Imbalanced EphB4/EphrinB2 Signaling Modulates Bone Resorption in Periodontitis Induced by Porphyromonas gingivalis.

Periodontitis, a chronic infectious disease in periodontal tissues, is characterized by an imbalance of alveolar bone resorption and remodeling, which eventually results in tooth loosening and even tooth loss. The etiology of periodontitis is polymicrobial synergy and dysbiosis, in which Porphyromonas gingivalis (P. gingivalis) is one of the primary pathogens responsible for periodontitis progression. The interplay of EphrinB2/EphB4 is crucial for osteoblast-osteoclast communication during bone remodeling and healing. This study investigates the mechanism of EphB4/EphrinB2 transduction modulating osteogenesis inhibition and bone resorption in periodontitis induced by P. gingivalis. An in vivo model of chronic periodontitis provoked by P. gingivalis was constructed, the inflammation and bone resorption were evaluated. The expression of EphB4 and EphrinB2 proteins in periodontal tissues was detected, which was also evaluated, respectively, in osteoblasts and osteoclasts infected with P. gingivalis in vitro. Then, a simulated coculture model of osteoblasts and osteoclasts was established to activate the forward and reverse pathways of EphB4/EphrinB2 with P. gingivalis infection. This study showed that P. gingivalis infection promoted alveolar bone resorption in rats and enhanced EphB4 and EphrinB2 expression in periodontal tissues. EphB4 and molecules associated with osteogenesis in osteoblasts infected with P. gingivalis were inhibited, while EphrinB2 and osteoclast differentiation-related markers in osteoclasts were activated. In conclusion, this study suggested that EphB4/EphrinB2 proteins were involved in alveolar bone remodeling in the process of periodontitis induced by P. gingivalis infection. Moreover, attenuated EphB4/EphrinB2 with P. gingivalis infection weakened osteoblast activity and enhanced osteoclast activity.

Abnormal amyloid precursor protein processing in periodontal tissue in a murine model of periodontitis induced by Porphyromonas gingivalis.

OBJECTIVE: The study aimed to investigate the change of amyloid precursor protein (APP) processing and amyloid ß (Aß) metabolites in linking periodontitis to Alzheimer's disease (AD). BACKGROUND: Aß is one of the main pathological features of AD, and few studies have discussed changes in its expression in peripheral tissues or analyzed the relationship between the peripheral imbalance of Aß production and clearance. METHODS: A murine model of periodontitis was established by oral infection with Porphyromonas gingivalis (P. gingivalis). Micro-computed tomography (Micro-CT) was used to observe the destruction of the alveolar bone. Nested quantitative polymerase chain reaction (qPCR) was used to measure small quantities of P.gingivalis DNA in different tissues. Behavioral experiments were performed to measure cognitive function in the mice. The mRNA levels of TNF-α, IL-6, IL-8, RANKL, OPG, APP695, APP751, APP770, and BACE1 in the gingival tissues or cortex were detected by RT-PCR. The levels of Aß1-40 and Aß1-42 in gingival crevicular fluid (GCF) and plasma were tested by ELISA. RESULTS: P. gingivalis oral infection was found to cause alveolar bone resorption and impaired learning and memory. P.gingivalis DNA was detected in the gingiva, blood and cortex of the P.gingivalis group by nested qPCR (p < .05). The mRNA expression of TNF-α, IL-6, IL-8, RANKL/OPG, and BACE1 in the gingival tissue was significantly higher than that in the control group (p < .05). Similarly, upregulated mRNA levels of APP695 and APP770 were observed in the gingival tissuses and cortex of the P. gingivalis group (p < .05). The levels of Aß1-40 and Aß1-42 in the GCF and plasma of the P. gingivalis group were significantly higher than those in the control group (p < .05). CONCLUSION: P. gingivalis can directly invade the brain via hematogenous infection. The invasion of P. gingivalis could trigger an immune response and lead to an imbalance between Aß production and clearance in peripheral tissues, which may trigger an abnormal Aß metabolite in the brain, resulting in the occurrence and development of AD.

Porphyromonas gingivalis Lipopolysaccharide Damages Mucosal Barrier to Promote Gastritis-Associated Carcinogenesis.

