Metagenomic Analysis of Bacterial Microflora in Dental and Atherosclerotic Plaques of Patients With Internal Carotid Artery Stenosis.

Background: Internal carotid artery stenosis is primarily attributed to atherosclerosis in the carotid artery bifurcation. Previous studies have detected oral bacteria in atherosclerotic lesions, suggesting an association between oral bacteria and atherosclerosis. In this study, we compared the bacterial flora of the atherosclerotic plaque in the carotid artery and dental plaque of patients with internal carotid artery stenosis using 16S ribosomal RNA (16S rRNA) metagenomic sequencing. Methods: Fifty-four patients who underwent internal carotid endarterectomy for internal carotid artery stenosis at the Showa University Hospital between April 2016 and February 2018 were included. Polymerase chain reaction targeting the 16S rRNA gene detected bacterial DNA in the carotid plaques of 11 cases, of which only 5 could be further analyzed. Thereafter, DNA extracted from the carotid and oral plaques of these 5 cases were analyzed using metagenomic sequencing targeting 16S rRNA. In addition, their general condition and oral conditions were evaluated. The patients were classified into symptomatic and asymptomatic groups based on the presence or absence of symptoms of transient ischemic attack, and their bacterial flora was evaluated. Results: The results demonstrated that the microflora of carotid plaques (n = 5) contained bacterial species from 55 families and 78 genera. In addition, 86.5% of the bacteria detected in the carotid plaques were also detected in oral plaques. Cariogenic and periodontopathic bacteria accounted for 27.7% and 4.7% of the bacteria in the carotid plaques, respectively. Conclusions: These results suggest that oral bacteria are directly or indirectly involved in the pathogenesis of atherosclerosis. More extensive studies of oral commensal bacteria detected in extra-oral lesions are warranted to comprehensively investigate the role of oral bacteria in the pathogenesis of systemic diseases.

The Klebsiella mannose phosphotransferase system promotes proliferation and the production of extracellular polymeric substances from mannose, facilitating adaptation to the host environment.

OBJECTIVES: Klebsiella spp., an opportunistic infectious organism, is commensal in the nasal and oral cavities of humans. Recently, it has been reported that oral Klebsiella spp. ectopically colonize the intestinal tract and induce the accumulation of intestinal Th1 cells. For oral bacteria to colonize the intestinal tract, they need to compete for nutrients with indigenous intestinal bacteria. Therefore, we focused on mannose, a mucus-derived sugar, and the mannose phosphotransferase system (PTS) (ManXYZ), a mechanism for mannose uptake, in terms of their effects on intestinal colonization and immune responses to Klebsiella spp. METHODS: We generated a Klebsiella manXYZ-deficient strain and investigated whether the utilization of intestinal mucus-derived sugars is associated with the growth. Furthermore, we examine the virulence of this organism in the mouse intestinal tract, especially the ability to colonize the host using competition assay. RESULTS: We found that Klebsiella ManXYZ is a PTS that specifically takes up mannose and glucosamine. Through ManXYZ, mannose was used for bacterial growth and the upregulated production of extracellular polymeric substances. In vivo competition assays showed that mannose metabolism promoted intestinal colonization. However, ManXYZ was not involved in Th1 and Th17 induction in the intestinal tract. CONCLUSION: The fundamental roles of ManXYZ were to ensure that mannose, which is present in the host, is made available for bacterial growth, to promote the production of extracellular polymeric substances, thus facilitating bacterial adaptation to the host environment.

Metagenomic analysis of oral plaques and aortic valve tissues reveals oral bacteria associated with aortic stenosis.

