Integration of metalloproteome and immunoproteome reveals a tight link of iron-related proteins with COVID-19 pathogenesis and immunity.

Increasing clinical data show that the imbalance of host metallome is closely associated with different kinds of disease, however, the intrinsic mechanisms of action of metals in immunity and pathogenesis of disease remain largely undefined. There is lack of multiplexed profiling system to integrate the metalloproteome-immunoproteome information at systemic level for exploring the roles of metals in immunity and disease pathogenesis. In this study, we build up a metal-coding assisted multiplexed proteome assay platform for serum metalloproteomic and immunoproteomic profiling. By taking COVID-19 as a showcase, we unbiasedly uncovered the most evident modulation of iron-related proteins, i.e., Ft and Tf, in serum of severe COVID-19 patients, and the value of Ft/Tf could work as a robust biomarker for COVID-19 severity stratification, which overtakes the well-established clinical risk factors (cytokines). We further uncovered a tight association of transferrin with inflammation mediator IL-10 in COVID-19 patients, which was proved to be mainly governed by the monocyte/macrophage of liver, shedding light on new pathophysiological and immune regulatory mechanisms of COVID-19 disease. We finally validated the beneficial effects of iron chelators as anti-viral agents in SARS-CoV-2-infected K18-hACE2 mice through modulation of iron dyshomeostasis and alleviating inflammation response. Our findings highlight the critical role of liver-mediated iron dysregulation in COVID-19 disease severity, providing solid evidence on the involvement of iron-related proteins in COVID-19 pathophysiology and immunity.

N-acyl homoserine lactones lactonase est816 suppresses biofilm formation and periodontitis in rats mediated by Aggregatibacter actinomycetemcomitans.

Aims: The current study aimed to explore the adjuvant therapeutic effect of N-acyl homoserine lactones (AHLs)-lactonase est816 on Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) biological behaviors and periodontitis progression. Methods: The inhibitory properties of est816 were detected by live/dead bacterial staining, scanning electron microscope (SEM), crystal-violet staining and reverse-transcription quantitative PCR (RT-qPCR). Biocompatibility of est816 on human gingival fibroblasts (HGFs) and human gingival epithelial cells (HGEs) was evaluated by CCK8 and ELISA. The ligature-induced periodontitis model was established in rats. Micro computed tomography and immunohistochemical and histological staining served to evaluate the effect of est816 on the prevention of periodontitis in vivo. Results: est816 significantly attenuated biofilm formation, reduced the mRNA expression of cytolethal distending toxin, leukotoxin and poly-N-acetyl glucosamine (PNAG) and downregulated expressions of interleukin-6 and tumor necrosis factor-α with low cell toxicity. In vivo investigations revealed est816 decreased alveolar bone resorption, suppressed matrix metalloproteinase-9 expression and increased osteoprotegerin expression. Conclusion: est816 inhibited A. actinomycetemcomitans biofilm formation and virulence release, resulting in anti-inflammation and soothing of periodontitis in rats, indicating that est816 could be investigated in further research on periodontal diseases.

Human bone marrow mesenchymal stem cell-derived extracellular vesicles restore Th17/Treg homeostasis in periodontitis via miR-1246.

T-cell-mediated immunity is crucial in the immunopathology of periodontitis. The restoration of the homeostasis between the T helper cell 17 (Th17) and regulatory T cell (Treg) subsets by extracellular vesicles (EVs) obtained from human bone marrow stem cells (hBMSCs) promotes new bone formation and suppresses inflammation. Uncovering the functions of hBMSC-derived EVs in the immune microenvironment of periodontal tissue and their underlying regulatory mechanisms may shed new light on developing potential cell-free immunotherapies for periodontal regeneration. Here, we reported that the Th17/Treg ratio elevated in peripheral blood from periodontitis patients. Furthermore, we found that hBMSC-derived EVs could reduce the Th17/Treg ratio in CD4+ T cells from periodontitis patients in vitro and ameliorate conditions of experimental periodontitis in mice. Additionally, by investigating the differentially expressed miRNAs and target genes in EVs from hBMSCs stimulated with Porphyromonas gingivalis LPS using miRNA sequencing, we found that EV-miR-1246 is highly effective at downregulating the ratio of Th17/Treg in vitro. Mechanistically, EV-miR-1246 suppressed expression of its potential target angiotensin-converting enzyme 2 (ACE2) and increased the p-Yes-associated protein (YAP)1/YAP1 ratio in CD4+ T cells. Our results indicated that hBMSC-derived EVs improve periodontitis via miR-1246, consequently downregulating Th17/Treg ratio, and represented a promising therapeutic target for precision treatment in periodontitis.