BACKGROUND: Recent epidemiological studies suggested correlation between gastric cancer (GC) and periodontal disease. AIMS: We aim to clarify involvement of lipopolysaccharide of Porphyromonas gingivalis (Pg.), one of the red complex periodontal pathogens, in the GC development. METHODS: To evaluate barrier function of background mucosa against the stimulations, we applied biopsy samples from 76 patients with GC using a Ussing chamber system (UCs). K19-Wnt1/C2mE transgenic (Gan) mice and human GC cell-lines ± THP1-derived macrophage was applied to investigate the role of Pg. lipopolysaccharide in inflammation-associated carcinogenesis. RESULTS: In the UCs, Pg. lipopolysaccharide reduced the impedance of metaplastic and inflamed mucosa with increases in mRNA expression of toll-like receptor (TLR) 2, tumor necrosis factor (TNF) α, and apoptotic markers. In vitro, Pg. lipopolysaccharide promoted reactive oxidative stress (ROS)-related apoptosis as well as activated TLR2-ß-catenin-signaling on MKN7, and it increased the TNFα production on macrophages, respectively. TNFα alone activated TLR2-ß-catenin-signaling in MKN7, while it further increased ROS and TNFα in macrophages. Under coculture with macrophages isolated after stimulation with Pg. lipopolysaccharide, ß-catenin-signaling in MKN7 was activated with an increase in supernatant TNFα concentration, both of which were decreased by adding a TNFα neutralization antibody into the supernatant. In Gan mice with 15-week oral administration of Pg. lipopolysaccharide, tumor enlargement with ß-catenin-signaling activation were observed with an increase in TNFα with macrophage infiltration. CONCLUSIONS: Local exposure of Pg. lipopolysaccharide may increase ROS on premalignant gastric mucosa to induce apoptosis-associated barrier dysfunction and to secrete TNFα from activated macrophages, and both stimulation of Pg. lipopolysaccharide and TNFα might activate TLR2-ß-catenin-signaling in GC.

Porphyromonas gingivalis infection in the oral cavity is associated with elevated galactose-deficient IgA1 and increased nephritis severity in IgA nephropathy.

BACKGROUND: The relationship between the major periodontal bacteria, Porphyromonas gingivalis, and the pathogenesis of IgA nephropathy (IgAN)-particularly with respect to galactose-deficient IgA1 (Gd-IgA1)-has not been fully elucidated. METHODS: Saliva samples from 30 IgAN patients and 44 patients with chronic kidney disease (CKD) were subjected to analysis of P. gingivalis status via polymerase chain reaction using a set of P. gingivalis-specific primers. The associations between P. gingivalis presence and clinical parameters, including plasma Gd-IgA1, were analyzed in each group. RESULTS: Compared with the CKD group, the IgAN group demonstrated significantly higher plasma Gd-IgA1 levels (p < 0.05). Compared with the P. gingivalis-negative subgroup, the P. gingivalis-positive subgroup exhibited significantly higher plasma Gd-IgA1 levels in both IgAN and CKD patients (p < 0.05). Additionally, among IgAN patients, the P. gingivalis-positive subgroup displayed significantly higher plasma Gd-IgA1 and urine protein levels, compared with the P. gingivalis-negative subgroup (p < 0.05). With respect to renal biopsy findings, the frequencies of segmental glomerulosclerosis and tubular atrophy/interstitial fibrosis were significantly greater in the P. gingivalis-positive subgroup than in the P. gingivalis-negative subgroup, according to the Oxford classification of IgAN (p < 0.05). CONCLUSION: Our findings suggest an association between the presence of P. gingivalis in the oral cavity and the pathogenesis of IgAN, mediated by increased levels of Gd-IgA1.

Corydalis saxicolaBunting total alkaloid eliminates Porphyromonas gingivalis strain 33277 internalized into macrophages by inhibition of TLR2.

OBJECTIVE: This study aimed to investigate the impact of Corydalis Saxicola Bunting Total Alkaloid (CSBTA) on Porphyromonas gingivalis internalization within macrophages and explore the potential role of Toll-Like Receptor 2 (TLR2) in this process. METHODS: We established a P. gingivalis internalization model in macrophages by treating P. gingivalis-infected macrophages (MOI=100:1) with 200 µg/mL metronidazole and 300 µg/mL gentamicin for 1 h. Subsequently, the model was exposed to CSBTA at concentrations of 0.02 g/L or 1 µg/mL Pam3CSK4. After a 6 h treatment, cell lysis was performed with sterile water to quantify bacterial colonies. The mRNA expressions of TLR2 and interleukin-8 (IL-8) in macrophages were analyzed using RT-qPCR, while their protein levels were assessed via Western blot and ELISA respectively. RESULTS: P. gingivalis could internalize into macrophages and enhance the expression of TLR2 and IL-8. Activation of TLR2 by Pam3CSK4 contributed to P. gingivalis survival within macrophages and increased TLR2 and IL-8 expression. Conversely, 0.02 g/L CSBTA effectively cleared intracellular P. gingivalis, achieving a 90 % clearance rate after 6 h. Moreover, it downregulated the expression of TLR2 and IL-8 induced by P. gingivalis. However, the inhibitory effect of CSBTA on the internalized P. gingivalis model was attenuated by Pam3CSK4. CONCLUSION: CSBTA exhibited the ability to reduce the presence of live intracellular P. gingivalis and lower IL-8 expression in macrophages, possibly by modulating TLR2 activity.

