citrullinated peptides autoantibody is an important marker for diagnosis and disease activity of RA. These findings, as well as various results obtained by other researchers, strongly suggest that EBV is directly involved in the pathogenesis of RA, a typical autoimmune disease.

Reactivation of latent HIV-1 infection by the periodontopathic bacterium Porphyromonas gingivalis involves histone modification.

Latently infected cells harbor the HIV-1 proviral DNA genome primarily integrated into heterochromatin, allowing the persistence of transcriptionally silent proviruses. Hypoacetylation of histone proteins by histone deacetylases (HDAC) is involved in the maintenance of HIV-1 latency by repressing viral transcription. In addition, periodontal diseases, caused by polymicrobial subgingival bacteria including Porphyromonas gingivalis, are among the most prevalent infections of mankind. Here we demonstrate the effects of P. gingivalis on HIV-1 replication. This activity could be ascribable to the bacterial culture supernatant but not to other bacterial components such as fimbriae or LPS. We found that this HIV-1-inducing activity was recovered in the lower molecular mass (<3 kDa) fraction of the culture supernatant. We also demonstrated that P. gingivalis produces high concentrations of butyric acid, acting as a potent inhibitor of HDACs and causing histone acetylation. Chromatin immunoprecipitation assays revealed that the corepressor complex containing HDAC1 and AP-4 was dissociated from the HIV-1 long terminal repeat promoter upon stimulation with bacterial culture supernatant concomitantly with the association of acetylated histone and RNA polymerase II. We thus found that P. gingivalis could induce HIV-1 reactivation via chromatin modification and that butyric acid, one of the bacterial metabolites, is responsible for this effect. These results suggest that periodontal diseases could act as a risk factor for HIV-1 reactivation in infected individuals and might contribute to the systemic dissemination of the virus.

Analysis of the Interaction Between HIV and Periodontopathic Bacteria That Reactivates HIV Replication in Latently Infected Cells.

The acquired immunodeficiency syndrome (AIDS) pandemic caused by the human immunodeficiency virus (HIV) is a major global health concern affecting 38 million people worldwide. HIV gene expression is the major determinant of the rate of viral replication leading to the progression of AIDS. The persistence of cellular reservoirs of HIV proviruses, despite prolonged treatment with antiretroviral drugs, represents the main obstacle preventing the eradication of HIV. Epigenetic silencing by histone deacetylase (HDAC) contributes to maintaining HIV transcriptional latency. However, the mechanism of the switch from latency to full HIV replication is unknown. HIV infection and antiretroviral treatment or a combination of both contribute to a higher incidence and severity of periodontitis. Periodontopathic bacteria such as Porphyromonas gingivalis and Fusobacterium nucleatum produce high concentrations of butyric acid, which strongly inhibit HDAC, indicating that periodontitis may mediate the reactivation of HIV replication. Here we describe a stepwise protocol for analyzing HIV reactivation by periodontal pathogens. However, the experiments using HIV requires BSL3 containment, making it difficult to handle HIV in dentistry. Therefore, we present an experimental method using cell lines latently infected with HIV.

Relationship between the oral cavity and respiratory diseases: Aspiration of oral bacteria possibly contributes to the progression of lower airway inflammation.

The global population is aging, and elderly people have a higher incidence of lower airway diseases owing to decline in swallowing function, airway ciliary motility, and overall immunity associated with aging. Furthermore, lower airway diseases in the elderly tend to have a high mortality rate. Their prevention is important for extending healthy life expectancy and improving the quality of life of each individual. In recent years, the relationship between "chronic periodontitis and oral bacteria, especially the periodontopathic ones" and "respiratory diseases" (e.g., pneumonia, chronic obstructive pulmonary disease, and influenza) has become clear. In addition, the association of several periodontal pathogens with the onset and aggravation of coronavirus disease 2019 (COVID-19) is also being reported. In support of these findings, oral health management has shown to reduce deaths from pneumonia and prevent influenza in nursing homes and inpatient wards. This has led to clinical and multidisciplinary cooperation between physicians and dentists, among others. However, to date, the mechanisms by which "chronic periodontitis and oral bacteria" contribute to lower airway diseases have not been well understood. Clarifying these mechanisms will lead to a theoretical basis for answering the question, "Why is oral health management effective in preventing lower airway diseases?"

Porphyromonas gingivalis enhances pneumococcal adhesion to human alveolar epithelial cells by increasing expression of host platelet-activating factor receptor.

Streptococcus pneumoniae causes pneumonia by infecting the alveolar epithelium via binding to host receptors, such as the platelet-activating factor receptor (PAFR). Although chronic periodontitis has been identified as a pneumonia risk factor, how periodontopathic bacteria cause pneumonia is not known. We found that S. pneumoniae adhered to PAFR expressed on A549 human alveolar epithelial cells stimulated by Porphyromonas gingivalis culture supernatant, and this was abrogated by a PAFR-specific inhibitor. Among the major virulence factors of P. gingivalis [lipopolysaccharide (LPS), fimbriae and gingipains (Rgps and Kgp)], PAFR expression and pneumococcal adhesion were executed in an Rgp-dependent manner. LPS and fimbriae did not induce PAFR expression. Hence, our findings suggest that P. gingivalis enhances pneumococcal adhesion to human alveoli by inducing PAFR expression and that gingipains are responsible for this.

S-PRG Filler Eluate Induces Oxidative Stress in Oral Microorganism: Suppression of Growth and Pathogenicity, and Possible Clinical Application.