Proteomic and single-cell analysis shed new light on the anti-inflammatory role of interferonß in chronic periodontitis.

Periodontitis, a condition that results in periodontal attachment loss and alveolar bone resorption, contributes to the global burden of oral disease. The underlying mechanism of periodontitis involves the dysbiosis and dyshomeostasis between host and oral microbes, among which the macrophage is one of the major innate immune cell players, producing interferon ß (IFNß) in response to bacterial infection. The objective of this research was to examine the interaction of macrophages with periodontitis and the role and mechanism of IFNß on macrophages. IFNß has been shown to have the potential to induce the differentiation of M1 to M2 macrophages, which are stimulated by low levels of lipopolysaccharide (LPS). Additionally, IFNß has been demonstrated to promote the production of ISG15 by macrophages, which leads to the inhibition of the innate immune response. Moreover, our investigation revealed that IFNß has the potential to augment the secretion of ISG15 and its downstream cytokine, IL10, in LPS-stimulated macrophages. Single-cell analysis was conducted on the gingival tissues of patients with periodontitis, which revealed a higher proportion of macrophages in the periodontitis-diseased tissue and increased expression of IFNß, ISG15, and IL10. Gene Set Enrichment Analysis indicated that bacterial infection was associated with upregulation of IFNß, ISG15, and IL10. Notably, only IL10 has been linked to immunosuppression, indicating that the IFNß-ISG15-IL10 axis might promote an anti-inflammatory response in periodontitis through IL10 expression. It is also found that macrophage phenotype transitions in periodontitis involve the release of higher levels of IFNß, ISG15, and IL10 by the anti-inflammatory M2 macrophage phenotype compared to the pro-inflammatory M1 phenotype and myeloid-derived suppressor cells (MDSCs). This implies that the IFNß-induced production of IL10 might be linked to the M2 macrophage phenotype. Furthermore, cell communication analysis demonstrated that IL10 can promote fibroblast proliferation in periodontal tissues via STAT3 signaling.

Metal-coding assisted serological multi-omics profiling deciphers the role of selenium in COVID-19 immunity.

Uncovering how host metal(loid)s mediate the immune response against invading pathogens is critical for better understanding the pathogenesis mechanism of infectious disease. Clinical data show that imbalance of host metal(loid)s is closely associated with the severity and mortality of COVID-19. However, it remains elusive how metal(loid)s, which are essential elements for all forms of life and closely associated with multiple diseases if dysregulated, are involved in COVID-19 pathophysiology and immunopathology. Herein, we built up a metal-coding assisted multiplexed serological metallome and immunoproteome profiling system to characterize the links of metallome with COVID-19 pathogenesis and immunity. We found distinct metallome features in COVID-19 patients compared with non-infected control subjects, which may serve as a biomarker for disease diagnosis. Moreover, we generated the first correlation network between the host metallome and immunity mediators, and unbiasedly uncovered a strong association of selenium with interleukin-10 (IL-10). Supplementation of selenium to immune cells resulted in enhanced IL-10 expression in B cells and reduced induction of proinflammatory cytokines in B and CD4+ T cells. The selenium-enhanced IL-10 production in B cells was confirmed to be attributable to the activation of ERK and Akt pathways. We further validated our cellular data in SARS-CoV-2-infected K18-hACE2 mice, and found that selenium supplementation alleviated SARS-CoV-2-induced lung damage characterized by decreased alveolar inflammatory infiltrates through restoration of virus-repressed selenoproteins to alleviate oxidative stress. Our approach can be readily extended to other diseases to understand how the host defends against invading pathogens through regulation of metallome.

