Experimental Inoculation of Aggregatibacter actinomycetemcomitans and Streptococcus gordonii and Its Impact on Alveolar Bone Loss and Oral and Gut Microbiomes.

Oral bacteria are implicated not only in oral diseases but also in gut dysbiosis and inflammatory conditions throughout the body. The periodontal pathogen Aggregatibacter actinomycetemcomitans (Aa) often occurs in complex oral biofilms with Streptococcus gordonii (Sg), and this interaction might influence the pathogenic potential of this pathogen. This study aims to assess the impact of oral inoculation with Aa, Sg, and their association (Aa+Sg) on alveolar bone loss, oral microbiome, and their potential effects on intestinal health in a murine model. Sg and/or Aa were orally administered to C57Bl/6 mice, three times per week, for 4 weeks. Aa was also injected into the gingiva three times during the initial experimental week. After 30 days, alveolar bone loss, expression of genes related to inflammation and mucosal permeability in the intestine, serum LPS levels, and the composition of oral and intestinal microbiomes were determined. Alveolar bone resorption was detected in Aa, Sg, and Aa+Sg groups, although Aa bone levels did not differ from that of the SHAM-inoculated group. Il-1ß expression was upregulated in the Aa group relative to the other infected groups, while Il-6 expression was downregulated in infected groups. Aa or Sg downregulated the expression of tight junction genes Cldn 1, Cldn 2, Ocdn, and Zo-1 whereas infection with Aa+Sg led to their upregulation, except for Cldn 1. Aa was detected in the oral biofilm of the Aa+Sg group but not in the gut. Infections altered oral and gut microbiomes. The oral biofilm of the Aa group showed increased abundance of Gammaproteobacteria, Enterobacterales, and Alloprevotella, while Sg administration enhanced the abundance of Alloprevotella and Rothia. The gut microbiome of infected groups showed reduced abundance of Erysipelotrichaceae. Infection with Aa or Sg disrupts both oral and gut microbiomes, impacting oral and gut homeostasis. While the combination of Aa with Sg promotes Aa survival in the oral cavity, it mitigates the adverse effects of Aa in the gut, suggesting a beneficial role of Sg associations in gut health.

Oral infection with periodontal pathogens induced chronic obstructive pulmonary disease-like lung changes in mice.

BACKGROUND: Epidemiological studies have demonstrated that periodontitis is an independent risk factor for chronic obstructive pulmonary disease (COPD). However, the mechanism underlying the association between these two diseases remains unclear. The lung microbiota shares similarities with the oral microbiota, and there is growing evidence to suggest that the lung microbiome could play a role in the pathogenesis of COPD. This study aimed to investigate whether periodontal pathogens could contribute to the pathogenesis of COPD in a mouse model. METHODS: We established mouse models with oral infection by typical periodontal pathogens, porphyromonas gingivalis (Pg group) or fusobacterium nucleatum (Fn group), over a three-month period. Mice that did not receive oral infection were set as the control group (C group). We assessed the level of alveolar bone resorption, lung function, and histological changes in the lungs of the mice. Additionally, we measured the levels of inflammatory factors and tissue damage associated factors in the lung tissues. RESULTS: Lung function indices, including airway resistance, peak inspiratory/expiratory flow and expiratory flow-50%, were significantly reduced in the Fn group compared to the C group. Additionally, histological examination revealed an increased number of inflammatory cells and bullae formation in the lung tissue sections of the Fn group. Meanwhile, levels of inflammatory factors such as IL-1ß, IL-6, IFN-γ, and TNF-α, as well as tissue damage associated factors like matrix metalloproteinase-8 and neutrophil elastase, were significantly elevated in the lung tissue of the Fn group in comparison to the C group. The Pg group also showed similar but milder lung changes compared to the Fn group. Pg or Fn could be detected in the lungs of both oral infected groups. CONCLUSION: The results indicated that oral periodontal pathogens infection could induce COPD-like lung changes in mice, and they may play a biological role in the association between periodontitis and COPD.

Streptococcus gordonii Supragingival Bacterium Oral Infection-Induced Periodontitis and Robust miRNA Expression Kinetics.

