Cinnamaldehyde Protects against P. gingivalis Induced Intestinal Epithelial Barrier Dysfunction in IEC-6 Cells via the PI3K/Akt-Mediated NO/Nrf2 Signaling Pathway.

Porphyromonas gingivalis (Pg), a Gram-negative oral pathogen, promotes and accelerates periodontitis-associated gut disorders. Intestinal epithelial barrier dysfunction is crucial in the pathogenesis of intestinal and systemic diseases. In this study, we sought to elucidate the protective role of cinnamaldehyde (CNM, an activator of Nrf2) against P. gingivalis (W83) and Pg-derived lipopolysaccharide (Pg-LPS) induced intestinal epithelial barrier dysfunction via antioxidative mechanisms in IEC-6 cells. IEC-6 (ATCC, CRL-1592) cells were pretreated with or without CNM (100 µM), in the presence or absence of P. gingivalis (strain W83, 109 MOI) or Pg-LPS (1, 10, and 100 µg/mL), respectively, between 0-72 h time points by adopting a co-culture method. Intestinal barrier function, cytokine secretion, and intestinal oxidative stress protein markers were analyzed. P. gingivalis or Pg-LPS significantly (p < 0.05) increased reactive oxygen species (ROS) and malondialdehyde (MDA) levels expressing oxidative stress damage. Pg-LPS, as well as Pg alone, induces inflammatory cytokines via TLR-4 signaling. Furthermore, infection reduced Nrf2 and NAD(P)H quinone dehydrogenase 1 (NQO1). Interestingly, inducible nitric oxide synthase (iNOS) protein expression significantly (p < 0.05) increased with Pg-LPS or Pg infection, with elevated levels of nitric oxide (NO). CNM treatment suppressed both Pg- and Pg-LPS-induced intestinal oxidative stress damage by reducing ROS, MDA, and NO production. Furthermore, CNM treatment significantly upregulated the expression of tight junction proteins via increasing the phosphorylation levels of PI3K/Akt/Nrf2 suppressing inflammatory cytokines. CNM protected against Pg infection-induced intestinal epithelial barrier dysfunction by activating the PI3K/Akt-mediated Nrf2 signaling pathway in IEC-6 cells.

P. gingivalis in oral-prostate axis exacerbates benign prostatic hyperplasia via IL-6/IL-6R pathway.

BACKGROUND: Benign prostatic hyperplasia (BPH) is the most common disease in elderly men. There is increasing evidence that periodontitis increases the risk of BPH, but the specific mechanism remains unclear. This study aimed to explore the role and mechanism of the key periodontal pathogen Porphyromonas gingivalis (P. gingivalis) in the development of BPH. METHODS: The subgingival plaque (Sp) and prostatic fluid (Pf) of patients with BPH concurrent periodontitis were extracted and cultured for 16S rDNA sequencing. Ligature-induced periodontitis, testosterone-induced BPH and the composite models in rats were established. The P. gingivalis and its toxic factor P. gingivalis lipopolysaccharide (P.g-LPS) were injected into the ventral lobe of prostate in rats to simulate its colonization of prostate. P.g-LPS was used to construct the prostate cell infection model for mechanism exploration. RESULTS: P. gingivalis, Streptococcus oralis, Capnocytophaga ochracea and other oral pathogens were simultaneously detected in the Pf and Sp of patients with BPH concurrent periodontitis, and the average relative abundance of P. gingivalis was found to be the highest. P. gingivalis was detected in both Pf and Sp in 62.5% of patients. Simultaneous periodontitis and BPH synergistically aggravated prostate histological changes. P. gingivalis and P.g-LPS infection could induce obvious hyperplasia of the prostate epithelium and stroma (epithelial thickness was 2.97- and 3.08-fold that of control group, respectively), and increase of collagen fibrosis (3.81- and 5.02-fold that of control group, respectively). P. gingivalis infection promoted prostate cell proliferation, inhibited apoptosis, and upregulated the expression of inflammatory cytokines interleukin-6 (IL-6; 4.47-fold), interleukin-6 receptor-α (IL-6Rα; 5.74-fold) and glycoprotein 130 (gp130; 4.47-fold) in prostatic tissue. P.g-LPS could significantly inhibit cell apoptosis, promote mitosis and proliferation of cells. P.g-LPS activates the Akt pathway through IL-6/IL-6Rα/gp130 complex, which destroys the imbalance between proliferation and apoptosis of prostate cells, induces BPH. CONCLUSION: P. gingivalis was abundant in the Pf of patients with BPH concurrent periodontitis. P. gingivalis infection can promote BPH, which may affect the progression of BPH via inflammation and the Akt signaling pathway.

