Porphyromonas gingivalis infection alters microRNA composition in extracellular vesicles.

OBJECTIVES: Periodontitis, commonly associated with Porphyromonas gingivalis (Pg), involves intricate alterations of oral intercellular interactions, in which extracellular vesicles (EVs) play a pivotal role. The understanding of the miRNA profiles in the EVs derived from Pg-infected cells (Pg-EVs) remains incomplete despite acknowledging their importance in intercellular communication during periodontitis. Therefore, our objective was to identify and characterize the miRNAs enriched in Pg-EVs. METHODS: Microarray analysis was conducted to examine the miRNA profiles in the EVs derived from Pg-infected THP-1 cells. We compared the identified miRNAs with those upregulated in the EVs after stimulation with LPS. Additionally, we explored how inhibiting TLR signaling during Pg infection affects the transcription of specific miRNAs. We investigated the unique sequence motifs specific to the miRNAs concentrated in Pg-EVs. RESULTS: The levels of eleven miRNAs, including miR-155, were increased in Pg-EVs compared with those elevated after LPS stimulation. The Pg-induced miR-155 upregulation via TLR2 but not TLR4 signaling suggests the influence of TLR signaling on the miRNA composition of EVs. Furthermore, the miRNAs upregulated in Pg-EVs contained AGAGGG and GRGGSGC sequence motifs. CONCLUSIONS: Our findings demonstrate that Pg-induced alterations in EV-containing miRNA composition occur in a TLR4-independent manner. Notably, the concentrated miRNAs in Pg-EVs harbor specific motifs with a high G + C content within their sequences. The upregulation of specific miRNAs in EVs under infectious conditions suggests the influence of both innate immune receptor signals and miRNA sequence characteristics.

Porphyromonas gingivalis (W83) Infection Induces Alzheimer's Disease-Like Pathophysiology in Obese and Diabetic Mice.

BACKGROUND: Periodontal disease(s) and metabolic illnesses negatively impact the quality of life and, eventually mental health. OBJECTIVE: This study investigated the effect of Porphyromonas gingivalis (W83) oral infection on the development of Alzheimer's disease (AD) pathophysiology in a wild-type obese, diabetic (db/db) mouse model. METHODS: The db/db mice were either orally infected with P. gingivalis and Fusobacterium nucleatum or sham infected for 16 weeks. The presence of amyloid-ß (Aß) and neurofibrillary tangles (NFTs) were assessed using a silver impregnation technique and subsequently by immunohistochemistry for tau and neuroinflammation. The mRNA abundance of a panel of 184 genes was performed using quantitative real-time PCR, and the differentially expressed genes were analyzed by Ingenuity Pathway Analysis. RESULTS: While no Aß plaques and NFTs were evident by silver impregnation, immunohistochemistry (glial cell markers) of the P. gingivalis-infected mice tissue sections exhibited neuroinflammation in the form of reactive microglia and astrocytes. Anti-tau immunopositivity, in addition to cells, was prominent in thickened axons of hippocampal CA neurons. The mRNA abundance of crucial genes in the insulin signaling pathway (INSR, IGF1, IRS, IDE, PIK3R, SGK1, GYS, GSK3B, AKT1) were upregulated, potentially exacerbating insulin resistance in the brain by P. gingivalis oral infection. Increased mRNA abundance of several kinases, membrane receptors, transcription factors, and pro-inflammatory mediators indicated hyperactivation of intracellular cascades with potential for tau phosphorylation and Aß release in the same infection group. CONCLUSION: P. gingivalis W83 infection of db/db mice provides a disease co-morbidity model with the potential to reproduce AD pathophysiology with induced periodontal disease.

hTERT-immortalized gingival fibroblasts respond to cytokines but fail to mimic primary cell responses to Porphyromonas gingivalis.