OBJECTIVES: Bacteria derived from the oral cavity enter the bloodstream and cause the onset of various systemic diseases, including heart valve disease. However, information on the oral bacteria involved in aortic stenosis is limited. MATERIALS AND METHODS: We comprehensively analyzed the microbiota in aortic valve tissues collected from aortic stenosis patients using metagenomic sequencing and investigated the relationships between the valve microbiota, the oral microbiota, and oral cavity conditions. RESULTS: Metagenomic analysis revealed the presence of 629 bacterial species in five oral plaques and 15 aortic valve clinical specimens. Patients were classified into two groups (A and B) according to their aortic valve microbiota composition using principal coordinate analysis. Examination of the oral conditions of the patients showed no difference in the decayed/missing/filled teeth index. Bacteria in group B tend to be associated with severe disease, and the number of bacteria on the dorsum of the tongue and the positive rate of bleeding during probing were significantly higher in this group than in group A. The pathophysiology of aortic stenosis may be related to the presence of oral bacteria such as Streptococcus oralis and Streptococcus sanguinis following bacteremia. CONCLUSIONS: Systemic inflammation in severe periodontitis may be driven by the oral microbiota, supporting the indirect (inflammatory) association between oral bacteria and aortic stenosis. CLINICAL RELEVANCE: Appropriate oral hygiene management may contribute to the prevention and treatment of aortic stenosis.

Oral commensal bacterial flora is responsible for peripheral differentiation of neutrophils in the oral mucosa in the steady state.

OBJECTIVES: Commensal bacteria in the host body play a fundamental role in the differentiation and maintenance of the immune system. Studies on intestinal immunity have revealed that, under steady-state conditions, microflora have an important role in the maintenance of health. However, the role of oral commensal bacteria on the oral immune system is still unclear. Here, we clarify the interactions between commensal bacteria and the oral mucosal immune system under steady-state conditions. METHODS: We used germ-free mice that had never been exposed to bacteria and conventional mice grown with normal bacterial flora. Oral cells were isolated from the oral mucosa, stained with specific antibodies, and analyzed by flow cytometry. For the detection of myeloperoxidase and intracellular cytokines, oral cells were stimulated with N-formyl-methionine-leucyl-phenylalanine and phorbol 12-myristate 13-acetate/ionomycin, respectively. RESULTS: We found that the oral mucosa harbored more neutrophils in germ-free mice than in conventional mice. However, the majority of neutrophils in the germ-free oral mucosa exhibited an immature phenotype. Other immune cells, including macrophages, T cells, and B cells, in the oral mucosa of germ-free mice showed similar differentiation to those in conventional mice. These results indicate that in the steady-state oral mucosa, the normal commensal flora promote the peripheral differentiation of neutrophils. CONCLUSIONS: The presence of commensal flora is critical for the development of adequate immune system in the oral mucosa.

Oral mitis group streptococci reduce infectivity of influenza A virus via acidification and H2O2 production.

Members of the mitis group streptococci are the most abundant inhabitants of the oral cavity and dental plaque. Influenza A virus (IAV), the causative agent of influenza, infects the upper respiratory tract, and co-infection with Streptococcus pneumoniae is a major cause of morbidity during influenza epidemics. S. pneumoniae is a member of mitis group streptococci and shares many features with oral mitis group streptococci. In this study, we investigated the effect of viable Streptococcus oralis, a representative member of oral mitis group, on the infectivity of H1N1 IAV. The infectivity of IAV was measured by a plaque assay using Madin-Darby canine kidney cells. When IAV was incubated in growing culture of S. oralis, the IAV titer decreased in a time- and dose-dependent manner and became less than 100-fold, whereas heat-inactivated S. oralis had no effect. Other oral streptococci such as Streptococcus mutans and Streptococcus salivarius also reduced the viral infectivity to a lesser extent compared to S. oralis and Streptococcus gordonii, another member of the oral mitis group. S. oralis produces hydrogen peroxide (H2O2) at a concentration of 1-2 mM, and its mutant deficient in H2O2 production showed a weaker effect on the inactivation of IAV, suggesting that H2O2 contributes to viral inactivation. The contribution of H2O2 was confirmed by an inhibition assay using catalase, an H2O2-decomposing enzyme. These oral streptococci produce short chain fatty acids (SCFA) such as acetic acid as a by-product of sugar metabolism, and we also found that the inactivation of IAV was dependent on the mildly acidic pH (around pH 5.0) of these streptococcal cultures. Although inactivation of IAV in buffers of pH 5.0 was limited, incubation in the same buffer containing 2 mM H2O2 resulted in marked inactivation of IAV, which was similar to the effect of growing S. oralis culture. Taken together, these results reveal that viable S. oralis can inactivate IAV via the production of SCFAs and H2O2. This finding also suggests that the combination of mildly acidic pH and H2O2 at low concentrations could be an effective method to inactivate IAV.