Mapping the substrate-binding subsite specificity of a Porphyromonas gingivalis Tpr peptidase.

Calcium-dependent peptidases of the calpain family are widespread in eukaryotes but uncommon in prokaryotes. A few bacterial calpain homologs have been discovered but none of them have been characterized in detail. Here we present an in-depth substrate specificity analysis of the bacterial calpain-like peptidase Tpr from Porphyromonas gingivalis. Using the positional scanning hybrid combinatorial substrate library method, we found that the specificity of Tpr peptidase differs substantially from the papain family of cysteine proteases, showing a strong preference for proline residues at positions P2 and P3. Such a degree of specificity indicates that this P. gingivalis cell-surface peptidase has a more sophisticated role than indiscriminate protein degradation to generate peptide nutrients, and may fulfil virulence-related functions such as immune evasion.

MiR-499 enhances Porphyromonas gingivalis LPS-induced inflammatory response in macrophages by targeting NRIP1 via JAK/STAT pathway.

MicroRNA functions as an important part of the activity and development of immune cells. miR-499 has been demonstrated to play a significant role in the activity and development of immune cells. The precise mechanism by which miR-499 regulates the inflammatory response, however, remains unclear. This study was aimed to examine the role of microRNA miR-499 in the regulation of the inflammatory response in macrophages. RAW 264.7 macrophages were used as a cell model. The levels of miR-499 were measured in Porphyromonas gingivalis LPS-stimulated macrophages using qRT-PCR, and the levels of inflammatory cytokines (IL-6, IL-1ß, and TNF-α) were determined using both qRT-PCR and ELISA. StarBase was used to predict the binding sites between NRIP1 and miR-499, and the mRNA expression of NRIP1 was measured using qRT-PCR. The regulation of inflammatory factors controlled by miR-499 was also evaluated by using miR-499 inhibitor and sh-NRIP1. The activation of the JAK/STAT pathway was determined using western blotting to measure the levels of phosphorylated JAK2 and STAT1. Porphyromonas gingivalis LPS caused a high expression of miR-499, which promoted the inflammatory response in macrophages. miR-499 targeted the NRIP1 3' UTR and regulated the mRNA expression of inflammatory cytokines, including IL-6, IL-1ß, and TNF-α. The positive correlation between miR-499 and the expression of inflammatory factors and the negative correlation between NRIP1 and miR-499 suggests that the regulation of inflammatory factors controlled by miR-499 was associated with NRIP1. The phosphorylated proteins of the JAK/STAT pathway (p-JAK2 and p-STAT1) were activated by miR-499 through its regulation of NRIP1. These findings suggest that miR-499 regulates the P. gingivalis LPS-induced inflammatory response in macrophages and activates the JAK/STAT pathway through the regulation of NRIP1.

Hispidulin Inhibits the Vascular Inflammation Triggered by Porphyromonas gingivalis Lipopolysaccharide.

Hispidulin is a natural bioactive flavonoid that has been studied for its potential therapeutic properties, including its anti-inflammatory, antioxidant, and neuroprotective effects. The aim of this study was to explore whether hispidulin could inhibit the endothelial inflammation triggered by Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS). The adhesion of monocytes to the vascular endothelium was evaluated through in vitro and ex vivo monocyte adhesion assays. We analyzed the migration of monocytes across the endothelial layer using a transmigration assay. The results showed that treatment with hispidulin decreased the P. gingivalis LPS-induced adhesion of monocytes to endothelial cells and their migration by suppressing the P. gingivalis LPS-triggered expression of intercellular adhesion molecule-1 (ICAM-1) through downregulating nuclear factor-қB (NF-қB). In addition, hispidulin inhibited P. gingivalis LPS-induced mitogen-activated protein kinases (MAPKs) and AKT in endothelial cells. Altogether, the results indicate that hispidulin suppresses the vascular inflammation induced by P. gingivalis LPS. Mechanistically, it prevents the adhesion of monocytes to the vascular endothelium and migration and inhibits NF-қB, MAPKs, and AKT signaling in endothelial cells.

Production and properties of adhesin-free gingipain proteinase RgpA.

