Associations of Porphyromonas gingivalis Infection and Low Beclin1 Expression With Clinicopathological Parameters and Survival of Esophageal Squamous Cell Carcinoma Patients.

Purpose: The present study focused on exploring the associations of Porphyromonas gingivalis (P. gingivalis) infection and low Beclin1 expression with clinicopathological parameters and survival of esophageal squamous cell carcinoma (ESCC) patients, so as to illustrate its clinical significance and prognostic value. Methods: Immunohistochemistry (IHC) was used to detect P. gingivalis infection status and Beclin1 expression in 370 ESCC patients. The chi-square test was adopted to illustrate the relationship between categorical variables, and Cohen's kappa coefficient was used for correlation analysis. Kaplan-Meier survival curves with the log-rank test were used to analyse the correlation of P. gingivalis infection and low Beclin1 expression with survival time. The effects of P. gingivalis infection and Beclin1 downregulation on the proliferation, migration and antiapoptotic abilities of ESCC cells in vitro were detected by Cell Counting Kit-8, wound healing and flow cytometry assays. For P. gingivalis infection of ESCC cells, cell culture medium was replaced with antibiotic-free medium when the density of ESCC cells was 70-80%, cells were inoculated with P. gingivalis at a multiplicity of infection (MOI) of 10. Result: P. gingivalis infection was negatively correlated with Beclin1 expression in ESCC tissues, and P. gingivalis infection and low Beclin1 expression were associated with differentiation status, tumor invasion depth, lymph node metastasis, clinical stage and prognosis in ESCC patients. In vitro experiments confirmed that P. gingivalis infection and Beclin1 downregulation potentiate the proliferation, migration and antiapoptotic abilities of ESCC cells (KYSE150 and KYSE30). Our results provide evidence that P. gingivalis infection and low Beclin1 expression were associated with the development and progression of ESCC. Conclusion: Long-term smoking and alcohol consumption causes poor oral and esophageal microenvironments and ESCC patients with these features were more susceptible to P. gingivalis infection and persistent colonization, and exhibited lower Beclin1 expression, worse prognosis and more advanced clinicopathological features. Our findings indicate that effectively eliminating P. gingivalis colonization and restoring Beclin1 expression in ESCC patients may contribute to preventation and targeted treatment, and yield new insights into the aetiological research on ESCC.

The effects of periodontitis associated microbiota on the development of oral squamous cell carcinoma.

Epidemiological data have shown that periodontal bacterial infection, periodontitis, and oral squamous cell carcinoma have close relationship on the disease progress and risk. However, the specific role of periodontal microbes and their mechanism in the development of oral squamous cell carcinoma is not yet clear. In our previous work, metagenomic Illumina Mi-seq analysis was used to identify tstructure and abundance of periodontital microbiome. Accoding to the results, we used Porphyromonas.spp. and Fusobacterium.spp. as the periodontitis positive microbiota; Neisseria.spp and Corynebacterium.spp as periodontitis negative microbiota (their average relative abundance were >5%). These representative strains of the above genus were used to infect OSCC cells to explore their effect on tumor cell biology behavior, and detect the expression level of the gene in related to inflammation, migration, invasion and cell cycle. We find that periodontitis positive correlated microbiota had a promoting effect on the development of oral squamous cell carcinoma in vitro by regulating mRNA and protein expression of IL-6, IL-8, MMP-9 and Cyclin-D1. Periodontitis negative correlated microbiota had suppression effect on the development of oral squamous cell carcinoma in vitro analysis.

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.

Invasion of Gingival Epithelial Cells by Porphyromonas gingivalis.

Porphyromonas gingivalis is a major pathogen responsible for severe and chronic manifestations of periodontal disease, which is one of the most common infectious disorders of humans. Although human gingival epithelium prevents intrusions by periodontal bacteria, P. gingivalis is able to invade gingival epithelial cells. To study the dynamics and the fate of intracellular P. gingivalis, confocal laser scanning microscopy (CLSM) is a method of choice. Information gained with CLSM contains not only the number of P. gingivalis associated with gingival epithelial cells but also the bacterial localization on/inside the host cells, morphological change of host cells, and physical interaction between the bacteria and host organelle. In this chapter, we describe the protocols for microscopy techniques to morphologically study gingival epithelial cells infected by P. gingivalis.

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.

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.

Periodontal pathogens promote cancer aggressivity via TLR/MyD88 triggered activation of Integrin/FAK signaling that is therapeutically reversible by a probiotic bacteriocin.