Melatonin suppresses the antiviral immune response to EMCV infection through intracellular ATP deprivation caused by mitochondrial fragmentation.

Melatonin, a sleep hormone derived from the pineal gland, has an anti-inflammatory effect on the immune system in addition to modulating the brain nervous system. Previous studies have shown that melatonin suppresses signaling pathways downstream of multiple pattern recognition receptors on the innate immune cells during pathogen infection, but the specific mechanism of suppression has not been well understood. Using an encephalomyocarditis virus (EMCV) infection model in macrophages, we investigated the effects of melatonin on the antiviral response in innate immunity and found that melatonin attenuated the uptake of viral particles into macrophages. Furthermore, melatonin suppressed cytoskeletal regulation by decreasing ATP production by mitochondria. Finally, in an in vivo infection experiment, we also found that melatonin administration partially exacerbated the infection in the mouse brain. These results suggest that melatonin may have an inhibitory effect on excessive inflammation by suppressing cytoskeletal regulation in the innate immune system, but also suggest that suppression of inflammation may lead to insufficient protection against EMCV infection in vivo.

Oral mitis group streptococci: A silent majority in our oral cavity.

Members of the oral mitis group streptococci including Streptococcus oralis, Streptococcus sanguinis, and Streptococcus gordonii are the most abundant inhabitants of human oral cavity and dental plaque, and have been implicated in infectious complications such as bacteremia and infective endocarditis. Oral mitis group streptococci are genetically close to Streptococcus pneumoniae; however, they do not produce cytolysin (pneumolysin), which is a key virulence factor of S. pneumoniae. Similar to S. pneumoniae, oral mitis group streptococci possess several cell surface proteins that bind to the cell surface components of host mammalian cells. S. sanguinis expresses long filamentous pili that bind to the matrix proteins of host cells. The cell wall-anchored nuclease of S. sanguinis contributes to the evasion of the neutrophil extracellular trap by digesting its web-like extracellular DNA. Oral mitis group streptococci produce glucosyltransferases, which synthesize glucan (glucose polymer) from sucrose of dietary origin. Neuraminidase (NA) is a virulent factor in oral mitis group streptococci. Influenza type A virus (IAV) relies on viral NA activity to release progeny viruses from infected cells and spread the infection, and NA-producing oral streptococci elevate the risk of IAV infection. Moreover, oral mitis group streptococci produce hydrogen peroxide (H2 O2 ) as a by-product of sugar metabolism. Although the concentrations of streptococcal H2 O2 are low (1-2 mM), they play important roles in bacterial competition in the oral cavity and evasion of phagocytosis by host macrophages and neutrophils. In this review, we intended to describe the diverse pathogenicity of oral mitis group streptococci.

Novel antibody assessment method for microbial compositional alteration in the oral cavity.

