Discovery of SQSTM1/p62-dependent P-bodies that regulate the NLRP3 inflammasome.

Autophagy and ribonucleoprotein granules, such as P-bodies (PBs) and stress granules, represent vital stress responses to maintain cellular homeostasis. SQSTM1/p62 phase-separated droplets are known to play critical roles in selective autophagy; however, it is unknown whether p62 can exist as another form in addition to its autophagic droplets. Here, we found that, under stress conditions, including proteotoxicity, endotoxicity, and oxidation, autophagic p62 droplets are transformed to a type of enlarged PBs, termed p62-dependent P-bodies (pd-PBs). p62 phase separation is essential for the nucleation of pd-PBs. Mechanistically, pd-PBs are triggered by enhanced p62 droplet formation upon stress stimulation through the interactions between p62 and DDX6, a DEAD-box ATPase. Functionally, pd-PBs recruit the NLRP3 inflammasome adaptor ASC to assemble the NLRP3 inflammasome and induce inflammation-associated cytotoxicity. Our study shows that p62 droplet-to-PB transformation acts as a stress response to activate the NLRP3 inflammasome process, suggesting that persistent pd-PBs lead to NLRP3-dependent inflammation toxicity.

Evaluating clinical utility of subgingival and salivary endotoxin activity levels as periodontal biomarkers.

Objectives: The use of periodontal biomarkers for identification and monitoring of unique patient populations could foster better stratification of at-risk groups, increase access to treatment for those most in need, facilitate preventive measures and improve personalised care plans. The aim of this study was to examine the diagnostic and prognostic utility of oral lipopolysaccharides as bacterially-derived periodontal biomarkers. Methods: Periodontal parameters were recorded, and saliva and subgingival plaque samples were collected at the beginning of the study from periodontally healthy volunteers and periodontitis patients, and three months after completion of conventional periodontal treatment in the periodontitis group. Endotoxin activity in the samples was measured using the recombinant factor C assay. Associations between clinical periodontal parameters and subgingival and salivary endotoxin activities were analysed using a multivariate regression model, while the ROC curve was applied to estimate the sensitivity, specificity and c-statistics for salivary and subgingival endotoxin activities as diagnostic biomarkers for periodontitis. Results: Significant correlations were found between subgingival endotoxin activities, probing pocket depth and periodontal diagnosis, which were independent from patients' age, gender and smoking status. In addition, subgingival endotoxin levels had high specificity and sensitivity in detecting periodontal health and disease (0.91 and 0.85 respectively). Salivary endotoxin activity was positively associated with periodontal diagnosis, mean probing pocket depth, percentages of sites over 4 mm and full mouth bleeding score. However, it was inferior in discriminating patients with stable periodontium from those with periodontitis (sensitivity = 0.69, specificity = 0.61) compared to subgingival endotoxin activity. Conclusions: Subgingival endotoxin activity has good diagnostic and prognostic values as a site-specific periodontal biomarker and is not influenced by the patient's age, gender or smoking status. In contrast, salivary endotoxin activity, as a patient-level biomarker, is dependent on patient's age, has poorer diagnostic and prognostic capability, but shows good correlations with disease susceptibility and both its extent and severity.

Comparative analysis of total salivary lipopolysaccharide chemical and biological properties with periodontal status.

OBJECTIVE: Clinical manifestations of Gram-negative bacteria mediated diseases can be influenced by how the host senses their major microbe-associated molecular pattern, the cell wall lipopolysaccharide (LPS). Keystone periodontal pathogens can produce a heterogeneous population of LPS molecules, with strikingly different host-microbiome interactions and immune outcomes. DESIGN: Structure-function correlations of salivary LPS extracts in patients with periodontitis before and after periodontal treatment and healthy volunteers were analysed by comparing its lipid A and carbohydrate chain chemical structure and evaluating its endotoxin activity and inflammatory potential. RESULTS: Salivary LPS extracts from periodontitis patients were characterised by high m/z lipid A mass-spectrometry peaks, corresponding to over-acylated and phosphorylated lipid A ions and by a combination of rough and smooth LPS carbohydrate moieties. In contrast, gingival health was defined by the predominance of low m/z lipid A peaks, consistent with under-acylated and hypo-phosphorylated lipid A molecular signatures, with long and intermediate carbohydrate chains as determined by silver staining. Total, diseased salivary LPS extracts were stronger inducers of the recombinant factor C assay and triggered significantly higher levels of TNF-α, IL-8 and IP-10 production in THP-1 cells, compared to almost immunosilent healthy samples. Interestingly, salivary LPS architecture, endotoxin activity, and inflammatory potential were well conserved after periodontal therapy and showed similarities to diseased samples. CONCLUSIONS: This study sheds new light on molecular pathogenic mechanisms of oral dysbiotic communities and indicates that the regulation of LPS chemical structure is an important mechanism that drives oral bacteria-host immune system interactions into either a symbiotic or pathogenic relationship.

Subgingival lipid A profile and endotoxin activity in periodontal health and disease.

OBJECTIVES: Regulation of lipopolysaccharide (LPS) chemical composition, particularly its lipid A domain, is an important, naturally occurring mechanism that drives bacteria-host immune system interactions into either a symbiotic or pathogenic relationship. Members of the subgingival oral microbiota can critically modulate host immuno-inflammatory responses by synthesizing different LPS isoforms. The objectives of this study were to analyze subgingival lipid A profiles and endotoxin activities in periodontal health and disease and to evaluate the use of the recombinant factor C assay as a new, lipid A-based biosensor for personalized, point-of-care periodontal therapy. MATERIALS AND METHODS: Subgingival plaque samples were collected from healthy individuals and chronic periodontitis patients before and after periodontal therapy. Chemical composition of subgingival lipid A moieties was determined by ESI-Mass Spectrometry. Endotoxin activity of subgingival LPS extracts was assessed using the recombinant factor C assay, and their inflammatory potential was examined in THP-1-derived macrophages by measuring TNF-α and IL-8 production. RESULTS: Characteristic lipid A molecular signatures, corresponding to over-acylated, bi-phosphorylated lipid A isoforms, were observed in diseased samples. Healthy and post-treatment samples were characterized by lower m/z peaks, related to under-acylated, hypo-phosphorylated lipid A structures. Endotoxin activity levels and inflammatory potentials of subgingival LPS extracts from periodontitis patients were significantly higher compared to healthy and post-treatment samples. CONCLUSIONS: This is the first study to consider structure-function-clinical implications of different lipid A isoforms present in the subgingival niche and sheds new light on molecular pathogenic mechanisms of subgingival biofilm communities. CLINICAL RELEVANCE: Subgingival endotoxin activity (determined by lipid A chemical composition) could be a reliable, bacterially derived biomarker and a risk assessment tool for personalized periodontal care.