Comparative analysis of serum and saliva samples using Raman spectroscopy: a high-throughput investigation in patients with polycystic ovary syndrome and periodontitis.

BACKGROUND: Polycystic ovary syndrome (PCOS) and periodontitis significantly affect women's oral and systemic health worldwide, and yet increase the risk of cardiovascular-metabolic diseases like diabetes and coronary heart disease. Regarding the PCOS-periodontitis connection, whether sex hormones, metabolic and inflammatory mediators could account for the underlying linking mechanism needs to be further investigated. This case-control study evaluated the hormonal, metabolic and inflammatory profiles in PCOS and non-PCOS subjects with various periodontal conditions, via assessing serum and saliva samples by Raman spectroscopy. METHODS: A total of 66 females with PCOS and 22 systemically healthy female volunteers were recruited in a single hospital. Full-mouth periodontal examination was undertaken for identifying the subjects with periodontal health, gingivitis or periodontitis. The datasets of sex hormones and metabolic indicators were retrieved from the hospital information system. Both serum and saliva samples were collected for detecting inflammatory mediators and Raman spectroscopic assessment. The subjects were categorized into four groups according to their conditions of PCOS and periodontitis for Raman spectroscopic analysis. Partial least squares discriminant analysis was performed to examine the inter-group differences in Raman spectra. RESULTS: PCOS patients exhibited greater mean probing depth (P < 0.05) and higher serum levels of triglycerides (P < 0.05) and matrix metalloproteinase-8 (P < 0.05) than those in non-PCOS participants. Both probing depth and triglyceride level were positively correlated with PCOS (P < 0.05). There was a significant difference in mean Raman spectra of saliva samples among the four groups with different conditions of PCOS and periodontitis (P < 0.05), while no significant inter-group difference existed in serum samples. CONCLUSIONS: The present study shows that periodontal condition may affect the biomolecular profiles of Raman spectra in serum and saliva of PCOS patients. It underscores the importance of the collaborative teamwork of dentists and gynecologists for enhancing women's oral health, general wellbeing and quality of life.

Maternal periodontal diseases affect the leukocyte profiles of umbilical cord blood: A cohort study.

AIM: This study evaluated the connection of periodontal status with the leukocyte profiles of maternal peripheral blood (MPB) and umbilical cord blood (UCB). MATERIALS AND METHODS: Ninety-nine pregnant females were recruited, and their data were collected via questionnaire and from medical records, including demographics, systemic conditions, complete blood count (CBC) and C-reaction protein (CRP) level in MPB. Full-mouth periodontal assessment was performed. CBC and CRP levels in UCB were measured after parturition. RESULTS: All subjects and their neonates were generally healthy. 30.3% of the participants presented with periodontal health condition, whereas 69.7% had different severities of periodontal diseases. The counts/percentages of eosinophils and monocytes in UCB from the subjects with periodontal diseases elevated, and the percentage of neutrophils decreased referencing to that from the counterparts (p < 0.05). There were positive correlations for total leukocyte count, neutrophils and lymphocytes counts/percentages in MPB and UCB among the periodontally healthy subjects (r > 0.4, p < 0.05), but such findings did not exist in those with periodontal diseases. Moreover, periodontal diseases independently accounted for the counts/percentages of neutrophils and eosinophils in UCB after controlling confounders in four testing models (ANCOVA, p < 0.05). CONCLUSION: Maternal periodontal diseases could to some extent disturb the leukocyte profiles of umbilical cord blood.

Rapid synthesis of bismuth-organic frameworks as selective antimicrobial materials against microbial biofilms.