The Arg-specific gingipains of Porphyromonas gingivalis RgpA and RgpB have 97% identical sequences in their catalytic domains yet their propeptides are only 76% identical. RgpA isolates as a proteinase-adhesin complex (HRgpA) which hinders direct kinetic comparison of RgpAcat as a monomer with monomeric RgpB. We tested modifications of rgpA identifying a variant that enabled us to isolate histidine-tagged monomeric RgpA (rRgpAH). Kinetic comparisons between rRgpAH and RgpB used benzoyl-L-Arg-4-nitroanilide with and without cysteine and glycylglycine acceptor molecules. With no glycylglycine, values of Km, Vmax, kcat and kcat/Km for each enzyme were similar, but with glycylglycine Km decreased, Vmax increased and kcat increased ~ twofold for RgpB but ~ sixfold for rRgpAH. The kcat/Km for rRgpAH was unchanged whereas that of RgpB more than halved. Recombinant RgpA propeptide inhibited rRgpAH and RgpB with Ki 13 nM and 15 nM Ki respectively slightly more effectively than RgpB propeptide which inhibited rRgpAH and RgpB with Ki 22 nM and 29 nM respectively (p < 0.0001); a result that may be attributable to the divergent propeptide sequences. Overall, the data for rRgpAH reflected observations previously made by others using HRgpA, indicating rRgpAH fidelity and confirming the first production and isolation of functional affinity tagged RgpA.

Human ß-defensins are correlated with the immune infiltration and regulated by vitamin D3 in periodontitis.

OBJECTIVE: Exploring the correlation between human ß-defensins (HBDs) and immune infiltration in periodontitis, and whether it is regulated by vitamin D3 . BACKGROUND: The human body produces essential antimicrobial peptides called HBDs, which are associated with periodontitis. There is a strong link between periodontal tissue destruction and the immune cell infiltration. Moreover, vitamin D3 has been reported to regulate the expression of immune cell chemokines. However, the relationship between vitamin D3 , HBDs, and immune infiltration in periodontitis remains to be investigated. METHODS: The Gene Expression Omnibus database was accessed to obtain transcriptomic information of gingival samples taken from periodontitis patients. The expression value of HBD-2 and HBD-3 was calculated. Additionally, using the online program ImmuCellAl, 10 immune cells were scored for immune infiltration in the high-HBDs-expression group and the low-HBDs-expression group, separately. After that, transcriptome sequencing was done based on human gingival fibroblasts that had received vitamin D3 treatment. Furthermore, hGFs were treated by vitamin D3 , tumor necrosis factor-α (TNF-α), and Porphyromonas gingivalis lipopolysaccharide (Pg-LPS). The expressions of HBD-2, HBD-3, interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1) were detected. To seek the potential mechanism, CYP27A1 siRNA was employed to reduce the expression of CYP27A1, and nuclear factor-gene binding protein 65 (NF-κB p65) was examined. RESULTS: In GSE10334, the expressions of HBD-2 and HBD-3 were down-regulated in periodontitis group. Meanwhile, monocyte, macrophage, and CD4_T cell were less infiltrated in low-HBD-2-expression group, while less Gamma-delta T-cell infiltration was found in low-HBD-3-expression group. Transcriptome sequencing found that 21 genes were significantly expressed, of which the function was enriched in response to bacterial origin and TNF signal pathway. Vitamin D3 could significantly up-regulate the expression of HBD-2 and HBD-3, which could be controlled by knocking down CYP27A1 mRNA expression. With prolonged vitamin D3 stimulation, the expression of HBD-2 and HBD-3 increased. TNF-α/Pg-LPS could significantly increase the expression of HBD-2, HBD-3, IL-8, MCP-1, and p65, all of which were reduced by vitamin D3 . CONCLUSION: HBDs are correlated with immune infiltration in periodontitis. Vitamin D3 inhibits the expression of HBDs and chemokines induced by TNF-α/Pg-LPS, possibly through NF-κB pathway, in human gingival fibroblasts.

Lipopolysaccharides from Porphyromonas endodontalis and Porphyromonas gingivalis promote angiogenesis via Toll-like-receptors 2 and 4 pathways in vitro.