Comprehensive comparative analysis of the periodontal pathogen Porphyromonas gingivalis: exploring the pan-genome, the reconstruction of the gene regulatory network and genome-scale metabolic network.

Porphyromonas gingivalis is a nonmotile, obligate anaerobic, Gram-negative bacterium known for its association with periodontal disease and its involvement in systemic diseases such as atherosclerosis, cardiovascular disease, colon cancer, and Alzheimer's disease. This bacterium produces several virulence factors, including capsules, fimbriae, lipopolysaccharides, proteolytic enzymes, and hemagglutinins. A comparative genomic analysis revealed the open pangenome of P. gingivalis and identified complete type IV secretion systems in strain KCOM2805 and almost complete type VI secretion systems in strains KCOM2798 and ATCC49417, which is a new discovery as previous studies did not find the proteins involved in secretion systems IV and VI. Conservation of some virulence factors between different strains was observed, regardless of their genetic diversity and origin. In addition, we performed for the first time a reconstruction analysis of the gene regulatory network, identifying transcription factors and proteins involved in the regulatory mechanisms of bacterial pathogenesis. In particular, QseB regulates the expression of hemagglutinin and arginine deaminase, while Rex may suppress the release of gingipain through interactions with PorV and the formatum/nitrate transporter. Our study highlights the central role of conserved virulence factors and regulatory pathways, particularly QseB and Rex, in P. gingivalis and provides insights into potential therapeutic targets.

About a Possible Impact of Endodontic Infections by Fusobacterium nucleatum or Porphyromonas gingivalis on Oral Carcinogenesis: A Literature Overview.

Periodontitis is linked to the onset and progression of oral squamous cell carcinoma (OSCC), an epidemiologically frequent and clinically aggressive malignancy. In this context, Fusobacterium (F.) nucleatum and Porphyromonas (P.) gingivalis, two bacteria that cause periodontitis, are found in OSCC tissues as well as in oral premalignant lesions, where they exert pro-tumorigenic activities. Since the two bacteria are present also in endodontic diseases, playing a role in their pathogenesis, here we analyze the literature searching for information on the impact that endodontic infection by P. gingivalis or F. nucleatum could have on cellular and molecular events involved in oral carcinogenesis. Results from the reviewed papers indicate that infection by P. gingivalis and/or F. nucleatum triggers the production of inflammatory cytokines and growth factors in dental pulp cells or periodontal cells, affecting the survival, proliferation, invasion, and differentiation of OSCC cells. In addition, the two bacteria and the cytokines they induce halt the differentiation and stimulate the proliferation and invasion of stem cells populating the dental pulp or the periodontium. Although most of the literature confutes the possibility that bacteria-induced endodontic inflammatory diseases could impact on oral carcinogenesis, the papers we have analyzed and discussed herein recommend further investigations on this topic.

Persistent infection with Porphyromonas gingivalis increases the tumorigenic potential of human immortalised oral epithelial cells through ZFP36 inhibition.