Recently, it has been demonstrated that dysbiosis, an alteration in commensal microflora composition, is intimately involved in the onset of a variety of diseases. It is becoming increasingly evident that the composition of commensal microflora in the oral cavity is closely connected to oral diseases, such as periodontal disease, and systemic diseases, such as inflammatory bowel disease. Next-generation sequencing techniques are used as a method to examine changes in bacterial flora, but additional analytical methods to assess bacterial flora are needed to understand bacterial activity in more detail. In addition, the oral environment is unique because of the role of secretory antibodies contained in saliva in the formation of bacterial flora. The present study aimed to develop a new method for evaluating the compositional change of microbiota using flow cytometry (FCM) with specific antibodies against the bacterial surface antigen, as well as salivary antibodies. Using specific antibodies against Streptococcus mutans, a causative agent of dental caries, and human IgA, bacterial samples from human saliva were analyzed via FCM. The results showed that different profiles could be obtained depending on the oral hygiene status of the subjects. These results suggest that changes in the amount and type of antibodies that bind to oral bacteria may be an indicator for evaluating abnormalities in the oral flora. Therefore, the protocol established in this report could be applied as an evaluation method for alterations in the oral microbiota.

Particle Size Analysis in Aerosol-Generating Dental Procedures Using Laser Diffraction Technique.

The global outbreak of coronavirus disease 2019 (COVID-19) has raised concerns about the risk of airborne infection during dental treatment. Aerosol-generating dental procedures (AGDP) produce droplets and aerosols, but the details of the risks of COVID-19 transmission in AGDP are not well-understood. By discriminating between droplets and aerosols, we devised a method to measure particle size using laser diffraction analysis and evaluated aerosols generated from dental devices for providing a basis for proper infection control procedures. The droplets and aerosols generated from dental devices were characterized by multimodal properties and a wide range of droplet sizes, with the majority of droplets larger than 50 µm. AGDP emitted few aerosols smaller than 5 µm, which are of concern for pulmonary infections due to airborne transmission. In addition, the use of extraoral suction was found to prevent the spread of aerosols from high-speed dental engines. This study suggests that the risk of aerosol infections is considerably limited in regular dental practice and that current standard precautions, such as mainly focusing on protection against droplet and contact infections, are sufficient. While several cases of airborne transmission of COVID-19 in general clinics and emergency hospitals have been reported, cluster outbreaks in dental clinics have not yet been reported, which may indicate that AGDP does not pose a significant threat in contributing to the spread of SARS-CoV-2.

Streptococcal H2O2 inhibits IgE-triggered degranulation of RBL-2H3 mast cell/basophil cell line by inducing cell death.

Mast cells and basophils are central players in allergic reactions triggered by immunoglobulin E (IgE). They have intracellular granules containing allergic mediators (e.g., histamine, serotonin, inflammatory cytokines, proteases and ß-hexosaminidase), and stimulation by IgE-allergen complex leads to the release of such allergic mediators from the granules, that is, degranulation. Mast cells are residents of mucosal surfaces, including those of nasal and oral cavities, and play an important role in the innate defense system. Members of the mitis group streptococci such as Streptococcus oralis, are primary colonizers of the human oral cavity. They produce hydrogen peroxide (H2O2) as a by-product of sugar metabolism. In this study, we investigated the effects of streptococcal infection on RBL-2H3 mast cell/basophil cell line. Infection by oral streptococci did not induce degranulation of the cells. Stimulation of the RBL-2H3 cells with anti-dinitrophenol (DNP) IgE and DNP-conjugated human serum albumin triggers degranulation with the release of ß-hexosaminidase. We found that S. oralis and other mitis group streptococci inhibited the IgE-triggered degranulation of RBL-2H3 cells. Since mitis group streptococci produce H2O2, we examined the effect of S. oralis mutant strain deficient in producing H2O2, and found that they lost the ability to suppress the degranulation. Moreover, H2O2 alone inhibited the IgE-induced degranulation. Subsequent analysis suggested that the inhibition of degranulation was related to the cytotoxicity of streptococcal H2O2. Activated RBL-2H3 cells produce interleukin-4 (IL-4); however, IL-4 production was not induced by streptococcal H2O2. Furthermore, an in vivo study using the murine pollen-induced allergic rhinitis model suggested that the streptococcal H2O2 reduces nasal allergic reaction. These findings reveal that H2O2 produced by oral mitis group streptococci inhibits IgE-stimulated degranulation by inducing cell death. Consequently, streptococcal H2O2 can be considered to modulate the allergic reaction in mucosal surfaces.

