Periodontitis, chronic liver diseases, and the emerging oral-gut-liver axis.

The liver carries out a wide range of functions ranging from the control of metabolites, nutrient storage, and detoxification to immunosurveillance. While inflammation is essential for the tissue remodeling and maintenance of homeostasis and normal liver physiology, constant exposure to dietary and microbial products creates a niche for potentially prolonged immune activation and unresolved inflammation in susceptible host. Failure to restrain inflammation can lead to development of chronic liver diseases characterized by fibrosis, cirrhosis and eventually liver failure. The liver maintains close interactions with numerous organs which can influence its metabolism and physiology. It is also known that oral cavity microenvironment can influence the physiological conditions of other organs and emerging evidence implicates that this could be true for the liver as well. Presence of chronic inflammation and dysbiotic microbiota is a common feature leading to clinical pathology both in periodontitis and chronic liver diseases (CLDs). In fact, known CLDs appear to have some relationship with periodontitis, which impacts the onset or progression of these conditions in a bidirectional crosstalk. In this review, we explore the emerging association between oral-gut-liver axis focusing on periodontitis and common CLDs including nonalcoholic fatty liver disease, chronic viral hepatitis, liver cirrhosis, and hepatocellular cancer. We highlight the immune pathways and oral microbiome interactions which can link oral cavity and liver health and offer perspectives for future research.

A20 Orchestrates Inflammatory Response in the Oral Mucosa through Restraining NF-κB Activity.

Deregulated immune response to a dysbiotic resident microflora within the oral cavity leads to chronic periodontal disease, local tissue destruction, and various systemic complications. To preserve tissue homeostasis, inflammatory signaling pathways involved in the progression of periodontitis must be tightly regulated. A20 (TNFAIP3), a ubiquitin-editing enzyme, has emerged as one of the key regulators of inflammation. Yet, the function of A20 in the oral mucosa and the biological pathways in which A20 mitigates periodontal inflammation remain elusive. Using a combination of in vivo and ex vivo disease models, we report in this study that A20 regulates inflammatory responses to a keystone oral bacterium, Porphyromonas gingivalis, and restrains periodontal inflammation through its effect on NF-κB signaling and cytokine production. Depletion of A20 using gene editing in human macrophage-like cells (THP-1) significantly increased cytokine secretion, whereas A20 overexpression using lentivirus infection dampened the cytokine production following bacterial challenge through modulating NF-κB activity. Similar to human cells, bone marrow-derived macrophages from A20-deficient mice infected with P. gingivalis displayed increased NF-κB activity and cytokine production compared with the cells isolated from A20-competent mice. Subsequent experiments using a murine ligature-induced periodontitis model showed that even a partial loss of A20 promotes an increased inflammatory phenotype and more severe bone loss, further verifying the critical function of A20 in the oral mucosa. Collectively, to our knowledge, these findings reveal the first systematic evidence of a physiological role for A20 in the maintenance of oral tissue homeostasis as a negative regulator of inflammation.

Periodontal therapy favorably modulates the oral-gut-hepatic axis in cirrhosis.

Cirrhosis is associated with a systemic proinflammatory milieu, endotoxemia, and gut dysbiosis. The oral cavity could be an additional source of inflammation. We aimed to determine the effect of periodontal therapy in cirrhosis through evaluating endotoxemia, inflammation, cognition, and quality of life (QOL). Age-matched cirrhotic and noncirrhotic subjects exhibiting chronic gingivitis and/or mild or moderate periodontitis underwent periodontal therapy with follow-up at 30 days. Saliva/stool for microbial composition and serum for Model for End-stage Liver Disease (MELD) score, endotoxin and lipopolysaccharide binding protein (LBP) and immune-inflammatory markers (IL-1ß; IL-6; histatins 1, 3, 5; and lysozyme) were collected at baseline and day 30. The cognitive function and QOL were also evaluated similarly. A separate group of cirrhotic patients were followed for the same duration without periodontal therapy. Cirrhotics, especially those with hepatic encephalopathy (HE), demonstrated improved dysbiosis in stool and saliva, and improved endotoxin, LBP, and salivary and serum inflammatory mediators following periodontal therapy. These parameters, which were higher in HE at baseline, became statistically similar posttherapy. Pretherapy vs. posttherapy QOL and cognition also improved in HE patients following oral interventions. On the other hand, LBP and endotoxin increased over time in cirrhotic patients not receiving therapy, but the rest of the parameters, including microbiota remained similar over time in the no-therapy group. This proof-of-concept study demonstrates that periodontal therapy in cirrhosis, especially in those with HE, is associated with improved oral and gut dysbiosis, systemic inflammation, MELD score, and cognitive function, which was not observed in those who did not receive therapy over the same time period. NEW & NOTEWORTHY Systematic periodontal therapy in cirrhotic outpatients improved endotoxemia, as well as systemic and local inflammation, and modulated salivary and stool microbial dysbiosis over 30 days. This was associated with improved quality of life and cognition in patients with prior hepatic encephalopathy. In a cirrhotic group that was not provided periodontal therapy, there was an increase in endotoxin and lipopolysaccharide binding protein in the same duration. The oral cavity could be an important underdefined source of inflammation in cirrhosis.