Streptococcus gordonii (S. gordonii, Sg) is one of the early colonizing, supragingival commensal bacterium normally associated with oral health in human dental plaque. MicroRNAs (miRNAs) play an important role in the inflammation-mediated pathways and are involved in periodontal disease (PD) pathogenesis. PD is a polymicrobial dysbiotic immune-inflammatory disease initiated by microbes in the gingival sulcus/pockets. The objective of this study is to determine the global miRNA expression kinetics in S. gordonii DL1-infected C57BL/6J mice. All mice were randomly divided into four groups (n = 10 mice/group; 5 males and 5 females). Bacterial infection was performed in mice at 8 weeks and 16 weeks, mice were euthanized, and tissues harvested for analysis. We analyzed differentially expressed (DE) miRNAs in the mandibles of S. gordonii-infected mice. Gingival colonization/infection by S. gordonii and alveolar bone resorption (ABR) was confirmed. All the S. gordonii-infected mice at two specific time points showed bacterial colonization (100%) in the gingival surface, and a significant increase in mandible and maxilla ABR (p < 0.0001). miRNA profiling revealed 191 upregulated miRNAs (miR-375, miR-34b-5p) and 22 downregulated miRNAs (miR-133, miR-1224) in the mandibles of S. gordonii-infected mice at the 8-week mark. Conversely, at 16 weeks post-infection, 10 miRNAs (miR-1902, miR-203) were upregulated and 32 miRNAs (miR-1937c, miR-720) were downregulated. Two miRNAs, miR-210 and miR-423-5p, were commonly upregulated, and miR-2135 and miR-145 were commonly downregulated in both 8- and 16-week-infected mice mandibles. Furthermore, we employed five machine learning (ML) algorithms to assess how the number of miRNA copies correlates with S. gordonii infections in mice. In the ML analyses, miR-22 and miR-30c (8-week), miR-720 and miR-339-5p (16-week), and miR-720, miR-22, and miR-339-5p (combined 8- and 16-week) emerged as the most influential miRNAs.

An update on periodontal inflammation and bone loss.

Periodontal disease is a chronic inflammatory condition that affects the supporting structures of the teeth, including the periodontal ligament and alveolar bone. Periodontal disease is due to an immune response that stimulates gingivitis and periodontitis, and its systemic consequences. This immune response is triggered by bacteria and may be modulated by environmental conditions such as smoking or systemic disease. Recent advances in single cell RNA-seq (scRNA-seq) and in vivo animal studies have provided new insight into the immune response triggered by bacteria that causes periodontitis and gingivitis. Dysbiosis, which constitutes a change in the bacterial composition of the microbiome, is a key factor in the initiation and progression of periodontitis. The host immune response to dysbiosis involves the activation of various cell types, including keratinocytes, stromal cells, neutrophils, monocytes/macrophages, dendritic cells and several lymphocyte subsets, which release pro-inflammatory cytokines and chemokines. Periodontal disease has been implicated in contributing to the pathogenesis of several systemic conditions, including diabetes, rheumatoid arthritis, cardiovascular disease and Alzheimer's disease. Understanding the complex interplay between the oral microbiome and the host immune response is critical for the development of new therapeutic strategies for the prevention and treatment of periodontitis and its systemic consequences.

MyD88 exacerbates inflammation-induced bone loss by modulating dynamic equilibrium between Th17/Treg cells and subgingival microbiota dysbiosis.