Dysregulation of Intestinal Microbiota Elicited by Food Allergy Induces IgA-Mediated Oral Dysbiosis.

Food allergy is a life-threatening response to specific foods, and microbiota imbalance (dysbiosis) in gut is considered a cause of this disease. Meanwhile, the host immune response also plays an important role in the disease. Notably, interleukin 33 (IL-33) released from damaged or necrotic intestinal epithelial cells facilitates IL-2-producing CD4 helper T (Th2) responses. However, causal relationships between the gut and oral dysbiosis and food allergy remain unknown. In this study, we analyzed effects of gut and oral dysbiosis on development of food allergy. A murine model of food allergy was established via ovalbumin (OVA) injection in BALB/c mice. Viable fecal bacteria were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). il33 expression in colon-26 mouse colon cells stimulated by isolated fecal bacteria was quantified by real-time PCR. Intestinal T cells from the mice were analyzed by flow cytometry. Salivary IgA levels were quantified by enzyme-linked immunosorbent assay (ELISA), and IgA-bound oral bacteria were detected by flow cytometry. Among fecal bacteria, the abundance of Citrobacter sp. increased in the feces of allergic mice and induced il33 expression in colon-26 cells. Orally administered Citrobacter koseri JCM1658 exacerbated systemic allergic symptoms and reduced intestinal Th17 cells. Salivary IgA and IgA-bound oral bacteria increased in the allergic mice. Based on the results described above, food allergy induced both gut and oral dysbiosis. Citrobacter sp. aggravated allergy symptoms by inducing IL-33 release from intestinal epithelial cells.

Effect of kinematic viscosity on the staining performance of caries detector dyes.

The relationship between performance of caries detector dyes and solvent molecular weight has been reported, but viscosity has not yet been investigated. Therefore, using extracted human teeth, we compared 6 experimental caries detector dyes with different viscosities to clarify the relationship between dye viscosity and staining performance in sclerotic dentin. Carious dentin started showing dentin staining when stained with caries detector dyes with kinematic viscosity≥263 mm2/s, showed no immunoreactivity for Streptococcus mutans on immunohistochemistry, and contained dentinal tubules free of debris on scanning electron microscopy. Samples stained using a caries detector dye with kinematic viscosity of 332 mm2/s contained calcification product-like debris in the dentinal tubules. These findings suggest that the caries detector dye has a threshold kinematic viscosity between 263 and 332 mm2/s at which sclerotic dentin and tissues beyond are not stained. The caries detector dye appears to control tooth dyeability based on solvent viscosity.

Influence of excess branched-chain amino acid uptake by Streptococcus mutans in human host cells.

Oral streptococci, including cariogenic bacterium Streptococcus mutans, comprise a large percentage of human supragingival plaque, which contacts both tooth surfaces and gingiva. Eukaryotic cells are able to take up macromolecules and particles, including bacteria, by endocytosis. Increasing evidence indicates endocytosis may be used as an entry process by bacteria. We hypothesized that some endocytosed bacteria might survive and obtain nutrients, such as amino acids, until they are killed. To verify this hypothesis, we focused on bacterial utilization of branched-chain amino acids (BCAAs; isoleucine, leucine and valine) in host cells. A branched-chain aminotransferase, IlvE (EC 2.6.1.42), has been suggested to play an important role in internal synthesis of BCAAs in S. mutans UA159. Therefore, we constructed an ilvE-deficient S. mutans 109c strain and confirmed that it had similar growth behavior as reported previously. 14C radioactive leucine uptake assays showed that ilvE-deficient S. mutans took up more leucine both inside and outside of host cells. We further clarified that a relative decrease of BCAAs in host cells caused enhanced endocytic and autophagic activity. In conclusion, S. mutans is endocytosed by host cells and may survive and obtain nutrients, such as BCAAs, inside the cells, which might affect cellular functions of host cells.