Antibiotic resistance is a global public health threat, and urgent actions should be undertaken for developing alternative antimicrobial strategies and approaches. Notably, bismuth drugs exhibit potent antimicrobial effects on various pathogens and promising efficacy in tackling SARS-CoV-2 and related infections. As such, bismuth-based materials could precisely combat pathogenic bacteria and effectively treat the resultant infections and inflammatory diseases through a controlled release of Bi ions for targeted drug delivery. Currently, it is a great challenge to rapidly and massively manufacture bismuth-based particles, and yet there are no reports on effectively constructing such porous antimicrobial-loaded particles. Herein, we have developed two rapid approaches (i.e., ultrasound-assisted and agitation-free methods) to synthesizing bismuth-based materials with ellipsoid- (Ellipsoids) and rod-like (Rods) morphologies respectively, and fully characterized physicochemical properties. Rods with a porous structure were confirmed as bismuth metal-organic frameworks (Bi-MOF) and aligned with the crystalline structure of CAU-17. Importantly, the formation of Rods was a 'two-step' crystallization process of growing almond-flake-like units followed by stacking into the rod-like structure. The size of Bi-MOF was precisely controlled from micro-to nano-scales by varying concentrations of metal ions and their ratio to the ligand. Moreover, both Ellipsoids and Rods showed excellent biocompatibility with human gingival fibroblasts and potent antimicrobial effects on the Gram-negative oral pathogens including Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Fusobacterium nucleatum. Both Ellipsoids and Rods at 50 â€‹µg/mL could disrupt the bacterial membranes, and particularly eliminate P. gingivalis biofilms. This study demonstrates highly efficient and facile approaches to synthesizing bismuth-based particles. Our work could enrich the administration modalities of metallic drugs for promising antibiotic-free healthcare.

Periodontitis links to concurrent metabolic disorders and abnormal liver function in pregnant women.

OBJECTIVE: This cross-sectional study investigated the association of periodontitis with the metabolic status and hepatic function in pregnant women. MATERIALS AND METHODS: Full-mouth periodontal conditions, metabolic profiles, and hepatic function were assessed in 219 self-reported healthy pregnant females. The association of periodontal status with the systemic parameters was evaluated by parametric and non-parametric tests, and multivariate logistic regression analysis. RESULTS: Overall, periodontal status was positively associated with the metabolic profiles and hepatic function test results. The subjects with periodontitis exhibited higher levels of body mass index (BMI) (p < 0.01) and serum aspartate transaminase (AST) (p < 0.05), elevated diastolic blood pressure (DBP) (p < 0.05), and lower levels of high-density lipoprotein cholesterol (p < 0.05) than those of the counterparts. The periodontitis severity was strongly correlated with BMI and AST levels, and the extent of periodontal inflammation was related to DBP (p < 0.01). The periodontitis patients at 34-36 gestational weeks showed higher blood pressure and AST levels than those of non-periodontitis subjects (p < 0.05). CONCLUSION: Our findings on the notable links of periodontitis to concurrent metabolic disorders and abnormal liver function in pregnant women highlight the need of proactive integration of regular periodontal screening and healthcare in maternal programs for promoting optimal health and wellbeing of mothers-to-be and newborns.

Diagnostic performance of the AAP/EFP classification and the CDC/AAP case definition among pregnant women and a practical screening tool for maternal periodontal diseases.

BACKGROUND AND OBJECTIVE: There is a limited number of studies on the performance assessment of the 2017 AAP/EFP classification and the CDC/AAP case definition among pregnant females. This study evaluated the agreement between these two systems and explored a practical tool for screening maternal periodontal diseases by general dentists. MATERIALS AND METHODS: Totally, 204 systemically healthy females at different phases of pregnancy underwent a full-mouth periodontal examination. Demographic characteristics, lifestyles, and systemic conditions were recorded. Referring to the CDC/AAP definition, the diagnostic performance of the AAP/EFP classification was evaluated by the area under the ROC curve (AUC) and statistical tests (e.g., Youden's index and kappa coefficient). Additionally, a modified scoring system of the FDI Periodontal Diseases Chairside Guide (FDI-CG) was formulated with the addition of pregnancy for testing accordingly. RESULTS: Overall, there were 22.1% of the participants in early phase of pregnancy (7-13 weeks) and 77.9% in late phase (34-36 weeks). The majority of them were below 35 years and non-smokers without gestational diabetes. Notably, 30.9% of subjects presented with Moderate/Severe periodontitis (CDC/AAP), and 35.8% with Stages II-IV periodontitis (AAP/EFP). Referring to the CDC/AAP definition, the AUC, Youden's index, and κ of the AAP/EFP classification were 0.979, 0.890, and 92.9%, respectively. The modified FDI-CG system improved the AUC (0.815), Youden's index (63.0%), and κ (0.544) with reference to the original one. CONCLUSIONS: This study shows that the AAP/EFP classification is in high agreement with the CDC/AAP definition among the pregnant women. The phases of pregnancy-integrated FDI scoring system may serve as a convenient screening tool for maternal periodontal diseases in general dental practice.

