Elimination of Porphyromonas gingivalis inhibits liver fibrosis and inflammation in NASH.

AIM: Non-alcoholic steatohepatitis (NASH) is a critical liver disease showing potential progression to liver cirrhosis/cancer. Previously, we had reported that odontogenic infection of Porphyromonas gingivalis (P. gingivalis), a major periodontal pathogen, exacerbates fibrosis in NASH through the production of fibrosis mediators such as transforming growth factor-ß1 (TGF-ß1) and galectin-3. In this study, we determined the effects of therapeutic interventions using antibiotics on NASH progression induced by P. gingivalis odontogenic infection. MATERIALS AND METHODS: To eliminate P. gingivalis infection, the macrolide antibiotic [azithromycin (AZM)] was applied locally and/or systemically to a high-fat-diet-induced NASH mouse model with P. gingivalis odontogenic infection. After treatment with AZM, liver and periodontal tissues were analysed with focus on inflammation markers such as tumour necrosis factor-α (TNF-α)/Tnf-α and interleukin-1ß (IL-1ß)/Il-1ß, and fibrosis markers such as galectin-3, phosphorylated Smad2 (pSmad2; key signalling molecule of TGF-ß1), and the number of hepatic crown-like structures (hCLSs). Further, Non-alcoholic Fatty Liver Disease Activity Score (NAS), a common histological scoring system, and fibrosis area were evaluated. RESULTS: P. gingivalis odontogenic infection significantly increased the expression of Tnf-α, Il-1ß, galectin-3, and pSmad2, the number of hCLSs, and NAS score, whereas the elimination of P. gingivalis odontogenic infection, especially local with or without systemic application, significantly inhibited them. CONCLUSION: This study suggests that elimination of P. gingivalis odontogenic infection inhibited NASH progression induced by the infection.

Central mucoepidermoid carcinoma arising from glandular odontogenic cyst confirmed by analysis of MAML2 rearrangement: A case report.

Central mucoepidermoid carcinoma (MEC) poses a diagnostic challenge because of its rarity and histological overlap with glandular odontogenic cyst (GOC). In MEC of both salivary glands and jaws, MAML2 arrangement has been well known as the specific gene alteration. We report a case of central MEC arising from GOC diagnosed by MAML2 fusion gene. A 57-year-old male presented a multilocular cystic lesion in left molar region of the mandible. Histopathologically, multiple cysts lined by thin cuboidal or non-keratinized squamous epithelium with small duct-like structures, mucous cells and ciliated cells were present. It was diagnosed as GOC. The recurrent lesion after nine years showed the proliferation of many cystic and solid nests composed of epidermoid, mucous and intermediated cells. Nested PCR revealed CRTC3-MAML2 fusion gene in the recurrent lesion, but not in the primary one. Similarly, MAML-2 rearrangement by FISH analysis was positive in the recurrent lesion, while negative for the primary one, thus confirming the diagnosis of central MEC arising from GOC. Analysis of MAML2 rearrangement can be used as a supportive evidence to distinguish central MEC from GOC.

Cyclic pressure-induced cytokines from gingival fibroblasts stimulate osteoclast activity: Clinical implications for alveolar bone loss in denture wearers.

Purpose The involvement of oral mucosa cells in mechanical stress-induced bone resorption is unclear. The aim of this study was to investigate the effects of cyclic pressure-induced cytokines from oral mucosal cells (human gingival fibroblasts: hGFs) on osteoclast activity in vitro.Methods Cyclic pressure at 50 kPa, which represents high physiologic occlusal force of dentures on the molar area, was applied to hGFs. NFAT-reporter stable RAW264.7 preosteoclasts (NFAT/Luc-RAW cells) were cultured in conditioned medium collected from hGF cultures under cyclic pressure or static conditions. NFAT activity and osteoclast formation were determined by luciferase reporter assay and TRAP staining, respectively. Cyclic pressure-induced cytokines in hGF culture were detected by ELISA, real-time RT-PCR, and cytokine array analyses.Results Conditioned media from hGFs treated with 48 hours of cyclic pressure significantly induced NFAT activity and increased multinucleated osteoclast formation. Furthermore, the cyclic pressure significantly increased the bone resorption activity of RAW264.7 cells. Cyclic pressure significantly increased the expression of major inflammatory cytokines including IL-1ß/IL-1ß, IL-6/IL-6, IL-8/IL-8 and MCP-1/CCL2 in hGFs compared to hGFs cultured under static conditions, and it suppressed osteoprotegerin (OPG/OPG) expression. A cytokine array detected 12 cyclic pressure-induced candidates. Among them, IL-8, decorin, MCP-1 and ferritin increased, whereas IL-28A and PDGF-BB decreased, NFAT activation of NFAT/Luc-RAW cells.Conclusions These results suggest that cyclic pressure-induced cytokines from hGFs promote osteoclastogenesis, possibly including up-regulation of IL-1ß, IL-6, IL-8 and MCP-1, and down-regulation of OPG. These findings introduce the possible involvement of GFs in mechanical stress-induced alveolar ridge resorption, such as in denture wearers.