Streptococcus sanguinis Noncoding cia-Dependent Small RNAs Negatively Regulate Expression of Type IV Pilus Retraction ATPase PilT and Biofilm Formation.

Small noncoding RNAs (sRNAs) have been identified as important regulators of gene expression in various cellular processes. cia-dependent small RNAs (csRNAs), a group of sRNAs that are controlled by the two-component regulatory system CiaRH, are widely conserved in streptococci, but their targets have been identified only in Streptococcus pneumoniaeStreptococcus sanguinis, a pioneer colonizer of teeth and one of the most predominant bacteria in the early oral biofilm, has been shown to have six csRNAs. Using computational target prediction and the luciferase reporter assay, we identified pilT, a constituent of the type IV pilus operon, as a negative regulatory target for one of the csRNAs, namely, csRNA1-1, in S. sanguinis RNA-RNA electrophoretic mobility shift assay using a nucleotide exchange mutant of csRNA1-1 revealed that csRNA1-1 binds directly to pilT mRNA. In addition, csRNA1-1 and csRNA1-2, a putative gene duplication product of csRNA1-1 that is tandemly located in the S. sanguinis genome, negatively regulated S. sanguinis biofilm formation. These results suggest the involvement of csRNAs in the colonization step of S. sanguinis.

Caveolin-1, a binding protein of CD26, is essential for the anti-inflammatory effects of dipeptidyl peptidase-4 inhibitors on human and mouse macrophages.

We previously reported that inhibition of dipeptidyl peptidase (DPP)-4, the catalytic site of CD26, prevents atherosclerosis in animal models through suppression of inflammation; however, the underlying molecular mechanisms have not been fully elucidated. Caveolin-1 (Cav-1), a major structural protein of caveolae located on the surface of the cellular membrane, has been reported to modulate inflammatory responses by binding to CD26 in T cells. In this study, we investigated the role of Cav-1 in the suppression of inflammation mediated by the DPP-4 inhibitor, teneligliptin, using mouse and human macrophages. Mouse peritoneal macrophages were isolated from Cav-1+/+ and Cav-1-/- mice after stimulation with 3% thioglycolate. Inflammation was induced by the toll-like receptor (TLR)4 agonist, lipopolysaccharide (LPS), isolated from Escherichia coli. The expression of pro-inflammatory cytokines was determined using reverse transcription-polymerase chain reaction. Co-expression of Cav-1 and CD26 was detected using immunohistochemistry in both mouse and human macrophages. Teneligliptin treatment (10 nmol/L) suppressed the LPS-induced expression of interleukin (IL)-6 (70%) and tumor necrosis factor-α (37%) in peritoneal macrophages isolated from Cav-1+/+ mice. However, teneligliptin did not have any effect on the macrophages from Cav-1-/- mice. In human monocyte/macrophage U937 cells, teneligliptin treatment suppressed LPS-induced expression of pro-inflammatory cytokines in a dose-dependent manner (1-10 nmol/L). These anti-inflammatory effects of teneligliptin were mimicked by gene knockdown of Cav-1 or CD26 using small interfering RNA transfection. Furthermore, neutralization of these molecules using an antibody against CD26 or Cav-1 also showed similar suppression. Teneligliptin treatment specifically inhibited TLR4 and TLR5 agonist-mediated inflammatory responses, and suppressed LPS-induced phosphorylation of IL-1 receptor-associated kinase 4, a downstream molecule of TLR4. Next, we determined whether teneligliptin could directly inhibit the physical interaction between Cav-1 and CD26 using the Biacore system. Binding of CD26 to Cav-1 protein was detected. Unexpectedly, teneligliptin also bound to Cav-1, but did not interfere with CD26-Cav-1 binding, suggesting that teneligliptin competes with CD26 for binding to Cav-1. In conclusion, we demonstrated that Cav-1 is a target molecule for DPP-4 inhibitors in the suppression of TLR4-mediated inflammation in mouse and human macrophages.

