Porphyromonas gingivalis Lipopolysaccharide Damages Mucosal Barrier to Promote Gastritis-Associated Carcinogenesis.

BACKGROUND: Recent epidemiological studies suggested correlation between gastric cancer (GC) and periodontal disease. AIMS: We aim to clarify involvement of lipopolysaccharide of Porphyromonas gingivalis (Pg.), one of the red complex periodontal pathogens, in the GC development. METHODS: To evaluate barrier function of background mucosa against the stimulations, we applied biopsy samples from 76 patients with GC using a Ussing chamber system (UCs). K19-Wnt1/C2mE transgenic (Gan) mice and human GC cell-lines ± THP1-derived macrophage was applied to investigate the role of Pg. lipopolysaccharide in inflammation-associated carcinogenesis. RESULTS: In the UCs, Pg. lipopolysaccharide reduced the impedance of metaplastic and inflamed mucosa with increases in mRNA expression of toll-like receptor (TLR) 2, tumor necrosis factor (TNF) α, and apoptotic markers. In vitro, Pg. lipopolysaccharide promoted reactive oxidative stress (ROS)-related apoptosis as well as activated TLR2-ß-catenin-signaling on MKN7, and it increased the TNFα production on macrophages, respectively. TNFα alone activated TLR2-ß-catenin-signaling in MKN7, while it further increased ROS and TNFα in macrophages. Under coculture with macrophages isolated after stimulation with Pg. lipopolysaccharide, ß-catenin-signaling in MKN7 was activated with an increase in supernatant TNFα concentration, both of which were decreased by adding a TNFα neutralization antibody into the supernatant. In Gan mice with 15-week oral administration of Pg. lipopolysaccharide, tumor enlargement with ß-catenin-signaling activation were observed with an increase in TNFα with macrophage infiltration. CONCLUSIONS: Local exposure of Pg. lipopolysaccharide may increase ROS on premalignant gastric mucosa to induce apoptosis-associated barrier dysfunction and to secrete TNFα from activated macrophages, and both stimulation of Pg. lipopolysaccharide and TNFα might activate TLR2-ß-catenin-signaling in GC.

Guanylate-binding protein 5-mediated cell-autonomous immunity suppresses inflammation in dental pulpitis: An in vitro study.

AIM: Guanylate-binding protein 5 (GBP5) is an interferon (IFN)-inducible GTPase that plays a crucial role in the cell-autonomous immune response against microbial infections. In this study, we investigated the immunoregulatory role of GBP5 in the pathogenesis of dental pulpitis. METHODOLOGY: Gene-set enrichment analysis (GSEA) was utilized to evaluate the IFN-γ signalling pathway, and the differential expression of GBP mRNA in normal versus inflamed dental pulp tissues was screened, based on Gene Expression Omnibus (GEO) datasets associated with pulpitis. Both normal pulp tissues and inflamed pulp tissues were used for experiments. The expression of IFNs and GBPs was determined by qRT-PCR. Immunoblotting and double immunofluorescence were performed to examine the cellular localization of GBP5 in dental pulp tissues. For the functional studies, IFN-γ priming or lentivirus vector-delivered shRNA was used to, respectively, overexpress or knock down endogenous GBP5 expression in human dental pulp stem cells (HDPSCs). Subsequently, LPS was used to stimulate HDPSCs (overexpressing or with knocked-down GBP5) to establish an in vitro model of inflammation. qRT-PCR and ELISA were employed to examine the expression of proinflammatory cytokines (IL-6, IL-8 and IL-1ß) and cyclooxygenase 2 (COX2). Every experiment has three times of biological replicates and three technical replicates, respectively. Statistical analysis was performed using the Student's t-test and one-way ANOVA, and a p-value of <.05 was considered statistically significant. RESULTS: GSEA analysis based on the GEO dataset revealed a significant activation of the IFN-γ signalling pathway in the human pulpitis group. Among the human GBPs evaluated, GBP5 was selectively upregulated in inflamed dental pulp tissues and predominantly expressed in dental pulp cells. In vitro experiments demonstrated that IFN-γ robustly induced the expression of GBP5 in HDPSCs. Knockdown of GBP5 expression in HDPSCs significantly amplified the LPS-induced upregulation of inflammatory mediators (IL-6, IL-8, IL-1ß and COX2) both with and without IFN-γ priming. CONCLUSION: Our findings demonstrated that GBP5 partook in the pathogenesis of dental pulpitis. The involvement of GBP5 in pulpitis appeared to coordinate the regulation of inflammatory cytokines. Knockdown of GBP5 contributed to the exacerbation of LPS-mediated inflammation.