In periodontitis, gingival fibroblasts (GFs) interact with and respond to oral pathogens, significantly contributing to perpetuation of chronic inflammation and tissue destruction. The aim of this study was to determine the usefulness of the recently released hTERT-immortalized GF (TIGF) cell line for studies of host-pathogen interactions. We show that TIGFs are unable to upregulate expression and production of interleukin (IL)-6, IL-8 and prostaglandin E2 upon infection with Porphyromonas gingivalis despite being susceptible to adhesion and invasion by this oral pathogen. In contrast, induction of inflammatory mediators in TNFα- or IL-1ß-stimulated TIGFs is comparable to that observed in primary GFs. The inability of TIGFs to respond directly to P. gingivalis is caused by a specific defect in Toll-like receptor-2 (TLR2) expression, which is likely driven by TLR2 promoter hypermethylation. Consistently, TIGFs fail to upregulate inflammatory genes in response to the TLR2 agonists Pam2CSK4 and Pam3CSK4. These results identify important limitations of using TIGFs to study GF interaction with oral pathogens, though these cells may be useful for studies of TLR2-independent processes. Our observations also emphasize the importance of direct comparisons between immortalized and primary cells prior to using cell lines as models in studies of any biological processes.

Molecular Strategies Underlying Porphyromonas gingivalis Virulence.

The anaerobic Gram-negative bacterium Porphyromonas gingivalis is considered the keystone of periodontitis diseases, a set of inflammatory conditions that affects the tissues surrounding the teeth. In the recent years, the major virulence factors exploited by P. gingivalis have been identified and characterized, including a cocktail of toxins, mainly proteases called gingipains, which promote gingival tissue invasion. These effectors use the Sec pathway to cross the inner membrane and are then recruited and transported across the outer membrane by the type IX secretion system (T9SS). In P. gingivalis, most secreted effectors are attached to anionic lipopolysaccharides (A-LPS), and hence form a virulence coat at the cell surface. P. gingivalis produces additional virulence factors to evade host immune responses, such as capsular polysaccharide, fimbriae and outer membrane vesicles. In addition to periodontitis, it is proposed that this broad repertoire of virulence factors enable P. gingivalis to be involved in diverse human diseases such as rheumatoid arthritis, and neurodegenerative, Alzheimer, and cardiovascular disorders. Here, we review the major virulence determinants of P. gingivalis and discuss future directions to better understand their mechanisms of action.

Frequency of Porphyromonas gingivalis and fimA genotypes in patients with periodontitis and systemic lupus erythematosus.

OBJECTIVE: The objective of this study was to determine and compare the distribution of fimA genotypes of Porphyromonas gingivalis (P. gingivalis) in systemic lupus erythematosus (SLE) patients compared with control subjects. MATERIAL AND METHODS: This observational cross-sectional study included 281 patients divided into two groups. Group 1 (G1) consisted of 162 control subjects (30-54 years old) and, group 2 (G2) included 119 subjects (10-69 years old) diagnosed with SLE. The presence of P. gingivalis was detected by PCR. DNA sequences in acquired plaque samples were identified using P. gingivalis specific sequences and further analyzed to differentiate their fimA genotypes using six sets of fimA genotype-specific primers. RESULTS: The presence of periodontitis (PE) was similar in both groups; similar measurements were obtained regarding clinical attachment loss (CAL) (G1 1.76 ± 0.72 vs. G2 1.95 ± 0.76). G2 showed the highest frequency of P. gingivalis (94.95%). FimA genotype II is considered the most virulent and, was the most frequently found in the SLE group (53.09%). CONCLUSION: The genotypes associated with PE are more frequently detected in SLE, which could make them susceptible to develop PE.

Mechanisms of vascular damage by systemic dissemination of the oral pathogen Porphyromonas gingivalis.