The association between Porphyromonas gingivalis infection and oral squamous cell carcinoma (OSCC) has been established by numerous epidemiological studies. However, the underlying mechanism specific to this connection remains unclear. By bioinformatical analysis, we identified ZFP36 as a potentially significant co-expressed gene in both the OSCC gene database and the persistent infection model of P. gingivalis. To further investigate the role of ZFP36, we established a cell model that human immortalized oral epithelial cells (HIOECs) that were sustainedly infected by P. gingivalis (MOI = 1) for a duration of 30 weeks. Our findings indicated that sustained infection with P. gingivalis inhibited the expression of ZFP36 protein and induced changes in the biological behaviour of HIOECs. The mechanism investigation demonstrated the potential role of ZFP36 in regulating the cancer-related biological behaviour of HIOECs. Subsequent studies revealed that highly expressed CCAT1 could serve as a molecular scaffold in the formation of the ZFP36/CCAT1/MK2 complex. This complex formation enhanced the binding abundance of MK2 and ZFP36, thereby promoting the inhibition of ZFP36 protein phosphorylation. To summarize, low expression of ZFP36 protein under persistent P. gingivalis infection enhances the cancer-related biological behaviour of HIOECs.

Gingipains may be one of the key virulence factors of Porphyromonas gingivalis to impair cognition and enhance blood-brain barrier permeability: An animal study.

AIM: Blood-brain barrier (BBB) disorder is one of the early findings in cognitive impairments. We have recently found that Porphyromonas gingivalis bacteraemia can cause cognitive impairment and increased BBB permeability. This study aimed to find out the possible key virulence factors of P. gingivalis contributing to the pathological process. MATERIALS AND METHODS: C57/BL6 mice were infected with P. gingivalis or gingipains or P. gingivalis lipopolysaccharide (P. gingivalis LPS group) by tail vein injection for 8 weeks. The cognitive behaviour changes in mice, the histopathological changes in the hippocampus and cerebral cortex, the alternations of BBB permeability, and the changes in Mfsd2a and Cav-1 levels were measured. The mechanisms of Ddx3x-induced regulation on Mfsd2a by arginine-specific gingipain A (RgpA) in BMECs were explored. RESULTS: P. gingivalis and gingipains significantly promoted mice cognitive impairment, pathological changes in the hippocampus and cerebral cortex, increased BBB permeability, inhibited Mfsd2a expression and up-regulated Cav-1 expression. After RgpA stimulation, the permeability of the BBB model in vitro increased, and the Ddx3x/Mfsd2a/Cav-1 regulatory axis was activated. CONCLUSIONS: Gingipains may be one of the key virulence factors of P. gingivalis to impair cognition and enhance BBB permeability by the Ddx3x/Mfsd2a/Cav-1 axis.

Porphyromonas gingivalis bacteremia increases the permeability of the blood-brain barrier via the Mfsd2a/Caveolin-1 mediated transcytosis pathway.

Bacteremia induced by periodontal infection is an important factor for periodontitis to threaten general health. P. gingivalis DNA/virulence factors have been found in the brain tissues from patients with Alzheimer's disease (AD). The blood-brain barrier (BBB) is essential for keeping toxic substances from entering brain tissues. However, the effect of P. gingivalis bacteremia on BBB permeability and its underlying mechanism remains unclear. In the present study, rats were injected by tail vein with P. gingivalis three times a week for eight weeks to induce bacteremia. An in vitro BBB model infected with P. gingivalis was also established. We found that the infiltration of Evans blue dye and Albumin protein deposition in the rat brain tissues were increased in the rat brain tissues with P. gingivalis bacteremia and P. gingivalis could pass through the in vitro BBB model. Caveolae were detected after P. gingivalis infection in BMECs both in vivo and in vitro. Caveolin-1 (Cav-1) expression was enhanced after P. gingivalis infection. Downregulation of Cav-1 rescued P. gingivalis-enhanced BMECs permeability. We further found P. gingivalis-gingipain could be colocalized with Cav-1 and the strong hydrogen bonding between Cav-1 and arg-specific-gingipain (RgpA) were detected. Moreover, P. gingivalis significantly inhibited the major facilitator superfamily domain containing 2a (Mfsd2a) expression. Mfsd2a overexpression reversed P. gingivalis-increased BMECs permeability and Cav-1 expression. These results revealed that Mfsd2a/Cav-1 mediated transcytosis is a key pathway governing BBB BMECs permeability induced by P. gingivalis, which may contribute to P. gingivalis/virulence factors entrance and the subsequent neurological impairments.