Epithelial-to-Mesenchymal Transition Antagonizes Response to Targeted Therapies in Lung Cancer by Suppressing BIM.

Purpose: Epithelial-to-mesenchymal transition (EMT) confers resistance to a number of targeted therapies and chemotherapies. However, it has been unclear why EMT promotes resistance, thereby impairing progress to overcome it.Experimental Design: We have developed several models of EMT-mediated resistance to EGFR inhibitors (EGFRi) in EGFR-mutant lung cancers to evaluate a novel mechanism of EMT-mediated resistance.Results: We observed that mesenchymal EGFR-mutant lung cancers are resistant to EGFRi-induced apoptosis via insufficient expression of BIM, preventing cell death despite potent suppression of oncogenic signaling following EGFRi treatment. Mechanistically, we observed that the EMT transcription factor ZEB1 inhibits BIM expression by binding directly to the BIM promoter and repressing transcription. Derepression of BIM expression by depletion of ZEB1 or treatment with the BH3 mimetic ABT-263 to enhance "free" cellular BIM levels both led to resensitization of mesenchymal EGFR-mutant cancers to EGFRi. This relationship between EMT and loss of BIM is not restricted to EGFR-mutant lung cancers, as it was also observed in KRAS-mutant lung cancers and large datasets, including different cancer subtypes.Conclusions: Altogether, these data reveal a novel mechanistic link between EMT and resistance to lung cancer targeted therapies. Clin Cancer Res; 24(1); 197-208. ©2017 AACR.

Increased expression of toll-like receptors 2 and 9 is associated with reduced DNA methylation in spontaneous preterm labor.

The cause of spontaneous preterm labor (sPTL) is not known, but it could be due to epigenetic alterations that increase the sensitivity of decidual tissue to inflammatory stimuli. We collected decidual tissue from women at term not in labor (TNL), women at term in labor (TL), and women with sPTL. Illumina Infinium HumanMethylation450 BeadChip analysis revealed significantly reduced DNA methylation for TLR-2 and TLR-9 in sPTL as compared to TL. Immunohistochemical staining documented significantly increased expression of TLR-2 and TLR-9 in decidual tissue of women with sPTL as compared to TL or TNL. TLR expression was not present in decidual cells, but localized to tissue leukocytes as revealed by staining for CD14, a macrophage antigen, and neutrophil elastase. Microarray analysis of inflammatory genes to assess innate immune response demonstrated marked increases in expression of inflammatory cytokines and chemokines in women with TL as compared to TNL. However, when sPTL was compared to TL, there was a further increase in inflammatory cytokines, and a remarkable increase in neutrophil chemokines. These results suggest that epigenetic mechanisms could play a role in increasing leukocyte infiltration, and increasing the sensitivity of decidual tissue to inflammatory stimuli that could precipitate labor prematurely.

Interplay of Toll-Like Receptor 9, Myeloid Cells, and Deubiquitinase A20 in Periodontal Inflammation.

Toll-like receptor 9 (TLR9)-deficient (TLR9-/-) mice are resistant to periodontitis, a disease characterized by a dysbiotic microbiota and deregulated immune response and resulting in tooth loss and various systemic conditions. However, the mechanisms and biological pathways by which TLR9 instigates periodontal inflammation are yet to be identified. In a ligature-induced model of periodontitis, we demonstrate that TLR9-/- mice exhibited significantly less alveolar bone loss than their wild-type (WT) counterparts. Consistent with the disease phenotype, gingival tissues showed significantly more inflammatory cell infiltration in the WT ligated but not in the TLR9-/- ligated mice compared to the unligated controls. The peritoneal infection model using Porphyromonas gingivalis, a keystone pathogen for periodontitis, revealed reduced neutrophils in TLR9-/- mice on day 1 postinfection compared to the levels in WT mice. Transcriptomics analyses showed increased expression of A20 (tumor necrosis factor alpha [TNF-α]-induced protein 3 [TNFAIP3]), an inhibitor of the NF-κB pathway and a negative regulator of TLR signaling, in ligated TLR9-/- mouse gingival tissues compared to its expression in the WT. Ex vivo, TLR9-/- bone marrow-derived macrophages produced more A20 than WT cells following P. gingivalis challenge. Clinically, A20 was modestly upregulated in human gingival tissue specimens from chronic periodontitis patients, further confirming the biological relevance of A20 in periodontal inflammation. We conclude that TLR9 modulates periodontal disease progression at both the cellular and molecular level and identify A20 as a novel downstream signaling molecule in the course of periodontal inflammation. Understanding the regulation of the TLR9 signaling pathway and the involvement of A20 as a limiting factor of inflammation will uncover alternative therapeutic targets to treat periodontitis and other chronic inflammatory diseases.