Porphyromonas gingivalis lipopolysaccharide affects the angiogenic function of endothelial progenitor cells via Akt/FoxO1 signaling.

AIMS: Endothelial progenitor cells (EPCs) function as the angiogenic switch of many physiological and pathological conditions. We aimed to investigate the effects of Porphyromonas gingivalis lipopolysaccharide on the angiogenic capacity of EPCs and delineate the underlying mechanisms. MATERIALS AND METHODS: EPCs were isolated from human umbilical blood. CCK-8 assay was undertaken to analyze the cell viability. The migration and tube formation capacity were assessed by wound healing and tube formation, respectively. The protein expression of Akt/p-Akt, endothelial nitric oxide synthase (eNOS)/p-eNOS, and Forkhead box O1 (FoxO1)/p-FoxO1 was determined by Western blot. The intracellular localization of FoxO1 was evaluated by immunofluorescent staining. RESULTS: P. gingivalis LPS at 10 µg/ml significantly increased the viability (10.9 ± 2.9%), migration (16.3 ± 3.1%), and tube formation (38.6 ± 5.5%) of EPCs, along with increased phosphorylation of Akt, eNOS, and FoxO1. Mechanistically, Akt inhibition by specific inhibitor wortmannin and FoxO1 forced expression by adenovirus transfection in EPCs markedly attenuated the P. gingivalis LPS-induced eNOS activation, tube formation, and migration. Moreover, P. gingivalis LPS-induced phosphorylation and nuclear exclusion of FoxO1 were blunted by Akt inhibition. CONCLUSIONS: The present study suggests that P. gingivalis LPS could affect the angiogenic function of EPCs through the Akt/FoxO1 signaling. The current findings may shed light on the clinical association of periodontitis with aberrant angiogenesis seen in atherosclerotic plaque rupture.

RNA Sequencing Reveals the Upregulation of FOXO Signaling Pathway in Porphyromonas gingivalis Persister-Treated Human Gingival Epithelial Cells.

Porphyromonas gingivalis as the keystone periodontopathogen plays a critical role in the pathogenesis of periodontitis, and crucially accounts for inflammatory comorbidities such as cardiovascular disease and Alzheimer's disease. We recently identified the existence of P. gingivalis persisters and revealed the unforeseen perturbation of innate response in human gingival epithelial cells (HGECs) due to these noxious persisters. Herein, RNA sequencing revealed how P. gingivalis persisters affected the expression profile of cytokine genes and related signaling pathways in HGECs. Results showed that metronidazole-treated P. gingivalis persisters (M-PgPs) impaired the innate host defense of HGECs, in a similar fashion to P. gingivalis. Notably, over one thousand differentially expressed genes were identified in HGECs treated with M-PgPs or P. gingivalis with reference to the controls. Gene Ontology and KEGG pathway analysis demonstrated significantly enriched signaling pathways, such as FOXO. Importantly, the FOXO1 inhibitor rescued the M-PgP-induced disruption of cytokine expression. This study suggests that P. gingivalis persisters may perturb innate host defense, through the upregulation of the FOXO signaling pathway. Thus, the current findings could contribute to developing new approaches to tackling P. gingivalis persisters for the effective control of periodontitis and P. gingivalis-related inflammatory comorbidities.

Eradication of Porphyromonas gingivalis Persisters Through Colloidal Bismuth Subcitrate Synergistically Combined With Metronidazole.