A Polymethyl Methacrylate-Based Acrylic Dental Resin Surface Bound with a Photoreactive Polymer Inhibits Accumulation of Bacterial Plaque.

PURPOSE: The aim of this study was to examine the ability of a poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butylmethacrylate-co-2-methacryloyloxyethyloxy-p-azidobenzoate) (PMBPAz) coating on polymethyl methacrylate (PMMA)-based dental resin to inhibit bacterial plaque formation, as well as the polymer's durability against water soaking and chemical exposure. MATERIALS AND METHODS: Successful application of PMBPAz on PMMA surfaces was confirmed by x-ray photoelectron spectroscopy (XPS) and measuring the static air contact angle in water. The anti-adhesive effects to bacterial plaque were evaluated using Streptococcus mutans biofilm formation assay. The mechanical and chemical durabilities of the PMBPAz coating on the PMMA surfaces were examined using soaking and immersion tests, respectively. RESULTS: XPS signals for phosphorus and nitrogen atoms and hydrophilic status on PMMA surfaces treated with PMBPAz were observed, indicating the presence of the polymer on the substrates. The treated PMMA surfaces showed significant inhibition of S mutans biofilm formation compared to untreated surfaces. The PMBPAz coating was preserved after water soaking and chemical exposure. In addition, water soaking did not decrease the ability of treated PMMA to inhibit biofilm formation compared to treated PMMA specimens not subjected to water soaking. CONCLUSION: This study suggests that PMBPAz coating may represent a useful modification to PMMA surfaces for inhibiting denture plaque accumulation.

Microarray and gene co-expression analysis reveals that melatonin attenuates immune responses and modulates actin rearrangement in macrophages.

Melatonin produced by the pineal gland suppresses inflammatory responses in innate immune cells. However, the mechanism of how melatonin affects inflammatory gene regulation remains unclear. Here we performed comprehensive microarray analysis combined with transcription factor binding site (TFBS) analysis using LPS-induced mouse macrophages to investigate the effect of melatonin treatment. The results showed that melatonin preferentially downregulated interferon regulatory factors (IRFs) and signal transducers and activators of transcription (STATs) related signaling. The results also showed that melatonin strongly suppressed virus infection related gene expression. Furthermore, TFBS analysis implicated that melatonin downregulated the binding activity of hypoxia inducible factors (HIFs), following destabilizing actin cytoskeleton which are indispensable for induction of the TRIF-dependent signaling pathway. Indeed, it was demonstrated that melatonin treatment caused impaired phagocytosis in macrophages. Thus, melatonin regulates inflammatory responses by inhibiting specific subsets of transcription factors (TFs) by disrupting actin dynamics in the macrophage.

Microarray analysis of macrophage response to infection with Streptococcus oralis reveals the immunosuppressive effect of hydrogen peroxide.

Oral streptococci including mitis group streptococci are commensal residents and are also the first to colonize the oral cavity. However, various species of these oral streptococci have the potential to invade the host and occasionally lead to severe infectious disease such as cardiovascular diseases. Oral streptococci have close interactions with the host immune system including macrophages at the oral mucosal surface. One notable common trait of oral streptococcus including Streptococcus oralis (S. oralis) is the production of hydrogen peroxide (H2O2). Using a comprehensive microarray approach, we sought to understand the innate immune response profiling affected by H2O2 production from oral streptococci. We compared the gene expression patterns of macrophages infected with S. oralis wild type (WT) and streptococcal pyruvate oxidase knockout (SpxB-KO), a strain that does not produce H2O2. We found that H2O2 from S. oralis suppressed proinflammatory gene expression such as TNF-α, that is induced in response to infection, and activated the cellular stress genes such as Egr-1 in response to oxidative stress. A comparative gene ontology analysis of S. oralis WT and SpxB-KO strains revealed that during infection, down regulated genes were closely related to the processes involved in the host defense reaction and up regulated genes were related with the cellular stress responses. Using qPCR analysis, we also confirmed the same pattern of expression changes such as TNF-α, IL-6 and Egr-1. Furthermore, supernatant from SpxB-KO could not suppress the expression of TNF-α in macrophages stimulated with LPS. These findings suggested that H2O2 production from S. oralis leads to the suppression of inflammatory responses and NF-κB signaling pathways in macrophages as well as the induction of the oxidative stress response. We concluded that streptococcal H2O2 production has the beneficial effects of modulating the innate immune response, thereby stabilizing streptococcal colonization at the mucosal surface and even in the bloodstream leading to cardiovascular disease after invasion, in addition to the commensal role to compete other bacterial species as initial colonizer at oral cavity.