Toll-Like Receptor 9-Mediated Inflammation Triggers Alveolar Bone Loss in Experimental Murine Periodontitis.

Chronic periodontitis is a local inflammatory disease induced by a dysbiotic microbiota and leading to destruction of the tooth-supporting structures. Microbial nucleic acids are abundantly present in the periodontium, derived through release after phagocytic uptake of microbes and/or from biofilm-associated extracellular DNA. Binding of microbial DNA to its cognate receptors, such as Toll-like receptor 9 (TLR9), can trigger inflammation. In this study, we utilized TLR9 knockout (TLR9(-/-)) mice and wild-type (WT) controls in a murine model of Porphyromonas gingivalis-induced periodontitis and report the first in vivo evidence that TLR9 signaling mediates the induction of periodontal bone loss. P. gingivalis-infected WT mice exhibited significantly increased bone loss compared to that in sham-infected WT mice or P. gingivalis-infected TLR9(-/-) mice, which were resistant to bone loss. Consistent with this, the expression levels of interleukin 6 (IL-6), tumor necrosis factor (TNF), and receptor-activator of nuclear factor kappa B ligand (RANKL) were significantly elevated in the gingival tissues of the infected WT mice but not in infected TLR9(-/-) mice compared to their levels in controls. Ex vivo studies using splenocytes and bone marrow-derived macrophages revealed significantly diminished cytokine production in TLR9(-/-) cells relative to the cytokine production in WT cells in response to P. gingivalis, thereby implicating TLR9 in inflammatory responses to this organism. Intriguingly, compared to the cytokine production in WT cells, TLR9(-/-) cells exhibited significantly decreased proinflammatory cytokine production upon challenge with lipopolysaccharide (LPS) (TLR4 agonist) or Pam3Cys (TLR2 agonist), suggesting possible cross talk between TLR9, TLR4, and TLR2. Collectively, our results provide the first proof-of-concept evidence implicating TLR9-triggered inflammation in periodontal disease pathogenesis, thereby identifying a new potential therapeutic target to control periodontal inflammation.

Association of increased levels of fibrinogen and the -455G/A fibrinogen gene polymorphism with chronic periodontitis.

BACKGROUND: Fibrinogen is one of the acute-phase proteins whose levels are elevated during periodontal disease. Recent studies suggest that excessive fibrinogen production might play a role in upregulating host immune responses. In addition, there is a relationship between the -455G/A polymorphism (HaeIII) in the 5' flanking region of the beta-fibrinogen gene promoter and increased fibrinogen levels. In this study, we investigated the distribution of the -455G/A polymorphism and the relationship of this specific genotype to fibrinogen levels in periodontitis patients. METHODS: In order to assess the -455G/A polymorphism, restriction fragment length polymorphism (RFLP) analysis with HaeIII enzyme was performed in the promoter region of the beta-fibrinogen gene. This was carried out on 79 chronic periodontitis patients as compared to 75 periodontally healthy subjects, matched to age, gender, and race. Fibrinogen levels were determined by the radial immunodiffusion assay (RID). RESULTS: The frequency of homozygocity for the rare allele of the beta-fibrinogen gene (H2H2) was 13% for the periodontitis patients and 3% for the control group (P = 0.01). The distributions of H1H1 and H1H2 genotypes were 48% and 39% in the patient group and 70% and 27% in the control group, respectively. Chi-square analysis indicated that the distribution of these genotypes between the 2 groups was significantly different (P = 0.01). Fibrinogen levels were significantly higher in the patient group (2,496.5 mg/l +/- 105) compared to the control group (2,250.0 mg/l +/- 118.3) after adjusting for age, gender, and smoking status (P = 0.04). Consistent with previous reports, in our study population, those subjects with the H2H2 genotype had significantly higher fibrinogen levels (3,005.7 mg/l +/- 182.5) compared to subjects with the H1H1 genotype (2,325.0 mg/l +/- 91.6) or H1H2 genotype (2,438.0 mg/l +/- 117.4) (P = 0.001). Furthermore, the H1H2 and H2H2 genotypes were found at a higher frequency among periodontitis patients than controls. The odds ratios (OR) for these genotypes were 3.26 (95% confidence interval [CI]: 1.25 to 8.53) for the H1H2 genotype and 6.41 (95% CI: 1.15 to 35.83) for the H2H2 genotype as compared to individuals with the H1H1 genotype, after adjusting for age, gender, and smoking status. CONCLUSIONS: The results indicate that a higher percentage of chronic periodontitis patients exhibit genotypes associated with higher plasma fibrinogen levels than healthy individuals. Furthermore, periodontitis patients have significantly higher fibrinogen levels compared to healthy individuals. The presence of H1H2 or H2H2 genotypes as well as elevated fibrinogen levels, in conjunction with other factors, may put individuals at higher risk of having periodontal disease, or may result from periodontal infection-genetic interactions.