BACKGROUND: This study aimed to investigate the contribution of myeloid differentiation primary-response gene 88 (MyD88) on the differentiation of T helper type 17 (Th17) and regulatory T (Treg) cells and the emerging subgingival microbiota dysbiosis in Porphyromonas gingivalis-induced experimental periodontitis. METHODS: Alveolar bone loss, infiltrated inflammatory cells, immunostained cells for tartrate-resistant acid phosphatase (TRAP), the receptor activator of nuclear factor-kB ligand (RANKL), and osteoprotegerin (OPG) were quantified by microcomputerized tomography and histological staining between age- and sex-matched homozygous littermates (wild-type [WT, Myd88+/+] and Myd88-/- on C57BL/6 background). The frequencies of Th17 and Treg cells in cervical lymph nodes (CLNs) and spleen were determined by flow cytometry. Cytokine expression in gingival tissues, CLNs, and spleens were studied by quantitative polymerase chain reaction (qPCR). Analysis of the composition of the subgingival microbiome and functional annotation of prokaryotic taxa (FAPROTAX) analysis were performed. RESULTS: P. gingivalis-infected Myd88-/- mice showed alleviated bone loss, TRAP+ osteoclasts, and RANKL/OPG ratio compared to WT mice. A significantly higher percentage of Foxp3+CD4+ T cells in infected Myd88-/- CLNs and a higher frequency of RORγt+CD4+ T cells in infected WT mice was noted. Increased IL-10 and IL-17a expressions in gingival tissue at D14-D28 then declined in WT mice, whereas an opposite pattern was observed in Myd88-/- mice. The Myd88-/- mice exhibited characteristic increases in gram-positive species and species having probiotic properties, while gram-negative, anaerobic species were noted in WT mice. FAPROTAX analysis revealed increased aerobic chemoheterotrophy in Myd88-/- mice, whereas anaerobic chemoheterotrophy was noted in WT mice after P. gingivalis infection. CONCLUSIONS: MyD88 plays an important role in inflammation-induced bone loss by modulating the dynamic equilibrium between Th17/Treg cells and dysbiosis in P. gingivalis-induced experimental periodontitis.

Autoinducer-2 produced by oral microbial flora and alveolar bone loss in periodontitis.

OBJECTIVE: The aim of this study was to investigate the association between autoinducer-2 (AI-2) of oral microbial flora and the alveolar bone destruction in periodontitis to determine if AI-2 may have the potential that monitor periodontitis and predict bone loss. BACKGROUND: Plaque biofilm was the initiating factor of periodontitis and the essential factor of periodontal tissue destruction. The formation of biofilms depended on the complex regulation of the quorum sensing (QS) system, in which bacteria could sense changes in surrounding bacterial density by secreting the autoinducer (AI) to regulate the corresponding physiological function. Most oral bacteria also communicated with each other to form biofilms administrating the QS system, which implied that the QS system of periodontal pathogens was related to periodontitis, but the specific relationship was unknown. METHOD: We collected the gingival crevicular fluid (GCF) samples and measured the concentration of AI-2 in samples using the Vibrio harveyi BB180 bioluminescent-reporter system. To explore the interaction between AI-2 and bone metabolism, we utilized AI-2 purified from Fusobacterium nucleatum to investigate the impact of F. nucleatum AI-2 on osteoclast differentiation. Moreover, we constructed murine periodontitis models and multi-species biofilm models to study the association between AI-2 and periodontal disease progression. RESULTS: The AI-2 concentration in GCF samples increased along with periodontal disease progression (p < .0001). F. nucleatum AI-2 promoted osteoclast differentiation in a dose-dependent manner. In the periodontitis mice model, the CEJ-ABC distance in the F. nucleatum AI-2 treatment group was higher than that in the simple ligation group (p < .01), and the maxilla of the mice in the group exhibited significantly lower BMD and BV/TV values (p < .05). CONCLUSIONS: We demonstrated that the AI-2 concentration varied with the alveolar bone destruction in periodontitis, and it may have the potential for screening periodontitis. F. nucleatum AI-2 promoted osteoclast differentiation in a dose-dependent manner and aggravated bone loss.

Oral Lactobacillus zeae exacerbates the pathological manifestation of periodontitis in a mouse model.