AIM: Angiogenesis contributes to the development of apical periodontitis, periodontitis, and other oral pathologies; however, it remains unclear how this process is triggered. The aim was to evaluate whether lipopolysaccharide (LPS) from Porphyromonas endodontalis and Porphyromonas gingivalis induced angiogenesis-related effects in vitro via TLR2 and TLR4. METHODOLOGY: Porphyromonas endodontalis LPS (ATCC 35406 and clinical isolate) was purified with TRIzol, whereas P. gingivalis LPS was obtained commercially. The effects of the different LPS (24 h) in endothelial cell migration were analysed by Transwell assays, following quantification in an optical microscope (40×). The effects of LPS on FAK Y397 phosphorylation were assessed by Western blotting. Angiogenesis in vitro was determined in an endothelial tube formation assay (14 h) in Matrigel in the absence or presence of either LPS. IL-6 and VEGF-A levels were determined in cell supernatants, following 24 h treatment with LPS, and measured in multiplex bead immunoassay. The involvement of TLR2 and TLR4 was assessed with blocking antibodies. The statistical analysis was performed using STATA 12® (StataCorp LP). RESULTS: The results revealed that P. endodontalis LPS, but not P. gingivalis LPS, stimulated endothelial cell migration. Pre-treatment with anti-TLR2 and anti-TLR4 antibodies prevented P. endodontalis LPS-induced cell migration. P. endodontalis LPS promoted FAK phosphorylation on Y397, as observed by an increased p-FAK/FAK ratio. Both P. gingivalis and P. endodontalis LPS (ATCC 35406) induced endothelial tube formation in a TLR-2 and -4-dependent manner, as shown by using blocking antibodies, however, only TLR2 blocking decreased tube formation induced by P. endodontalis (clinical isolate). Moreover, all LPS induced IL-6 and VEGF-A synthesis in endothelial cells. TLR2 and TLR4 were required for IL-6 induction by P. endodontalis LPS (ATCC 35406), while only TLR4 was involved in IL-6 secretion by the other LPS. Finally, VEGF-A synthesis did not require TLR signalling. CONCLUSION: Porphyromonas endodontalis and P. gingivalis LPS induced angiogenesis via TLR2 and TLR4. Collectively, these data contribute to understanding the role of LPS from Porphyromonas spp. in angiogenesis and TLR involvement.

Interleukin-22 Inhibits Apoptosis of Gingival Epithelial Cells Through TGF-ß Signaling Pathway During Periodontitis.

Periodontitis is a chronic inflammatory disease characterized by the destruction of tooth-supporting tissues. The gingival epithelium is the first barrier of periodontal tissue against oral pathogens and harmful substances. The structure and function of epithelial lining are essential for maintaining the integrity of the epithelial barrier. Abnormal apoptosis can lead to the decrease of functional keratinocytes and break homeostasis in gingival epithelium. Interleukin-22 is a cytokine that plays an important role in epithelial homeostasis in intestinal epithelium, inducing proliferation and inhibiting apoptosis, but its role in gingival epithelium is poorly understood. In this study, we investigated the effect of interleukin-22 on apoptosis of gingival epithelial cells during periodontitis. Interleukin-22 topical injection and Il22 gene knockout were performed in experimental periodontitis mice. Human gingival epithelial cells were co-cultured with Porphyromonas gingivalis with interleukin-22 treatment. We found that interleukin-22 inhibited apoptosis of gingival epithelial cells during periodontitis in vivo and in vitro, decreasing Bax expression and increasing Bcl-xL expression. As for the underlying mechanisms, we found that interleukin-22 reduced the expression of TGF-ß receptor type II and inhibited the phosphorylation of Smad2 in gingival epithelial cells during periodontitis. Blockage of TGF-ß receptors attenuated apoptosis induced by Porphyromonas gingivalis and increased Bcl-xL expression stimulated by interleukin-22. These results confirmed the inhibitory effect of interleukin-22 on apoptosis of gingival epithelial cells and revealed the involvement of TGF-ß signaling pathway in gingival epithelial cell apoptosis during periodontitis.

Iron Deficiency Modulates Metabolic Landscape of Bacteroidetes Promoting Its Resilience during Inflammation.

Bacteria have to persist under low iron conditions in order to adapt to the nutritional immunity of a host. Since the knowledge of iron stimulon of Bacteroidetes is sparse, we examined oral (Porphyromonas gingivalis and Prevotella intermedia) and gut (Bacteroides thataiotaomicron) representatives for their ability to adapt to iron deplete and iron replete conditions. Our transcriptomics and comparative genomics analysis show that many iron-regulated mechanisms are conserved within the phylum. They include genes upregulated in low iron, as follows: fldA (flavodoxin), hmu (hemin uptake operon), and loci encoding ABC transporters. Downregulated genes were frd (ferredoxin), rbr (rubrerythrin), sdh (succinate dehydrogenase/fumarate reductase), vor (oxoglutarate oxidoreductase/dehydrogenase), and pfor (pyruvate:ferredoxin/flavodoxin oxidoreductase). Some genus-specific mechanisms, such as the sus of B. thetaiotaomicron coding for carbohydrate metabolism and the xusABC coding for xenosiderophore utilization were also identified. While all bacteria tested in our study had the nrfAH operon coding for nitrite reduction and were able to reduce nitrite levels present in culture media, the expression of the operon was iron dependent only in B. thetaiotaomicron. It is noteworthy that we identified a significant overlap between regulated genes found in our study and the B. thetaiotaomicron colitis study (W. Zhu, M. G. Winter, L. Spiga, E. R. Hughes et al., Cell Host Microbe 27:376-388, 2020, http://dx.doi.org/10.1016/j.chom.2020.01.010). Many of those commonly regulated genes were also iron regulated in the oral bacterial genera. Overall, this work points to iron being the master regulator enabling bacterial persistence in the host and paves the way for a more generalized investigation of the molecular mechanisms of iron homeostasis in Bacteroidetes. IMPORTANCE Bacteroidetes are an important group of anaerobic bacteria abundant both in the oral and gut microbiomes. Although iron is a required nutrient for most living organisms, the molecular mechanisms of adaptation to the changing levels of iron are not well known in this group of bacteria. We defined the iron stimulon of Bacteroidetes by examination of the transcriptomic response of Porphyromonas gingivalis and Prevotella intermedia (both belong to the oral microbiome) and Bacteroidetes thetaiotaomicron (belongs to the gut microbiome). Our results indicate that many of the iron-regulated operons are shared among the three genera. Furthermore, using bioinformatics analysis, we identified a significant overlap between our in vitro studies and transcriptomic data derived from a colitis study, thus underscoring the biological significance of our work. Defining the iron-dependent stimulon of Bacteroidetes can help to identify the molecular mechanisms of iron-dependent regulation as well as better understand the persistence of the anaerobes in the human host.