Several studies have shown a clear association between periodontal disease and increased risk of cardiovascular disease. Porphyromonas gingivalis (Pg), a key oral pathogen, and its cell surface-expressed gingipains, induce oedema in a zebrafish larvae infection model although the mechanism of these vascular effects is unknown. Here, we aimed to determine whether Pg-induced vascular damage is mediated by gingipains. In vitro, human endothelial cells from different vascular beds were invaded by wild-type (W83) but not gingipain-deficient (ΔK/R-ab) Pg. W83 infection resulted in increased endothelial permeability as well as decreased cell surface abundance of endothelial adhesion molecules PECAM-1 and VE-cadherin compared to infection with ΔK/R-ab. In agreement, when transgenic zebrafish larvae expressing fluorescently labelled PECAM-1 or VE-cadherin were systemically infected with W83 or ΔK/R-ab, a significant reduction in adhesion molecule fluorescence was observed specifically in endothelium proximal to W83 bacteria through a gingipain-dependent mechanism. Furthermore, this was associated with increased vascular permeability in vivo when assessed by dextran leakage microangiography. These data are the first to show that Pg directly mediates vascular damage in vivo by degrading PECAM-1 and VE-cadherin. Our data provide a molecular mechanism by which Pg might contribute to cardiovascular disease.

Characterization and regulation of osteoclast precursors following chronic Porphyromonas gingivalis infection.

Bone destruction in inflammatory osteolytic diseases including periodontitis is related to excessive activity of osteoclasts (OC), which originate from precursor cells of the myeloid lineage, termed osteoclast precursors (OCP). In contrast to ample knowledge that we currently have on mature OC, little is known about OCP and their regulation during bacterial infection. Therefore, this study aimed to identify and characterize OCP following chronic infection with a periodontal bacteria Porphyromonas gingivalis (Pg). We used a micro-osmotic pump to continually release Pg subcutaneously in a murine model. Two weeks after Pg infection, the frequency of CD11b+c-fms+Ly6Chi population is significantly elevated within the bone marrow, spleen and peripheral blood. In vitro and in vivo studies identified these cells as the OCP-containing population and Pg infection significantly enhanced the osteoclastogenic activity of these cells. Furthermore, mRNA sequencing analysis indicated a unique gene and pathway profile in CD11b+c-fms+Ly6Chi population following Pg infection, with changes in genes and pathways related to OC differentiation, cell proliferation and apoptosis, inflammatory response, phagocytosis and immunity, as well as antigen processing and presentation. Moreover, using IL-6 knockout mice, we found that IL-6 is important for Pg-induced accumulation of CD11b+c-fms+Ly6Chi population from the bone marrow and periphery. Our results provide new insights into the characterization and regulation of OCP following a chronic bacterial infection. This knowledge is relevant to the understanding of the pathogenesis of bacteria-induced bone loss, and to the identification of potential therapeutic targets of bone loss diseases.

Oral P. gingivalis impairs gut permeability and mediates immune responses associated with neurodegeneration in LRRK2 R1441G mice.

BACKGROUND: The R1441G mutation in the leucine-rich repeat kinase 2 (LRRK2) gene results in late-onset Parkinson's disease (PD). Peripheral inflammation and gut microbiota are closely associated with the pathogenesis of PD. Chronic periodontitis is a common type of peripheral inflammation, which is associated with PD. Porphyromonas gingivalis (Pg), the most common bacterium causing chronic periodontitis, can cause alteration of gut microbiota. It is not known whether Pg-induced dysbiosis plays a role in the pathophysiology of PD. METHODS: In this study, live Pg were orally administrated to animals, three times a week for 1 month. Pg-derived lipopolysaccharide (LPS) was used to stimulate mononuclear cells in vitro. The effects of oral Pg administration on the gut and brain were evaluated through behaviors, morphology, and cytokine expression. RESULTS: Dopaminergic neurons in the substantia nigra were reduced, and activated microglial cells were increased in R1441G mice given oral Pg. In addition, an increase in mRNA expression of tumor necrosis factor (TNF-α) and interleukin-1ß (IL-1ß) as well as protein level of α-synuclein together with a decrease in zonula occludens-1 (Zo-1) was detected in the colon in Pg-treated R1441G mice. Furthermore, serum interleukin-17A (IL-17A) and brain IL-17 receptor A (IL-17RA) were increased in Pg-treated R1441G mice. CONCLUSIONS: These findings suggest that oral Pg-induced inflammation may play an important role in the pathophysiology of LRRK2-associated PD.