Controlling the oral microbial flora is putatively thought to prevent not only oral diseases, but also systemic diseases caused by oral diseases. This study establishes the antibacterial effect of the novel bioactive substance "S-PRG filler" on oral bacteria. We examined the state of oxidative stress caused by the six types of ions released in eluate from the S-PRG filler in oral bacterial cells. Moreover, we investigated the effects of these ions on the growth and pathogenicity of Gram-positive and Gram-negative bacteria. We found that the released ions affected SOD amount and hydrogen peroxide in bacterial cells insinuating oxidative stress occurrence. In bacterial culture, growth inhibition was observed depending on the ion concentration in the medium. Additionally, released ions suppressed Streptococcus mutans adhesion to hydroxyapatite, S. oralis neuraminidase activity, and Porphyromonas gingivalis hemagglutination and gingipain activity in a concentration-dependent manner. From these results, it was suggested that the ions released from the S-PRG filler may suppress the growth and pathogenicity of the oral bacterial flora. This bioactive material is potentially useful to prevent the onset of diseases inside and outside of the oral cavity, which in turn may have possible applications for oral care and QOL improvement.

Novel antimicrobial denture adhesive containing S-PRG filler.

The antimicrobial effects of denture adhesives containing novel surface pre-reacted glass-ionomer (S-PRG) fillers were assessed. We prepared denture adhesives containing S-PRG (particle sizes: 1 and 3 µm; quantities: 5, 7.5, and 10 wt%). We evaluated acid buffering capacity, ion release, and antimicrobial effects of denture adhesives with and without S-PRG. Significantly higher pH changes were observed in 1 µm S-PRG adhesives than in 3 µm S-PRG adhesives. Adhesives containing 7.5 and 10 wt% S-PRG exhibited significantly higher ion release than adhesives with 5 wt% S-PRG. The 1µm-10wt% S-PRG denture adhesive exhibited significantly lower colony-forming units on the denture adhesive contact surface than in the control group; additionally, it exhibited excellent acid buffering capacity, ion release properties, and antimicrobial effect against C. albicans, C. glabrata, S. mutans, and A. naeslundii. Longer contact periods resulted in significantly lower adhesion of Candida albicans to the denture base resin treated with denture adhesive.

Porphyromonas gingivalis gingipains potentially affect MUC5AC gene expression and protein levels in respiratory epithelial cells.

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.

Exposure to Porphyromonas gingivalis Induces Production of Proinflammatory Cytokine via TLR2 from Human Respiratory Epithelial Cells.

Aspiration pneumonia is a major health problem owing to its high mortality rate in elderly people. The secretion of proinflammatory cytokines such as interleukin (IL)-8 and IL-6 by respiratory epithelial cells, which is induced by infection of respiratory bacteria such as Streptococcus pneumoniae, contributes to the onset of pneumonia. These cytokines thus play a key role in orchestrating inflammatory responses in the lower respiratory tract. In contrast, chronic periodontitis, a chronic inflammatory disease caused by the infection of periodontopathic bacteria, typically Porphyromonas gingivalis, is one of the most prevalent microbial diseases affecting humans globally. Although emerging evidence has revealed an association between aspiration pneumonia and chronic periodontitis, a causal relationship between periodontopathic bacteria and the onset of aspiration pneumonia has not been established. Most periodontopathic bacteria are anaerobic and are therefore unlikely to survive in the lower respiratory organs of humans. Therefore, in this study, we examined whether simple contact by heat-inactivated P. gingivalis induced proinflammatory cytokine production by several human respiratory epithelial cell lines. We found that P. gingivalis induced strong IL-8 and IL-6 secretion by BEAS-2B bronchial epithelial cells. P. gingivalis also induced strong IL-8 secretion by Detroit 562 pharyngeal epithelial cells but not by A549 alveolar epithelial cells. Additionally, Toll-like receptor (TLR) 2 but not TLR4 was involved in the P. gingivalis-induced proinflammatory cytokine production. Furthermore, P. gingivalis induced considerably higher IL-8 and IL-6 production than heat-inactivated S. pneumoniae. Our results suggest that P. gingivalis is a powerful inflammatory stimulant for human bronchial and pharyngeal epithelial cells and can stimulate TLR2-mediated cytokine production, thereby potentially contributing to the onset of aspiration pneumonia.

Aspiration of periodontopathic bacteria due to poor oral hygiene potentially contributes to the aggravation of COVID-19.

Coronavirus infectious disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was declared a pandemic in March 2020 by the World Health Organization. Periodontitis, one of the most prevalent diseases worldwide, leads to alveolar bone destruction and subsequent tooth loss, and develops due to pro-inflammatory cytokine production induced by periodontopathic bacteria. Periodontopathic bacteria are involved in respiratory diseases, including aspiration pneumonia and chronic obstructive pulmonary disease (COPD), and other systemic diseases, such as diabetes and cardiovascular disease. Patients with these diseases have an increased COVID-19 aggravation rate and mortality. Because aspiration of periodontopathic bacteria induces the expression of angiotensin-converting enzyme 2, a receptor for SARS-CoV-2, and production of inflammatory cytokines in the lower respiratory tract, poor oral hygiene can lead to COVID-19 aggravation. Conversely, oral care, including periodontal treatment, prevents the onset of pneumonia and influenza and the exacerbation of COPD. The reduced chance of receiving professional oral care owing to long-term hospitalization of patients with COVID-19 may increase the aggravation risk of infection in the lower respiratory tract. It can be hypothesized that periodontopathic bacteria are involved in the COVID-19 aggravation and therefore, the management of good oral hygiene potentially contributes to its prevention.