Asociación del hábito de fumar con la periodontitis crónica y la artritis reumatoide / Association of smoking with chronic periodontitis and rheumatoid arthritis

Introducción: Porphyromonas gingivalis es un microorganismo presente en las periodontitis, productor de la enzima peptidil arginina desminasa, inductora de la citrulinación de proteínas que convierte en antígenos, y que son reconocidos por los anticuerpos antipéptido cíclico citrulinados, marcadores específicos de la artritis reumatoide. Estudios clínicos y epidemiológicos relacionan el hábito de fumar con la periodontitis y la artritis reumatoide. Objetivo: Evaluar la asociación entre el hábito de fumar, la periodontitis crónica y la artritis reumatoide. Métodos: Se realizó un estudio observacional, analítico, de corte transversal, de casos y controles de pacientes con diagnóstico de artritis reumatoide tratados en el Centro de Reumatología y pacientes atendidos por medicina interna en el Hospital Clínico Quirúrgico 10 de octubre de La Habana, en el periodo entre septiembre del 2017 y mayo del 2019. Se estudiaron las variables edad, sexo, hábito de fumar y estado periodontal evaluado a través del índice de enfermedad periodontal de Russell y el nivel de inserción clínica. Para identificar la asociación entre variables se empleó la prueba de ji al cuadrado y el odds ratio. Se respetaron las legislaciones éticas. Resultados: En el estudio prevaleció el grupo de 35 a 44 años y el sexo femenino. El hábito de fumar predominó en los pacientes artríticos, con manifiesto incremento de la prevalencia y gravedad de la enfermedad periodontal. Conclusiones: El hábito de fumar incrementó el riesgo de periodontitis crónica en ambos grupos, y con menos intensidad de riesgo en la artritis reumatoide.

Introduction: Porphyromonas gingivalis is a microorganism present in periodontitis, producer of the enzyme peptidyl arginine deminase that induces citrullination of proteins, turning them into antigens, which are recognized by anti-citrullinated cyclic peptide antibodies, specific markers of rheumatoid arthritis. Clinical and epidemiological studies link smoking with periodontitis and rheumatoid arthritis. Objective: To evaluate the association between smoking, the presence of chronic periodontitis and rheumatoid arthritis. Methods: An observational, analytical, cross-sectional study of cases and controls of patients with a diagnosis of rheumatoid arthritis treated at the Rheumatology Center and patients treated by Internal Medicine in 10 de Octubre Surgical- Clinic Hospital in Havana, between September 2017 and May 2019. The variables were: age, sex, smoking habit and periodontal status evaluated through the Russell Periodontal Disease Index and Level of Clinical Insertion. For the association and relationship between variables, the chi square and the odds ratio were used. Ethical legislation was respected. Results: In the study the group of 35 to 44 years old and the female sex prevailed. Smoking prevailed in arthritic patients with a remarkable increase in the prevalence and severity of periodontal disease. Conclusions: Smoking increased the risk of chronic periodontitis in both groups with less intensity of risk in rheumatoid arthritis.

Structural Model of a Porphyromonas gingivalis type IX Secretion System Shuttle Complex.

Porphyromonas gingivalis is a gram-negative oral anaerobic pathogen and is one of the key causative agents of periodontitis. P. gingivalis utilises a range of virulence factors, including the cysteine protease RgpB, to drive pathogenesis and these are exported and attached to the cell surface via the type IX secretion system (T9SS). All cargo proteins possess a conserved C-terminal signal domain (CTD) which is recognised by the T9SS, and the outer membrane ß-barrel protein PorV (PG0027/LptO) can interact with cargo proteins as they are exported to the bacterial surface. Using a combination of solution nuclear magnetic resonance (NMR) spectroscopy, biochemical analyses, machine-learning-based modelling and molecular dynamics (MD) simulations, we present a structural model of a PorV:RgpB-CTD complex from P. gingivalis. This is the first structural insight into CTD recognition by the T9SS and shows how the conserved motifs in the CTD are the primary sites that mediate binding. In PorV, interactions with extracellular surface loops are important for binding the CTD, and together these appear to cradle and lock RgpB-CTD in place. This work provides insight into cargo recognition by PorV but may also have important implications for understanding other aspects of type-IX dependent secretion.

