The Anti-Inflammatory Effect of SDF-1 Derived Peptide on Porphyromonas gingivalis Infection via Regulation of NLRP3 and AIM2 Inflammasome.

(1) Background: Peptides are appealing as pharmacological materials because they are easily produced, safe, and tolerable. Despite increasing gum-care awareness, periodontitis is still prevalent and is influenced by factors like high sugar consumption, smoking, and aging. Porphyromonas gingivalis is considered a major etiologic agent of periodontitis and activates the NLR family pyrin domain containing 3 (NLRP3) but is absent in melanoma 2 (AIM2) inflammasomes, resulting in pro-inflammatory cytokine release. (2) Methods: We examined the anti-inflammatory effects of 18 peptides derived from human stromal cell-derived factor-1 (SDF-1) on THP-1 macrophages. Inflammation was induced by P. gingivalis, and the anti-inflammatory effects were analyzed using molecular biological techniques. In a mouse periodontitis model, alveolar bone resorption was assessed using micro-CT. (3) Results: Of the 18 SDF-1-derived peptides, S10 notably reduced IL-1ß and TNF-α secretion. S10 also diminished the P. gingivalis-induced expression of NLRP3, AIM2, ASC (apoptosis-associated speck-like protein), caspase-1, and IL-1ß. Furthermore, S10 attenuated the enhanced TLR (toll-like receptor) signaling pathway and decreased the phosphorylation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs). In addition, S10 mitigated alveolar bone loss in our P. gingivalis-induced mouse model of periodontitis. (4) Conclusions: S10 suppressed TLR/NF-κB/NLRP3 inflammasome signaling and the AIM2 inflammasome in our P. gingivalis-induced murine periodontitis model, which suggests that it has potential use as a therapeutic treatment for periodontitis.

Microbial Profiles in Oral Lichen Planus: Comparisons with Healthy Controls and Erosive vs. Non-Erosive Subtypes.

Recent studies have begun exploring the potential involvement of microbiota in the pathogenesis of oral lichen planus (OLP), yet comprehensive investigations remain limited. Hence, this study aimed to compare the microbial profiles in saliva samples obtained from patients with OLP against those from healthy controls (HC), along with a comparison between erosive (E) and non-erosive (NE) OLP patients. Saliva samples were collected from 60 OLP patients (E: n = 25, NE: n = 35) and 30 HC individuals. Analysis revealed no significant differences in alpha diversity, as assessed by the Chao1 and Shannon index, across the three groups. However, Bray-Curtis distance analysis indicated a significant disparity in microbiome composition distribution between HC and E-OLP, as well as HC and NE-OLP groups. The six most abundant phyla observed across the groups were Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Fusobacteria, and Saccharibacteria (TM7). Notably, OLP groups exhibited a higher prevalence of Bacteroidetes. Prevotella emerged as the predominant genus in the OLP groups, while Capnocytophaga showed a relatively higher prevalence in E-OLP compared to NE-OLP. This study's findings indicate a notable difference in microbiota composition between HC and patients with OLP. Additionally, differences in the microbiome were identified between the E-OLP and NE-OLP groups. The increase in the proportion of certain bacterial species in the oral microbiome suggests that they may exacerbate the inflammatory response and act as antigens for OLP.

Comparison of the performance of MiSeq and NovaSeq in oral microbiome study.

Objective: Next generation sequencing is commonly used to characterize the microbiome structure. MiSeq is most commonly used to analyze the microbiome due to its relatively long read length. Illumina also introduced the 250 × 2 chip for NovaSeq. The purpose of this study was to compare the performance of MiSeq and NovaSeq in the context of oral microbiome study. Methods: Total read count, read quality score, relative bacterial abundance, community diversity, and correlation between two platforms were analyzed. Phylogenetic trees were analyzed for Streptococcus and periodontopathogens. Results: NovaSeq produced significantly more read counts and assigned more operational taxonomic units (OTUs) compared to MiSeq. Community diversity was similar between MiSeq and NovaSeq. NovaSeq were able to detect more unique OTUs compared to MiSeq. When phylogenetic trees were constructed for Streptococcus and periodontopathogens, both platforms detected OTUs for most of the clades. Conclusion: Taken together, while both MiSeq and NovaSeq platforms effectively characterize the oral microbiome, NovaSeq outperformed MiSeq in terms of read counts and detection of unique OTUs, highlighting its potential as a valuable tool for large scale oral microbiome studies.