Exploring potential of vaginal Lactobacillus isolates from South African women for enhancing treatment for bacterial vaginosis.

Antibiotics continue to be the standard-of-care for bacterial vaginosis (BV), although recurrence rates are high. Vaginal probiotics may improve durability of BV treatment, although few probiotics for vaginal health contain Lactobacillus spp. that commonly colonize the lower female genital tract. Characteristics of vaginal Lactobacillus strains from South African women were evaluated for their probiotic potential in vitro compared to strains from commercial vaginal products, including growth at varying pHs, ability to lower pH, produce D-/L-lactate and H2O2, influence growth of BV-associated Gardnerella vaginalis and Prevotella bivia, adherence to cervical cells and susceptibility to antibiotics. Fifty-seven Lactobacillus strains were purified from cervico-vaginal fluid, including L. crispatus, L. jensenii, L. gasseri, L. mucosae, and L. vaginalis. L crispatus strains grew better at pHs below 4.5 and lowered pH more effectively than other strains. Production of D-/L-lactate and H2O2 varied between Lactobacillus species and strains. Lactobacillus strains generally inhibited P. bivia more uniformly than G. vaginalis isolates. All vaginal Lactobacillus isolates were resistant to metronidazole while susceptibility to clindamycin varied. Furthermore, vaginal Lactobacillus strains tended to be broadly susceptible to penicillin, amoxicillin, rifampicin and rifabutin. Whole-genome-sequencing of five of the best-performing vaginal Lactobacillus strains confirmed their likely safety, due to antimicrobial resistance elements being largely absent, while putative intact prophages were present in the genomes of two of the five strains. Overall, vaginal Lactobacillus strains largely performed better in these in vitro assays than probiotic strains currently used in probiotics for vaginal health. Including the best-performing vaginal Lactobacillus isolates in a region-specific probiotic for vaginal health may result in improved BV treatment options.

Porphyromonas gingivalis and adverse pregnancy outcomes: a review on its intricate pathogenic mechanisms.

Porphyromonas gingivalis (P. gingivalis), a Gram-negative facultative anaerobe of the oral cavity, is associated with the onset of various adverse pregnancy outcomes. P. gingivalis is linked with the development of preeclampsia, preterm labour, spontaneous abortion, gestational diabetes, foetal growth restriction, and misconception. The unique virulence factors, surface adhesions, enzymes of P. gingivalis can directly injure and alter the morphology, microbiome the foetal and maternal tissues. P. gingivalis can even exaggerate the production of cytokines, free radicals and acute-phase proteins in the uterine compartment that increases the risk of myometrial contraction and onset of preterm labour. Although evidence confirms the presence of P. gingivalis in the amniotic fluid and placenta of women with poor pregnancy outcomes, the intricate molecular mechanisms by which P. gingivalis initiates various antenatal and postnatal maternal and foetal complications are not well explained in the literature. Therefore, the present review aims to comprehensively summarise and highlight the recent and unique molecular pathogenic mechanisms of P. gingivalis associated with adverse pregnancy outcomes.

Frontline Science: Characterization and regulation of osteoclast precursors following chronic Porphyromonas gingivalis infection.

