High glucose condition aggravates inflammatory response induced by Porphyromonas gingivalis in THP-1 macrophages via autophagy inhibition.

BACKGROUND: Porphyromonase gingivalis (P. gingivalis) is a type of bacteria that causes periodontitis, which is strongly correlated with systemic diseases such as diabetes. However, the effect of hyperglycemia on periodontitis are unclear. The present study examined the effects of high glucose levels on the response to P. gingivalis infection. RESULTS: The expression of P. gingivalis-induced interleukin-1ß (IL-1ß) and inflammasomes increased as the glucose concentration increased. High glucose conditions suppressed P. gingivalis-induced autophagy in human acute monocytic leukemia cell line (THP-1) macrophages. Zingerone increased autophagy and alleviated P. gingivalis-induced inflammatory response in THP-1 macrophages under high glucose conditions. In addition, P. gingivalis- induced inflammation in bone marrow-derived macrophages of diabetic mice was higher than in wild-type mice, but a zingerone treatment decreased the levels. Alveolar bone loss due to a P. gingivalis infection was significantly higher in diabetic mice than in wild-type mice. CONCLUSIONS: High-glucose conditions aggravated the inflammatory response to P. gingivalis infection by suppressing of autophagy, suggesting that autophagy induction could potentially to treat periodontitis in diabetes. Zingerone has potential use as a treatment for periodontal inflammation induced by P. gingivalis in diabetes patients.

Association of DOK3 and infiltrated tumor-associated macrophages with risk for the prognosis of Porphyromonas gingivalis-infected oral cancer: a 12-year data analysis of 200 patients from a tertiary teaching hospital, Urumqi, China.

BACKGROUND: While there is an understanding of the association between the expression of Porphyromonas gingivalis (P. gingivalis) and prognosis of oral squamous cell carcinoma (OSCC), significance specially to address the relevance between different immunohistochemical intensities of P. gingivalis and tumor-associated macrophages (TAMs) in OSCC tissue and related clinicopathologic characteristics has not been well investigated. The present study aimed to investigate the pathological features related to M2-TAM in P. gingivalis-infected OSCC and ascertain its clinical relevance with patients' prognosis. METHODS: A prospective cohort study was designed to comparatively analyze 200 patients from June 2008 to June 2020. Bioinformatics analyses were implemented to identify DOK3 as a key molecule and to appraise immunocyte infiltration using Gene Expression Omnibus and The Cancer Genome Atlas databases. Immunohistochemical evaluation was performed to analyze the association between the expression levels of P. gingivalis, DOK3, and M2-TAM and clinicopathological variables using Fisher's exact test or Pearson's chi-square test. Cox analysis was used to calculate hazard ratios (HR) with corresponding 95% confidence interval (CI) for various clinicopathological features. The Kaplan-Meier approach and log-rank test were used to plot the survival curves. RESULTS: The expression level of P. gingivalis was positively associated with DOK3 and M2-TAMs expression level (P < 0.001). Parameters, including body mass index, clinical stage, recurrence, tumor differentiation, and P. gingivalis, DOK3, and M2-TAM immunoexpression levels, affected the prognosis of patients with OSCC (all P < 0.05). In addition, P. gingivalis (HR = 1.674, 95%CI 1.216-4.142, P = 0.012), DOK3 (HR = 1.881, 95%CI 1.433-3.457, P = 0.042), and M2-TAM (HR = 1.649, 95%CI 0.824-3.082, P = 0.034) were significantly associated with the 10-year cumulative survival rate. CONCLUSIONS: Elevated expression of P. gingivalis and DOK3 indicates M2-TAM infiltration and unfavorable prognosis of OSCC, and could be considered as three novel independent risk factors for predicting the prognosis of OSCC.

Porphyromonas gingivalis promotes the progression of oral squamous cell carcinoma by stimulating the release of neutrophil extracellular traps in the tumor immune microenvironment.

