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.

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 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.

Interleukin-34 permits Porphyromonas gingivalis survival and NF-κB p65 inhibition in macrophages.

Interleukin-34 (IL-34) is a cytokine that supports the viability and differentiation of macrophages. An important cytokine for the development of epidermal immunity, IL-34, is present and plays a role in the immunity of the oral environment. IL-34 has been linked to inflammatory periodontal diseases, which involve innate phagocytes, including macrophages. Whether IL-34 can alter the ability of macrophages to effectively interact with oral microbes is currently unclear. Using macrophages derived from human blood monocytes with either the canonical cytokine colony-stimulating factor (CSF)1 or IL-34, we compared the ability of the macrophages to phagocytose, kill, and respond through the production of cytokines to the periodontal keystone pathogen Porphyromonas gingivalis. While macrophages derived from both cytokines were able to engulf the bacterium equally, IL-34-derived macrophages were much less capable of killing internalized P. gingivalis. Of the macrophage cell surface receptors known to interact with P. gingivalis, dendritic cell-specific intercellular adhesion molecule-grabbing nonintegrin was found to have the largest variation between IL-34- and CSF1-derived macrophages. We also found that upon interaction with P. gingivalis, IL-34-derived macrophages produced significantly less of the neutrophil chemotactic factor IL-8 than macrophages derived in the presence of CSF1. Mechanistically, we identified that the levels of IL-8 corresponded with P. gingivalis survival and dephosphorylation of the major transcription factor NF-κB p65. Overall, we found that macrophages differentiated in the presence of IL-34, a dominant cytokine in the oral gingiva, have a reduced ability to kill the keystone pathogen P. gingivalis and may be susceptible to specific bacteria-mediated cytokine modification.

Oral and Intestinal Bacterial Substances Associated with Disease Activities in Patients with Rheumatoid Arthritis: A Cross-Sectional Clinical Study.

Intestinal bacterial compositions of rheumatoid arthritis (RA) patients have been reported to be different from those of healthy people. Dysbiosis, imbalance of the microbiota, is widely known to cause gut barrier damage, resulting in an influx of bacteria and their substances into host bloodstreams in animal studies. However, few studies have investigated the effect of bacterial substances on the pathophysiology of RA. In this study, eighty-seven active RA patients who had inadequate responses to conventional synthetic disease-modifying antirheumatic drugs or severe comorbidities were analyzed for correlations between many factors such as disease activities, disease biomarkers, intestinal bacterial counts, fecal and serum lipopolysaccharide (LPS), LPS-binding protein (LBP), endotoxin neutralizing capacity (ENC), and serum antibacterial substance IgG and IgA antibody levels by multiple regression analysis with consideration for demographic factors such as age, sex, smoking, and methotrexate treatment. Serum LBP levels, fecal LPS levels, total bacteria counts, serum anti-LPS from Porphyromonas gingivalis (Pg-LPS) IgG antibody levels, and serum anti-Pg-LPS IgA antibody levels were selected for multiple regression analysis using Spearman's correlation analysis. Serum LBP levels were correlated with disease biomarker levels, such as erythrocyte sedimentation rate (p < 0.001), C-reactive protein (p < 0.001), matrix metalloproteinase-3 (p < 0.001), and IL-6 (p = 0.001), and were inversely correlated with hemoglobin (p = 0.005). Anti-Pg-LPS IgG antibody levels were inversely correlated with activity indices such as patient global assessments using visual analogue scale (VAS) (p = 0.002) and painVAS (p < 0.001). Total bacteria counts were correlated with ENC (p < 0.001), and inversely correlated with serum LPS (p < 0.001) and anti-Pg-LPS IgA antibody levels (p < 0.001). These results suggest that substances from oral and gut microbiota may influence disease activity in RA patients.

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.

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.

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.

Porphyromonas gingivalis Promotes Colorectal Carcinoma by Activating the Hematopoietic NLRP3 Inflammasome.

