The combination of experimental periodontitis and oral microbiota from periodontitis patients aggravates liver fibrosis in mice.

AIM: Periodontitis (PD) is the sixth most prevalent disease around the world and is involved in the development and progression of multiple systemic diseases. Previous studies have reported that PD may aggravate liver injuries. The objective of this study was to investigate whether and how PD affects liver fibrosis. MATERIALS AND METHODS: Ligature-induced PD (LIP) was induced in male C57/B6J mice, and sub-gingival plaques (PL) from patients with PD were applied to mouse teeth. Liver fibrosis was induced by carbon tetrachloride (CCl4 ) injection. The mice were randomly divided into six groups: Oil, Oil+LIP, Oil+LIP+PL, CCl4 , CCl4 +LIP, and CCl4 +LIP+PL. Alveolar bone resorption was evaluated by methylene blue staining. Hepatic function was analysed by serum alanine aminotransferase and hepatic hydroxyproline. Picrosirius red and α-smooth muscle actin (SMA) staining were used to evaluate the fibrotic area. RNA sequencing and quantitative RT-PCR were used to measure gene expression. Western blotting was used to measure protein levels. Flow cytometry was used to analyse the accumulation of immune cells. Mouse microbiota were analysed using 16S rRNA gene sequencing. RESULTS: Mice in the CCl4 +LIP+PL group displayed higher serum alanine aminotransferase and hepatic hydroxyproline as well as more Picrosirius red-positive and α-SMA-positive areas in liver samples than those of the CCl4 group, suggesting that PD (LIP+PL) aggravated CCl4 -induced hepatic dysfunction and liver fibrosis. Consistently, the expression of fibro-genic genes and the protein levels of transforming growth factor ß were much higher in the CCl4 +LIP+PL group than in the CCl4 group. Flow cytometry revealed that PD increased the accumulation of immune cells, including Kupffer cells, B cells, and Th17 cells, in the liver of mice with CCl4 treatment. PD also increased the expression of inflammatory genes and activated pro-inflammatory nuclear factor-kappa B pathway in the livers of CCl4 -injected mice. Moreover, PD altered both oral and liver microbiota in CCl4 -injected mice. CONCLUSIONS: PD aggravates CCl4 -induced hepatic dysfunction and fibrosis in mice, likely through the increase of inflammation and alteration of microbiota in the liver.

Periodontitis aggravates renal inflammatory response in a mouse model of renal fibrosis.

OBJECTIVES: To assess the effects of periodontitis on renal interstitial fibrosis in a mouse model. MATERIALS AND METHODS: Thirty C57BL/6 male mice were divided into control, periodontitis (PD), unilateral ureteral ligation (UUO) and PD+UUO groups. Unilateral ureteral ligation was performed 6 days after periodontitis. After 2 weeks, all mice were sacrificed, and samples were collected for the assessment of gene expression, immune cells, biochemical indicators and renal pathology. RESULTS: Expression of tumour necrosis factor-α, interleukin-1ß, and Ly6G in the kidneys in the PD+UUO group was significantly greater than in the UUO group. The percentage of CD11b+ Ly6G+ cells was significantly higher in the PD+UUO than in the UUO group. Fibrotic areas in the kidneys in the PD+UUO group were slightly, but not significantly, greater than those in the UUO group. Kidneys from the PD+UUO group showed markedly higher gene expression of matrix metalloproteinase-9, but not α-smooth muscle actin or collagen I, than those in the UUO group. There were no significant differences in blood urea nitrogen, serum creatinine and uric acid between the PD+UUO and UUO groups. CONCLUSIONS: Periodontitis increases the renal inflammatory response without showing a significant influence on renal interstitial fibrosis or renal function in the UUO mouse model.

Alterations of oral and gut viromes in hypertension and/or periodontitis.