IWP-2 modulates the immunomodulatory properties of human dental pulp stem cells in vitro.

AIM: To investigate the effect of IWP-2, Wnt inhibitor, on human dental pulp stem cells (hDPSCs) responses. METHODOLOGY: hDPSCs were isolated from human dental pulp tissues. Cells were treated with 25 µM IWP-2 for 24 h, and subsequently, the gene expression profile was examined using high-throughput RNA sequencing. The mRNA expression was analysed using qPCR. The effect of IWP-2 was investigated in both normal and LPS-induced hDPSCs (inflamed hDPSCs). CD4+ T cells and CD14+ monocyte-derived macrophages were cultured with conditioned media of IWP-2 treated hDPSCs to observe the immunosuppressive property. RESULTS: RNA sequencing indicated that IWP-2 significantly downregulated several KEGG pathways, including cytokine-cytokine receptor interaction, IL-17 signalling pathway, and TNF signalling pathway. In both normal and inflamed conditions, IWP-2 markedly upregulated TGFB1 mRNA expression while the mRNA expression of pro-inflammatory cytokines, TNFA, IL1B, IFNG, and IL6, was inhibited. In the inhibition experiment, the pretreatment with p38, MAPK, or PI3K inhibitors abolished the effects of IWP-2 in LPS-induced inflammation. In terms of immune cells, IWP-2-treated-inflamed hDPSCs conditioned media attenuated T cell proliferation and regulated regulatory T cell differentiation. In addition, the migratory property of macrophage was decreased after being exposed to IWP-2-treated inflamed hDPSCs conditioned media. CONCLUSION: IWP-2 suppressed inflammatory cytokine expression in both normal and inflamed hDPSCs. Moreover, hDPSCs exerted the immunosuppressive property after IWP-2 treatment. These results suggest the role of Wnt in inflammatory responses and immunomodulation in dental pulp tissues.

Osteopontin interacts with dendritic cells and macrophages in pulp inflammation: Comprehensive transcriptomic analysis and laboratory investigations.

AIM: To investigate novel diagnostic markers for pulpitis and validate by clinical samples from normal and inflamed pulp. To explore the relationship between diagnostic markers and immune cells or their phenotypes during pulp inflammation. METHODOLOGY: Two microarray datasets, GSE77459 and GSE92681, and identified differential expression genes were integrated. To understand immune features, gene functions, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Disease Ontology (DO) and ImmuneSigDB Gene Set Enrichment Analysis (GSEA) were analysed. For predictive purposes, machine learning techniques were applied to detect diagnostic markers. Immune infiltration in inflamed pulp was studied using CIBERSORT. The relationship between diagnostic markers and immune cells was investigated and validated their gene expression in clinical samples from the normal or inflamed pulp by qRT-PCR. Finally, the correlation between one marker, secreted phosphoprotein 1 (SPP1), encoding osteopontin (OPN), and dendritic cells (DCs)/macrophages was identified via HE staining and multiplex immunohistochemistry. An in vitro inflammatory dental pulp microenvironment model of THP-1 macrophages cocultured with dental pulp cells derived conditioned media (DPCs-CM) to investigate OPN production and macrophage phenotypes was established. RESULTS: Analysis revealed unique immunologic features in inflamed pulp. Three diagnostic markers for pulpitis: endothelin-1 (EDN1), SPP1, and purine nucleoside phosphorylase (PNP), and validated them using qRT-PCR were predicted. Multiplex immunohistochemistry demonstrated OPN co-localized with activated DCs and M2 macrophages during pulp inflammation. In vitro experiments showed that THP-1 macrophages produced the highest levels of OPN when stimulated with DPCs-CM derived from the 20 µg/mL LPS pre-conditioned group, suggesting an M2b-like phenotype by increasing surface marker CD86 and expression of IL6, TNFα, IL10, and CCL1 but not CCL17 and MerTK. Levels of CCL1 and IL10 elevated significantly in the macrophages' supernatant from the 20 µg/mL LPS pre-conditioned CM group. OPN was proven co-localizing with CD86 in the inflamed pulp by immunofluorescence. CONCLUSIONS: The current findings suggest that OPN can serve as a promising biomarker for pulpitis, correlated with DCs and macrophages. OPN+ macrophages in the inflamed pulp are associated with M2b-like phenotypes. These insights offer the potential for improved diagnosis and targeted therapy.