Porphyromonas gingivalis (P. gingivalis) is a keystone periodontal pathogen associated with various digestive cancers. However, whether P. gingivalis can promote colorectal cancer and the underlying mechanism associated with such promotion remains unclear. In this study, we found that P. gingivalis was enriched in human feces and tissue samples from patients with colorectal cancer compared with those from patients with colorectal adenoma or healthy subjects. Cohort studies demonstrated that P. gingivalis infection was associated with poor prognosis in colorectal cancer. P. gingivalis increased tumor counts and tumor volume in the ApcMin/+ mouse model and increased tumor growth in orthotopic rectal and subcutaneous carcinoma models. Furthermore, orthotopic tumors from mice exposed to P. gingivalis exhibited tumor-infiltrating myeloid cell recruitment and a proinflammatory signature. P. gingivalis promoted colorectal cancer via NLRP3 inflammasome activation in vitro and in vivo. NLRP3 chimeric mice harboring orthotopic tumors showed that the effect of NLRP3 on P. gingivalis pathogenesis was mediated by hematopoietic sources. Collectively, these data suggest that P. gingivalis contributes to colorectal cancer neoplasia progression by activating the hematopoietic NLRP3 inflammasome. SIGNIFICANCE: This study demonstrates that the periodontal pathogen P. gingivalis can promote colorectal tumorigenesis by recruiting myeloid cells and creating a proinflammatory tumor microenvironment. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/10/2745/F1.large.jpg.

Respiratory FimA-Specific Secretory IgA Antibodies Upregulated by DC-Targeting Nasal Double DNA Adjuvant Are Essential for Elimination of Porphyromonas gingivalis.

Our previous studies showed that a combination of a DNA plasmid encoding Flt3 ligand (pFL) and CpG oligodeoxynucleotides 1826 (CpG ODN) (FL/CpG) as a nasal adjuvant provoked antigen-specific immune responses. In this study, we investigated the efficacy of a nasal vaccine consisting of FimA as the structural subunit of Porphyromonas gingivalis (P. gingivalis) fimbriae and FL/CpG for the induction of FimA-specific antibody (Ab) responses and their protective roles against nasal and lung infection by P. gingivalis, a keystone pathogen in the etiology of periodontal disease. C57BL/6 mice were nasally immunized with recombinant FimA (rFimA) plus FL/CpG three times at weekly intervals. As a control, mice were given nasal rFimA alone. Nasal washes (NWs) and bronchoalveolar lavage fluid (BALF) of mice given nasal rFimA plus FL/CpG resulted in increased levels of rFimA-specific secretory IgA (SIgA) and IgG Ab responses when compared with those in controls. Significantly increased numbers of CD8- or CD11b-expressing mature-type dendritic cells (DCs) were detected in the respiratory inductive and effector tissues of mice given rFimA plus FL/CpG. Additionally, significantly upregulated Th1/Th2-type cytokine responses by rFimA-stimulated CD4+ T cells were noted in the respiratory effector tissues. When mice were challenged with live P. gingivalis via the nasal route, mice immunized nasally with rFimA plus FL/CpG inhibited P. gingivalis colonization in the nasal cavities and lungs. In contrast, controls failed to show protection. Of interest, when IgA-deficient mice given nasal rFimA plus FL/CpG were challenged with nasal P. gingivalis, the inhibition of bacterial colonization in the respiratory tracts was not seen. Taken together, these results show that nasal FL/CpG effectively enhanced DCs and provided balanced Th1- and Th2-type cytokine response-mediated rFimA-specific IgA protective immunity in the respiratory tract against P. gingivalis. A nasal administration with rFimA and FL/CpG could be a candidate for potent mucosal vaccines for the elimination of inhaled P. gingivalis in periodontal patients.

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.

Natural Killer-Like B Cells Secreting Interleukin-18 Induces a Proinflammatory Response in Periodontitis.