The microbiota plays an important role in both hypertension (HTN) and periodontitis (PD), and PD exacerbates the development of HTN by oral and gut microbiota. Previous studies have focused on exploring the importance of the bacteriome in HTN and PD but overlooked the impact of the virome, which is also a member of the microbiota. We collected 180 samples of subgingival plaques, saliva, and feces from a cohort of healthy subjects (nHTNnPD), subjects with HTN (HTNnPD) or PD (PDnHTN), and subjects with both HTN and PD (HTNPD). We performed metagenomic sequencing to assess the roles of the oral and gut viromes in HTN and PD. The HTNnPD, PDnHTN, and HTNPD groups all showed significantly distinct beta diversity from the nHTNnPD group in saliva. We analyzed alterations in oral and gut viral composition in HTN and/or PD and identified significantly changed viruses in each group. Many viruses across three sites were significantly associated with blood pressure and other clinical parameters. Combined with these clinical associations, we found that Gillianvirus in subgingival plaques was negatively associated with HTN and that Torbevirus in saliva was positively associated with HTN. We found that Pepyhexavirus from subgingival plaques was indicated to be transferred to the gut. We finally evaluated viral-bacterial transkingdom interactions and found that viruses and bacteria may cooperate to affect HTN and PD. Correspondingly, HTN and PD may synergize to improve communications between viruses and bacteria.IMPORTANCEPeriodontitis (PD) and hypertension (HTN) are both highly prevalent worldwide and cause serious adverse outcomes. Increasing studies have shown that PD exacerbates HTN by oral and gut microbiota. Previous studies have focused on exploring the importance of the bacteriome in HTN and PD but overlooked the impact of the virome, even though viruses are common inhabitants in humans. Alterations in oral and gut viral diversity and composition contribute to diseases. The present study, for the first time, profiled the oral and gut viromes in HTN and/or PD. We identified key indicator viruses and their clinical implications in HTN and/or PD. We also investigated interactions between viruses and bacteria. This work improved the overall understanding of the viromes in HTN and PD, providing vital insights into the role of the virome in the development of HTN and PD.

Oral pathogens exacerbate Parkinson's disease by promoting Th1 cell infiltration in mice.

BACKGROUND: Parkinson's disease (PD) is a common chronic neurological disorder with a high risk of disability and no cure. Periodontitis is an infectious bacterial disease occurring in periodontal supporting tissues. Studies have shown that periodontitis is closely related to PD. However, direct evidence of the effect of periodontitis on PD is lacking. Here, we demonstrated that ligature-induced periodontitis with application of subgingival plaque (LIP-SP) exacerbated motor dysfunction, microglial activation, and dopaminergic neuron loss in 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice. RESULTS: The 16S rRNA gene sequencing revealed that LIP-SP induced oral and gut dysbiosis. Particularly, Veillonella parvula (V. parvula) and Streptococcus mutans (S. mutans) from oral ligatures were increased in the fecal samples of MPTP + LIP-SP treated mice. We further demonstrated that V. parvula and S. mutans played crucial roles in LIP-SP mediated exacerbation of motor dysfunction and neurodegeneration in PD mice. V. parvula and S. mutans caused microglial activation in the brain, as well as T helper 1 (Th1) cells infiltration in the brain, cervical lymph nodes, ileum and colon in PD mice. Moreover, we observed a protective effect of IFNγ neutralization on dopaminergic neurons in V. parvula- and S. mutans-treated PD mice. CONCLUSIONS: Our study demonstrates that oral pathogens V. parvula and S. mutans necessitate the existence of periodontitis to exacerbate motor dysfunction and neurodegeneration in MPTP-induced PD mice. The underlying mechanisms include alterations of oral and gut microbiota, along with immune activation in both brain and peripheral regions. Video Abstract.

Alleviate Periodontitis and Its Comorbidity Hypertension using a Nanoparticle-Embedded Functional Hydrogel System.