Porphyromonas gingivalis-odontogenic infection is the potential risk for progression of nonalcoholic steatohepatitis-related neoplastic nodule formation.

Porphyromonas gingivalis (P.g.), a major periodontal pathogen is a known risk factor for various systemic diseases. However, the relationship between P.g. and nonalcoholic steatohepatitis (NASH)-related hepatocellular carcinoma (HCC) is unclear. Thus, we aimed to elucidate whether P.g.-odontogenic infection promotes NASH-related HCC development/progression and to clarify its mechanism. Using high-fat diet (HFD)-induced NASH mouse model, P.g. was infected odontogenically. After 60 weeks of infection, tumor profiles were examined. Chow diet (CD) groups were also prepared at 60 weeks. Nodule formation was only seen in HFD-mice. P.g.-odontogenic infection significantly increased the mean nodule area (P = 0.0188) and tended to promote histological progression score after 60 weeks (P = 0.0956). Interestingly, P.g. was detected in the liver. HFD-P.g. (+) showed numerous TNF-α positive hepatic crown-like structures and 8-OHdG expression in the non-neoplastic liver. In P.g.-infected hepatocytes, phosphorylation of integrin ß1 signaling molecules (FAK/ERK/AKT) was upregulated in vitro. In fact, total AKT in the liver of HFD-P.g. (+) was higher than that of HFD-P.g. (-). P.g.-infected hepatocytes showed increased cell proliferation and migration, and decreased doxorubicin-mediated apoptosis. Integrin ß1 knockdown inhibited these phenotypic changes. P.g.-odontogenic infection may promote the progression of neoplastic nodule formation in an HFD-induced NASH mouse model via integrin signaling and TNF-α induced oxidative DNA damage.

Odontogenic infection by Porphyromonas gingivalis exacerbates fibrosis in NASH via hepatic stellate cell activation.

Odontogenic infection of Porphyromonas gingivalis (P.g.), a major periodontal pathogen, exacerbates pathological progression of non-alcoholic steatohepatitis (NASH). In this study, we aimed to clarify the detailed mechanism in which P.g. induced hepatic stellate cells (HSCs; key effector cells in liver fibrosis) activation. In the liver of high fat diet-induced NASH mouse model with P.g. odontogenic infection, immunolocalization of P.g. was detected. The number of hepatic crown-like structure, which was macrophage aggregation and related to liver fibrosis, was drastically increased and fibrosis area was also increased through upregulating immunoexpression of Phosphorylated Smad2 (key signaling molecule of TGF-ß1) and Galectin-3. P.g.-secreted trypsin-like enzyme [gingipain; an activator of protease-activated receptor 2 (PAR2)] stimulated HSC proliferation and differentiation through Smad and ERK signaling induced by TGF-ß1 produced from HSCs with P.g.-infection. Further, Galectin-3 produced from HSCs with P.g. infection and P.g.-derived LPS/lipoprotein stimulation stabilized TGFß-receptor II resulting in increasing sensitivity for TGF-ß1, finally leading to HSC differentiation via activating Smad and ERK signaling. In addition to them, hepatocytes (main component cells of liver) contributed to HSC activation through TGF-ß1 and Galectin-3 production in paracrine manner. Collectively, P.g.-odontogenic infection exacerbates fibrosis of NASH by HSC activation through TGF-ß1 and Gal-3 production from HSCs and hepatocytes.

Galectin-3 Plays an Important Role in Preterm Birth Caused by Dental Infection of Porphyromonas gingivalis.

Dental infection is risk for preterm birth (PTB) through unclear mechanisms. We established a dental infection-induced PTB mouse model, in which Porphyromonas gingivalis (P.g.) induced PTB by 2 days. We analysed pathogenic factors contributing to PTB and their effects on trophoblasts in vitro. TNF-α, IL-8, and COX-2 were upregulated in P.g.-infected placenta. Galectin-3 (Gal-3), an immune regulator, was significantly upregulated in placenta, amniotic fluid, and serum. In vitro, P.g.-lipopolysaccharide (P.g.-LPS) increased TNF-α and Gal-3 in trophoblasts via NF-κB/MAPK signalling. Gal-3 inhibition significantly downregulated P.g.-LPS-induced TNF-α production. TNF-α upregulated Gal-3. Gal-3 also increased cytokines and Gal-3 through NF-κB/MAPK signalling. Moreover, Gal-3 suppressed CD-66a expression at the maternal-foetal interface. Co-stimulation with Gal-3 and P.g.-LPS upregulated cytokine levels, while Gal-3 plus Aggregatibacter actinomycetemcomitans (A.a.)- or Escherichia coli (E. coli)-LPS treatment downregulated them, indicating the critical role of Gal-3 especially in P.g. dental infection-induced PTB. P.g.-dental infection induced PTB, which was associated with Gal-3-dependent cytokine production. New therapies and/or diagnostic systems targeting Gal-3 may reduce PTB.