Microbial persisters enable the development of certain intrinsic strategies for survival with extreme tolerance to multiple antimicrobials. Porphyromonas gingivalis is considered to be the "keystone" periodontopathogen. Indeed, periodontitis, as a highly common inflammatory disease, is the major cause of severe tooth loss and edentulism in adults globally, and yet it is crucially involved in various systemic comorbidities like diabetes. We have recently revealed P. gingivalis persisters-induced perturbation of immuno-inflammatory responses and effective suppression of this key pathogen by bismuth drugs. This study further explored novel approaches to eradicating P. gingivalis persisters through synergistic combination of colloidal bismuth subcitrate (CBS) with traditional antibiotics. P. gingivalis (ATCC 33277) cells in planktonic and biofilm states were cultured to stationary phase, and then treated with metronidazole (100 mg/L), amoxicillin (100 mg/L), CBS, (100 µM) and combinations of these medications, respectively. Persister survival rate was calculated by colony-forming unit. Cell viability and cytotoxicity of CBS were assessed in human gingival epithelial cells (HGECs). Notably, CBS combined with metronidazole enabled the effective eradication of P. gingivalis persisters in planktonic mode, and nearly eliminated their existence in biofilm mode. Importantly, CBS exhibited no effects on the viability of HGECs, along with minimal cytotoxicity (<5%) even at a high concentration (400 µM). This pioneering study shows that P. gingivalis persisters could be well eliminated via the synergistic combination of CBS with metronidazole. Our findings may contribute to developing novel approaches to tackling periodontitis and inflammatory systemic comorbidities.

mRNA and long non-coding RNA expression profiling of human periodontal ligament cells under tension loading.

OBJECTIVE: This study explored the expression profiles of messenger RNAs (mRNAs) and long non-coding RNAs (lncRNAs) in human periodontal ligament (PDL) cells subjected to tensile loading. METHODS: PDL cells were isolated from the teeth of five healthy individuals, cultured and then exposed to tensile loading. RNA sequencing was performed to explore the mRNA and lncRNA expression profiles with or without tensile loading. Differential expression, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted to reveal enriched biological functions and signal transduction pathways. Quantitative polymerase chain reaction (qPCR) was performed to validate the expression of specific mRNAs and lncRNAs associated with the enriched pathways. RESULTS: Tensile loading significantly enhanced the osteogenic potential of PDL cells. Overall, 1438 mRNAs (860 up- and 578 down-regulated) and 195 lncRNAs (107 up- and 88 down-regulated) were differentially expressed (adjusted P-value <0.05) in the tensile loading group versus the control group. GO and KEGG analyses of the differentially expressed genes indicated significant enrichment in osteogenesis-related biological processes and intracellular signal transduction pathways (e.g. the PI3K-Akt pathway), respectively. The qPCR analysis validated the expression levels of five selected mRNAs (EGFR, FGF5, VEGFA, HIF1A, and FOXO1) and three selected lncRNAs (CYTOR, MIR22HG, and SNHG3). LIMITATION: Further studies are warranted to validate the mechanisms regulating tension-induced bone remodelling in PDL cells and potential regulation by the identified lncRNAs. CONCLUSION: The notably altered mRNA and lncRNA expression profiles in PDL cells under tensile loading enhance our mechanistic understanding of tension-induced osteogenesis.

Metallodrug ranitidine bismuth citrate suppresses SARS-CoV-2 replication and relieves virus-associated pneumonia in Syrian hamsters.