A distinctive subgingival microbiome in patients with periodontitis and Alzheimer's disease compared with cognitively unimpaired periodontitis patients.

AIM: Periodontitis is caused by dysbiosis of oral microbes and is associated with increased cognitive decline in Alzheimer's disease (AD), and recently, a potential functional link was proposed between oral microbes and AD. We compared the oral microbiomes of patients with or without AD to evaluate the association between oral microbes and AD in periodontitis. MATERIALS AND METHODS: Periodontitis patients with AD (n = 15) and cognitively unimpaired periodontitis patients (CU) (n = 14) were recruited for this study. Each patient underwent an oral examination and neuropsychological evaluation. Buccal, supragingival and subgingival plaque samples were collected, and microbiomes were analysed by next-generation sequencing. Alpha diversity, beta diversity, linear discriminant analysis effect size, analysis of variance-like differential expression analysis and network analysis were used to compare group oral microbiomes. RESULTS: All 29 participants had moderate to severe periodontitis. Group buccal and supragingival samples were indistinguishable, but subgingival samples demonstrated significant alpha and beta diversity differences. Differential analysis showed subgingival samples of the AD group had higher prevalence of Atopobium rimae, Dialister pneumosintes, Olsenella sp. HMT 807, Saccharibacteria (TM7) sp. HMT 348 and several species of Prevotella than the CU group. Furthermore, subgingival microbiome network analysis revealed a distinct, closely connected network in the AD group comprised of various Prevotella spp. and several anaerobic bacteria. CONCLUSIONS: A unique microbial composition was discovered in the subgingival region in the AD group. Specifically, potential periodontal pathogens were found to be more prevalent in the subgingival plaque samples of the AD group. These bacteria may possess a potential to worsen periodontitis and other systemic diseases. We recommend that AD patients receive regular, careful dental check-ups to ensure proper oral hygiene management.

Comparative Analysis of Primers Used for 16S rRNA Gene Sequencing in Oral Microbiome Studies.

Recent advances in genomic technologies have enabled more in-depth study of the oral microbiome. In this study, we compared the amplicons generated by primers targeting different sites of the 16S rRNA gene found in the Human Oral Microbiome Database (HOMD). Six sets of primer targeting V1-V2, V1-V3, V3-V4, V4-V5, V5-V7 and V6-V8 regions of 16S rRNA were tested via in silico simulation. Primers targeting the V1-V2, V3-V4, and V4-V5 regions generated more than 90% of the original input sequences. Primers targeting the V1-V2 and V1-V3 regions exhibited a low number of mismatches and unclassified sequences at the taxonomic level, but there were notable discrepancies at the species level. Phylogenetic tree comparisons showed primers targeting the V1-V2 and V3-V4 regions showed performances similar to primers targeting the whole 16s RNA region in terms of separating total oral microbiomes and periodontopathogens. In an analysis of clinical oral samples, V1-V2 primers showed superior performance for identifying more taxa and had better resolution sensitivity for Streptococcus than V3-V4 primers. In conclusion, primers targeting the V1-V2 region of 16S rRNA showed the best performance for oral microbiome studies. In addition, the study demonstrates the need for careful PCR primer selections.

The oral microbiome of implant-abutment screw holes compared with the peri-implant sulcus and natural supragingival plaque in healthy individuals.

PURPOSE: An implant-supported prosthesis consists of an implant fixture, an abutment, an internal screw that connects the abutment to the implant fixture, and the upper prosthesis. Numerous studies have investigated the microorganisms present on the implant surface, surrounding tissues, and the subgingival microflora associated with peri-implantitis. However, there is limited information regarding the microbiome within the internal screw space. In this study, microbial samples were collected from the supragingival surfaces of natural teeth, the peri-implant sulcus, and the implant-abutment screw hole, in order to characterize the microbiome of the internal screw space in healthy subjects. METHODS: Samples were obtained from the supragingival region of natural teeth, the peri-implant sulcus, and the implant screw hole in 20 healthy subjects. DNA was extracted, and the V3-V4 region of the 16S ribosomal RNA was sequenced for microbiome analysis. Alpha diversity, beta diversity, linear discriminant analysis effect size (LEfSe), and network analysis were employed to compare the characteristics of the microbiomes. RESULTS: We observed significant differences in beta diversity among the samples. Upon analyzing the significant taxa using LEfSe, the microbial composition of the implant-abutment screw hole's microbiome was found to be similar to that of the other sampling sites' microbiomes. Moreover, the microbiome network analysis revealed a unique network complexity in samples obtained from the implant screw hole compared to those from the other sampling sites. CONCLUSIONS: The bacterial composition of the biofilm collected from the implant-abutment screw hole exhibited significant differences compared to the supra-structure of the implant. Therefore, long-term monitoring and management of not only the peri-implant tissue but also the implant screw are necessary.