Dental Infection of Porphyromonas gingivalis Induces Preterm Birth in Mice.

BACKGROUND: Epidemiological studies have revealed a link between dental infection and preterm birth or low birth weight (PTB/LBW), however, the underlying mechanisms remain unclear. Progress in understanding the associated mechanisms has been limited in part by lack of an animal model for chronic infection-induced PTB/LBW, mimicking pregnancy under conditions of periodontitis. We aimed to establish a mouse model of chronic periodontitis in order to investigate the link between periodontitis and PTB/LBW. METHODS: To establish chronic inflammation beginning with dental infection, we surgically opened mouse (female, 8 weeks old) 1st molar pulp chambers and directly infected with w83 strain Porphyromonas gingivalis (P.g.), a keystone periodontal pathogen. Mating was initiated at 6 wks post-infection, by which time dental granuloma tissue had developed and live P.g. was cultured from extracted tooth root, which serves as a persistent source of P.g. The gestational day (gd) and birth weight were recorded during for P.g.-infected and control mice, and serum and placental tissues were collected at gd 15 to evaluate the systemic and local conditions during pregnancy. RESULTS: Dental infection with P.g. significantly increased circulating TNF-α (2.5-fold), IL-17 (2-fold), IL-6 (2-fold) and IL-1ß (2-fold). The P.g.-infected group delivered at gd 18.25 vs. gd 20.45 in the non-infected control (NC) group (p < 0.01), and pups exhibited LBW compared to controls (p < 0.01). P.g. was localized to placental tissues by immunohistochemistry and PCR, and defects in placental tissues of P.g. infected mice included premature rupture of membrane, placental detachment, degenerative changes in trophoblasts and endothelial cells, including necrotic areas. P.g. infection caused significantly increased numbers of polymorphonuclear leukocytes (PMNLs) and macrophages in placental tissues, associated with increased local expression of pro-inflammatory mediators including TNF-α and COX-2. Further placental tissue damage was indicated in P.g. infected mice by decreased CD-31 in endothelial cells, increased expression of 8OHdG, an indicator of oxidative DNA damage, and cleaved caspase-3, a marker of apoptosis. In vitro, P.g. lipopolysaccharide significantly increased expression of COX-2, IL-8 and TNF-α, in HTR-8 trophoblasts in an NF-κB-dependent fashion. CONCLUSIONS: Our novel mouse model supports previous epidemiological studies signifying dental infection as predisposing factor for PTB/LBW. We demonstrate PTB and LBW in infected mice, translocation of P.g to placental tissues, increased circulating and local pro-inflammatory markers, and the capability of P.g. LPS to directly induce cytokine production in trophoblasts, in vitro. These findings further underscore the importance of local and systemic infections and inflammation during pregnancy and suggest that prevention and/or elimination of dental infections such as marginal or periapical periodontitis before pregnancy may have a beneficial effect on PTB/LBW.

Infection with Porphyromonas gingivalis exacerbates endothelial injury in obese mice.

BACKGROUND: A number of studies have revealed a link between chronic periodontitis and cardiovascular disease in obese patients. However, there is little information about the influence of periodontitis-associated bacteria, Porphyromonas gingivalis (Pg), on pathogenesis of atherosclerosis in obesity. METHODS: In vivo experiment: C57BL/6J mice were fed with a high-fat diet (HFD) or normal chow diet (CD), as a control. Pg was infected from the pulp chamber. At 6 weeks post-infection, histological and immunohistochemical analysis of aortal tissues was performed. In vitro experiment: hTERT-immortalized human umbilical vein endothelial cells (HuhT1) were used to assess the effect of Pg/Pg-LPS on free fatty acid (FFA) induced endothelial cells apoptosis and regulation of cytokine gene expression. RESULTS: Weaker staining of CD31 and increased numbers of TUNEL positive cells in aortal tissue of HFD mice indicated endothelial injury. Pg infection exacerbated the endothelial injury. Immunohistochemically, Pg was detected deep in the smooth muscle of the aorta, and the number of Pg cells in the aortal wall was higher in HFD mice than in CD mice. Moreover, in vitro, FFA treatment induced apoptosis in HuhT1 cells and exposure to Pg-LPS increased this effect. In addition, Pg and Pg-LPS both attenuated cytokine production in HuhT1 cells stimulated by palmitate. CONCLUSIONS: Dental infection of Pg may contribute to pathogenesis of atherosclerosis by accelerating FFA-induced endothelial injury.