BACKGROUND: The aim of this study was to investigate the impact of Porphyromonas gingivalis (P. gingivalis) on the progression of oral squamous cell carcinoma (OSCC) through neutrophil extracellular traps (NETs) in the tumor immune microenvironment. METHODS: The expression of NETs-related markers was identified through immunohistochemistry, immunofluorescence, and Western blotting in different clinical stages of OSCC samples. The relationship between NETs-related markers and clinicopathological characteristics in 180 samples was analyzed using immunohistochemistry data. Furthermore, the ability to predict the prognosis of OSCC patients was determined by ROC curve analysis and survival analysis. The effect of P. gingivalis on the release of NETs was identified through immunofluorescence and immunohistochemistry, both in vitro and in vivo. CAL27 and SCC25 cell lines were subjected to NETs stimulation to elucidate the influence of NETs on various cellular processes, including cell proliferation, migration, invasion, and metastasis in vitro. Furthermore, the impact of NETs on the growth and metastatic potential of OSCC was assessed using in vivo models involving tumor-bearing mice and tumor metastasis mouse models. RESULTS: Immunochemistry analysis revealed a significant correlation between the NETs-related markers and clinical stage, living status as well as TN stage. P. gingivalis has demonstrated its ability to effectively induce the release of NETs both in vivo and in vitro. NETs have the potential to facilitate cell migration, invasion, and colony formation. Moreover, in vivo experiments have demonstrated that NETs play a pivotal role in promoting tumor metastasis. CONCLUSION: High expression of NETs-related markers demonstrates a strong correlation with the progression of OSCC. Inhibition of the NETs release process stimulated by P. gingivalis and targeted NETs could potentially open up a novel avenue in the field of immunotherapy for patients afflicted with OSCC.

Differential Response of Human Dendritic Cells upon Stimulation with Encapsulated or Non-Encapsulated Isogenic Strains of Porphyromonas gingivalis.

During periodontitis, the extracellular capsule of Porphyromonas gingivalis favors alveolar bone loss by inducing Th1 and Th17 patterns of lymphocyte response in the infected periodontium. Dendritic cells recognize bacterial antigens and present them to T lymphocytes, defining their activation and polarization. Thus, dendritic cells could be involved in the Th1 and Th17 response induced against the P. gingivalis capsule. Herein, monocyte-derived dendritic cells were obtained from healthy individuals and then stimulated with different encapsulated strains of P. gingivalis or two non-encapsulated isogenic mutants. Dendritic cell differentiation and maturation were analyzed by flow cytometry. The mRNA expression levels for distinct Th1-, Th17-, or T-regulatory-related cytokines and transcription factors, as well as TLR2 and TLR4, were assessed by qPCR. In addition, the production of IL-1ß, IL-6, IL-23, and TNF-α was analyzed by ELISA. The encapsulated strains and non-encapsulated mutants of P. gingivalis induced dendritic cell maturation to a similar extent; however, the pattern of dendritic cell response was different. In particular, the encapsulated strains of P. gingivalis induced higher expression of IRF4 and NOTCH2 and production of IL-1ß, IL-6, IL-23, and TNF-α compared with the non-encapsulated mutants, and thus, they showed an increased capacity to trigger Th1 and Th17-type responses in human dendritic cells.

Porphyromonas gingivalis Promotes Immunoevasion of Oral Cancer by Protecting Cancer from Macrophage Attack.