Periodontitis and hypertension often occur as comorbidities, which need to be treated at the same time. To resolve this issue, a controlled-release composite hydrogel approach is proposed with dual antibacterial and anti-inflammatory activities as a resolution to achieve the goal of co-treatment of comorbidities. Specifically, chitosan (CS) with inherent antibacterial properties is cross-linked with antimicrobial peptide (AMP)-modified polyethylene glycol (PEG) to form a dual antibacterial hydrogel (CS-PA). Subsequently, curcumin loaded into biodegradable nanoparticles (CNP) are embedded in the hydrogel exhibiting high encapsulation efficiency and sustained release to achieve long-term anti-inflammatory activities. In a mouse model of periodontitis complicated with hypertension, CS-PA/CNP is applied to gingival sulcus and produced an optimal therapeutic effect on periodontitis and hypertension simultaneously. The therapeutic mechanisms are deeply studied and indicated that CS-PA/CNP exerted excellent immunoregulatory effects by suppressing the accumulation of lymphocytes and myeloid cells and enhanced the antioxidant capacity and thus the anti-inflammatory capacity of macrophages through the glutathione metabolism pathway. In conclusion, CS-PA/CNP has demonstrated its superior therapeutic effects and potential clinical translational value in the co-treatment of periodontitis and hypertension, and also serves as a drug delivery platform to provide combinatorial therapeutic options for periodontitis with complicated pathogenesis.

Periodontitis exacerbates atherosclerosis through Fusobacterium nucleatum-promoted hepatic glycolysis and lipogenesis.

AIMS: Positive associations between periodontitis (PD) and atherosclerosis have been established, but the causality and mechanisms are not clear. We aimed to explore the causal roles of PD in atherosclerosis and dissect the underlying mechanisms. METHODS AND RESULTS: A mouse model of PD was established by ligation of molars in combination with application of subgingival plaques collected from PD patients and then combined with atherosclerosis model induced by treating atheroprone mice with a high-cholesterol diet (HCD). PD significantly aggravated atherosclerosis in HCD-fed atheroprone mice, including increased en face plaque areas in whole aortas and lesion size at aortic roots. PD also increased circulating levels of triglycerides and cholesterol, hepatic levels of cholesterol, and hepatic expression of rate-limiting enzymes for lipogenesis. Using 16S ribosomal RNA (rRNA) gene sequencing, Fusobacterium nucleatum was identified as the most enriched PD-associated pathobiont that is present in both the oral cavity and livers. Co-culture experiments demonstrated that F. nucleatum directly stimulated lipid biosynthesis in primary mouse hepatocytes. Moreover, oral inoculation of F. nucleatum markedly elevated plasma levels of triglycerides and cholesterol and promoted atherogenesis in HCD-fed ApoE-/- mice. Results of RNA-seq and Seahorse assay indicated that F. nucleatum activated glycolysis, inhibition of which by 2-deoxyglucose in turn suppressed F. nucleatum-induced lipogenesis in hepatocytes. Finally, interrogation of the molecular mechanisms revealed that F. nucleatum-induced glycolysis and lipogenesis by activating PI3K/Akt/mTOR signalling pathway in hepatocytes. CONCLUSIONS: PD exacerbates atherosclerosis and impairs lipid metabolism in mice, which may be mediated by F. nucleatum-promoted glycolysis and lipogenesis through PI3K/Akt/mTOR signalling in hepatocytes. Treatment of PD and specific targeting of F. nucleatum are promising strategies to improve therapeutic effectiveness of hyperlipidaemia and atherosclerosis.

Roles of oral microbiota and oral-gut microbial transmission in hypertension.