INTRODUCTION: The worldwide prevalence of periodontitis is considerably high, and its pathogenic mechanisms must be investigated and understood in order to improve clinical treatment outcomes and reduce the disease prevalence and burden. The exacerbation of the host immune system induced by oral microbial dysbiosis and the subsequent tissue destruction are the hallmarks of the periodontitis. However, the oral bacteria involved in periodontitis are not fully understood. We used the Oxford Nanopore Technologies (ONT) sequencing system to analyze metagenomic information in subgingival dental plaque from periodontitis and non-periodontitis patients. The number of Lactobacillus zeae (L. zeae) in the periodontitis patients was 17.55-fold higher than in the non-periodontitis patients, suggesting that L. zeae is a novel periodontitis-associated pathogen. Although several Lactobacillus species are used in vivo as probiotics to treat periodontitis and compete with Porphyromonas gingivalis (P. gingivalis), the roles of L. zeae in periodontitis progression, and the relationship between L. zeae and P. gingivalis needs to be investigated. METHODS: Both L. zeae and P. gingivalis were inoculated in the ligature-implant site of periodontitis mice. We collected mouse gingival crevicular fluid to analyze inflammatory cytokine secretion using a multiplex assay. Intact or sliced mouse maxilla tissue was used for micro-computed tomography analysis or hematoxylin and eosin staining, immunohistochemistry, and tartrate-resistant acid phosphatase staining to evaluate alveolar bone loss, neutrophil infiltration, and osteoclast activation, respectively. RESULTS: We observed that L. zeae competed with P. gingivalis, and it increased inflammatory cytokine secretion at the ligature-implant site. Similar to P. gingivalis, L. zeae promoted ligature-induced neutrophile infiltration, osteoclast activation, and alveolar bone loss. DISCUSSION: We, therefore, concluded that L. zeae accelerated the progression of periodontitis in the ligature-induced periodontitis mouse model.

The neutrophil-osteogenic cell axis promotes bone destruction in periodontitis.

The immune-stromal cell interactions play a key role in health and diseases. In periodontitis, the most prevalent infectious disease in humans, immune cells accumulate in the oral mucosa and promote bone destruction by inducing receptor activator of nuclear factor-κB ligand (RANKL) expression in osteogenic cells such as osteoblasts and periodontal ligament cells. However, the detailed mechanism underlying immune-bone cell interactions in periodontitis is not fully understood. Here, we performed single-cell RNA-sequencing analysis on mouse periodontal lesions and showed that neutrophil-osteogenic cell crosstalk is involved in periodontitis-induced bone loss. The periodontal lesions displayed marked infiltration of neutrophils, and in silico analyses suggested that the neutrophils interacted with osteogenic cells through cytokine production. Among the cytokines expressed in the periodontal neutrophils, oncostatin M (OSM) potently induced RANKL expression in the primary osteoblasts, and deletion of the OSM receptor in osteogenic cells significantly ameliorated periodontitis-induced bone loss. Epigenomic data analyses identified the OSM-regulated RANKL enhancer region in osteogenic cells, and mice lacking this enhancer showed decreased periodontal bone loss while maintaining physiological bone metabolism. These findings shed light on the role of neutrophils in bone regulation during bacterial infection, highlighting the novel mechanism underlying osteoimmune crosstalk.

Nicotinamide employs a starvation strategy against Porphyromonas gingivalis virulence by inhibiting the heme uptake system and gingipain activities.

Periodontitis is a common oral bacterial infection characterized by inflammatory responses. Its high prevalence lowers the quality of life for individuals and increases the global economic and disease burden. As microorganisms in dental plaque are responsible for this oral disease, antibacterial drug treatments are effective strategies for preventing and treating periodontitis. In this study, we investigated the inhibitory effect of nicotinamide (NAM), a vitamin B3 derivative, on the growth and virulence of Porphyromonas gingivalis, a key member of the red complex. Our findings revealed that NAM inhibited bacterial growth and gingipain activities, which played a dominant role in protein hydrolysis and heme acquisition. NAM decreased hemagglutination and hemolysis abilities and changed hemin and hemoglobin binding capacities, controlling bacterial infection through a starvation strategy by blocking access to growth-essential nutrients from the outside and reducing bacterial virulence. Several experiments in an animal model showed the effectiveness of NAM in preventing alveolar bone loss and reducing inflammatory cell infiltration, shedding light on its potential therapeutic applicability.

Luteolin inhibits Porphyromonas gingivalis growth and alleviates alveolar bone destruction in experimental murine periodontitis.