Epidemiological studies reveal significant associations between periodontitis and oral cancer. However, knowledge about the contribution of periodontal pathogens to oral cancer and potential regulatory mechanisms involved is limited. Previously, we showed that nisin, a bacteriocin and commonly used food preservative, reduced oral cancer tumorigenesis and extended the life expectancy in tumor-bearing mice. In addition, nisin has antimicrobial effects on key periodontal pathogens. Thus, the purpose of this study was to test the hypothesis that key periodontal pathogens (Porphyromonas gingivalis, Treponema denticola, and Fusobacterium nucleatum) promote oral cancer via specific host-bacterial interactions, and that bacteriocin/nisin therapy may modulate these responses. All three periodontal pathogens enhanced oral squamous cell carcinoma (OSCC) cell migration, invasion, tumorsphere formation, and tumorigenesis in vivo, without significantly affecting cell proliferation or apoptosis. In contrast, oral commensal bacteria did not affect OSCC cell migration. Pathogen-enhanced OSCC cell migration was mediated via integrin alpha V and FAK activation, since stably blocking alpha V or FAK expression abrogated these effects. Nisin inhibited these pathogen-mediated processes. Further, Treponema denticola induced TLR2 and 4 and MyD88 expression. Stable suppression of MyD88 significantly inhibited Treponema denticola-induced FAK activation and abrogated pathogen-induced migration. Together, these data demonstrate that periodontal pathogens contribute to a highly aggressive cancer phenotype via crosstalk between TLR/MyD88 and integrin/FAK signaling. Nisin can modulate these pathogen-mediated effects, and thus has therapeutic potential as an antimicrobial and anti-tumorigenic agent.

Porphyromonas gingivalis promotes progression of esophageal squamous cell cancer via TGFß-dependent Smad/YAP/TAZ signaling.

Microbial dysbiosis in the upper digestive tract is linked to an increased risk of esophageal squamous cell carcinoma (ESCC). Overabundance of Porphyromonas gingivalis is associated with shorter survival of ESCC patients. We investigated the molecular mechanisms driving aggressive progression of ESCC by P. gingivalis. Intracellular invasion of P. gingivalis potentiated proliferation, migration, invasion, and metastasis abilities of ESCC cells via transforming growth factor-ß (TGFß)-dependent Drosophila mothers against decapentaplegic homologs (Smads)/Yes-associated protein (YAP)/Transcriptional coactivator with PDZ-binding motif (TAZ) activation. Smads/YAP/TAZ/TEA domain transcription factor1 (TEAD1) complex formation was essential to initiate downstream target gene expression, inducing an epithelial-mesenchymal transition (EMT) and stemness features. Furthermore, P. gingivalis augmented secretion and bioactivity of TGFß through glycoprotein A repetitions predominant (GARP) up-regulation. Accordingly, disruption of either the GARP/TGFß axis or its activated Smads/YAP/TAZ complex abrogated the tumor-promoting role of P. gingivalis. P. gingivalis signature genes based on its activated effector molecules can efficiently distinguish ESCC patients into low- and high-risk groups. Targeting P. gingivalis or its activated effectors may provide novel insights into clinical management of ESCC.

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.

Exploring the role of oral microorganisms in the pathogenesis of mucositis by assessing their impact on metabolic activity and reproductive capacity of epithelial cells in vitro.

PURPOSE: Clinical and in vitro studies showed selected oral microorganisms to be related to delayed wound healing and ulcerative oral mucositis. However, it is not known whether this effect is due to reduced metabolism and/or the reduced reproductive capacity of epithelial cells. Therefore, we studied the influence of the oral microorganisms Porphyromonas gingivalis, Candida glabrata, and Candida kefyr on cell metabolism and reproductive capacity of oral epithelial cells, aimed to further unravel the pathogenesis of oral mucositis. METHODS: Oral epithelial cells were exposed to different concentrations of P. gingivalis, C. glabrata, and C. kefyr as mono-infections or mixed together. An MTT assay was performed to determine the effect on cell metabolism. A clonogenic assay was used to study the effect on the reproductive capacity of oral epithelial cells. RESULTS: The metabolism of oral epithelial cells was reduced when the microorganisms were present in high concentrations: P. gingivalis at a multiplicity of infection (MOI) of 1000 and the Candida spp. at MOI 100. No statistical difference was observed in the ability of a single epithelial cell to grow into a colony of cells between control and P. gingivalis, C. glabrata, and C. kefyr, independent of the concentrations and combinations used. CONCLUSION: P. gingivalis, C. glabrata, and C. kefyr lowered the metabolic activity of oral epithelial cells in high concentrations, yet they did not influence the reproductive capacity of epithelial cells. Their impact on ulcerative oral mucositis is likely due to an effect on the migration, proliferation, and metabolism of epithelial cells.

Enhanced dual function of osteoclast precursors following calvarial Porphyromonas gingivalis infection.