INTRODUCTION: Considerable evidence has linked periodontitis (PD) to hypertension (HTN), but the nature behind this connection is unclear. Dysbiosis of oral microbiota leading to PD is known to aggravate different systematic diseases, but the alteration of oral microbiota in HTN and their impacts on blood pressure (BP) remains to be discovered. OBJECTIVES: To characterize the alterations of oral and gut microbiota and their roles in HTN. METHODS: We performed a cross-sectional (95 HTN participants and 39 controls) and a 6-month follow-up study (52 HTN participants and 26 controls) to analyze the roles of oral and gut microbiota in HTN. Saliva, subgingival plaques, and feces were collected for 16S rRNA gene sequencing or metagenomic analysis. C57BL/6J mice were pretreated with antibiotics to deplete gut microbiota, and then transplanted with human saliva by gavage to test the impacts of abnormal oral-gut microbial transmission on HTN. RESULTS: BP in participants with PD was higher than no PD in both cross-sectional and follow-up cohort. Relative abundances of 14 salivary genera, 15 subgingival genera and 10 gut genera significantly altered in HTN and those of 7 salivary genera, 12 subgingival genera and 6 gut genera significantly correlated with BP. Sixteen species under 5 genera were identified as oral-gut transmitters, illustrating the presence of oral-gut microbial transmission in HTN. Veillonella was a frequent oral-gut transmitter stably enriched in HTN participants of both cross-sectional and follow-up cohorts. Saliva from HTN participants increased BP in hypertensive mice. Human saliva-derived Veillonella successfully colonized in mouse gut, more abundantly under HTN condition. CONCLUSIONS: PD and oral microbiota are strongly associated with HTN, likely through oral-gut transmission of microbes. Ectopic colonization of saliva-derived Veillonella in the gut may aggravate HTN. Therefore, precise manipulations of oral microbiota and/or oral-gut microbial transmission may be useful strategies for better prevention and treatment of HTN.

A Mouse Periodontitis Model With Humanized Oral Bacterial Community.

Increasing evidence suggests that periodontitis, characterized by oral dysbiosis, is a critical player in the progression of multiple systemic diseases in humans. However, there is still a lack of a proper mouse model of periodontitis with the colonization of human periodontitis-associated bacteria. We here established a new mouse periodontitis model by combining ligation of the second molars with application of subgingival plaques from periodontitis patients. Using 16S rRNA gene sequencing and Taxonomic classification, we found that human periodontitis-associated bacteria efficiently colonized in the mouse model and were enriched in both ligature silk and mouse saliva. Furthermore, the well-recognized periodontal pathogens including Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, and Tannerella forsythia were enriched in the new model, but not in ligature-induced periodontitis model or Sham mice. The human periodontitis-associated bacteria potently aggravated mouse periodontitis, as demonstrated by more severe bone resorption and higher expression of inflammatory and osteoclastogenesis genes. In summary, the new mouse periodontitis model paves the way for studying human periodontitis-associated bacteria in oral diseases and systemic diseases.

Biomimetic Hydroxyapatite Nanorods Promote Bone Regeneration via Accelerating Osteogenesis of BMSCs through T Cell-Derived IL-22.

Manipulations of morphological properties of nanobiomaterials have been demonstrated to modulate the outcome of osteoimmunomodulation and eventually osteogenesis through innate immune response. However, the functions and mechanisms of adaptive immune cells in the process of nanobiomaterials-mediated bone regeneration have remained unknown. Herein, we developed bone-mimicking hydroxyapatite (HAp) nanorods with different aspect ratios as model materials to investigate the impacts of the nanoshape features on osteogenesis and to explore the underlying mechanisms focusing on the functions of T cells and T cell-derived cytokines. HAp nanorods with different aspect ratios (HAp-0, HAp-30, and HAp-100) were implanted into mouse mandibular defect models. Micro-CT and hematoxylin and eosin staining demonstrated that HAp-100 had the best osteogenic effects. Flow cytometry analysis revealed that HAp-100 increased the percentage of T cells in injured mandibles. The osteogenic effects of HAp-100 were significantly blunted in injured mandibles of TCRß-/- mice. The Luminex xMAP assay and ELISA showed that HAp-100 induced a marked increase of interleukin (IL)-22 in injured mandibles. In cultured T cells, HAp-100 manifested the best capacity to induce the production of IL-22. Conditioned media from HAp-100-primed T cells promoted osteogenesis and JAK1/STAT3 activation in bone marrow stromal cells, all of which were abolished by neutralizing antibodies against IL-22. In summary, bone-mimicking HAp nanorods with different aspect ratios could regulate osteogenesis through modulation of T cells and IL-22 in the bone regeneration process. These findings provided insights for mediation of the immune response of T cells by nanomaterials on osteogenesis and strategies for designing biomaterials with osteoimmunomodulative functions.