Periodontal disease is a major oral infectious disease that destroys alveolar bones and causes tooth loss. Porphyromonas gingivalis is a key pathogen that plays a crucial role in periodontitis. In our previous study on the anti-P. gingivalis activity of flavonoid, luteolin, a major flavonoid in edible plants, inhibited the proteolytic activity of gingipains, the major virulence factor in P. gingivalis. This study demonstrated luteolin in vitro and in vivo anti-bacterial activities. Thus, luteolin inhibits planktonic growth and biofilm formation in P. gingivalis. Furthermore, oral administration of luteolin alleviated maxillary alveolar bone resorption (ABR) in murine periodontitis induced by P. gingivalis infection. These results indicate that luteolin may be a potential therapeutic compound that targets P. gingivalis by hindering its growth, biofilm formation, and ABR in the oral cavity.

Oral biofilm dysbiosis during experimental periodontitis.

OBJECTIVES: We have previously characterized the main osteoimmunological events that occur during ligature periodontitis. This study aims to determine the polymicrobial community shifts that occur during disease development. METHODS: Periodontitis was induced in C57BL/6 mice using the ligature-induced periodontitis model. Healthy oral mucosa swabs and ligatures were collected every 3 days from 0 to 18 days post-ligature placement. Biofilm samples were evaluated by 16SrRNA gene sequencing (Illumina MiSeq) and QIIME. Time-course changes were determined by relative abundance, diversity, and rank analyses (PERMANOVA, Bonferroni-adjusted). RESULTS: Microbial differences between health and periodontal inflammation were observed at all phylogenic levels. An evident microbial community shift occurred in 25 genera during the advancement of "gingivitis" (3-6 days) to periodontitis (9-18 days). From day 0 to 18, dramatic changes were identified in Streptococcus levels, with an overall decrease (54.04%-0.02%) as well an overall increase of Enterococcus and Lactobacillus (23.7%-73.1% and 10.1%-70.2%, respectively). Alpha-diversity decreased to its lowest at 3 days, followed by an increase in diversity as disease advancement. Beta-diversity increased after ligature placement, indicating that bone loss develops in response to a greater microbial variability (p = 0.001). Levels of facultative and strict anaerobic bacteria augmented over the course of disease progression, with a total of eight species significantly different during the 18-day period. CONCLUSION: The data supports that murine gingival inflammation and alveolar bone loss develop in response to microbiome shifts. Bacterial diversity increased during progression to bone loss. These findings further support the utilization of the periodontitis ligature model for microbial shift analysis under different experimental conditions.

Interleukin-33 Deficiency Exacerbates Bone Loss Associated with Porphyromonas Gingivalis-Induced Experimental Periodontitis in Female Mice.

BACKGROUND/AIMS: Interleukin 33 (IL-33) plays a significant role in immunity but its role in bone physiology and periodontitis needs to be further investigated. The aim of this study was to decipher the contribution of IL-33 to bone homeostasis under physiological conditions, and to alveolar bone loss associated with experimental periodontitis (EP) in IL-33 knockout (KO) mice and their wildtype (WT) littermates. METHODS: The bone phenotype of IL-33 KO mice was studied in the maxilla, femur, and fifth lumbar vertebra by micro-computed tomography (micro-CT). EP was induced by a ligature soaked with the periopathogen Porphyromonas gingivalis (Pg) around a maxillary molar. Alveolar bone loss was quantified by micro-CT. The resorption parameters were assessed via toluidine blue staining on maxillary sections. In vitro osteoclastic differentiation assays using bone marrow cells were performed with or without lipopolysaccharide from Pg (LPS-Pg). RESULTS: First, we showed that under physiological conditions, IL-33 deficiency increased the trabecular bone volume/total volume ratio (BV/TV) of the maxillary bone in male and female mice, but not in the femur and fifth lumbar vertebra, suggesting an osteoprotective role for IL-33 in a site-dependent manner. The severity of EP induced by Pg-soaked ligature was increased in IL-33 KO mice but in female mice only, through an increase in the number of osteoclasts. Moreover, osteoclastic differentiation from bone marrow osteoclast progenitors in IL-33-deficient female mice is enhanced in the presence of LPS-Pg. CONCLUSION: Taken together, our data demonstrate that IL-33 plays a sex-dependent osteoprotective role both under physiological conditions and in EP with Pg.

Application of Ginsenoside Rd in Periodontitis With Inhibitory Effects on Pathogenicity, Inflammation, and Bone Resorption.