Critical roles of PU.1/cathepsin S activation in regulating inflammatory responses of macrophages in periodontitis.

OBJECTIVE: To determine the critical roles of PU.1/cathepsin S activation in regulating inflammatory responses of macrophages during periodontitis. BACKGROUND: Cathepsin S (CatS) is a cysteine protease and exerts important roles in the immune response. Elevated CatS has been found in the gingival tissues of periodontitis patients and is involved in alveolar bone destruction. However, the underlying mechanism of CatS-driven IL-6 production in periodontitis remains unclear. METHODS: Western blot was applied to measure mature cathepsin S(mCatS) and IL-6 expression in gingival tissues from periodontitis patients and RAW264.7 cells exposed to lipopolysaccharide from Porphyromonas gingivalis (P.g. LPS). Immunofluorescence was applied to confirm the localization of PU.1, and CatS in the gingival tissues of periodontitis patients. ELISA was performed to determine IL-6 production by the P.g. LPS-exposed RAW264.7 cells. Knockdown by shRNA was used to determine the effects of PU.1 on p38/ nuclear factor (NF)-κB activation, mCatS expression and IL-6 production in RAW264.7 cells. RESULTS: The expressions mCatS and IL-6 were significantly upregulated in gingival macrophages. In cultured RAW264.7 cells, increased mCatS and IL-6 protein paralleled the activation of p38 and NF-κB after exposure to P.g. LPS. CatS knockdown by shRNA significantly decreased P.g. LPS-induced IL-6 expression and p38/NF-κB activation. PU.1 was significantly increased in P.g. LPS-exposed RAW264.7 cells, and PU.1 knockdown dramatically abolished the P.g. LPS-induced upregulation of mCatS and IL-6 and the activation of p38 and NF-κB. Furthermore, PU.1 and CatS colocalized in macrophages within the gingival tissues of periodontitis patients. CONCLUSION: PU.1-dependent CatS drives IL-6 production in macrophages by activating p38 and NF-κB in periodontitis.

Effect of Hesperidin on Barrier Function and Reactive Oxygen Species Production in an Oral Epithelial Cell Model, and on Secretion of Macrophage-Derived Inflammatory Mediators during Porphyromonas gingivalis Infection.

Porphyromonas gingivalis is a periodontopathogenic bacterium that can adhere to and colonize periodontal tissues, leading to an inflammatory process, and, consequently, tissue destruction. New therapies using flavonoids, such as hesperidin, are being studied, and their promising properties have been highlighted. The aim of this study was to evaluate the effect of hesperidin on the epithelial barrier function, reactive oxygen species (ROS) production, and on the inflammatory response caused by P. gingivalis in in vitro models. The integrity of the epithelial tight junctions challenged by P. gingivalis was determined by monitoring the transepithelial electrical resistance (TER). P. gingivalis adherence to a gingival keratinocyte monolayer and a basement membrane model were evaluated by a fluorescence assay. A fluorometric assay was used to determine the ROS production in gingival keratinocytes. The level of pro-inflammatory cytokines and matrix metalloproteinases (MMPs) secretion was evaluated by ELISA; to assess NF-κB activation, the U937-3xjB-LUC monocyte cell line transfected with a luciferase reporter gene was used. Hesperidin protected against gingival epithelial barrier dysfunction caused by P. gingivalis and reduced the adherence of P. gingivalis to the basement membrane model. Hesperidin dose-dependently inhibited P. gingivalis-mediated ROS production by oral epithelial cells as well as the secretion of IL-1ß, TNF-α, IL-8, MMP-2, and MMP-9 by macrophages challenged with P. gingivalis. Additionally, it was able to attenuate NF-κB activation in macrophages stimulated with P. gingivalis. These findings suggest that hesperidin has a protective effect on the epithelial barrier function, in addition to reducing ROS production and attenuating the inflammatory response associated with periodontal disease.