The relationship of Porphyromonas gingivalis and oral squamous cell carcinoma (OSCC) has been studied for several years. Previous studies have focused on the direct effect of P. gingivalis on the activities of primary epithelial cells and OSCC cells. However, the immune system is responsible for mediating cancer development, whether P. gingivalis can affect oral cancer immunity has seldom been explored to date. In this study, we investigated the role of P. gingivalis in the immunoevasion of OSCC. We evaluated the effect of P. gingivalis on the phagocytosis of Cal-27 cells (OSCC cell line) by bone marrow-derived macrophages in vitro and studied the effect of P. gingivalis on the growth of OSCC and the polarization of tumor-associated macrophages in vivo. We found that P. gingivalis was able to inhibit the phagocytosis of Cal-27 cells by macrophages, and membrane-component molecules of P. gingivalis, such as proteins, were speculated to be the effector components. In addition, sustained infection with antibiotics-inactivated P. gingivalis promoted OSCC growth in mice and induced the polarization of macrophages into M2 tumor-associated macrophages, which mainly display protumor properties. Transcriptome analysis and quantitative RT-PCR revealed that P. gingivalis infection upregulated the expression of genes encoding protumor molecules in Cal-27 cells (suprabasin, IL-1R2, and CD47) and in macrophages (IL-1α, CCL-3, and CCL-5). Our in vitro and in vivo data suggest that P. gingivalis can promote immunoevasion of oral cancer by protecting cancer from macrophage attack. To our knowledge, the present study reveals a novel mechanism by which P. gingivalis promotes OSCC development.

Porphyromonas gingivalis lipopolysaccharide induces interleukin-6 and c-c motif chemokine ligand 2 expression in cultured hCMEC/D3 human brain microvascular endothelial cells.

OBJECTIVE: This paper describes the effect of Porphyromonas gingivalis (P gingivalis) lipopolysaccharide (LPS) on the expression of interleukin-6 (IL-6) and C-C motif chemokine ligand 2 (CCL2) in cultured hCMEC/D3 human brain microvascular endothelial cells. BACKGROUND: P gingivalis is one of the important pathogens in periodontitis, and periodontitis is a risk factor for brain disorders including cerebrovascular diseases and Alzheimer's disease. However, the mechanisms underlying the pathogenesis of P gingivalis-mediated brain diseases are incompletely understood. Effects of P gingivalis LPS on brain endothelial cells are not known well. METHODS: The hCMEC/D3 human brain microvascular endothelial cells were cultured and treated with P gingivalis LPS. The expression of IL-6 and CCL2 mRNA and protein was examined using quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Effect of inhibitors of Toll-like receptor (TLR) 2, TLR4, nuclear factor-κB (NF-κB), p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) was also investigated. Phosphorylation of NF-κB p65, p38 MAPK and JNK was examined using Western blotting. RESULTS: P gingivalis LPS-induced mRNA and protein expression of IL-6 and CCL2 in hCMEC/D3 cells in a concentration-dependent manner at the concentration of 0.5-50 µg/mL. Maximal mRNA expression of IL-6 and CCL2 was found 2 and 4 hours after stimulation, respectively. Induction of IL-6 and CCL2 by P gingivalis LPS was almost completely inhibited by pretreatment of cells with TLR4 inhibitor but not by TLR2 inhibitor. Treatment of cells with P gingivalis LPS for up to 2 hours induced phosphorylation of NF-κB p65, p38 MAPK and JNK. IL-6 induction was decreased by pretreatment of cells with NF-κB inhibitor SN50 or p38 MAPK inhibitor SB203580, while CCL2 induction was reduced by SN50 or JNK inhibitor SP600125. CONCLUSIONS: IL-6 and CCL2 produced upon P gingivalis LPS stimulation may contribute to the inflammatory reactions in brain endothelial cells and subsequent neurological disorders such as cerebrovascular and Alzheimer's diseases.

Sphingolipid-Containing Outer Membrane Vesicles Serve as a Delivery Vehicle To Limit Macrophage Immune Response to Porphyromonas gingivalis.