Simultaneous Analysis of Bacterial and Fungal Communities in Oral Samples from Intubated Patients in Intensive Care Unit.

Intubated patients in intensive care units (ICUs) too frequently contract ventilator-associated pneumonia or Candida infections. Oropharyngeal microbes are believed to play an important etiologic role. This study was undertaken to determine whether next-generation sequencing (NGS) can be used to simultaneously analyze bacterial and fungal communities. Buccal samples were collected from intubated ICU patients. Primers targeting the V1-V2 region of bacterial 16S rRNA and the internal transcribed spacer 2 (ITS2) region of fungal 18S rRNA were used. V1-V2, ITS2, or mixed V1-V2/ITS2 primers were used to prepare an NGS library. Bacterial and fungal relative abundances were comparable for V1-V2, ITS2, or mixed V1-V2/ITS2 primers, respectively. A standard microbial community was used to adjust the relative abundances to theoretical abundance, and NGS and RT-PCR-adjusted relative abundances showed a high correlation. Using mixed V1-V2/ITS2 primers, bacterial and fungal abundances were simultaneously determined. The constructed microbiome network revealed novel interkingdom and intrakingdom interactions, and the simultaneous detection of bacterial and fungal communities using mixed V1-V2/ITS2 primers enabled analysis across two kingdoms. This study provides a novel approach to simultaneously determining bacterial and fungal communities using mixed V1-V2/ITS2 primers.

Microbial profiling of peri-implantitis compared to the periodontal microbiota in health and disease using 16S rRNA sequencing.

PURPOSE: The objective of this study was to analyze the microbial profile of individuals with peri-implantitis (PI) compared to those of periodontally healthy (PH) subjects and periodontitis (PT) subjects using Illumina sequencing. METHODS: Buccal, supragingival, and subgingival plaque samples were collected from 109 subjects (PH: 30, PT: 49, and PI: 30). The V3-V4 region of 16S rRNA was sequenced and analyzed to profile the plaque microbiota. RESULTS: Microbial community diversity in the PI group was higher than in the other groups, and the 3 groups showed significantly separated clusters in the buccal samples. The PI group showed different patterns of relative abundance from those in the PH and PT groups depending on the sampling site at both genus and phylum levels. In all samples, some bacterial species presented considerably higher relative abundances in the PI group than in the PH and PT groups, including Anaerotignum lactatifermentans, Bacteroides vulgatus, Faecalibacterium prausnitzii, Olsenella uli, Parasutterella excrementihominis, Prevotella buccae, Pseudoramibacter alactolyticus, Treponema parvum, and Slackia exigua. Network analysis identified that several well-known periodontal pathogens and newly recognized bacteria were closely correlated with each other. CONCLUSIONS: The composition of the microbiota was considerably different in PI subjects compared to PH and PT subjects, and these results could shed light on the mechanisms involved in the development of PI.

Assessing the effect of antihypertensives on plaque microbiota in patients with periodontitis and hypertension using 16S rRNA sequencing: A cross-sectional study.

BACKGROUND: Periodontitis is initiated or accelerated by dysbiosis of oral microorganisms. When hypertension is accompanied in periodontitis patients, changes of oral microbiota occur. Since there are no reports on antihypertensives, we assessed their effect on the oral microbial profiles of patients with periodontitis. METHODS: This study involved 95 participants divided into two groups: those with periodontitis and hypertension (P_HT), and those with periodontitis and taking medications for hypertension (P_mHT). Plaque samples were collected from the buccal, supragingival, and subgingival sites of the oral cavities of these patients. DNA was extracted, and the V3-V4 region of the 16S ribosomal RNA was sequenced and analyzed. RESULTS: The P_HT and P_mHT groups were similar with respect to the alpha- and beta-diversity as well as the dominant phyla and genera, but differed in the relative abundance of bacterial species (85 species). In the P_mHT group, the relative abundance of major periodontal pathogens was greatly increased. In particular, Tannerella forsythia, Treponema denticola, and Fretibacterium fastidiosum increased nearly three times in the linear discriminant analysis score in the supragingival plaque. Also, there was an increase in the relative abundance of Prevotella spp., associated with periodontitis and nitrate reduction, which was also evident in the supragingival plaque. CONCLUSIONS: These findings indicate that antihypertensives induce dysbiotic changes in the oral microbiota of patients with periodontitis, which are associated with increases in the relative abundance of periodontal pathogens. Therefore, more active periodontal treatment and supportive periodontal therapy are required in patients taking antihypertensives.