BACKGROUND AND OBJECTIVE: Excessive osteoclast activity is a major characteristic of pathogenic bone loss in inflammatory bone diseases including periodontitis. However, beyond the knowledge that osteoclasts are differentiated from the monocyte/macrophage lineage and share common ancestry with macrophages and DC, the nature and function of osteoclast precursors are not completely understood. Furthermore, little is known about how osteoclast precursors respond to bacterial infection in vivo. We have previously demonstrated in vitro that the periodontal pathogen Porphyromonas gingivalis (Pg) plays a biphasic role on the receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclast differentiation. In this study, we investigated the in vivo effect of Pg infection on the regulation of osteoclast precursors, using a mouse calvarial infection model. METHODS AND RESULTS: C57BL/6 wild-type and the myeloid differentiation factor 88 knockout (MyD88-/- ) mice were infected with Pg by calvarial injection. Local and systemic bone loss, and the number and function of CD11b+ c-fms+ cells from bone marrow and spleen were analyzed. Our results show that Pg infection induces localized inflammatory infiltration and osteoclastogenesis, as well as increased number and osteoclastogenic potential of CD11b+ c-fms+ osteoclast precursors in the bone marrow and periphery. We also show that CD11b+ c-fms+ RANK+ and CD11b+ c-fms+ RANK- are precursors with similar osteoclastogenic and pro-inflammatory potentials. In addition, CD11b+ c-fms+ cells exhibit an antigen-specific T-cell immune-suppressive activity, which are increased with Pg infection. Moreover, we demonstrate that MyD88 is involved in the regulation of osteoclast precursors upon Pg infection. CONCLUSIONS: In this study, we demonstrate an enhanced dual function of osteoclast precursors following calvarial Pg infection. Based on our findings, we propose the following model: Pg infection increases a pool of precursor cells that can be shunted toward osteoclast formation at the infection/inflammation sites, while at the same time dampening host immune responses, which is beneficial for the persistence of infection and maintenance of the characteristic chronic nature of periodontitis. Understanding the nature, function, and regulation of osteoclast precursors will be helpful for identifying therapeutic interventions to aid in the control and prevention of inflammatory bone loss diseases including periodontitis.

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.

Pathogenesis of Important Virulence Factors of Porphyromonas gingivalis via Toll-Like Receptors.

Periodontitis is a common intraoral infection and is inextricably linked to systemic diseases. Recently, the regulation between host immunologic response and periodontal pathogens has become a hotspot to explain the mechanism of periodontitis and related systemic diseases. Since Porphyromonas gingivalis (P. gingivalis) was proved as critical periodontal pathogen above all, researches focusing on the mechanism of its virulence factors have received extensive attention. Studies have shown that in the development of periodontitis, in addition to the direct release of virulent factors by periodontal pathogens to destroy periodontal tissues, over-low or over-high intrinsic immune and inflammatory response mediated by Toll-like receptors (TLRs) can lead to more lasting destruction of periodontal tissues. It is very necessary to sort out how various cytopathic factors of P. gingivalis mediate inflammation and immune responses between the host through TLRs so as to help precisely prevent, diagnose, and treat periodontitis in clinic. This review summarizes the role of three most widely studied pathogenic factors produced by P. gingivalis (lipopolysaccharide, gingipains, pili) and their interactions with TLRs at the cellular and molecular level in the progress of periodontitis.

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.

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.

Monocyte-macrophage activation is associated with nonalcoholic fatty liver disease and liver fibrosis in HIV monoinfection independently of the gut microbiome and bacterial translocation.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is common among people living with HIV. There are limited data available on the pathophysiology of NAFLD and the development of fibrosis in this population. OBJECTIVES: The aim of this study was to investigate the association of bacterial translocation, adipose tissue dysfunction, monocyte activation and gut dysbiosis in patients with HIV monoinfection and NAFLD. METHODS: Cases with biopsy-proven NAFLD and HIV monoinfection were age and sex-matched to HIV-positive and HIV-negative controls. Markers of bacterial translocation [lipopolysaccharide-binding protein (LBP), bacterial DNA and lipopolysaccharide (LPS)], adipose tissue dysfunction (leptin, adiponectin) and monocyte activation (sCD14 and sCD163) were measured by ELISA. Hepatic patterns of macrophage activation were explored with immunohistochemistry. 16 s rRNA sequencing was performed with stool. RESULTS: Thirty-three cases were included (≥F2 fibrosis n = 16), matched to HIV-positive (n = 29) and HIV-negative (n = 17) controls. Cases with NAFLD were more obese (BMI 31.0 ±â€Š4.4 vs. 24.1 ±â€Š2.8 kg/m, P < 0.001) and had significantly increased levels of sCD14, sCD163 and higher leptin to adiponectin ratio vs. HIV-positive controls. Cases with ≥F2 verses < F2 fibrosis had increased sCD14 (1.4 ±â€Š0.4 vs. 1.1 ±â€Š0.3 µg/ml, P = 0.023) and sCD163 (1.0 ±â€Š0.3 vs. 0.8 ±â€Š0.3 µg/ml, P = 0.060), which correlated with waist circumference (sCD14 P = 0.022, sCD163 P = 0.011). Immunohistochemistry showed increased hepatic portal macrophage clusters in patients with fibrosis. No markers of bacterial translocation or changes to the microbiome were associated with NAFLD or fibrosis. CONCLUSION: NAFLD fibrosis stage in HIV monoinfected patients is associated with monocyte activation in the context of obesity, which may be independent of bacterial translocation and gut microbiome.