SGK1 negatively regulates inflammatory immune responses and protects against alveolar bone loss through modulation of TRAF3 activity.

Serum- and glucocorticoid-regulated kinase 1 (SGK1) is a serine/threonine kinase that plays important roles in the cellular stress response. While SGK1 has been reported to restrain inflammatory immune responses, the molecular mechanisms involved remain elusive, especially in oral bacteria-induced inflammatory milieu. Here, we found that SGK1 curtails Porphyromonas gingivalis-induced inflammatory responses through maintaining levels of tumor necrosis factor receptor-associated factor (TRAF) 3, thereby suppressing NF-κB signaling. Specifically, SGK1 inhibition significantly enhances production of proinflammatory cytokines, including tumor necrosis factor α, interleukin (IL)-6, IL-1ß, and IL-8 in P. gingivalis-stimulated innate immune cells. The results were confirmed with siRNA and LysM-Cre-mediated SGK1 KO mice. Moreover, SGK1 deletion robustly increased NF-κB activity and c-Jun expression but failed to alter the activation of mitogen-activated protein kinase signaling pathways. Further mechanistic data revealed that SGK1 deletion elevates TRAF2 phosphorylation, leading to TRAF3 degradation in a proteasome-dependent manner. Importantly, siRNA-mediated traf3 silencing or c-Jun overexpression mimics the effect of SGK1 inhibition on P. gingivalis-induced inflammatory cytokines and NF-κB activation. In addition, using a P. gingivalis infection-induced periodontal bone loss model, we found that SGK1 inhibition modulates TRAF3 and c-Jun expression, aggravates inflammatory responses in gingival tissues, and exacerbates alveolar bone loss. Altogether, we demonstrated for the first time that SGK1 acts as a rheostat to limit P. gingivalis-induced inflammatory immune responses and mapped out a novel SGK1-TRAF2/3-c-Jun-NF-κB signaling axis. These findings provide novel insights into the anti-inflammatory molecular mechanisms of SGK1 and suggest novel interventional targets to inflammatory diseases relevant beyond the oral cavity.

Pasteurized Akkermansia muciniphila reduces periodontal and systemic inflammation induced by Porphyromonas gingivalis in lean and obese mice.

AIM: The aim of this study was to evaluate the effect of the administration of pasteurized Akkermansia muciniphila and Amuc_1100 on periodontal destruction in lean and obese mice and to determine the impact of the mode of administration. MATERIALS AND METHODS: Porphyromonas gingivalis-associated experimental periodontitis was induced in lean and obese mice. After 3 weeks, live, pasteurized A. muciniphila or Amuc_1100 was administered by oral or gastric gavage for three additional weeks. Moreover, an evaluation of the interaction between A. muciniphila and P. gingivalis was performed by RNA-sequencing, and cytokines secretion was measured in exposed macrophages. RESULTS: Oral administration of live, pasteurized A. muciniphila or Amuc_1100 significantly decreased P. gingivalis-induced periodontal destruction and inflammatory infiltrate in lean and obese mice and contributed to the reduction of the plasma level of TNF-α and to the increase of IL-10. The co-culture of A. muciniphila and P. gingivalis induced an increased expression of genes linked to the synthesis of monobactam-related antibiotics in A. muciniphila, while a decrease of the gingipains and type IX secretion system was observed in P. gingivalis. In P. gingivalis-infected macrophages, pasteurized A. muciniphila decreased TNF-α and increased IL-10 levels. CONCLUSIONS: Pasteurized A. muciniphila can counteract P. gingivalis-associated periodontal destruction.