Periodontitis is a worldwide oral disease induced by the interaction of subgingival bacteria and host response and is characterized by local inflammation, bone resorption, and tooth loss. Ginsenoside Rd (Rd) is a biologically active component derived from Panax ginseng and has been demonstrated to exert antibacterial and anti-inflammatory activities. This study aims to investigate the inhibitory efficiency of Rd towards Porphyromonas gingivalis (P. gingivalis), periodontal inflammatory response, and osteoclastogenesis in vitro and to further validate the results in a mouse periodontitis model, thus, evaluate the potential effects of Rd on the control and prevention of periodontitis. According to the results, Rd exerted excellent antibacterial activities against planktonic P. gingivalis, along with attenuating P. gingivalis virulence and inhibiting its biofilms. Meanwhile, the inflammatory cytokine production and osteoclastogenesis were remarkably inhibited by Rd both in vitro and in vivo. Furthermore, Rd efficiently ameliorated the subgingival P. gingivalis abundance and suppressed the alveolar bone resorption in vivo as well. In conclusion, Rd has the potential to be developed as a promising medication in the control and prevention of periodontitis.

Treponema denticola Induces Alzheimer-Like Tau Hyperphosphorylation by Activating Hippocampal Neuroinflammation in Mice.

Alzheimer's disease (AD) is the most common type of dementia. Tau hyperphosphorylation and amyloid ß (Aß) deposition are the key pathological hallmarks of AD. Recent studies have shown that periodontitis is a significant risk factor for AD. The periodontal pathogen Porphyromonas gingivalis and its virulence factors have been shown to initiate and promote the hallmark pathologies and behavioral symptoms of AD. A possible link between Treponema denticola, another main periodontal pathogen, and AD has been reported. However, the role of T. denticola in AD pathogenesis is still unclear, and whether T. denticola and P. gingivalis exert a synergistic effect to promote AD development needs to be further studied. In this study, we investigated whether oral infection with T. denticola caused tau hyperphosphorylation in the hippocampi of mice and explored the underlying mechanisms. Orally administered T. denticola induced alveolar bone resorption, colonized brain tissues, and increased the activity of the phosphokinase GSK3ß by activating neuroinflammation in the hippocampus, thus promoting the hyperphosphorylation of the tau protein at Ser396, Thr181, and Thr231 in mice. An in vitro study with BV2 and N2a cell models of T. denticola invasion also verified the role of this pathogen in tau phosphorylation. T. denticola and P. gingivalis were not found to exert a synergistic effect on tau phosphorylation. In summary, these findings provide new insight into the important role of T. denticola in AD pathogenesis, providing biological connections between periodontal diseases and AD.

Sublingual immunization with recombinant GroEL plus CpG-ODN inhibits Porphyromonas gingivalis-induced inflammation and alveolar bone loss.

It has been reported that GroEL, a heat shock protein (HSP) produced by the representative periodontopathogenic bacterium, Porphyromonas gingivalis, induces inflammation-induced osteoclastogenesis and promotes alveolar bone resorption. In this study, we demonstrated the efficacy of a mucosal vaccine targeting GroEL against bone resorption induced by P. gingivalis. Female BALB/c mice received sublingual CpG oligodeoxynucleotide as an adjuvant with recombinant GroEL (rGroEL) prior to P. gingivalis exposure. Animals were euthanized 30 days after P. gingivalis inoculation. Sublingual immunization (SLI) with rGroEL elicited significant rGroEL-specific serum immunoglobulin (Ig)G and salivary IgA antibody (Ab) responses, and these responses were sustained for approximately 1 year. Interestingly, 10-fold more GroEL-specific IgA Ab-producing cells were detected in the submandibular glands (SMGs) than in the spleen. Antigen (Ag)-specific cells isolated from the spleen and SMGs induced significantly higher levels of IFN-γ expression after Ag restimulation in vitro. Flow cytometry illustrated that the frequency of CD11b+ dendritic cells with enhanced expression of CD80, CD86, CD40, and major histocompatibility complex II molecules was significantly increased in the SMGs. Furthermore, SLI with rGroEL significantly suppressed P. gingivalis-induced alveolar bone resorption and P. gingivalis-stimulated tumor necrosis factor-α, interleukin-6, and HSP60 expression in the gingiva. These findings suggest that SLI with rGroEL and CpG oligodeoxynucleotide is a beneficial strategy for preventing periodontal disease, mainly by presenting Ags in the oral region and inducing antibody production in the mucosal and systemic systems.