Natural killer-like B (NKB) cells, which are newly identified immune subsets, reveal a critical immunoregulatory property in the eradication of microbial infection via the secretion of interleukin (IL)-18. For the first time, this study investigated the role of NKB cells in secreting IL-18 in the pathogenesis of periodontitis. In this study, NKB cells' percentage and IL-18 concentration in peripheral blood and periodontium in periodontitis patients was measured using flow cytometry and ELISA. The role of IL-18 in regulating periodontal inflammation was examined in a Porphyromonas gingivalis (P. gingivalis)-induced periodontitis murine model. Peripheral and periodontal-infiltrating CD3-CD19+NKp46+ NKB cells, which were the main source of IL-18, were elevated and correlated with attachment loss in periodontitis patients. In vitro IL-18 stimulation promoted proinflammatory cytokine production in periodontal ligament cells. P. gingivalis infection induced elevation of IL-18 receptor in periodontium in a periodontitis murine model. IL-18 neutralization not only suppressed P. gingivalis-induced alveolar bone resorption, but also inhibited recruitment of antigen-non-specific inflammatory cells into the periodontium, probably via dampening expressions of cytokines, chemokines, and matrix metalloproteinases. NKB cells secreting IL-18 appeared to be an important mediator in the inflammatory response following intraoral P. gingivalis infection. These findings might be relevant to the development of immunotherapies for periodontitis.

Intranasal Vaccine Study Using Porphyromonas gingivalis Membrane Vesicles: Isolation Method and Application to a Mouse Model.

Bacteria release spherical nanobodies, known as membrane vesicles (MVs), during various growth phases. MVs have been gaining recognition as structurally stable vehicles in the last two decades because they deliver a wide range of antigens, virulence factors, and immunomodulators to the host. These functions suggest not only the possible contribution of MVs to pathogenicity but also the potential applicability of low-dose MVs for use as vaccines. Here, we describe a series of methods for isolating MVs of Porphyromonas gingivalis, which is an important species among periodontopathic bacteria. The present chapter also introduces a mouse model of intranasal immunization using MVs from P. gingivalis.

In-vivo imaging revealed antigen-directed gingival B10 infiltration in experimental periodontitis.

Our previous study demonstrated that IL-10 secreting B (B10) cells alleviate inflammation and bone loss in experimental periodontitis. The purpose of this study is to determine whether antigen-specificity is required for the local infiltration of B10 cells. Experimental periodontitis was induced in the recipient mice by placement of silk ligature with or without the presence of live Porphyromonas gingivalis (P. gingivalis). Donor mice were pre-immunized by intraperitoneal (IP) injection of formalin-fixed P. gingivalis, or PBS as non-immunized control. Spleen B cells were purified and treated with LPS and CpG for 48 h to expand the B10 population in vitro. Fluorescence-labelled B10 cells were transferred into the recipient mice by tail vein injection and were tracked on day 0, 3, 5 and 10 using IVIS Spectrum in vivo imaging system. The number of B10 cells and P. gingivalis-binding B cells were significantly increased after in vitro treatment of LPS and CpG. On day 5, the fluorescence intensity in gingival tissues was the highest in mice transferred with B10 cells from pre-immunized donor mice. Gingival expression of IL-6, TNF-α, RANKL/OPG ratio and periodontal bone loss in recipient mice were significantly reduced, and the expression of IL-10 and the number of CD19+ B cells were significantly increased after pre-immunized B10 cell transfer in the presence of antigen, compared to those with non-immunized B10 cell transfer or no antigen presence. This study suggests that antigen specificity dictate the local infiltration of B10 cells into periodontal tissue and these antigen-specific B10 cells promote anti-inflammatory responses.

Perturbation of the gut microbiome by Prevotella spp. enhances host susceptibility to mucosal inflammation.