Bone destruction in inflammatory osteolytic diseases including periodontitis is related to excessive activity of osteoclasts (OC), which originate from precursor cells of the myeloid lineage, termed osteoclast precursors (OCP). In contrast to ample knowledge that we currently have on mature OC, little is known about OCP and their regulation during bacterial infection. Therefore, this study aimed to identify and characterize OCP following chronic infection with a periodontal bacteria Porphyromonas gingivalis (Pg). We used a microosmotic pump to continually release Pg subcutaneously in a murine model. Two weeks after Pg infection, the frequency of CD11b+c-fms+Ly6Chi population is significantly elevated within the bone marrow, spleen, and peripheral blood. In vitro and in vivo studies identified these cells as the OCP-containing population and Pg infection significantly enhanced the osteoclastogenic activity of these cells. Furthermore, mRNA sequencing analysis indicated a unique gene and pathway profile in CD11b+c-fms+Ly6Chi population following Pg infection, with changes in genes and pathways related to OC differentiation, cell proliferation and apoptosis, inflammatory response, phagocytosis, and immunity, as well as antigen processing and presentation. Moreover, using IL-6 knockout mice, we found that IL-6 is important for Pg-induced accumulation of CD11b+c-fms+Ly6Chi population from the bone marrow and periphery. Our results provide new insight into the characterization and regulation of OCP following a chronic bacterial infection. This knowledge is relevant to the understanding of the pathogenesis of bacteria-induced bone loss, and to the identification of potential therapeutic targets of bone loss diseases.

Transcription modulation by CDK9 regulates inflammatory genes and RIPK3-MLKL-mediated necroptosis in periodontitis progression.

Cyclin-dependent kinase 9 (CDK9), one crucial molecule in promoting the transition from transcription pausing to elongation, is a critical modulator of cell survival and death. However, the pathological function of CDK9 in bacterial inflammatory diseases has never been explored. CDK9 inhibition or knock-down attenuated Porphyromonas gingivalis-triggered inflammatory gene expression. Gene-expression microarray analysis of monocytes revealed that knock-down of CDK9 not only affected inflammatory responses, but also impacted cell death network, especially the receptor-interacting protein kinase 3 (RIPK3)-mixed lineage kinase domain-like (MLKL)-mediated necroptosis after P. gingivalis infection. Inhibition of CDK9 significantly decreased necroptosis with downregulation of both MLKL and phosphorylated MLKL. By regulating caspase-8 and cellular FLICE inhibitory protein (cFLIP), key molecules in regulating cell survival and death, CDK9 affected not only the classic RIPK1-RIPK3-mediated necroptosis, but also the alternate TIR-domain-containing adapter-inducing interferon-ß-RIPK3-mediated necroptosis. CDK9 inhibition dampened pro-inflammatory gene production in the acute infection process in the subcutaneous chamber model in vivo. Moreover, CDK9 inhibition contributed to the decreased periodontal bone loss and inflammatory response induced by P. gingivalis in the periodontal micro-environment. In conclusion, by modulating the RIPK3-MLKL-mediated necroptosis, CDK9 inhibition provided a novel mechanism to impact the progress of bacterial infection in the periodontal milieu.

Distinct gene expression characteristics in epithelial cell-Porphyromonas gingivalis interactions by integrating transcriptome analyses.

Porphyromonas gingivalis is a pivotal periodontal pathogen, and the epithelial cells serve as the first physical barrier to defend the host from bacterial attack. Within this host-bacteria interaction, P. gingivalis can modify the host immune reaction and adjust the gene expression, which is associated with periodontitis pathogenesis and developing strategies. Herein, a meta-analysis was made to get the differential gene expression profiles in epithelial cells with or without P. gingivalis infection. The network-based meta-analysis program for gene expression profiling was used. Both the gene ontology analysis and the pathway enrichment analysis of the differentially expressed genes were conducted. Our results determined that 290 genes were consistently up-regulated in P. gingivalis infected epithelial cells. 229 gene ontology biological process terms of up-regulated genes were discovered, including "negative regulation of apoptotic process" and "positive regulation of cell proliferation/migration/angiogenesis". In addition to the well-known inflammatory signaling pathways, the pathway associated with a transcriptional misregulation in cancer has also been increased. Our findings indicated that P. gingivalis benefited from the survival of epithelial cells, and got its success as a colonizer in oral epithelium. The results also suggested that infection of P. gingivalis might contribute to oral cancer through chronic inflammation. Negative regulation of the apoptotic process and transcriptional misregulation in cancer pathway are important contributors to the cellular physiology changes during infection development, which have particular relevance to the pathogenesis and progressions of periodontitis, even to the occurrence of oral cancer.