Neuropeptide Y: a potential theranostic biomarker for diabetic peripheral neuropathy in patients with type-2 diabetes.

BACKGROUND: Diabetic peripheral neuropathy (DPN), the most common microvascular complication of type-2 diabetes mellitus (T2DM), results in nontraumatic lower-limb amputations. When DPN is not detected early, disease progression is irreversible. Thus, biomarkers for diagnosing DPN are needed. METHODS: We analyzed three data sets of T2DM DPN: two for mouse models (GSE70852 and GSE34889) and one for a human model (GSE24290). We found common differentially expressed genes (DEGs) in the two mouse data sets and validated them in the human data set. To identify the phenotypic function of the DEGs, we overexpressed them in zebrafish embryos. Clinical information and serum samples of T2DM patients with and without DPN were obtained from the Korea Biobank Network. To assess the plausibility of DEGs as biomarkers of DPN, we performed an enzyme-linked immunosorbent assay. RESULTS: Among the DEGs, only NPY and SLPI were validated in the human data set. As npy is conserved in zebrafish, its mRNA was injected into zebrafish embryos, and it was observed that the branches of the central nervous system became thicker and the number of dendritic branches increased. Baseline characteristics between T2DM patients with and without DPN did not differ, except for the sex ratio. The mean serum NPY level was higher in T2DM patients with DPN than in those without DPN (p = 0.0328), whereas serum SLPI levels did not differ (p = 0.9651). CONCLUSION: In the pathogenesis of DPN, NPY may play a protective role in the peripheral nervous system and may be useful as a biomarker for detecting T2DM DPN.

Molecular subgroup of periodontitis revealed by integrated analysis of the microbiome and metabolome in a cross-sectional observational study.

Background: Periodontitis (PT) is a multifactorial, chronic inflammatory disease that can have heterogeneous clinical presentations. The oral microbiome and its metabolites have been implicated as the causes and regulators of PT pathogenesis. In this study, we assessed the oral microbiome and its metabolome in PT patients to clarify the interactions between the microbiome and its metabolites.Methods: A total of 112 subjects were recruited. Buccal and supragingival samples were collected for microbiome analysis. Saliva samples were collected for metabolomic analyses. Microbiome and metabolome data were analyzed and further integrated for combined analysis using various bioinformatics approaches.Results: Oral metabolomic analysis identified 28 metabolites distinguishing the healthy (H) and PT groups. PT group were further clustered into two subgroups (PT_G1 and PT_G2) depending on metabolite profiles. Oral microbiome analysis revealed discriminatory bacterial species in the H, PT_G1, and PT_G2 microbiota. Interestingly, PT_G2 had significantly higher concentration of short chain fatty acids and higher abundance of pathogenic bacteria. Integrated analysis of the microbiome and metabolome showed close association.Conclusion: Our results provide evidence of a close interplay between the oral microbiome and metabolome. Multi-omics approach including microbiome and microbe-associated metabolites may serve as diagnostic biomarkers and enhance treatment prediction in periodontal disease.

Washing- and Separation-Free Electrochemical Detection of Porphyromonas gingivalis in Saliva for Initial Diagnosis of Periodontitis.