Dental Stem Cells and Lipopolysaccharides: A Concise Review.

Dental tissue stem cells (DTSCs) are well known for their multipotent capacity and regenerative potential. They also play an important role in the immune response of inflammatory processes derived from caries lesions, periodontitis, and gingivitis. These oral diseases are triggered by toxins known as lipopolysaccharides (LPS) produced by gram-negative bacteria. LPS present molecular patterns associated with pathogens and are recognized by Toll-like receptors (TLRs) in dental stem cells. In this review, we describe the effect of LPS on the biological behavior of DTSCs. We also focus on the molecular sensors, signaling pathways, and emerging players participating in the interaction of DTSCs with lipopolysaccharides. Although the scientific advances generated provide an understanding of the immunomodulatory potential of DTSCs, there are still new reflections to explore with regard to their clinical application in the treatment of oral inflammatory diseases.

Proanthocyanidins Ameliorate LPS-Inhibited Osteogenesis of PDLSCs by Restoring Lysine Lactylation.

Periodontitis is a bacteria-induced inflammatory disease characterized by the progressive destruction of periodontal supporting tissues. Periodontal ligament stem cells (PDLSCs) are capable of differentiating into osteoblasts, which is an important stem cell source for endogenous periodontal tissue regeneration. Lysine lactylation (Kla) is a novel post-translational modification of proteins that is recently thought to be associated with osteogenic differentiation. Here, we found that lactylation levels are reduced both in the periodontal tissue of rats with periodontitis and lipopolysaccharide (LPS)-stimulated human PDLSCs. Proanthocyanidins were able to promote the osteogenesis of inflamed PDLSCs by restoring lactylation levels. Mechanistically, proanthocyanidins increased lactate production and restored the lactylation levels of PDLSCs, which recovered osteogenesis of inflamed PDLSCs via the Wnt/ß-catenin pathway. These results provide evidence on how epigenetic regulation by pharmacological agents influence the osteogenic phenotype of stem cells and the process of periodontal tissue repair. Our current study highlights the valuable potential of natural product proanthocyanidins in the regenerative engineering of periodontal tissues.

Vesicular Messages from Dental Biofilms for Neutrophils.

The encounter between dental biofilm and neutrophils in periodontitis remains elusive, although it apparently plays a crucial role in the periodontal pathology and constitutes a key topic of periodontology. Dental biofilm and neutrophils were isolated from orally healthy persons and patients with periodontitis. We investigated biofilm and its particle-shedding phenomenon with electron microscopy and nanoparticle tracking analysis (NTA); biofilm shedding-neutrophil interactions were examined ex vivo with epi-fluorescence microscopy. For this purpose, we used acellular dental biofilm shedding, purified lipopolysaccharide (LPS), and phorbol 12-myristate 13-acetate (PMA) as activators, and the interleukin 8 receptor beta (CXCR2) inhibitor and the anti-interleukin 8 receptor alpha (CXCR1) antibody as modulators. The shedding of acellular dental biofilms overwhelmingly consists of bacterial extracellular vesicles (BEVs). The latter induced the moderate formation of neutrophil extracellular traps (NETs) in orally healthy subjects and a strong formation in patients with periodontitis. A CXCR2 inhibitor and an anti-CXCR1 antibody had a minor effect on NET formation. Neutrophils from patients with periodontitis exhibited NET hyper-responsiveness. BEVs were stronger inducers of NET formation than purified LPS and PMA. A plateau of neutrophil responsiveness is reached above the age of 40 years, indicating the abrupt switch of maladaptive trained immunity (TI) into the activated modus. Our results suggest that dental biofilms consist of and disseminate immense amounts of outer membrane vesicles (OMVs), which initiate NET formation via a non-canonical cytosolic LPS/caspase-4/11/Gasdermin D pathway. This modus of NET formation is independent of neutrophil elastase (NE), myeloperoxidase (MPO), peptidylarginine deiminase 4 (PAD4), and toll-like receptors (TLR). In periodontitis, the hyper-responsiveness of neutrophils to BEVs and the increased NET formation appear to be a consequence of TI.

Unveiling Therapeutic Potential: Targeting Fusobacterium nucleatum's Lipopolysaccharide Biosynthesis for Endodontic Infections-An In Silico Screening Study.