Sphingolipids (SLs) are essential structural components of mammalian cell membranes. Our group recently determined that the oral anaerobe Porphyromonas gingivalis delivers its SLs to host cells and that the ability of P. gingivalis to synthesize SLs limits the elicited host inflammatory response during cellular infection. As P. gingivalis robustly produces outer membrane vesicles (OMVs), we hypothesized that OMVs serve as a delivery vehicle for SLs, that the SL status of the OMVs may impact cargo loading to OMVs, and that SL-containing OMVs limit elicited host inflammation similar to that observed by direct bacterial challenge. Transwell cell culture experiments determined that in comparison to the parent strain W83, the SL-null mutant elicited a hyperinflammatory immune response from THP-1 macrophage-like cells with elevated tumor necrosis factor alpha (TNF-α), interleukin 1ß (IL-1ß), and IL-6. Targeted assessment of Toll-like receptors (TLRs) identified elevated expression of TLR2, unchanged TLR4, and elevated expression of the adaptor molecules MyD88 and TRIF (Toll/IL-1 receptor domain-containing adaptor-inducing beta interferon) by SL-null P. gingivalis No significant differences in gingipain activity were observed in our infection models, and both strains produced OMVs of similar sizes. Using comparative two-dimensional gel electrophoresis, we identified differences in the protein cargo of the OMVs between parent and SL-null strain. Importantly, use of purified OMVs recapitulated the cellular inflammatory response observed in the transwell system with whole bacteria. These findings provide new insights into the role of SLs in P. gingivalis OMV cargo assembly and expand our understanding of SL-OMVs as bacterial structures that modulate the host inflammatory response.

Lipopolysaccharide- TLR-4 Axis regulates Osteoclastogenesis independent of RANKL/RANK signaling.

BACKGROUND: Lipopolysaccharide (LPS) is an endotoxin and a vital component of gram-negative bacteria's outer membrane. During gram-negative bacterial sepsis, LPS regulates osteoclast differentiation and activity, in addition to increasing inflammation. This study aimed to investigate how LPS regulates osteoclast differentiation of RAW 264.7 cells in vitro. RESULTS: Herein, we revealed that RAW cells failed to differentiate into mature osteoclasts in vitro in the presence of LPS. However, differentiation occurred in cells primed with receptor activator of nuclear factor-kappa-Β ligand (RANKL) for 24 h and then treated with LPS for 48 h (henceforth, denoted as LPS-treated cells). In cells treated with either RANKL or LPS, an increase in membrane levels of toll-like receptor 4 (TLR4) receptor was observed. Mechanistically, an inhibitor of TLR4 (TAK-242) reduced the number of osteoclasts as well as the secretion of tumor necrosis factor (TNF)-α in LPS-treated cells. RANKL-induced RAW cells secreted a very basal level TNF-α. TAK-242 did not affect RANKL-induced osteoclastogenesis. Increased osteoclast differentiation in LPS-treated osteoclasts was not associated with the RANKL/RANK/OPG axis but connected with the LPS/TLR4/TNF-α tumor necrosis factor receptor (TNFR)-2 axis. We postulate that this is because TAK-242 and a TNF-α antibody suppress osteoclast differentiation. Furthermore, an antibody against TNF-α reduced membrane levels of TNFR-2. Secreted TNF-α appears to function as an autocrine/ paracrine factor in the induction of osteoclastogenesis independent of RANKL. CONCLUSION: TNF-α secreted via LPS/TLR4 signaling regulates osteoclastogenesis in macrophages primed with RANKL and then treated with LPS. Our findings suggest that TLR4/TNF-α might be a potential target to suppress bone loss associated with inflammatory bone diseases, including periodontitis, rheumatoid arthritis, and osteoporosis.

Porphyromonas gingivalis induces penetration of lipopolysaccharide and peptidoglycan through the gingival epithelium via degradation of junctional adhesion molecule 1.

Porphyromonas gingivalis is a major pathogen in severe and chronic manifestations of periodontal disease, which is one of the most common infections of humans. A central feature of P. gingivalis pathogenicity is dysregulation of innate immunity at the gingival epithelial interface; however, the molecular basis underlying P. gingivalis-dependent abrogation of epithelial barrier function remains unknown. Gingival epithelial cells express junctional adhesion molecule (JAM1), a tight junction-associated protein, and JAM1 homodimers regulate epithelial barrier function. Here we show that Arg-specific or Lys-specific cysteine proteases (gingipains) secreted by P. gingivalis can specifically degrade JAM1 at K134 and R234 in gingival epithelial cells, resulting in permeability of the gingival epithelium to 40 kDa dextran, lipopolysaccharide (LPS), and proteoglycan (PGN). A P. gingivalis strain lacking gingipains was impaired in degradation of JAM1. Knockdown of JAM1 in monolayer cells and a three-dimensional multilayered tissue model also increased permeability to LPS, PGN, and gingipains. Inversely, overexpression of JAM1 in epithelial cells prevented penetration by these agents following P. gingivalis infection. Our findings strongly suggest that P. gingivalis gingipains disrupt barrier function of stratified squamous epithelium via degradation of JAM1, allowing bacterial virulence factors to penetrate into subepithelial tissues.