Indirect detection of Porphyromonas gingivalis in saliva, based on proteolytic cleavage by an Arg-specific gingipain (Arg-gingipain), has traditionally been used for simple, initial diagnosis of periodontitis. To accurately detect P. gingivalis using a point-of-care format, development of a simple biosensor that can measure the exact concentration of P. gingivalis is required. However, electrochemical detection in saliva is challenging due to the presence of various interfering electroactive species in different concentrations. Here, we report a washing- and separation-free electrochemical biosensor for sensitive detection of P. gingivalis in saliva. Glycine-proline-arginine conjugated with 4-aminophenol (AP) was used as an electrochemical substrate for a trypsin-like Arg-gingipain, and glycylglycine was used to increase the Arg-gingipain activity. The electrochemical signal of AP was increased using electrochemical-chemical (EC) redox cycling involving an electrode, AP, and tris(2-carboxyethyl)phosphine, and the electrochemical charge signal was corrected using the initial charge obtained before a 15 min incubation period. The EC redox cycling combined with the matrix-corrected signal facilitated a high and reproducible signal without requiring washing and separation steps. The proteolytic cleavage of the electrochemical substrate was specific to P. gingivalis. The calculated detection limit for P. gingivalis in artificial saliva was 5 × 105 colony-forming units/mL, and the concentration of P. gingivalis in human saliva could be measured. The developed biosensor can be used as an initial diagnosis method to distinguish between healthy people and patients with periodontal diseases.

Prognostic role of high cathepsin D expression in breast cancer: a systematic review and meta-analysis.

BACKGROUND: High cathepsin D has been associated with poor prognosis in breast cancer; however, the results of many studies are controversial. Here, we assessed the association between high cathepsin D levels and worse breast cancer prognosis by conducting a meta-analysis. METHODS: A comprehensive search strategy was used to search relevant literature in PUBMED and EMBASE by September 2018. The meta-analysis was performed in Review Manager 5.3 using hazard ratios (HRs) with 95% confidence intervals (CIs). RESULTS: A total of 15,355 breast cancer patients from 26 eligible studies were included in this meta-analysis. Significant associations between elevated high cathepsin D and poor overall survival (OS) (HR = 1.61, 95% CI: 1.35-1.92, p < 0.0001) and disease-free survival (DFS) (HR = 1.52, 95% CI: 1.31-2.18, p < 0.001) were observed. In the subgroup analysis for DFS, high cathepsin D was significantly associated with poor prognosis in node-positive patients (HR = 1.38, 95% CI: 1.25-1.71, p < 0.00001), node-negative patients (HR = 1.78, 95% CI: 1.39-2.27, p < 0.0001), early stage patients (HR = 1.73, 95% CI: 1.34-2.23, p < 0.0001), and treated with chemotherapy patients (HR = 1.60, 95% CI: 1.21-2.12, p < 0.001). Interestingly, patients treated with tamoxifen had a low risk of relapse when their cathepsin D levels were high (HR = 0.71, 95% CI: 0.52-0.98, p = 0.04) and a high risk of relapse when their cathepsin D levels were low (HR = 1.50, 95% CI: 1.22-1.85, p = 0.0001). CONCLUSIONS: Our meta-analysis suggests that high expression levels of cathepsin D are associated with a poor prognosis in breast cancer. Based on our subgroup analysis, we believe that cathepsin D can act as a marker for poor breast cancer prognosis and also as a therapeutic target for breast cancer.

Identification of Potential Oral Microbial Biomarkers for the Diagnosis of Periodontitis.

Periodontitis is a chronic and multifactorial inflammatory disease that can lead to tooth loss. At present, the diagnosis for periodontitis is primarily based on clinical examination and radiographic parameters. Detecting the periodontal pathogens at the subgingival plaque requires skilled professionals to collect samples. Periodontal pathogens are also detected on various mucous membranes in patients with periodontitis. In this study, we characterized the oral microbiome profiles from buccal mucosa and supragingival space in a total of 272 healthy subjects as a control group, and periodontitis patients as a disease group. We identified 13 phyla, 193 genera, and 527 species and determined periodontitis-associated taxa. Porphyromonas gingivalis, Tannerella forsythia, Treponema denticolar, Filifactor alocis, Porphyromonas endodontalis, Fretibacterium fastiosum and Peptostreptococcus species were significantly increased in both the buccal mucosa and the supragingival space in periodontitis patients. The identified eight periodontitis-associated bacterial species were clinically validated in an independent cohort. We generated the prediction model based on the oral microbiome profiles using five machine learning algorithms, and validated its capability in predicting the status of patients with periodontitis. The results showed that the oral microbiome profiles from buccal mucosa and supragingival space can represent the microbial composition of subgingival plaque and further be utilized to identify potential microbial biomarkers for the diagnosis of periodontitis. Besides, bacterial community interaction network analysis found distinct patterns associated with dysbiosis in periodontitis. In summary, we have identified oral bacterial species from buccal and supragingival sites which can predict subgingival bacterial composition and can be used for early diagnosis of periodontitis. Therefore, our study provides an important basis for developing easy and noninvasive methods to diagnose and monitor periodontitis.