Specific RANK Cytoplasmic Motifs Drive Osteoclastogenesis.

Upon receptor activator of NF-κB ligand (RANKL) binding, RANK promotes osteoclast formation through the recruitment of tumor necrosis factor (TNF) receptor-associated factors (TRAFs). In vitro assays identified two RANK intracellular motifs that bind TRAFs: PVQEET560-565 (Motif 2) and PVQEQG604-609 (Motif 3), which potently mediate osteoclast formation in vitro. To validate the in vitro findings, we have generated knock-in (KI) mice harboring inactivating mutations in RANK Motifs 2 and 3. Homozygous KI (RANKKI/KI ) mice are born at the predicted Mendelian frequency and normal in tooth eruption. However, RANKKI/KI mice exhibit significantly more trabecular bone mass than age- and sex-matched heterozygous KI (RANK+/KI ) and wild-type (RANK+/+ ) counterparts. Bone marrow macrophages (BMMs) from RANKKI/KI mice do not form osteoclasts when they are stimulated with macrophage colony-stimulating factor (M-CSF) and RANKL in vitro. RANKL is able to activate the NF-κB, ERK, p38, and JNK pathways in RANKKI/KI BMMs, but it cannot stimulate c-Fos or NFATc1 in the RANKKI/KI cells. Previously, we showed that RANK signaling plays an important role in Porphyromonas gingivalis (Pg)-mediated osteoclast formation by committing BMMs into the osteoclast lineage. Here, we show that RANKL-primed RANKKI/KI BMMs are unable to differentiate into osteoclasts in response to Pg stimulation, indicating that the two RANK motifs are required for Pg-induced osteoclastogenesis. Mechanistically, RANK Motifs 2 and 3 facilitate Pg-induced osteoclastogenesis by stimulating c-Fos and NFATc1 expression during the RANKL pretreatment phase as well as rendering c-Fos and NFATc1 genes responsive to subsequent Pg stimulation. Cell-penetrating peptides (CPPs) conjugated with RANK segments containing Motif 2 or 3 block RANKL- and Pg-mediated osteoclastogenesis. The CPP conjugates abrogate RANKL-stimulated c-Fos and NFATc1 expression but do not affect RANKL-induced activation of NF-κB, ERK, p38, JNK, or Akt signaling pathway. Taken together, our current findings demonstrate that RANK Motifs 2 and 3 play pivotal roles in osteoclast formation in vivo and mediate Pg-induced osteoclastogenesis in vitro.

Heat-killed Candida albicans augments synthetic bacterial component-induced proinflammatory cytokine production.

Candida albicans can enhance the invasion of oral epithelial cells by Porphyromonas gingivalis, although the fungus is not a periodontal pathogen. In this study, we investigated whether C. albicans augments proinflammatory cytokine production by mouse macrophage-like J774.1 cells incubated with synthetic bacterial components. Mouse macrophage-like J774.1 cells, mouse primary splenocytes, human THP-1 cells, and A549 cells were pretreated with or without heat-killed C. albicans (HKCA) or substitutes for C. albicans cell wall components in 96-well flat-bottomed plates. Cells were then washed and incubated with Pam3CSK4, a Toll-like receptor (TLR) 2 ligand, or lipid A, a TLR4 ligand. Culture supernatants were analyzed by ELISA for secreted IL-6, MCP-1, TNF-α, and IL-8. HKCA augmented TLR ligand-induced proinflammatory cytokine production by J774.1 cells, mouse splenocytes, and THP-1 cells, but not A549 cells. However, IL-6, MCP-1, and TNF-α production induced by Pam3CSK4 or lipid A was not augmented when cells were pretreated with curdlan, a dectin-1 ligand, or mannan, a dectin-2 ligand. In contrast, pretreatment of cells with TLR ligands upregulated the production of IL-6 and TNF-α, but not MCP-1, induced by Pam3CSK4 or lipid A. The results suggest that C. albicans augments synthetic bacterial component-induced cytokine production by J774.1 cells via the TLR pathway, but not the dectin-1 or dectin-2 pathway.