Molecular Mechanisms Leading from Periodontal Disease to Cancer.

Periodontitis is prevalent in half of the adult population and raises critical health concerns as it has been recently associated with an increased risk of cancer. While information about the topic remains somewhat scarce, a deeper understanding of the underlying mechanistic pathways promoting neoplasia in periodontitis patients is of fundamental importance. This manuscript presents the literature as well as a panel of tables and figures on the molecular mechanisms of Porphyromonas gingivalis and Fusobacterium nucleatum, two main oral pathogens in periodontitis pathology, involved in instigating tumorigenesis. We also present evidence for potential links between the RANKL-RANK signaling axis as well as circulating cytokines/leukocytes and carcinogenesis. Due to the nonconclusive data associating periodontitis and cancer reported in the case and cohort studies, we examine clinical trials relevant to the topic and summarize their outcome.

A quantitative framework reveals traditional laboratory growth is a highly accurate model of human oral infection.

Bacterial behavior and virulence during human infection is difficult to study and largely unknown, as our vast knowledge of infection microbiology is primarily derived from studies using in vitro and animal models. Here, we characterize the physiology of Porphyromonas gingivalis, a periodontal pathogen, in its native environment using 93 published metatranscriptomic datasets from periodontally healthy and diseased individuals. P. gingivalis transcripts were more abundant in samples from periodontally diseased patients but only above 0.1% relative abundance in one-third of diseased samples. During human infection, P. gingivalis highly expressed genes encoding virulence factors such as fimbriae and gingipains (proteases) and genes involved in growth and metabolism, indicating that P. gingivalis is actively growing during disease. A quantitative framework for assessing the accuracy of model systems showed that 96% of P. gingivalis genes were expressed similarly in periodontitis and in vitro midlogarithmic growth, while significantly fewer genes were expressed similarly in periodontitis and in vitro stationary phase cultures (72%) or in a murine abscess infection model (85%). This high conservation in gene expression between periodontitis and logarithmic laboratory growth is driven by overall low variance in P. gingivalis gene expression, relative to other pathogens including Pseudomonas aeruginosa and Staphylococcus aureus Together, this study presents strong evidence for the use of simple test tube growth as the gold standard model for studying P. gingivalis biology, providing biological relevance for the thousands of laboratory experiments performed with logarithmic phase P. gingivalis Furthermore, this work highlights the need to quantitatively assess the accuracy of model systems.

Inflammasome dysregulation in human gingival fibroblasts in response to periodontal pathogens.

OBJECTIVE: Uncontrolled production of Interleukin-1ß (IL-1ß), a major proinflammatory cytokine, is associated with tissue destruction in periodontal disease. IL-1ß production is controlled by inflammasomes which are multiprotein regulatory complexes. The current study aimed to elucidate potential regulatory pathways by monitoring the effects of periodontal pathogens Fusobacterium nucleatum (Fn) and Porphyromonas gingivalis (Pg) on inflammasomes and their regulators in human gingival fibroblasts (HGFs) in vitro. METHODS: HGFs were exposed to Fn and Pg alone or in combination for 24 hr at a multiplicity of infection of 100, ±30 min exposure with 5 mM adenosine triphosphate (ATP) incubation. Gene expression of NLRP3 and AIM2, inflammasome regulatory proteins POP1, CARD16 and TRIM16, and inflammasome components ASC and CASPASE 1, and IL-1ß, were evaluated by RT-PCR. Pro- and mature IL-1ß levels were monitored intracellularly by immunocytochemistry and extracellularly by ELISA. RESULTS: Fn + ATP significantly upregulated NLRP3, AIM2, IL-1ß, ASC, and CASPASE 1; however, it downregulated POP1 and TRIM16. Pg + ATP downregulated NLRP3, ASC, POP1, but upregulated IL-1ß and CARD16. Pg + Fn+ATP significantly upregulated AIM2, IL-1ß and CARD16, and downregulated POP1, TRIM16, and CASPASE 1. Pg + ATP exposure significantly increased pro- and mature IL-1ß production. CONCLUSION: Bacterial exposure with ATP may deregulate IL-1ß by dysregulating inflammasomes and their regulators in HGFs.