SARS-CoV-2 is causing a pandemic of COVID-19, with high infectivity and significant mortality1. Currently, therapeutic options for COVID-19 are limited. Historically, metal compounds have found use as antimicrobial agents, but their antiviral activities have rarely been explored. Here, we test a set of metallodrugs and related compounds, and identify ranitidine bismuth citrate, a commonly used drug for the treatment of Helicobacter pylori infection, as a potent anti-SARS-CoV-2 agent, both in vitro and in vivo. Ranitidine bismuth citrate exhibited low cytotoxicity and protected SARS-CoV-2-infected cells with a high selectivity index of 975. Importantly, ranitidine bismuth citrate suppressed SARS-CoV-2 replication, leading to decreased viral loads in both upper and lower respiratory tracts, and relieved virus-associated pneumonia in a golden Syrian hamster model. In vitro studies showed that ranitidine bismuth citrate and its related compounds exhibited inhibition towards both the ATPase (IC50 = 0.69 µM) and DNA-unwinding (IC50 = 0.70 µM) activities of the SARS-CoV-2 helicase via an irreversible displacement of zinc(II) ions from the enzyme by bismuth(III) ions. Our findings highlight viral helicase as a druggable target and the clinical potential of bismuth(III) drugs or other metallodrugs for the treatment of SARS-CoV-2 infection.

Metronidazole-Treated Porphyromonas gingivalis Persisters Invade Human Gingival Epithelial Cells and Perturb Innate Responses.

Periodontitis as a biofilm-associated inflammatory disease is highly prevalent worldwide. It severely affects oral health and yet closely links to systemic diseases like diabetes and cardiovascular disease. Porphyromonas gingivalis as a "keystone" periodontopathogen drives the shift of microbe-host symbiosis to dysbiosis and critically contributes to the pathogenesis of periodontitis. Persisters represent a tiny subset of biofilm-associated microbes highly tolerant to lethal treatment of antimicrobials, and, notably, metronidazole-tolerant P. gingivalis persisters have recently been identified by our group. This study further explored the interactive profiles of metronidazole-treated P. gingivalis persisters (M-PgPs) with human gingival epithelial cells (HGECs). P. gingivalis cells (ATCC 33277) at stationary phase were treated with a lethal dosage of metronidazole (100 µg/ml, 6 h) for generating M-PgPs. The interaction of M-PgPs with HGECs was assessed by microscopy, flow cytometry, cytokine profiling, and quantitative PCR (qPCR). We demonstrated that the overall morphology and ultracellular structure of M-PgPs remained unchanged. Importantly, M-PgPs maintained the capabilities to adhere to and invade HGECs. Moreover, M-PgPs significantly suppressed proinflammatory cytokine expression in HGECs at a level comparable to that seen with the untreated P. gingivalis cells, through the thermosensitive components. The present report reveals that P. gingivalis persisters induced by lethal treatment of antibiotics were able to maintain their capabilities to adhere to and invade human gingival epithelial cells and to perturb the innate host responses. Novel strategies and approaches need to be developed for tackling P. gingivalis and favorably modulating the dysregulated immunoinflammatory responses for oral/periodontal health and general well-being.

Bismuth drugs tackle Porphyromonas gingivalis and attune cytokine response in human cells.

Periodontitis is the leading cause of severe tooth loss and edentulism in adults worldwide and is closely linked to systemic conditions such as diabetes and cardiovascular disease. Porphyromonas gingivalis is the key pathogen in periodontitis. Herein, we provided the first evidence that bismuth drugs suppress P. gingivalis in its planktonic, biofilm, and intracellular states. In total, 42 bismuth-associated proteins were identified including its major virulent factors (e.g., gingipains, hemagglutinin HagA, and fimbriae). Bismuth perturbed its iron acquisition, disturbed the energy metabolism and virulence, and deactivated multiple key enzymes (e.g., superoxide dismutase and thioredoxins). Moreover, bismuth inhibited its biofilm formation and disrupted the 3-day matured biofilms. Notably, the internalized P. gingivalis in various human cells (e.g., human gingival epithelium progenitors, HGEPs) was oppressed by bismuth but not the commonly used antibiotic metronidazole. Importantly, bismuth drugs enabled the counteraction of immuno-inflammatory responses in different host cells perturbed by P. gingivalis. The production of IL-6 and IL-8 attenuated by P. gingivalis in both of native and IL-1ß-stimulated HGEPs was restored, while the bacterium-enhanced expression of IL-6, IL-1ß, and TNFα in THP-1 macrophages was alleviated. This proof-of-concept study brings prospects for the potential reposition of the routinely used anti-Helicobacter pylori bismuth drugs to better manage inflammatory diseases such as periodontitis and P. gingivalis-related complex systemic disorders.