Complex microbial communities have been reported to be involved in endodontic infections. The microorganisms invade the dental pulp leading to pulpitis and initiating pulp inflammation. Fusobacterium nucleatum is a dominant bacterium implicated in both primary and secondary endodontic infections. Drugs targeting the molecular machinery of F. nucleatum will minimize pulp infection. LpxA and LpxD are early acyltransferases involved in the formation of lipid A, a major component of bacterial membranes. The identification of leads which exhibit preference towards successive enzymes in a single pathway can also prevent the development of bacterial resistance. A stringent screening strategy utilizing physicochemical and pharmacokinetic parameters along with a virtual screening approach identified two compounds, Lomefloxacin and Enoxacin, with good binding affinity towards the early acyltransferases LpxA and LpxD. Lomefloxacin and Enoxacin, members of the fluoroquinolone antibiotic class, exhibit wide-ranging activity against diverse bacterial strains. Nevertheless, their effectiveness in the context of endodontic treatment requires further investigation. This study explored the potential of Lomefloxacin and Enoxacin to manage endodontic infections via computational analysis. Moreover, the compounds identified herein serve as a foundation for devising novel combinatorial libraries with enhanced efficacy for endodontic therapeutic strategies.

Toll-like receptor and C-type lectin receptor agonists attenuate osteogenic differentiation in human dental pulp stem cells.

BACKGROUND: This study aimed to investigate the effects of various toll-like receptor (TLR) and C-type lectin receptor (CLR) ligands on osteogenic differentiation in human dental pulp stem cells (hDPSCs). METHODS: hDPSCs were cultured and treated with various concentrations (0.01, 0.1, 1.0, and 10 µg/mL) of TLR or CLR agonists (PG-LPS, E.coli LPS, poly(I:C), Pam3CSK4, Furfurman, and Zymosan). Cell viability was determined by MTT assay. The effects of TLR and CLR agonists on osteogenic differentiation of hDPSCs were measured by alkaline phosphatase (ALP) activity, Alizarin Red S staining, and Von Kossa staining. In addition, the mRNA expression of osteogenesis-related genes (ALP, COL1A1, RUNX2, OSX, OCN and DMP1) was examined by RT-qPCR. A non-parametric analysis was employed for the statistical analyses. The statistically significant difference was considered when p < 0.05. RESULTS: Treatment with TLR and CLR agonists was associated with an increase in hDPSCs' colony-forming unit ability. Compared with the control group, TLR and CLR agonists significantly inhibited the osteogenic differentiation of hDPSCs by decreasing the ALP activity, mineralised nodule formation, and mRNA expression levels of osteogenesis-related genes (ALP, COL1A1, RUNX2, OSX, OCN and DMP1). The inhibition of TRIF but not Akt signalling rescued the effects of TLR and CLR agonist attenuating hDPSCs' mineralisation. CONCLUSIONS: The activation of TLRs or CLRs exhibited an inhibitory effect on osteogenic differentiation of hDPSCs via the TRIF-dependent signalling pathway.

Biomimetic asymmetric bacterial membranes incorporating lipopolysaccharides.

Gram-negative bacteria are equipped with a cell wall that contains a complex matrix of lipids, proteins, and glycans, which form a rigid layer protecting bacteria from the environment. Major components of this outer membrane are the high-molecular weight and amphiphilic lipopolysaccharides (LPSs). They form the extracellular part of a heterobilayer with phospholipids. Understanding LPS properties within the outer membrane is therefore important to develop new antimicrobial strategies. Model systems, such as giant unilamellar vesicles (GUVs), provide a suitable platform for exploring membrane properties and interactions. However, LPS molecules contain large polysaccharide parts that confer high water solubility, which makes LPS incorporation in artificial membranes difficult; this hindrance is exacerbated for LPS with long polysaccharide chains, i.e., the smooth LPS. Here, a novel emulsification step of the inverted emulsion method is introduced to incorporate LPS in the outer or the inner leaflet of GUVs, exclusively. We developed an approach to determine the LPS content on individual GUVs and quantify membrane asymmetry. The asymmetric membranes with outer leaflet LPS show incorporations of 1-16 mol % smooth LPS (corresponding to 16-79 wt %), while vesicles with inner leaflet LPS reach coverages of 2-7 mol % smooth LPS (28-60 wt %). Diffusion coefficient measurements in the obtained GUVs showed that increasing LPS concentrations in the membranes resulted in decreased diffusivity.