Salivary biomarkers in association with periodontal parameters and the periodontitis risk haplotype.

Genetic factors play a role in periodontitis. Here we examined whether the risk haplotype of MHC class III region BAT1-NFKBIL1-LTA and lymphotoxin-α polymorphisms associate with salivary biomarkers of periodontal disease. A total of 455 individuals with detailed clinical and radiographic periodontal health data were included in the study. A 610 K genotyping chip and a Sequenom platform were used in genotyping analyses. Phospholipid transfer protein activity, concentrations of lymphotoxin-α, IL-8 and myeloperoxidase, and a cumulative risk score (combining Porphyromonas gingivalis, IL-1ß and matrix metalloproteinase-8) were examined in saliva samples. Elevated IL-8 and myeloperoxidase concentrations and cumulative risk scores associated with advanced tooth loss, deepened periodontal pockets and signs of periodontal inflammation. In multiple logistic regression models adjusted for periodontal parameters and risk factors, myeloperoxidase concentration (odds ratio (OR); 1.37, P = 0.007) associated with increased odds for having the risk haplotype and lymphotoxin-α concentration with its genetic variants rs2857708, rs2009658 and rs2844482. In conclusion, salivary levels of IL-8, myeloperoxidase and cumulative risk scores associate with periodontal inflammation and tissue destruction, while those of myeloperoxidase and lymphotoxin-α associate with genetic factors as well.

Quorum sensing molecules regulate epithelial cytokine response and biofilm-related virulence of three Prevotella species.

Quorum sensing (QS) signaling regulates the motility, adhesion, and biofilm formation of bacteria, and at the same time activates immune response in eukaryotic organisms. We recently demonstrated that the QS molecule, dihydroxy-2, 3-pentanedione (DPD), and its analogs significantly inhibit estradiol-regulated virulence of Prevotella aurantiaca, one of the four species in the Prevotella intermedia group. Here, we examined the combined effects of estradiol and QS signaling on 1) cytokine response of human gingival keratinocytes (HMK) against whole cell extract (WCE) of P. intermedia, Prevotella nigrescens, and Prevotella pallens, and 2) biofilm formation of these three Prevotella species. All experiments were performed in the presence or absence of estradiol, and with different QS molecules: DPD and its analogs (ethyl-DPD, butyl-DPD, and isobutyl-DPD). Concentrations of interleukin (IL)-1ß, -6, and -8 were determined by the Luminex multiplex immunoassay, biofilm mass was quantitatively evaluated by measuring protein concentration via the Bradford method, and the microtopography of biofilms was assessed by scanning electron microscopy (SEM) imaging. Concentrations of IL-6 and IL-8 were elevated when HMK cells were incubated with estradiol and WCE of P. intermedia and P. nigrescens, but decreased when incubated with estradiol and WCE of P. pallens. Butyl-DPD neutralized the estradiol- and WCE-induced regulation of HMK interleukin expression and, at the same time, inhibited the biofilm formation of P. intermedia and P. nigrescens. SEM micrographs revealed a decrease in biofilm mass after application of butyl-DPD, which was most detectable among the P. intermedia ATCC 25611 and P. nigrescens ATCC 33563 and AHN 8293 strains. In conclusion, butyl-DPD analog is able to neutralize the WCE-induced epithelial cytokine response and, at the same time, to inhibit the biofilm formation of P. intermedia and P. nigrescens.

Identification of novel genes involved in gingival epithelial cells responding to Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis infections.