An Explorative Study on Monocyte Reprogramming in the Context of Periodontitis In Vitro and In Vivo.

Aims: Periodontitis is an independent risk factor for cardiovascular disease, but the mechanistic link is not fully understood. In atherosclerotic cardiovascular disease, monocytes can adopt a persistent hyperresponsive phenotype, termed trained immunity. We hypothesized that periodontitis-associated bacteria can induce trained immunity in monocytes, which subsequently accelerate atherosclerosis development. Materials and Methods: We combined in vitro experiments on human primary monocytes and in vivo techniques in patients with periodontitis to test this hypothesis. Adherent peripheral blood mononuclear cells (PBMCs) were transiently exposed in vitro to Porphyromonas gingivalis for 24 hours, and restimulated with lipopolysaccharide (LPS) or Pam3CysK4 (P3C) six days later, to measure interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) production. In an exploratory observational study, patients with severe periodontitis (63 ± 6 years, n=14) and control subjects with no-to-mild periodontitis (54 ± 10 years, n=14) underwent venipuncture and 2'-deoxy-2'-[18F]fluoro-D-glucose positron-emission-tomography ([18F]FDG PET/CT) scanning. Results: When adherent peripheral blood mononuclear cells (PBMCs) were transiently exposed in vitro to Porphyromonas gingivalis for 24 hours, and restimulated with LPS or P3C six days later, IL-6 and TNFα production was significantly increased (TNFα/P3C, p<0.01). Circulating leukocytes, IL-6 and interleukin-1 receptor antagonist (IL-1Ra) concentrations were generally higher in patients compared to controls (leukocytes: p<0.01; IL-6: p=0.08; IL-1Ra: p=0.10). Cytokine production capacity in PBMCs after 24h stimulation revealed no differences between groups. [18F]FDG PET/CT imaging showed a trend for increased [18F]FDG-uptake in the periodontium [mean standard uptake value (SUVmean), p=0.11] and in femur bone marrow (SUVmean, p=0.06), but no differences were observed for vascular inflammation. Positive correlations between severity of periodontitis, measured by The Dutch Periodontal Screening Index and pocket depth, with circulating inflammatory markers and tissue inflammation were found. Conclusions: P. gingivalis induces long-term activation of human monocytes in vitro (trained immunity). Patients with severe periodontitis did have signs of increased systemic inflammation and hematopoietic tissue activation. However, their circulating monocytes did not show a hyperresponsive phenotype. Together we suggest that trained immunity might contribute to local periodontal inflammation which warrants further investigation.

Extracellular vesicles of P. gingivalis-infected macrophages induce lung injury.

Periodontal diseases are common inflammatory diseases that are induced by infection with periodontal bacteria such as Porphyromonas gingivalis (Pg). The association between periodontal diseases and many types of systemic diseases has been demonstrated; the term "periodontal medicine" is used to describe how periodontal infection/inflammation may impact extraoral health. However, the molecular mechanisms by which the factors produced in the oral cavity reach multiple distant organs and impact general health have not been elucidated. Extracellular vesicles (EVs) are nano-sized spherical structures secreted by various types of cells into the tissue microenvironment, and influence pathophysiological conditions by delivering their cargo. However, a detailed understanding of the effect of EVs on periodontal medicine is lacking. In this study, we investigated whether EVs derived from Pg-infected macrophages reach distant organs in mice and influence the pathophysiological status. EVs were isolated from human macrophages, THP-1 cells, infected with Pg. We observed that EVs from Pg-infected THP-1 cells (Pg-inf EVs) contained abundant core histone proteins such as histone H3 and translocated to the lungs, liver, and kidneys of mice. Pg-inf EVs also induced pulmonary injury, including edema, vascular congestion, inflammation, and collagen deposition causing alveoli destruction. The Pg-inf EVs or the recombinant histone H3 activated the NF-κB pathway, leading to increase in the levels of pro-inflammatory cytokines in human lung epithelial A549 cells. Our results suggest a possible mechanism by which EVs produced in periodontal diseases contribute to the progression of periodontal medicine.