Human serum-derived exosomes modulate macrophage inflammation to promote VCAM1-mediated angiogenesis and bone regeneration.

During exogenous bone-graft-mediated bone defect repair, macrophage inflammation dictates angiogenesis and bone regeneration. Exosomes from different human cells have shown macrophage immunomodulation-mediated bone regeneration potential. However, the effect of human serum-derived exosomes (serum-Exo) on macrophage immunomodulation-mediated angiogenesis during bone defect repair has not been investigated yet. In this study, we explored the effects of serum-Exo on macrophage inflammation regulation-mediated angiogenesis during bone defect repair and preliminarily elucidated the mechanism. Healthy serum-Exo was isolated by ultracentrifugation. The effect of serum-Exo on LPS-induced M1 macrophage inflammation was analysed in vitro. The conditioned medium of serum-Exo-treated LPS-induced M1 macrophage (serum-Exo-treated M1 macrophage-CM) was used to culture human umbilical vein endothelial cells (HUVEC), and the effect on angiogenesis was analysed by western blot, qRT-PCR, etc. mRNA-sequencing of HUVECs was performed to identify deferentially expressed genes. Finally, the rat mandibular defect model was established and treated with Bio-Oss and Bio-Oss + Exo. The effect of the Bio-Oss + Exo combination on mandibular bone regeneration was observed by micro-computed tomography (micro-CT), haematoxylin and eosin (HE) staining, Masson staining, and immunohistochemical staining. Serum-Exo promoted the proliferation of RAW264.7 macrophages and reduced the expression of M1-related genes such as IL-6, IL-1ß, iNOS, and CD86. Serum-Exo-treated M1 macrophage-CM induced the proliferation, migration, and angiogenic differentiation of HUVEC, as well as the expression of H-type blood vessel markers CD31 and endomucin (EMCN), compared with M1 macrophage-CM. Moreover, higher expression of vascular endothelial adhesion factor 1 (VCAM1) in HUVEC cultured with serum-Exo-treated M1 macrophage-CM compared with M1 macrophages-CM. Inhibition of VCAM1 signalling abrogated the pro-angiogenic effect of serum-Exo-treated M1 macrophage-CM on HUVEC. Local administration of serum-Exo during mandibular bone defect repair reduced the number of M1 macrophages and promoted angiogenesis and osteogenesis. Collectively, our results demonstrate the macrophage inflammation regulation-mediated pro-angiogenic potential of serum-Exo during bone defect repair possibly via upregulation of VCAM1 signalling in HUVEC.

Polystyrene nanoplastics aggravates lipopolysaccharide-induced apoptosis in mouse kidney cells by regulating IRE1/XBP1 endoplasmic reticulum stress pathway via oxidative stress.

Nanoplastics (NPs) pollution poses a huge threat to the ecosystem and has become one of the environmental pollutants that have attracted much attention. There is increasing evidence that both oxidative stress and endoplasmic reticulum stress (ERS) are associated with polystyrene nanoplastics (PS-NPs) exposure. Lipopolysaccharide (LPS) has been shown to induce apoptotic damage in various tissues, but whether PS-NPs can aggravate LPS-induced apoptosis in mouse kidneys through oxidative stress-regulated inositol-requiring enzyme 1 (IRE1)/X-box binding protein 1 (XBP1) ERS pathway remains unclear. In this study, based on the establishment of in vitro and in vivo PS-NPs and LPS exposure models alone and in combination in mice and HEK293 cells, the effects and mechanisms of PS-NPs on LPS-induced renal cell apoptosis were investigated. The results showed that PS-NPs could aggravate LPS-induced apoptosis. PS-NPs/LPS can induce ERS through oxidative stress, activate the IRE1/XBP1 pathway, and promote the expression of apoptosis markers (Caspase-3 and Caspase-12). Kidney oxidative stress, ERS, and apoptosis in PS-NPs + LPS combined exposure group were more severe than those in the single exposure group. Interestingly, 4-phenylbutyric acid-treated HEK293 cells inhibited the expression of the IRE1/XBP1 ERS pathway and apoptotic factors in the PS-NPs + LPS combined exposure group. N-acetyl-L-cysteine effectively blocked the activation of the IRE1/XBP1 ERS pathway, suggesting that PS-NPs-induced oxidative stress is an early event that triggers ERS. Collectively, these results confirmed that PS-NPs aggravated LPS-induced apoptosis through the oxidative stress-induced IRE1/XBP1 ERS pathway. Our study provides new insights into the health threats of PS-NPs exposed to mammals and humans.