Role of Aggregatibacter actinomycetemcomitans-induced autophagy in inflammatory response.

BACKGROUND: Aggressive periodontitis is characterized by the early-onset and rapid progression of periodontal destruction and is closely associated with Aggregatibacter actinomycetemcomitans. Autophagy is a conserved process that is critical for removing damaged proteins, organelles, and even intracellular pathogens. Therefore, this study examined whether A. actinomycetemcomitans induces autophagy. In addition, the relationship among autophagy, bacterial internalization, and inflammatory molecules in periodontal aggressive inflammation was analyzed. METHODS: The expression of autophagy-related proteins in human gingival tissue and THP-1 cells was assessed by Western blot analysis. The formation of light chain 3 (LC3) puncta was examined by confocal microscopy. The degree of bacterial internalization into the cells was determined by the viable cell count. Phagocytosis and reactive oxygen species (ROS) production were measured using confocal microscopy and flow cytometry. RESULTS: When macrophages were infected with live A. actinomycetemcomitans, the autophagy influx was activated by the increase in LC3-II, autophagy-related gene 5/12, and Beclin-1 expression through the Toll-like receptors and extracellular signal-regulated kinase signaling pathways. The inhibition of A. actinomycetemcomitans-induced autophagy suppressed bacterial internalization via phagocytosis into the macrophages and increased interleukin (IL)-1ß production. Moreover, treatment with an ROS inhibitor inhibited these enhanced inflammatory responses. CONCLUSIONS: A. actinomycetemcomitans-induced autophagy promotes bacterial internalization by phagocytosis, which restricts the excessive inflammatory response by downregulating IL-1ß and ROS production in macrophages. Thus, A. actinomycetemcomitans-induced autophagy and its role in regulating the inflammatory response may play an important role in the aggressive periodontal inflammatory process, and be a target for the development of new periodontal therapies.

Catechin ameliorates Porphyromonas gingivalis-induced inflammation via the regulation of TLR2/4 and inflammasome signaling.

BACKGROUND: Porphyromonas gingivalis is a major periodontopathogen found in patients with chronic periodontitis that can lead to alveolar bone or tooth loss. Interleukin-1ß (IL-1ß), a proinflammatory cytokine, is most relevant to the pathogenesis of periodontitis. Catechin is one of the main polyphenol compounds found in green tea and possesses a range of health benefits. This study examined the anti-inflammatory effects of catechin in THP-1-derived macrophages infected with P. gingivalis as well as its effects on P. gingivalis-induced periodontitis in a mouse model. METHODS: The cytokine levels and relevant protein expression in THP-1 cells were measured using an enzyme-linked immunosorbent assay and Western blot analysis, respectively. An apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) pyroptosome formation was measured by confocal laser scanning microscopy. Micro-computed tomography was used to determine the level of bone loss induced by a P. gingivalis oral infection. RESULTS: Catechin attenuated the production of IL-1ß by inhibiting pro-IL-1ß expression via the downregulation of nuclear factor-κB, p38 mitogen-activated protein kinase, and Toll-like receptor signaling. In addition, catechin inhibited the activation of inflammasomes induced by P. gingivalis, but did not affect the growth of P. gingivalis. Catechin reduced the level of alveolar bone loss in a P. gingivalis-induced periodontitis mouse model. CONCLUSION: Catechin possesses anti-inflammatory properties by reducing the level of IL-1ß production, suggesting that it can potentially be used for the prevention and treatment of periodontal inflammation caused by P. gingivalis.

A built-in adjuvant-engineered mucosal vaccine against dysbiotic periodontal diseases.

Periodontitis is associated with a dysbiotic shift in the oral microbiome. Vaccine approaches to prevent microbial shifts from healthy to diseased state in oral biofilms would provide a fundamental therapeutic strategy against periodontitis. Since dental plaque formation is a polymicrobial and multilayered process, vaccines targeting single bacterial species would have limited efficacy in clinical applications. In this study, we developed a divalent mucosal vaccine consisting of a mixture of FlaB-tFomA and Hgp44-FlaB fusion proteins targeting virulence factors of inflammophilic bacteria Fusobacterium nucleatum and Porphyromonas gingivalis, respectively. Introduction of peptide linkers between FlaB and antigen improved the stability and immunogenicity of engineered vaccine antigens. The intranasal immunization of divalent vaccine induced protective immune responses inhibiting alveolar bone loss elicited by F. nucleatum and P. gingivalis infection. The built-in flagellin adjuvant fused to protective antigens enhanced antigen-specific antibody responses and class switch recombination. The divalent vaccine antisera recognized natural forms of surface antigens and reacted with diverse clinical isolates of Fusobacterium subspecies and P. gingivalis. The antisera inhibited F. nucleatum-mediated biofilm formation, co-aggregation of P. gingivalis and Treponema denticola, and P. gingivalis-host cell interactions. Taken together, the built-in adjuvant-engineered mucosal vaccine provides a technological platform for multivalent periodontitis vaccines targeting dysbiotic microbiome.