[The effect of chronic kidney disease on periodontitis-related microorganisms in mice].

PURPOSE: To explore the effect of chronic kidney disease on the composition of oral microbial community in mice and find the significant species. METHODS: Twenty C57BL/6 male mice were randomly divided into 4 groups: healthy control group (HC), periodontitis group (PD), chronic kidney disease group (CKD) and chronic kidney disease and periodontitis group (CKD+PD). The periodontitis model was created in the fourth week when the chronic kidney disease model was established, and then the mice were sacrificed in the sixth week. Histopathological analysis of the kidney was performed by H-E staining and Masson's trichrome staining. Alveolar bone resorption of maxilla was analyzed by micro-CT analysis. The third-generation full-length sequencing of 16SrRNA gene was used to analyze the composition of oral microbial community among groups. Statistical analysis was performed using SPSS 24.0 software package. RESULTS: There were significant differences in alveolar bone resorption, the richness of species and the overall composition of the microbial community among the four groups (P＜0.001). In CKD group, Streptococcus azizii had the most significant abundance. Escherichia coli was the most significantly different species among identifiable bacteria in CKD+PD group, while Staphylococcus lentus and Lactobacillus murinus were species with the most significant differences in HC group and PD group, respectively. CONCLUSIONS: The composition of the oral microbial community was significantly different among four groups with significant species.

Engineering DNA-Nanozyme Interfaces for Rapid Detection of Dental Bacteria.

Engineering biological interfaces represents a powerful means to improve the performance of biosensors. Here, we developed a DNA-engineered nanozyme interface for rapid and sensitive detection of dental bacteria. We employed DNA aptamer as both molecular recognition keys and adhesive substrates to functionalize the nanozyme. Utilizing different immobilization strategies and DNA designs, a range of DNA nanoscale biointerfaces were constructed to modulate enzymatic and biological properties of the nanozyme systems. These functional biointerfaces improved the accessibility of bacteria to the nanozyme surface, providing large signal change range at optimal DNA probe density. The DNA-functionalized nanozymes demonstrate a rapid, label-free, and highly sensitive direct colorimetric detection of Streptococcus mutans, with a detection limit of 12 CFU mL-1, as well as excellent discrimination from other dental bacteria. We demonstrate the use of this biological nanointerface for identifying dental bacteria in salivary samples, showing its potential in clinical prevention and diagnosis of dental diseases.

Impact of Graphene Exposure on Microbial Activity and Community Ecosystem in Saliva.

Graphene-based nanomaterials (GMs) are served as great promising agents for the prevention and therapy of infectious diseases. However, their dental applications remain to be evaluated, especially under the context of the oral microbial community. Here, we examined the exposure-response of salivary bacterial community to two types of GMs, that is, graphene oxide (GO) and GO-silver nanoparticles (AgNPs). Both GO and GO-AgNPs showed lethal effect against salivary bacteria in a concentration-dependent manner, and the antibacterial capacity of GO-AgNPs is superior to GO. Interestingly, the salivary bacterial community enhanced the tolerance to GMs as compared to homogeneous bacteria. High-throughput sequencing revealed that both 80 µg/mL GO and 20 µg/mL GO-AgNPs significantly altered the biodiversity of salivary bacterial community. Especially, they increased the relative abundance of Gram-positive bacteria compared to the untreated sample, notably Streptococcus, suggesting that the bacterial wall structure plays a critical role in resisting the damage of GMs. Although GMs could effectively limit the salivary bacterial activity and cause changes in bacterial community structure, they are not toxic to mammalian cell lines. We envision this study could provide novel insights into the application of GMs as "green antibiotics" in nanomedicine.