Astragaloside reduces toxic effect of periodontal ligament fibroblasts induced by lipopolysaccharide.

Periodontitis is a non-specific and chronic disease which is highly prevalent, resulting in inflammation and destruction of periodontal tissues. This study aims to explore the effect and mechanism of astragaloside on periodontitis. We used CCK-8, Western Blot, qPCR and flow cytometry to analyze cell viability, related protein and mRNA expression, and cell apoptosis. We found that AST could promote cell proliferation and reduce apoptosis induced by LPS. Besides, AST could alleviate the increased expression of COX-2 and ICAM-1 induced by LPS. MiR-26b-3P specifically targeted the 3' UTR of ICAM-1. These results indicate that AST reduces toxic effect of human periodontal ligament cells through regulating miR-26b-3P/ICAM-1, thus highlighting its protective role in periodontitis.

Human gingival mesenchymal stem cell-derived exosomes cross-regulate the Wnt/ß-catenin and NF-κB signalling pathways in the periodontal inflammation microenvironment.

AIM: To examine the immunomodulatory effect of exosomes originating from gingival mesenchymal stem cells (GMSC-Exo) on periodontal bone regeneration and its role in the regulation of the nuclear-factor kappaB (NF-κB) and Wnt/ß-catenin pathways in the periodontal inflammatory microenvironment. MATERIALS AND METHODS: First, periodontal ligament stem cells (PDLSCs) were treated with GMSC-Exo or Porphyromonas gingivalis-derived lipopolysaccharide (P.g-LPS) in vitro. Quantitative real-time PCR (qRT-PCR) and western blot were carried out to detect the expressions of osteogenic differentiation-related factors in cells. Further, PDLSCs were treated with P.g-LPS or inhibitors. The expression of NF-κB pathway-related factors as well as of Wnt/ß-catenin pathway-related factors were detected by qRT-PCR and western blot. RESULTS: GMSC-Exo treatment promoted the expression of osteogenic differentiation-related factors within PDLSCs in both normal and inflammatory environments. Further investigations showed that GMSC-Exo could also inhibit the P.g-LPS-induced activation of the NF-κB pathway, leading to the up-regulation of the Wnt/ß-catenin pathway. When the Wnt/ß-catenin signalling was blocked, the inhibitory effect of GMSC-Exo on the NF-κB pathway was abolished. CONCLUSIONS: GMSC-Exo could promote the osteogenic differentiation of PDLSCs. There could be mutually exclusive regulatory roles between the NF-κB and Wnt/ß-catenin signalling pathways in a periodontal inflammatory environment. GMSC-Exo exhibited an effective cross-regulation ability for both pathways.

Effect of mesencephalic astrocyte-derived neurotrophic factor on the inflammatory response in human gingival fibroblasts cells.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a unique member of the neurotrophic factor family residing in the endoplasmic reticulum, where it functions as a stress response protein maintaining endoplasmic reticulum homeostasis, in addition to being secreted extracellularly as a neurotrophic factor to bind with receptors to initiate intracellular signal transduction pathways. Interestingly, MANF has shown an important protective role in the inflammatory response of many diseases. In neural stem cells, pancreatic ß cells, and retinal cells, MANF can inhibit the inflammatory response, modulate the immune response, and promote tissue repair. However, the role of MANF in the periodontal inflammatory response remains unclear. In the present study, we used lipopolysaccharide (LPS) from Porphyromonas gingivalis (Pg) to establish a Pg-LPS-stimulated periodontal inflammatory model in human gingival fibroblasts cells (HGF-1) to investigate the role of MANF in vitro. We found that MANF could inhibit pro-inflammatory cytokine secretion, alleviate the endoplasmic reticulum stress response, promote cell survival, and inhibit cell apoptosis. Therefore, MANF might be a novel promising target for the treatment of periodontitis.

Lipoteichoic acid polymer length is determined by competition between free starter units.