Diverse microbial signatures within the intestinal microbiota have been associated with intestinal and systemic inflammatory diseases, but whether these candidate microbes actively modulate host phenotypes or passively expand within the altered microbial ecosystem is frequently not known. Here we demonstrate that colonization of mice with a member of the genus Prevotella, which has been previously associated to colitis in mice, exacerbates intestinal inflammation. Our analysis revealed that Prevotella intestinalis alters composition and function of the ecosystem resulting in a reduction of short-chain fatty acids, specifically acetate, and consequently a decrease in intestinal IL-18 levels during steady state. Supplementation of IL-18 to Prevotella-colonized mice was sufficient to reduce intestinal inflammation. Hence, we conclude that intestinal Prevotella colonization results in metabolic changes in the microbiota, which reduce IL-18 production and consequently exacerbate intestinal inflammation, and potential systemic autoimmunity.

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.

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.

Halofuginone functions as a therapeutic drug for chronic periodontitis in a mouse model.

Periodontitis is an inflammatory disease caused by host immune response, resulting in a loss of periodontium and alveolar bone. Immune cells, such as T cells and macrophages, play a critical role in the periodontitis onset. Halofuginone, a natural quinazolinone alkaloid, has been shown to possess anti-fibrosis, anti-cancer, and immunomodulatory properties. However, the effect of halofuginone on periodontitis has never been reported. In this study, a ligature-induced mice model of periodontitis was applied to investigate the potential beneficial effect of halofuginone on periodontitis. We demonstrated that the administration of halofuginone significantly reduced the expression levels of pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α) in vivo, and markedly suppressed immune cell infiltration into the infected sites. Furthermore, we also observed that halofuginone treatment blocked the T-helper 17 (Th17) cell differentiation in vivo and in vitro. We demonstrated for the first time that halofuginone alleviated the onset of periodontitis through reducing immune responses.

Alzheimer's Disease-Like Pathology Triggered by Porphyromonas gingivalis in Wild Type Rats Is Serotype Dependent.

Periodontal disease is a disease of tooth-supporting tissues. It is a chronic disease with inflammatory nature and infectious etiology produced by a dysbiotic subgingival microbiota that colonizes the gingivodental sulcus. Among several periodontal bacteria, Porphyromonas gingivalis (P. gingivalis) highlights as a keystone pathogen. Previous reports have implied that chronic inflammatory response and measurable bone resorption are observed in young mice, even after a short period of periodontal infection with P. gingivalis, which has been considered as a suitable model of experimental periodontitis. Also, encapsulated P. gingivalis strains are more virulent than capsular-defective mutants, causing an increased immune response, augmented osteoclastic activity, and accrued alveolar bone resorption in these rodent experimental models of periodontitis. Recently, P. gingivalis has been associated with Alzheimer's disease (AD) pathogenesis, either by worsening brain pathology in AD-transgenic mice or by inducing memory impairment and age-dependent neuroinflammation middle-aged wild type animals. We hypothesized here that the more virulent encapsulated P. gingivalis strains could trigger the appearance of brain AD-markers, neuroinflammation, and cognitive decline even in young rats subjected to a short periodontal infection exposure, due to their higher capacity of activating brain inflammatory responses. To test this hypothesis, we periodontally inoculated 4-week-old male Sprague-Dawley rats with K1, K2, or K4 P. gingivalis serotypes and the K1-isogenic non-encapsulated mutant (GPA), used as a control. 45-days after periodontal inoculations with P. gingivalis serotypes, rat´s spatial memory was evaluated for six consecutive days in the Oasis maze task. Following functional testing, the animals were sacrificed, and various tissues were removed to analyze alveolar bone resorption, cytokine production, and detect AD-specific biomarkers. Strikingly, only K1 or K2 P. gingivalis-infected rats displayed memory deficits, increased alveolar bone resorption, pro-inflammatory cytokine production, changes in astrocytic morphology, increased Aß1-42 levels, and Tau hyperphosphorylation in the hippocampus. None of these effects were observed in rats infected with the non-encapsulated bacterial strains. Based on these results, we propose that the bacterial virulence factors constituted by capsular polysaccharides play a central role in activating innate immunity and inflammation in the AD-like pathology triggered by P. gingivalis in young rats subjected to an acute experimental infection episode.