Triggering NETosis via protease-activated receptor (PAR)-2 signaling as a mechanism of hijacking neutrophils function for pathogen benefits.

Neutrophil-derived networks of DNA-composed extracellular fibers covered with antimicrobial molecules, referred to as neutrophil extracellular traps (NETs), are recognized as a physiological microbicidal mechanism of innate immunity. The formation of NETs is also classified as a model of a cell death called NETosis. Despite intensive research on the NETs formation in response to pathogens, the role of specific bacteria-derived virulence factors in this process, although postulated, is still poorly understood. The aim of our study was to determine the role of gingipains, cysteine proteases responsible for the virulence of P. gingivalis, on the NETosis process induced by this major periodontopathogen. We showed that NETosis triggered by P. gingivalis is gingipain dependent since in the stark contrast to the wild-type strain (W83) the gingipain-null mutant strain only slightly induced the NETs formation. Furthermore, the direct effect of proteases on NETosis was documented using purified gingipains. Notably, the induction of NETosis was dependent on the catalytic activity of gingipains, since proteolytically inactive forms of enzymes showed reduced ability to trigger the NETs formation. Mechanistically, gingipain-induced NETosis was dependent on proteolytic activation of protease-activated receptor-2 (PAR-2). Intriguingly, both P. gingivalis and purified Arg-specific gingipains (Rgp) induced NETs that not only lacked bactericidal activity but instead stimulated the growth of bacteria species otherwise susceptible to killing in NETs. This protection was executed by proteolysis of bactericidal components of NETs. Taken together, gingipains play a dual role in NETosis: they are the potent direct inducers of NETs formation but in the same time, their activity prevents P. gingivalis entrapment and subsequent killing. This may explain a paradox that despite the massive accumulation of neutrophils and NETs formation in periodontal pockets periodontal pathogens and associated pathobionts thrive in this environment.

GDF15 Supports the Inflammatory Response of PdL Fibroblasts Stimulated by P. gingivalis LPS and Concurrent Compression.

Periodontitis is characterized by bacterially induced inflammatory destruction of periodontal tissue. This also affects fibroblasts of the human periodontal ligaments (HPdLF), which play a coordinating role in force-induced tissue and alveolar bone remodeling. Excessive inflammation in the oral tissues has been observed with simultaneous stimulation by pathogens and mechanical forces. Recently, elevated levels of growth differentiation factor 15 (GDF15), an immuno-modulatory member of the transforming growth factor (TGFB) superfamily, were detected under periodontitis-like conditions and in force-stressed PdL cells. In view of the pleiotropic effects of GDF15 in various tissues, this study aims to investigate the role of GDF15 in P. gingivalis-related inflammation of HPdLF and its effect on the excessive inflammatory response to concurrent compressive stress. To this end, the expression and secretion of cytokines (IL6, IL8, COX2/PGE2, TNFα) and the activation of THP1 monocytic cells were analyzed in GDF15 siRNA-treated HPdLF stimulated with P. gingivalis lipopolysaccharides alone and in combination with compressive force. GDF15 knockdown significantly reduced cytokine levels and THP1 activation in LPS-stimulated HPdLF, which was less pronounced with additional compressive stress. Overall, our data suggest a pro-inflammatory role for GDF15 in periodontal disease and demonstrate that GDF15 partially modulates the force-induced excessive inflammatory response of PdLF under these conditions.

Uptake of Nanotitania by Gingival Epithelial Cells Promotes Inflammatory Response and Is Accelerated by Porphyromonas gingivalis Lipopolysaccharide: An In Vitro Study.