The involvement of CdhR in Porphyromonas gingivalis during nitric oxide stress.

Porphyromonas gingivalis, the causative agent of adult periodontitis, must gain resistance to frequent oxidative and nitric oxide (NO) stress attacks from immune cells in the periodontal pocket to survive. Previously, we found that, in the wild-type and under NO stress, the expression of PG1237 (CdhR), the gene encoding for a putative LuxR transcriptional regulator previously called community development and hemin regulator (CdhR), was upregulated 7.7-fold, and its adjacent gene PG1236 11.9-fold. Isogenic mutants P. gingivalis FLL457 (ΔCdhR::ermF), FLL458 (ΔPG1236::ermF), and FLL459 (ΔPG1236-CdhR::ermF) were made by allelic exchange mutagenesis to determine the involvement of these genes in P. gingivalis W83 NO stress resistance. The mutants were black pigmented and ß hemolytic and their gingipain activities varied with strains. FLL457 and FLL459 mutants were more sensitive to NO compared to the wild type, and complementation restored NO sensitivity to that of the wild type. DNA microarray analysis of FLL457 showed that approximately 2% of the genes were upregulated and over 1% of the genes downregulated under NO stress conditions compared to the wild type. Transcriptome analysis of FLL458 and FLL459 under NO stress showed differences in their modulation patterns. Some similarities were also noticed between all mutants. The PG1236-CdhR gene cluster revealed increased expression under NO stress and may be part of the same transcriptional unit. Recombinant CdhR showed binding activity to the predicted promoter regions of PG1459 and PG0495. Taken together, the data indicate that CdhR may play a role in NO stress resistance and be involved in a regulatory network in P. gingivalis.

Perturbation of mitochondrial dynamics links to the aggravation of periodontitis by diabetes.

Diabetes and periodontitis are prevalent diseases that considerably impact global economy and diabetes is a major risk factor of periodontitis. Mitochondrial dynamic alterations are involved in many diseases including diabetes and this study aims to evaluate their relevance with diabetes aggravated periodontitis. Sixty mice are randomly divided into 4 groups: control, periodontitis, diabetes and diabetic periodontitis. Periodontitis severity is evaluated by alveolar bone loss, inflammation and oxidative stress status. Mitochondrial structural and functional defects are evaluated by the mitochondrial fission/fusion events, mitochondrial reactive oxygen species (ROS) accumulation, complex activities and adenosine triphosphate (ATP) production. Advanced glycation end product (AGE) and Porphyromonas gingivalis are closely related to periodontitis occurrence and development. Human gingival fibroblast cells (HGF-1) are used to investigate the AGE role and lipopolysaccharide (LPS) from Porphyromonas gingivalis (P-LPS) in aggravating diabetic periodontitis by mitochondrial dynamic and function alterations. In vivo, diabetic mice with periodontitis show severe bone loss, increased inflammation and oxidative stress accumulation. Among mice with periodontitis, diabetic mice show worse mitochondrial dynamic perturbations than lean mice, along with fusion protein levels inducing more mitochondrial fission in gingival tissue. In vitro, AGEs and P-LPS co-treatment causes severe.

Zoledronic acid affects the process of Porphyromonas gingivalis infecting oral mucosal epithelial barrier: An in-vivo and in-vitro study.