Carbohydrate polymers exhibit incredible chemical and structural diversity, yet are produced by polymerases without a template to guide length and composition. As the length of carbohydrate polymers is critical for their biological functions, understanding the mechanisms that determine polymer length is an important area of investigation. Most Gram-positive bacteria produce anionic glycopolymers called lipoteichoic acids (LTA) that are synthesized by lipoteichoic acid synthase (LtaS) on a diglucosyl-diacylglycerol (Glc2DAG) starter unit embedded in the extracellular leaflet of the cell membrane. LtaS can use phosphatidylglycerol (PG) as an alternative starter unit, but PG-anchored LTA polymers are significantly longer, and cells that make these abnormally long polymers exhibit major defects in cell growth and division. To determine how LTA polymer length is controlled, we reconstituted Staphylococcus aureus LtaS in vitro. We show that polymer length is an intrinsic property of LtaS that is directly regulated by the identity and concentration of lipid starter units. Polymerization is processive, and the overall reaction rate is substantially faster for the preferred Glc2DAG starter unit, yet the use of Glc2DAG leads to shorter polymers. We propose a simple mechanism to explain this surprising result: free starter units terminate polymerization by displacing the lipid anchor of the growing polymer from its binding site on the enzyme. Because LtaS is conserved across most Gram-positive bacteria and is important for survival, this reconstituted system should be useful for characterizing inhibitors of this key cell envelope enzyme.

The effects of dimethyl fumarate on cytoplasmic LPS-induced noncanonical pyroptosis in periodontal ligament fibroblasts and dental pulp cells.

AIM: Pyroptosis is a type of inflammatory cell death and is related to pulpitis and apical periodontitis. In this study, the aim was to investigate how periodontal ligament fibroblasts (PDLFs) and dental pulp cells (DPCs) respond to pyroptotic stimuli and explore whether dimethyl fumarate (DMF) could block pyroptosis in PDLFs and DPCs. METHODOLOGY: Three methods (stimulation with lipopolysaccharide [LPS] plus nigericin, poly(dA:dT) transfection and LPS transfection) were used to induce pyroptosis in PDLFs and DPCs, two types of fibroblasts related to pulpitis and apical periodontitis. THP-1 cell was used as a positive control. Afterwards, PDLFs and DPCs were treated with or without DMF before inducing pyroptosis to examine the inhibitory effect of DMF. Pyroptotic cell death was measured by lactic dehydrogenase (LDH) release assays, cell viability assays, propidium iodide (PI) staining and flow cytometry. The expression levels of cleaved gasdermin D N-terminal (GSDMD NT), caspase-1 p20, caspase-4 p31 and cleaved PARP were examined by immunoblotting. Immunofluorescence analysis was used to detect the cellular distribution of GSDMD NT. RESULTS: Periodontal ligament fibroblasts and DPCs were more sensitive to cytoplasmic LPS-induced noncanonical pyroptosis than to canonical pyroptosis induced by stimulation with LPS priming plus nigericin or by poly(dA:dT) transfection. In addition, treatment with DMF attenuated cytoplasmic LPS-induced pyroptotic cell death in PDLFs and DPCs. Mechanistically, it was shown that the expression and plasma membrane translocation of GSDMD NT were inhibited in DMF-treated PDLFs and DPCs. CONCLUSIONS: This study indicates that PDLFs and DPCs are more sensitive to cytoplasmic LPS-induced noncanonical pyroptosis and that DMF treatment blocks pyroptosis in LPS-transfected PDLFs and DPCs by targeting GSDMD, suggesting DMF might be a promising drug for the management of pulpitis and apical periodontitis.

Regulatory Effects of Three-Dimensional Cultured Lipopolysaccharide-Pretreated Periodontal Ligament Stem Cell-Derived Secretome on Macrophages.

Phenotypic transformation of macrophages plays important immune response roles in the occurrence, development and regression of periodontitis. Under inflammation or other environmental stimulation, mesenchymal stem cells (MSCs) exert immunomodulatory effects through their secretome. It has been found that secretome derived from lipopolysaccharide (LPS)-pretreated or three-dimensional (3D)-cultured MSCs significantly reduced inflammatory responses in inflammatory diseases, including periodontitis, by inducing M2 macrophage polarization. In this study, periodontal ligament stem cells (PDLSCs) pretreated with LPS were 3D cultured in hydrogel (termed SupraGel) for a certain period of time and the secretome was collected to explore its regulatory effects on macrophages. Expression changes of immune cytokines in the secretome were also examined to speculate on the regulatory mechanisms in macrophages. The results indicated that PDLSCs showed good viability in SupraGel and could be separated from the gel by adding PBS and centrifuging. The secretome derived from LPS-pretreated and/or 3D-cultured PDLSCs all inhibited the polarization of M1 macrophages, while the secretome derived from LPS-pretreated PDLSCs (regardless of 3D culture) had the ability to promote the polarization of M1 to M2 macrophages and the migration of macrophages. Cytokines involved in the production, migration and polarization of macrophages, as well as multiple growth factors, increased in the PDLSC-derived secretome after LPS pretreatment and/or 3D culture, which suggested that the secretome had the potential to regulate macrophages and promote tissue regeneration, and that it could be used in the treatment of inflammation-related diseases such as periodontitis in the future.