Titanium is often used in the medical field and in dental implants due to its biocompatibility, but it has a high rate of leading to peri-implantitis, which progresses faster than periodontitis. Therefore, in the present study, the expression of cytokines from gingival epithelial cells by nanotitania was investigated, which is derived from titanium in the oral cavity, and the additional effect of Porphyromonasgingivalis (periodontopathic bacteria) lipopolysaccharide (PgLPS) was investigated. Ca9-22 cells were used as a gingival epithelial cell model and were cultured with nanotitania alone or with PgLPS. Cytokine expression was examined by reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. In addition, cellular uptake of nanotitania was observed in scanning electron microscopy images. The expression of interleukin (IL)-6 and IL-8 significantly increased in Ca9-22 cells by nanotitania treatment alone, and the expression was further increased by the presence of PgLPS. Nanotitania was observed to phagocytose Ca9-22 cells in a dose- and time-dependent manner. Furthermore, when the expression of IL-11, related to bone resorption, was investigated, a significant increase was confirmed by stimulation with nanotitania alone. Therefore, nanotitania could be associated with the onset and exacerbation of peri-implantitis, and the presence of periodontal pathogens may worsen the condition. Further clinical reports are needed to confirm these preliminary results.

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.

Bacterial citrullinated epitopes generated by Porphyromonas gingivalis infection-a missing link for ACPA production.

OBJECTIVES: Porphyromonas gingivalis (P.g.) is discussed to be involved in triggering self-reactive immune responses. The aim of this study was to investigate the autocitrullinated prokaryotic peptidylarginine deiminase (PPAD) from P.g. CH2007 (RACH2007-PPAD) from a rheumatoid arthritis (RA) patient and a synthetic citrullinated PPAD peptide (CPP) containing the main autocitrullination site as potential targets for antibody reactivity in RA and to analyse the possibility of citrullinating native human proteins by PPAD in the context of RA. METHODS: Recombinant RACH2007-PPAD was cloned and expressed in Escherichia coli. Purified RACH2007-PPAD and its enzymatic activity was analysed using two-dimensional electrophoresis, mass spectrometry, immunoblot and ELISA. Autoantibody response to different modified proteins and peptides was recorded and bioinformatically evaluated. RESULTS: RACH2007-PPAD was capable to citrullinate major RA autoantigens, such as fibrinogen, vimentin, hnRNP-A2/B1, histone H1 and multiple peptides, which identify a common RG/RGG consensus motif. 33% of RA patients (n=30) revealed increased reactivity for α-cit-RACH2007-PPAD before RA onset. 77% of RA patients (n=99) presented α-cit-specific signals to CPP amino acids 57-71 which were positively correlated to α-CCP2 antibody levels. Interestingly, 48% of the α-CPP-positives were rheumatoidfactor IgM/anti-citrullinated peptide/protein antibodies (ACPA)-negative. Anti-CPP and α-RACH2007-PPAD antibody levels increase with age. Protein macroarrays that were citrullinated by RACH2007-PPAD and screened with RA patient sera (n=6) and controls (n=4) uncovered 16 RACH2007-PPAD citrullinated RA autoantigens and 9 autoantigens associated with lung diseases. We showed that the α-CPP response could be an important determinant in parenchymal changes in the lung at the time of RA diagnosis (n=106; p=0.018). CONCLUSIONS: RACH2007-PPAD induced internal citrullination of major RA autoantigens. Anti-RACH2007-PPAD correlates with ACPA levels and interstitial lung disease autoantigen reactivity, supporting an infection-based concept for induction of ACPAs via enzymatic mimicry.

Porphyromonas gingivalis induces the production of interleukin-31 by human mast cells, resulting in dysfunction of the gingival epithelial barrier.