Loss of periodontal ligament fibroblasts by RIPK3-MLKL-mediated necroptosis in the progress of chronic periodontitis.

Periodontal homeostasis is maintained by the dynamic equilibrium between cell death, differentiation and proliferation of resident cells in the periodontal microenvironment. Loss of resident periodontal ligament fibroblasts (PDLFs) has been a major challenge in the periodontal treatment. This study aimed to investigate the exact role of necroptotic cell death in periodontal diseases. Elevated levels of receptor-interacting protein serine-threonine kinases -1 (RIPK1), phosphorylated RIPK3, mixed lineage kinase domain-like protein (MLKL), phosphorylated MLKL and FLIPL were observed in gingival tissues collected from patients with untreated chronic periodontitis; whereas no difference in caspase 8 was observed between the periodontitis and healthy control group. In contrast to the high incidence of necroptotic cell death in monocytes during live P. gingivalis infection with a low multiplicity of infection (MOI), necroptosis was only observed in PDLFs with a high MOI. Priming PDLFs with frozen thawed monocytes enhanced proinflammatory responses to P. gingivalis infection; moreover, frozen thawed monocytes stimulation triggered RIPK1, RIPK3 and MLKL-mediated-necroptotic cell death in PDLFs. These results indicated that RIPK3 and MLKL-mediated-necroptotic cell death participated in the pathogenesis of periodontitis, and DAMPs released from monocytes after P. gingivalis stimulation by necroptosis triggered not only inflammatory responses, but also necroptosis of PDLFs.

Late ascending aortic prosthesis infection by Porphyromonas pogonae: An unexpected infectious complication.

Late complications in ascending aortic surgeries are uncommon and may occur by infectious processes, usually caused by gram positive bacteria. We report a case of aortic prosthesis infection by Porphyromonas pogonae, an anaerobic gram-negative coccobacillus that can grow under microaerobic conditions, three years after ascending aortic reconstruction surgery.

Glycyrrhizic acid suppresses inflammation and reduces the increased glucose levels induced by the combination of Porphyromonas gulae and ligature placement in diabetic model mice.

Diabetic patients are at an increased risk of developing severe and progressive periodontitis. Periodontal disease also increases the severity of diabetes by enhancing insulin resistance. Therefore, the regulation of periodontal inflammation in diabetic patients may contribute to the control of both diseases. Glycyrrhizic acid exerts anti-inflammatory effects by inhibiting high mobility group box 1 (HMGB1). HMGB1, one of the ligands of the receptor for advanced glycation end products (RAGE), is a damage-associated molecular pattern and induces inflammatory cytokine production. In the present study, we examined the effects of glycyrrhizic acid on ligature- and Porphyromonas gulae infection-induced periodontitis as well as the involvement of the HMGB1-RAGE axis in diabetic model mice. The molars of diabetic model mice, established by feeding HFD32 to KK/TaJcl mice, were subjected to silk thread ligation and P. gulae was then intraorally applied in the presence or absence of glycyrrhizic acid given topically. The topical application of glycyrrhizic acid suppressed ligature/P. gulae-induced increases in interleukin (IL)-6 and tumor necrosis factor (TNF)-α at the mRNA level in the gingiva and at the protein level in serum. Furthermore, glycyrrhizic acid suppressed ligature/P. gulae-induced increases in serum amyloid A (SAA) in serum and fasting blood glucose levels. It also suppressed ligature/P. gulae-induced increases of HMGB1 and RAGE at the mRNA level in the gingiva and at the protein level in serum. A mouse anti-HMGB1-neutralizing antibody inhibited increases in serum glucose levels. In conclusion, topical treatments with glycyrrhizic acid may suppress periodontal and systemic inflammation and reduce blood glucose levels through the HMGB1-RAGE axis in diabetic mice.