Zoledronic acid (ZA), one of the commonly used bisphosphonates, is mainly used for bone-metabolic diseases. Studies proved that ZA has adverse effects on oral soft tissues. As the first line of innate immunity, the gingival epithelium could be infected by periodontal pathogens, which is a key process of the initiation of periodontal diseases. Yet, how ZA affects the periodontal pathogens infecting the epithelial barrier remains unclear. This study aimed to investigate the influences of ZA on the process of Porphyromonas gingivalis (P. gingivalis) infecting the gingival epithelial barrier via in-vitro and in-vivo experiments. In the in-vitro experiments, under the condition of different concentrations of ZA (0, 1, 10, and 100 µM), P. gingivalis was used to infect human gingival epithelial cells (HGECs). The infections were detected by transmission electron microscope and confocal laser scanning microscope. Besides, the internalization assay was applied to quantify the P. gingivalis, which infected the HGECs, in the different groups. To evaluate the expression levels of pro-inflammatory cytokines, including interleukin (IL)-1ß, IL-6, and IL-8, by infected HGECs, real-time quantitative reverse transcription-polymerase chain reactions were applied. In the in-vivo experiments, rats were given ZA solution (ZA group) or saline (control group) by tail intravenous injection for 8 weeks. Subsequently, we put ligatures around the maxillary second molars of all the rats and inoculated P. gingivalis to the gingiva every other day from day 1 to day 13. The rats were sacrificed on days 3, 7, and 14 for micro-CT and histological analyses. The in-vitro results manifested that the quantity of P. gingivalis that had infected HGECs increased with the ZA concentrations. Pro-inflammatory cytokines expression by HGECs were significantly increased by 100 µM ZA. In the in-vivo study, compared to the control group, more P. gingivalis was detected in the superficial layer of gingival epithelium in the ZA group. Besides, ZA significantly increased the expression level of IL-1ß on day 14 and IL-6 on days 7 and 14 in gingival tissues. These findings suggest that the oral epithelial tissues of patients who receive high-dose ZA treatment may be more susceptible to periodontal infections, resulting in severe inflammatory conditions.

AMPK/mTOR/p70S6K axis prevents apoptosis of Porphyromonas gingivalis-infected gingival epithelial cells via BadSer136 phosphorylation.

Epithelial disruption is the initiation of most infectious disease. Regulation of epithelium apoptosis may play a key role in balance the survival competition between resident bacteria and host cells. The role of the mTOR/p70S6K pathway in preventing apoptosis of human gingival epithelial cells (hGECs) infected with Porphyromonas gingivalis (Pg) was investigated in order to further understand the survival strategy of the epithelial cells in during Pg infecting. hGECs was challenged with Pg for 4, 12, and 24 h. Additionally, hGECs was pretreated with LY294002 (PI3K signaling inhibitor) or Compound C (AMPK inhibitor) for 12 h and exposed them to Pg for 24 h. Subsequently, apoptosis was detected using flow cytometry, and expression and activity of Bcl-2, Bad, Bax, PI3K, AKT, AMPK, mTOR, and p70S6K proteins were analyzed using western blotting. Pg-infecting did not increase apoptosis of hGECs; but the expression ratio of Bad to Bcl-2 was increased after infecting. In contrast, BadSer136 phosphorylation was promoted, accompanied by a significant reduction of mTOR/p70S6K and PI3K/AKT signaling, along with the upregulation of AMPKThr172 signaling. Morrover, the PI3K inhibitor LY294002 promoted Pg-mediated reduction of mTOR/p70S6K expression, and the increase of AMPK signaling and BadSer136 phosphorylation rate, eventually decreasing apoptosis. While Compound C inhibited Pg-mediated activation of AMPK and downregulation of mTOR/p70S6K signaling, significantly reduced the BadSer136 phosphorylation rate, thereby increasing apoptosis. Thus, hGECs prevent apoptosis via an inherent cellular-homeostasis, pro-survival mechanism during Pg infection, the AMPK/mTOR/p70S6K pathway helps prevent apoptosis in hGECs infected with Pg by regulating BadSer136 phosphorylation.

Alleviation of Porphyromonas gingivalis or Its Extracellular Vesicles Provoked Periodontitis and Cognitive Impairment by Lactobacillus pentosus NK357 and Bifidobacterium bifidum NK391.

Porphyromonas gingivalis (PG) is closely involved in the outbreak of periodontitis and cognitive impairment (CI). Herein, we examined the effects of anti-inflammatory Lactobacillus pentosus NK357 and Bifidobacterium bifidum NK391 on PG- or its extracellular vesicles (pEVs)-induced periodontitis and CI in mice. Oral administration of NK357 or NK391 significantly decreased PG-induced tumor necrosis factor (TNF)-α, receptor activator of nuclear factors κB (RANK), and RANK ligand (RANKL) expression, gingipain (GP)+lipopolysaccharide (LPS)+ and NF-κB+CD11c+ populations, and PG 16S rDNA level in the periodontal tissue. Their treatments also suppressed PG-induced CI -like behaviors, TNF-α expression and NF-κB-positive immune cells in the hippocampus and colon, while PG-suppressed hippocampal BDNF and N-methyl-D-aspartate receptor (NMDAR) expression increased. The combination of NK357 and NK391 additively alleviated PG- or pEVs-induced periodontitis, neuroinflammation, CI-like behaviors, colitis, and gut microbiota dysbiosis and increased PG- or pEVs-suppressed BDNF and NMDAR expression in the hippocampus. In conclusion, NK357 and NK391 may alleviate periodontitis and dementia by regulating NF-κB, RANKL/RANK, and BDNF-NMDAR signaling and gut microbiota.