Antimicrobial-induced oral dysbiosis exacerbates naturally occurring alveolar bone loss.

Periodontitis-mediated alveolar bone loss is caused by dysbiotic shifts in the commensal oral microbiota that upregulate proinflammatory osteoimmune responses. The study purpose was to determine whether antimicrobial-induced disruption of the commensal microbiota has deleterious effects on alveolar bone. We administered an antibiotic cocktail, minocycline, or vehicle-control to sex-matched C57BL/6T mice from age 6- to 12 weeks. Antibiotic cocktail and minocycline had catabolic effects on alveolar bone in specific-pathogen-free (SPF) mice. We then administered minocycline or vehicle-control to male mice reared under SPF and germ-free conditions, and we subjected minocycline-treated SPF mice to chlorhexidine oral antiseptic rinses. Alveolar bone loss was greater in vehicle-treated SPF versus germ-free mice, demonstrating that the commensal microbiota drives naturally occurring alveolar bone loss. Minocycline- versus vehicle-treated germ-free mice had similar alveolar bone loss outcomes, implying that antimicrobial-driven alveolar bone loss is microbiota dependent. Minocycline induced phylum-level shifts in the oral bacteriome and exacerbated naturally occurring alveolar bone loss in SPF mice. Chlorhexidine further disrupted the oral bacteriome and worsened alveolar bone loss in minocycline-treated SPF mice, validating that antimicrobial-induced oral dysbiosis has deleterious effects on alveolar bone. Minocycline enhanced osteoclast size and interface with alveolar bone in SPF mice. Neutrophils and plasmacytoid dendritic cells were upregulated in cervical lymph nodes of minocycline-treated SPF mice. Paralleling the upregulated proinflammatory innate immune cells, minocycline therapy increased TH 1 and TH 17 cells that have known pro-osteoclastic actions in the alveolar bone. This report reveals that antimicrobial perturbation of the commensal microbiota induces a proinflammatory oral dysbiotic state that exacerbates naturally occurring alveolar bone loss.

Episymbiotic Saccharibacteria suppresses gingival inflammation and bone loss in mice through host bacterial modulation.

Saccharibacteria (TM7) are obligate epibionts living on the surface of their host bacteria and are strongly correlated with dysbiotic microbiomes during periodontitis and other inflammatory diseases, suggesting they are putative pathogens. However, due to the recalcitrance of TM7 cultivation, causal research to investigate their role in inflammatory diseases is lacking. Here, we isolated multiple TM7 species on their host bacteria from periodontitis patients. These TM7 species reduce inflammation and consequential bone loss by modulating host bacterial pathogenicity in a mouse ligature-induced periodontitis model. Two host bacterial functions involved in collagen binding and utilization of eukaryotic sialic acid are required for inducing bone loss and are altered by TM7 association. This TM7-mediated downregulation of host bacterial pathogenicity is shown for multiple TM7/host bacteria pairs, suggesting that, in contrast to their suspected pathogenic role, TM7 could protect mammalian hosts from inflammatory damage induced by their host bacteria.

Spirulina maxima reduces inflammation and alveolar bone loss in Porphyromonas gingivalis-induced periodontitis.