Streptococcus mutans activates the AIM2, NLRP3 and NLRC4 inflammasomes in human THP-1 macrophages.

Streptococcus mutans (S. mutans), a major aetiologic agent of dental caries, is involved in systemic diseases, such as bacterial endocarditis, if it enters the bloodstream through temporary bacteraemia. Interleukin (IL)-1ß, a proinflammatory cytokine, is related to the host defences against pathogens, and its synthesis, maturation, and secretion are tightly regulated by the activation of the inflammasome, an inflammatory signalling complex. This study examined the signalling mechanism of IL-1ß secretion and the inflammasome pathway induced by S. mutans to explain the molecular mechanism through which systemic infection by oral streptococci can occur. After infection of THP-1 cells with S. mutans, the expression of inflammasome components was detected using various methods. S. mutans induced IL-1ß secretion via caspase-1 activation, and S. mutans-induced IL-1ß secretion required absent in melanoma (AIM2), NLR family pyrin domain-containing 3 (NLRP3) and NLR family CARD domain-containing 4 (NLRC4) inflammasome activation. In particular, the S. mutans-induced NLRP3 inflammasome was mediated by adenosine triphosphate (ATP) release, potassium depletion and lysosomal damage. Our study provides novel insight into the innate immune response against S. mutans infection.

Trans-cinnamic aldehyde inhibits Aggregatibacter actinomycetemcomitans-induced inflammation in THP-1-derived macrophages via autophagy activation.

BACKGROUND: Inflammation is an essential response against bacterial infection as a host defense mechanism, which can lead to tissue damage. Aggregatibacter actinomycetemcomitans (Aa) is major pathogen for aggressive periodontitis characterized by rapid destruction of periodontal tissue surrounding teeth. Trans-cinnamic aldehyde is a key bioactive compound of the cinnamon extracts, which has anti-inflammatory, antioxidant, antipyretic, antimicrobial, and anti-cancer properties. The objective of the present study was to investigate the anti-inflammatory effect of trans-cinnamic aldehyde against Aa infection in human THP-1 derived macrophages and on Aa-induced periodontitis in mice. METHODS: THP-1 cells were differentiated with phorbol 12-mystristate 13-acetate and were infected with live Aa. Trans-cinnamic aldehyde was pretreated 30 minutes before the bacterial infection. Cytokine production was determined by enzyme-linked immunosorbent assay (ELISA) and protein expressions were detected by Western blot analysis. Autophagosome formation was detected by Cyto-ID. Viable cell count was carried out to determine bacterial adhesion, internalization, and intracellular survival. Experimental periodontitis was induced by inoculating Aa orally to mice, and microcomputed tomography was used to evaluate bone loss. RESULTS: Pretreatment of trans-cinnamic aldehyde significantly inhibited Aa-stimulated release of tumor necrosis factor-α and interleukin (IL)-1ß. Pretreatment of trans-cinnamic aldehyde inhibited Aa-induced expression of TLR signaling pathway as well as the phosphorylation of JNK, p38, and nuclear factor (NF)-κB. Also, trans-cinnamic aldehyde treatment downregulated the expression of pro-IL-1ß, caspase-1, and inflammasome components. Trans-cinnamic aldehyde treatment significantly decreased intracellular survival of Aa. Moreover, the autophagosome formation and the expressions of autophagy markers including Beclin-1, ATG5, and LC3 were increased. Finally, trans-cinnamic aldehyde significantly inhibited bone loss in Aa-induced mouse periodontitis. CONCLUSIONS: Trans-cinnamic aldehyde inhibited Aa-stimulated expression of inflammatory responses and inhibited intracellular bacterial survival via autophagy activation. These results suggest that trans-cinnamic aldehyde may serve as an anti-inflammatory agent for aggressive periodontitis.