Porphyromonas gingivalis Outer Membrane Vesicles as the Major Driver of and Explanation for Neuropathogenesis, the Cholinergic Hypothesis, Iron Dyshomeostasis, and Salivary Lactoferrin in Alzheimer's Disease.

Porphyromonas gingivalis (Pg) is a primary oral pathogen in the widespread biofilm-induced "chronic" multi-systems inflammatory disease(s) including Alzheimer's disease (AD). It is possibly the only second identified unique example of a biological extremophile in the human body. Having a better understanding of the key microbiological and genetic mechanisms of its pathogenesis and disease induction are central to its future diagnosis, treatment, and possible prevention. The published literature around the role of Pg in AD highlights the bacteria's direct role within the brain to cause disease. The available evidence, although somewhat adopted, does not fully support this as the major process. There are alternative pathogenic/virulence features associated with Pg that have been overlooked and may better explain the pathogenic processes found in the "infection hypothesis" of AD. A better explanation is offered here for the discrepancy in the relatively low amounts of "Pg bacteria" residing in the brain compared to the rather florid amounts and broad distribution of one or more of its major bacterial protein toxins. Related to this, the "Gingipains Hypothesis", AD-related iron dyshomeostasis, and the early reduced salivary lactoferrin, along with the resurrection of the Cholinergic Hypothesis may now be integrated into one working model. The current paper suggests the highly evolved and developed Type IX secretory cargo system of Pg producing outer membrane vesicles may better explain the observed diseases. Thus it is hoped this paper can provide a unifying model for the sporadic form of AD and guide the direction of research, treatment, and possible prevention.

Periodontitis induces endothelial dysfunction in mice.

The treatment of periodontitis has numerous positive effects on established chronic health conditions, including cardiovascular disease and diabetes. However, ethical considerations do limit the establishment of human trials to investigate whether periodontitis promotes the early stages of chronic conditions. Therefore, the aim of this study was to investigate whether periodontitis induces endothelial dysfunction in hyperlipidemic apolipoprotein E gene-deficient (ApoE-/-) mice. Forty-five 8-week-old ApoE-/- mice were challenged by oral lavage with Porphyromonas gingivalis and Streptococcus gordonii for 4 weeks. A subgroup of animals (n = 15-17/group) was placed in a metabolic chamber immediately before euthanasia at 4 weeks to measure VO2/CO2 concentrations and voluntary locomotion. In infected and control animals alveolar bone levels were measured by x-ray imaging and endothelial function was determined by measuring endothelial-dependent vasorelaxation of aortic rings. The mRNA expression levels of serum amyloid A and tumor necrosis factor were determined in liver tissues by qRT PCR and protein concentrations in serum by ELISA. Caecal contents were analysed by sequencing to determine changes to the gut microbiota to investigate linkages between microbiome and systemic changes. The results showed that oral lavage of P. gingivalis and S. gordonii for 4 weeks, initiated periodontitis in ApoE-/- mice, similar to the human situation. The oral inflammation was accompanied by a significant increase in mRNA expression of pro-inflammatory mediators serum amyloid A1 and tumor necrosis factor in the liver. Mice with periodontitis also exhibited impaired endothelial-dependent vasorelaxation responses to acetylcholine. This systemic response was connected to increased energy expenditure, locomotion and respiratory quotient. No differences were detected in caecal microbiota between the infected and control animals. Overall, this is the first report that provide evidence that periodontitis induces endothelial dysfunction in mice. Other systemic responses observed in response to the local reaction need further investigation. The study suggests that early prevention of periodontitis may help limit the early stages of endothelial dysfunction that is linked to atherogenesis in humans.