Interleukin (IL)-31 is important for innate immunity in mucosal tissues and skin, and increased IL-31 expression participates in the pathogenesis of chronic inflammatory diseases affecting the skin, airways, lungs, and intestines. We investigated the contribution of mast cells to the induction of IL-31 production following infection with the periodontal pathogen, Porphyromonas gingivalis. We found that oral infection with P. gingivalis increased IL-31 expression in the gingival tissues of wild-type mice but not in those of mast cell-deficient mice. The P. gingivalis-induced IL-31 production by human mast cells occurred through the activation of the JNK and NF-κB signalling pathways and was dependent on the P. gingivalis lysine-specific protease gingipain-K. P. gingivalis infection induced IL-31 receptor α and oncostatin M receptor ß expression in human gingival epithelial cells. Notably, the P. gingivalis-induced IL-31 production by mast cells led to the downregulation of claudin-1, a tight junction molecule, in gingival epithelial cells, resulting in an IL-31-dependent increase in the paracellular permeability of the gingival epithelial barrier. These findings suggest that IL-31 produced by mast cells in response to P. gingivalis infection causes gingival epithelial barrier dysfunction, which may contribute to the chronic inflammation observed in periodontitis.

Is periodontitis a prognostic factor in order to indicate antibodies against citrullinated peptides in patients with rheumatoid arthritis?

OBJECTIVES: In this cross-sectional study we investigated antibody titres against cyclic citrullinated peptides derived from filaggrin (anti-CCP) and citrullinated α-enolase (anti-CEP-1) among patients with RA as a function of periodontal findings. METHODS: 107 patients with RA (median age 56 years, 75% females) were included. For periodontal diagnoses missing teeth, periodontal epithelial surface area, periodontal inflamed surface area and periodontal diagnosis according to the working group's guidelines of the Center for Disease Control and Prevention were determined. Subgingival bacterial DNA of five periodontopathic bacteria was assessed by PCR with sequence-specific oligonucleotides. Anti-CCP and anti-CEP-1 antibodies in plasma samples were investigated using enzyme-linked immunosorbent assays. Low resolution human leukocyte antigen (HLA) typing was carried out using PCR with sequence-specific primers. RESULTS: PESA was found associated with a low adjusted odds ratio for anti-CCP positivity (OR=1.002, p=0.040). All patients who were infected with Aggregatibacter actinomycetemcomitans were simultaneously anti-CCP positive (p=0.043). HLA-DRB1*13 lowered the adjusted odds ratio for anti-CCP (OR=0.073, p=0.002) and anti-CEP-1 (OR=0.068, p=0.018) positivity whereas HLA-DRB1*07 indicated a lower risk only for demonstrable anti-CCP antibodies (OR=0.079, p=0.004). HLA-DRB1*04 was associated with increased adjusted odds ratio for anti-CEP-1 positivity (OR=4.154, p=0.005) and the simultaneous proof of both investigated autoantibodies (OR=3.725, p=0.011). CONCLUSIONS: Among patients with RA periodontitis may be a minor risk factor for anti-CCP positivity. Our data first provide evidence that an infection with A. actinomycetemcomitans is associated with an increased formation of anti-CCP. HLA phenotype proved to be a significant risk indicator for both investigated antibodies.

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.

Novel synthetic peptide derived from Porphyromonas gingivalis Lys-gingipain detects IgG-mediated host response in periodontitis.

Porphyromonas gingivalis is a keystone pathogen in periodontitis. Analysis of the immunogenicity of its virulence factors may provide insight into the host response to this infection. The Kgp12 (IEDB Epitope ID 763561), an epitope of Lys-gingipain (Kgp) virulence factor from P. gingivalis ATCC 33277, elicits an immunoglobulin G (IgG) immunoreactivity with low cross-reactivity and, therefore, more specificity. The aim of the present study was to determine in silico the localization of Kgp12 within the protein and to evaluate the IgG host response to this novel Kgp peptide through its capacity to differentiate individuals with different periodontal status. Sera of 71 volunteers were tested by indirect ELISA to detect the IgG immunoreactivity specific to Kgp12, as well as to the protein HmuY and to the sonicated total extract of P. gingivalis ATCC33277, both used as gold standard. The participants had no systemic disease and were classified according to periodontal clinical parameters to comparison, firstly, into periodontitis (P) and without periodontitis (WP) groups and, secondly, into periodontitis (P), gingivitis (G) and clinically health (CH) ones. All the antigens tested, Kgp12 (p = 0.02), HmuY (p = 0.00) and P. gingivalis extract (p = 0.03), could differentiate P from WP groups considering IgG serum levels. P group also had higher IgG levels specific to Kgp12 (p = 0.03), HmuY (p < 0.01) and P. gingivalis extract (p = 0.01) when compared to G group. We conclude that the Kgp12 synthetic peptide was useful to detect the IgG-mediated host response signaling that it is a promising epitope to analyze the immunogenicity of P. gingivalis.