BACKGROUND: Periodontitis is a common oral disease characterized as inflammation on gingival tissue and alveolar bone resorption. Spirulina maxima has been reported to have anti-oxidative and anti-inflammatory effects on gastric ulcers. However, its effects on gingival inflammation and alveolar bone resorption of periodontitis have not been studied. PURPOSE: This study was designed to investigate the effects of S. maxima on the P. gingivalis-induced periodontitis and to elucidate its mechanism. METHODS: The phycocyanin contents in S. maxima were identified by high-performance liquid chromatography. 8-week old SD rats were induced periodontitis by inoculation with P. gingivalis for 14 days. The rats were then orally treated with S. maxima 100, 200, 400 mg/kg, or indomethacin (IND, positive control) 5 mg/kg for an additional 14 days. Inflammatory responses, expressions of collagenases in gingival tissue, osteoclast formation and activation, alveolar bone resorption, osteogenesis-related markers, and BMP2/Smad signaling in alveolar bone were measured. RESULTS: Pro-inflammatory cytokines such as TNF-α, IL-1ß, IL-6, and inflammatory transcription factor NF-κB were decreased in gingival tissue by S. maxima administration. Also, myeloperoxidase (MPO) activity and matrix metalloproteinase (MMPs) expression were decreased by S. maxima administration. Conversely, S. maxima increased IL-4, anti-inflammatory cytokine from Th2 cells. The osteoprotegerin (OPG) / receptor activator of NF-κB ligand (RANKL) expression ratio, which represents osteoclast-osteoblast balance, was increased in S. maxima-treated groups. The alveolar bone loss and the number of TRAP-positive osteoclast cells were also declined in S. maxima-treated groups while the osteoblasts count was increased. Besides, in S. maxima-treated groups, the osteogenesis-related factors were promoted and BMP-2/Smad pathway was up-regulated in a periodontitis condition. CONCLUSION: S. maxima reduces periodontitis induced by P. gingivalis through anti-inflammatory effect and resultant reduction in bone loss, suggesting that S. maxima might be a potential agent for treating periodontitis.

Impaired salivary SIgA antibodies elicit oral dysbiosis and subsequent induction of alveolar bone loss.

OBJECTIVE: Secreted IgA (SIgA) plays a central role in preventing bacterial and viral infections on mucosal surfaces by neutralizing toxins and viruses and inhibiting bacterial attachment to epithelial cells. However, the role of salivary SIgA antibodies (Abs) in regulating oral flora is still unknown. This study aimed to evaluate the association among oral bacteria, their metabolites and periodontitis in IgA-deficient (IgA KO) and wild-type (WT) control mice. METHODS: Microcomputed tomography (micro-CT) analysis was used to assess alveolar bone resorption as a development of periodontitis. The bacterial profiles of saliva were determined using the next-generation sequencing assays. Furthermore, the metabolites in saliva were measured and compared using CE-TOFMS. RESULTS: Salivary microbiota of IgA KO mice revealed a remarkably decreased frequency of Streptococcus, and increased percentages of Aggregatibacer, Actinobacillus, and Prevotella at the genus level when compared with those of WT. Compared to WT control mice of the same age, the level of alveolar bone loss was significantly increased in IgA KO mice, and infiltration of osteoclasts was found on the surface of the alveolar bone. The metabolome profile indicated that the metabolites of IgA KO mice had greater variability in carbon metabolic, urea cycle, and lipid pathways than WT mice. CONCLUSION: These results suggest that salivary SIgA plays an important role in regulating and maintaining normal oral microflora to prevent the development of periodontal disease.

Role of the RprY response regulator in P. gingivalis community development and virulence.

Porphyromonas gingivalis expresses a limited number of two-component systems, including RprY, an orphan response regulator which lacks a cognate sensor kinase. In this study, we examined cross-phosphorylation of RprY on tyrosine residues and its importance for RprY function. We show that RprY reacts with phosphotyrosine antibodies, and found that the tyrosine (Y) residue at position 41 is predicted to be solvent accessible. Loss of RprY increased the level of heterotypic community development with Streptococcus gordonii, and the community-suppressive function of RprY required Y41. Expression of the Mfa1 fimbrial adhesin was increased in the rprY mutant and in the mutant complemented with rprY containing a Y41F mutation. In a microscale thermophoresis assay, recombinant RprY protein bound to the promoter region of mfa1, and binding was diminished with RprY containing the Y41F substitution. RprY was required for virulence of P. gingivalis in a murine model of alveolar bone loss. Transcriptional profiling indicated that RprY can control the expression of genes encoding the type IX secretion system (T9SS) machinery and virulence factors secreted through the T9SS, including the gingipain proteases and peptidylarginine deiminase (PPAD). Collectively, these results establish the RprY response regulator as a component of the tyrosine phosphorylation regulon in P. gingivalis, which can independently control heterotypic community development through the Mfa1 fimbriae and virulence through the T9SS.