Induction of Antimicrobial Protein S100A15 Expression by Oral Microbial Pathogens Is Toll-like Receptors-Dependent Activation of c-Jun-N-Terminal Kinase (JNK), p38, and NF-κB Pathways.

The antimicrobial protein S100A15 belongs to the S100 family, which is differentially expressed in a variety of normal and pathological tissues. Although the function of S100A15 protein has been discussed in several studies, its induction and regulation in oral mucosa, so far, are largely unknown. In this study, we demonstrate that S100A15 is induced by the stimulation of oral mucosa with gram- or gram+ bacterial pathogens, as well as with the purified membrane components, namely lipopolysaccharides (LPS) and lipoteichoic acid (LTA). The stimulation of the human gingival fibroblast (GF) and the human mouth epidermal carcinoma (KB) cell lines with either gram- or gram+ bacterial pathogens or their purified membrane components (LPS and LTA) results in the activation of NF-κB, apoptosis-regulating kinase1 (ASK1), and MAP kinase signaling pathways including, c-Jun N-terminal kinase (JNK) and p38 together with their physiological substrates AP-1 and ATF-2, respectively. Inhibition of S100A15 by antibodies-mediated Toll-like receptor 4 (TLR4) or Toll-like receptor 2 (TLR2) neutralization reveals the induction of S100A15 protein by LPS/gram- bacterial pathogens to be TLR4- dependent mechanism, whereas induction by LTA/gram+ bacterial pathogens to be TLR2- dependent mechanism. Pre-treatment of GF and KB cells with JNK (SP600125), p38 (SB-203580), or NF-κB (Bay11-7082) specific inhibitors further demonstrates the importance of JNK, p38 and NF-κB pathways in the regulation of gram-/gram+ bacterial pathogen-induced S100A15 expression. Our data provide evidence that S100A15 is induced in cancer and non-cancer oral mucosa-derived cell lines by gram-/gram+ bacterial pathogens and provide insight into the molecular mechanisms by which gram- and gram+ bacterial pathogens induce S100A15 expression in the oral mucosa.

Pomegranate Peel Extract Differently Modulates Gene Expression in Gingiva-Derived Mesenchymal Stromal Cells under Physiological and Inflammatory Conditions.

Pomegranate has shown a favorable effect on gingivitis/periodontitis, but the mechanisms involved are poorly understood. The aim of this study was to test the effect of pomegranate peel extract (PoPEx) on gingiva-derived mesenchymal stromal cells (GMSCs) under physiological and inflammatory conditions. GMSC lines from healthy (H) and periodontitis (P) gingiva (n = 3 of each) were established. The lines were treated with two non-toxic concentrations of PoPEX (low-10; high-40 µg/mL), with or without additional lipopolysaccharide (LPS) stimulation. Twenty-four genes in GMSCs involved in different functions were examined using real-time polymerase chain reaction (RT-PCR). PoPEx (mostly at higher concentrations) inhibited the basal expression of IL-6, MCP-1, GRO-α, RANTES, IP-10, HIF-1α, SDF-1, and HGF but increased the expression of IL-8, TLR3, TGF-ß, TGF-ß/LAP ratio, IDO-1, and IGFB4 genes in H-GMSCs. PoPEx increased IL-6, RANTES, MMP3, and BMP2 but inhibited TLR2 and GRO-α gene expression in P-GMSCs. LPS upregulated genes for proinflammatory cytokines and chemokines, tissue regeneration/repair (MMP3, IGFBP4, HGF), and immunomodulation (IP-10, RANTES, IDO-1, TLR3, COX-2), more strongly in P-GMSCs. PoPEx also potentiated most genes' expression in LPS-stimulated P-GMSCs, including upregulation of osteoblastic genes (RUNX2, BMP2, COL1A1, and OPG), simultaneously inhibiting cell proliferation. In conclusion, the modulatory effects of PoPEx on gene expression in GMSCs are complex and dependent on applied concentrations, GMSC type, and LPS stimulation. Generally, the effect is more pronounced in inflammation-simulating conditions.