Candida albicans Shields the Periodontal Killer Porphyromonas gingivalis from Recognition by the Host Immune System and Supports the Bacterial Infection of Gingival Tissue.

Candida albicans is a pathogenic fungus capable of switching its morphology between yeast-like cells and filamentous hyphae and can associate with bacteria to form mixed biofilms resistant to antibiotics. In these structures, the fungal milieu can play a protective function for bacteria as has recently been reported for C. albicans and a periodontal pathogen-Porphyromonas gingivalis. Our current study aimed to determine how this type of mutual microbe protection within the mixed biofilm affects the contacting host cells. To analyze C. albicans and P. gingivalis persistence and host infection, several models for host-biofilm interactions were developed, including microbial exposure to a representative monocyte cell line (THP1) and gingival fibroblasts isolated from periodontitis patients. For in vivo experiments, a mouse subcutaneous chamber model was utilized. The persistence of P. gingivalis cells was observed within mixed biofilm with C. albicans. This microbial co-existence influenced host immunity by attenuating macrophage and fibroblast responses. Cytokine and chemokine production decreased compared to pure bacterial infection. The fibroblasts isolated from patients with severe periodontitis were less susceptible to fungal colonization, indicating a modulation of the host environment by the dominating bacterial infection. The results obtained for the mouse model in which a sequential infection was initiated by the fungus showed that this host colonization induced a milder inflammation, leading to a significant reduction in mouse mortality. Moreover, high bacterial counts in animal organisms were noted on a longer time scale in the presence of C. albicans, suggesting the chronic nature of the dual-species infection.

The Distinct Immune-Stimulatory Capacities of Porphyromonas gingivalis Strains 381 and ATCC 33277 Are Determined by the fimB Allele and Gingipain Activity.

The Porphyromonas gingivalis strain ATCC 33277 (33277) and 381 genomes are nearly identical. However, strain 33277 displays a significantly diminished capacity to stimulate host cell Toll-like receptor 2 (TLR2)-dependent signaling and interleukin-1ß (IL-1ß) production relative to 381, suggesting that there are strain-specific differences in one or more bacterial immune-modulatory factors. Genomic sequencing identified a single nucleotide polymorphism in the 33277 fimB allele (A→T), creating a premature stop codon in the 33277 fimB open reading frame relative to the 381 fimB allele. Gene exchange experiments established that the 33277 fimB allele reduces the immune-stimulatory capacity of this strain. Transcriptome comparisons revealed that multiple genes related to carboxy-terminal domain (CTD) family proteins, including the gingipains, were upregulated in 33277 relative to 381. A gingipain substrate degradation assay demonstrated that cell surface gingipain activity is higher in 33277, and an isogenic mutant strain deficient for the gingipains exhibited an increased ability to induce TLR2 signaling and IL-1ß production. Furthermore, 33277 and 381 mutant strains lacking CTD cell surface proteins were more immune-stimulatory than the parental wild-type strains, consistent with an immune-suppressive role for the gingipains. Our data show that the combination of an intact fimB allele and limited cell surface gingipain activity in P. gingivalis 381 renders this strain more immune-stimulatory. Conversely, a defective fimB allele and high-level cell surface gingipain activity reduce the capacity of P. gingivalis 33277 to stimulate host cell innate immune responses. In summary, genomic and transcriptomic comparisons identified key virulence characteristics that confer divergent host cell innate immune responses to these highly related P. gingivalis strains.