Streptococcus oralis Induces Lysosomal Impairment of Macrophages via Bacterial Hydrogen Peroxide.

Streptococcus oralis, an oral commensal, belongs to the mitis group of streptococci and occasionally causes opportunistic infections, such as bacterial endocarditis and bacteremia. Recently, we found that the hydrogen peroxide (H2O2) produced by S. oralis is sufficient to kill human monocytes and epithelial cells, implying that streptococcal H2O2 is a cytotoxin. In the present study, we investigated whether streptococcal H2O2 impacts lysosomes, organelles of the intracellular digestive system, in relation to cell death. S. oralis infection induced the death of RAW 264 macrophages in an H2O2-dependent manner, which was exemplified by the fact that exogenous H2O2 also induced cell death. Infection with either a mutant lacking spxB, which encodes pyruvate oxidase responsible for H2O2 production, or Streptococcus mutans, which does not produce H2O2, showed less cytotoxicity. Visualization of lysosomes with LysoTracker revealed lysosome deacidification after infection with S. oralis or exposure to H2O2, which was corroborated by acridine orange staining. Similarly, fluorescent labeling of lysosome-associated membrane protein-1 gradually disappeared during infection with S. oralis or exposure to H2O2 The deacidification and the following induction of cell death were inhibited by chelating iron in lysosomes. Moreover, fluorescent staining of cathepsin B indicated lysosomal destruction. However, treatment of infected cells with a specific inhibitor of cathepsin B had negligible effects on cell death; instead, it suppressed the detachment of dead cells from the culture plates. These results suggest that streptococcal H2O2 induces cell death with lysosomal destruction and then the released lysosomal cathepsins contribute to the detachment of the dead cells.

Potential effect of cationic liposomes on interactions with oral bacterial cells and biofilms.

CONTEXT: Although oral infectious diseases have been attributed to bacteria, drug treatments remain ineffective because bacteria and their products exist as biofilms. Cationic liposomes have been suggested to electrostatically interact with the negative charge on the bacterial surface, thereby improving the effects of conventional drug therapies. However, the electrostatic interaction between oral bacteria and cationic liposomes has not yet been examined in detail. OBJECTIVE: The aim of the present study was to examine the behavior of cationic liposomes and Streptococcus mutans in planktonic cells and biofilms. MATERIALS AND METHODS: Liposomes with or without cationic lipid were prepared using a reverse-phase evaporation method. The zeta potentials of conventional liposomes (without cationic lipid) and cationic liposomes were -13 and 8 mV, respectively, and both had a mean particle size of approximately 180 nm. We first assessed the interaction between liposomes and planktonic bacterial cells with a flow cytometer. We then used a surface plasmon resonance method to examine the binding of liposomes to biofilms. We confirmed the binding behavior of liposomes with biofilms using confocal laser scanning microscopy. RESULTS: The interactions between cationic liposomes and S. mutans cells and biofilms were stronger than those of conventional liposomes. Microscopic observations revealed that many cationic liposomes interacted with the bacterial mass and penetrated the deep layers of biofilms. DISCUSSION AND CONCLUSION: In this study, we demonstrated that cationic liposomes had higher affinity not only to oral bacterial cells, but also biofilms than conventional liposomes. This electrostatic interaction may be useful as a potential drug delivery system to biofilms.