The role of citrate, lactate and transferrin in determining titanium release from surgical devices into human serum.

The presence of ionic titanium in the serum of patients with titanium implants is currently unexplained. This is presumed due to corrosion, and yet the serum titanium concentration measured in patients is far greater than that predicted by its solubility. The binding of titanium ion as Ti(IV) to human transferrin (hTF) in serum indicates that Ti(IV) ions interact with human physiology. This is an intriguing finding since there is currently no known role for titanium ions in human physiology. Thus, understanding the factors that determine in vivo titanium ion release is relevant to further understanding this metal's interactions with human biochemistry. The present study sought to determine the extent of titanium ion release of into human serum in vitro, and the role of citrate, lactate and hTF in this process. It was found that, when surgical devices of commercially pure titanium were placed into human serum, citrate and lactate concentrations were the prime determinants of titanium release. Crystallography revealed Ti(IV) bound to hTF in the presence of citrate alone, signalling that citrate can act as an independent ligand for Ti(IV) binding to hTF. Based on these findings, a two-stage process of titanium ion release into human serum that is dependent upon both citrate and hTF is proposed to explain the ongoing presence of titanium ion in human subjects with implanted titanium devices.

Tracking iron-associated proteomes in pathogens by a fluorescence approach.

Porphyromonas gingivalis is a key oral anaerobic bacterium involved in human periodontitis, which may affect up to 15% adults worldwide. Using the membrane permeable fluorescent probe Fe-TRACER, we identified 17 iron-associated proteins in Porphyromonas gingivalis. We demonstrated the specific binding of the probe towards iron-associated proteins using transferrin as an example and provided an X-ray structure of the fluorescent probe-bound transferrin. Our study provides a basis for the understanding of iron homeostasis in pathogens, and our approach based on the integration of fluorescence imaging with proteomics and bioinformatics can be readily extended to mine other metalloproteomes in microbials.

Shear stress inhibits IL-17A-mediated induction of osteoclastogenesis via osteocyte pathways.

Interleukin (IL)-17 is crucial to osteoclast differentiation and activation. Osteocytes support osteoclast formation and are thought to orchestrate bone remodeling in response to fluid flow. The contribution of IL-17 to osteocyte-related bone resorption remains unclear. Here, we used the osteocyte-like MLO-Y4 cell line to examine the role of IL-17 and fluid flow in osteoclastogenesis. It was the first time to demonstrate that IL-17A promoted MLO-Y4 cell proliferation, enhanced expression of receptor activator of nuclear factor κ-B ligand (RANKL) and tumor necrosis factor-α (TNF-α), and induced osteoclastogenesis when MLO-Y4 cells were co-cultured with bone marrow-derived macrophage (BMM) cells. Additionally, shear stress upregulated osteoprotegerin expression in osteocytes, downregulated the effect of IL-17A on RANKL and TNF-α expression, and attenuated IL-17A-activated osteoclastic differentiation in the co-culture system of MLO-Y4 and BMM cells. Furthermore, we explored the signaling pathways that potentially mediate these effects in osteocytes, and found that the extracellular signal-regulated kinase (ERK)1/2 and signal transducer and activator of transcription (STAT3) pathways were suppressed by IL-17A but induced by fluid flow. EphA2 signaling enhances osteoclastogenesis in osteocytes, and the intercellular reversed EphA2-ephrinA2 signaling from osteocytes to BMM play an important role in IL-17A-dependent osteoclastic differentiation. EphB4 signaling inhibits osteoclastogenesis in osteocytes, and the intercellular reversed EphB4-ephrinB2 signaling from osteocytes to BMM could inhibit IL-17A-dependent osteoclastic differentiation. The current findings suggest that IL-17A as a promoter of bone resorption and fluid shear stress critically regulate bone remodeling via osteocyte-specific signaling pathways. IL-17 modulation-based approaches may be developed as a novel therapeutic strategy for enhancing bone remodeling efficiency and stability.