OBJECTIVE: This study aimed to identify the differentially expressed genes (DEGs) in gingiva epithelial cells responding to Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis infections using bioinformatics method. STUDY DESIGN: GSE9723 dataset was downloaded from Gene Expression Omnibus, and DEGs between the infected cells and controls were identified using unpaired t-test. Overlapping DEGs in responding to Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis infections were extracted. Protein-protein interaction networks were constructed and functional modules were isolated using Molecular Complex Detection algorithm. Key genes in protein-protein interaction network and Molecular Complex Detection modules were subjected to functional enrichment analyses. In addition, the transcriptional factors were predicted. RESULTS: A total of 533 co-up-regulated and 202 co-down-regulated genes were identified. The up-regulated genes, including IL6, CCL19, EDN1, ADCY9, and BCL2 and the down-regulated genes, including CCNB1, PLK1, and CCNA2 were the key genes in the protein-protein interaction network and modules. They were intensively enriched in chemokine signaling pathway, calcium signaling pathway and cell cycle. Finally, two transcriptional factors, E12 and NRSF, targeting to the up-regulated genes and one transcriptional factor, NRP1, targeting the down-regulated genes, were predicted. CONCLUSIONS: CCNB1, PLK1, and CCNA2 might play important roles in the response of host epithelial cells to Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis.

Kavain Reduces Porphyromonas gingivalis-Induced Adipocyte Inflammation: Role of PGC-1α Signaling.

A link between obesity and periodontitis has been suggested because of compromised immune response and chronic inflammation in obese patients. In this study, we evaluated the anti-inflammatory properties of Kavain, an extract from Piper methysticum, on Porphyromonas gingivalis-induced inflammation in adipocytes with special focus on peroxisome proliferation-activated receptor γ coactivator α (PGC-1α) and related pathways. The 3T3-L1 mouse preadipocytes and primary adipocytes harvested from mouse adipose tissue were infected with P. gingivalis, and inflammation (TNF-α; adiponectin/adipokines), oxidative stress, and adipogenic marker (FAS, CEBPα, and PPAR-γ) expression were measured. Furthermore, effect of PGC-1α knockdown on Kavain action was evaluated. Results showed that P. gingivalis worsens adipocyte dysfunction through increase of TNF-α, IL-6, and iNOS and decrease of PGC-1α and adiponectin. Interestingly, although Kavain obliterated P. gingivalis-induced proinflammatory effects in wild-type cells, Kavain did not affect PGC-1α-deficient cells, strongly advocating for Kavain effects being mediated by PGC-1α. In vivo adipocytes challenged with i.p. injection of P. gingivalis alone or P. gingivalis and Kavain displayed the same phenotype as in vitro adipocytes. Altogether, our findings established anti-inflammatory and antioxidant effects of Kavain on adipocytes and emphasized protective action against P. gingivalis-induced adipogenesis. The use of compounds such as Kavain offer a portal to potential therapeutic approaches to counter chronic inflammation in obesity-related diseases.

Distribution of Porphyromonas gingivalis fimA genotypes in patients affected by rheumatoid arthritis and periodontitis.

OBJECTIVE: To determine and compare the distribution of Porphyromonas gingivalis fimA genotypes in patients affected by Rheumatoid arthritis (RA) and periodontitis (PE). MATERIALS AND METHODS: This study involved 394 subjects divided into four groups, RA, PE, RA and PE and healthy subjects. PE was diagnosed by using clinical attachment loss (CAL) and probing depth (PD) indexes. Presence of P. gingivalis and its genotypes was identified by polymerase chain reaction in subgingival biofilm. RESULTS: P. gingivalis was more frequent in patients with RA (82.69%), and fimA II genotype was the most frequent in all groups, especially in PE/RA (76.71%). There was statistical difference (p < .05) regarding the frequency of P. gingivalis genotypes such as fimA Ib, II and III. CONCLUSIONS: Distribution of P. gingivalis fimA II genotypes was different among groups, it could play a critical role in the presence of PE in RA patients.