Pg LPS Research Articles

The Effect of Total Glucosides of Peony on Lipopolysaccharide Induced Monocyte Immune Inflammation

This study aimed to explore the effect of total glucosides of peony (TGP) on the lipopolysaccharide induced secretion of IL-1, IL-6 and TNF-α in monocytes. Human peripheral blood was used to isolate and culture monocytes, with 25μg/mL Porphyromonas gingivalis lipopolysaccharide (Pg LPS) as a stimulating factor, then we observed the inhibit influence of 1, 5, 10, 20, 50μg/mL concentrations of TGP on the secretion of IL-1, IL-6, and TNF-α. Enzyme-linked immunosorbent assay (ELISA) was used on the concentration determination of IL-1, IL-6, and TNF-α. This study discovered that TGP had a significant inhibitory effect on Pg LPS induced monocyte secretion of IL-1 and IL-6, and the inhibitory effect on IL-1 was enhanced with the increase of TGP concentration. However, when the TGP reached a certain concentration, the inhibitory effect on IL-6 weakens. No effect was observed on the inhibitory effect of TNF-α. The results of the experiments showed that TGP have a significant inhibitory effect on the secretion of IL-1 and IL-6 by monocytes induced by Pg LPS within a certain concentration range, which might be one of its mechanisms in treating periodontitis.

Development and applications of peri-implantitis mouse models.

Peri-implantitis is one of the most common complications of implants. However, its pathogenesis has not been clarified. In recent years, mouse models are gradually being used in the study of peri-implantitis. This review aims to summarize the methods used to induce peri-implantitis in mice and their current applications. Articles of peri-implantitis mouse models were collected. We analyzed the various methods of inducing peri-implantitis and their application in different areas. Most researchers have induced peri-implantitis by silk ligatures. Some others have induced peri-implantitis by Pg gavage and LPS injection. Current applications of peri-implantitis mouse models are in the following areas: investigation of pathogenesis and exploration of new interventions, comparison of peri-implantitis with periodontitis, the interaction between systemic diseases and peri-implantitis, etc. CONCLUSION: Silk ligature for 2-4 weeks, Pg gavage for 6 weeks, and LPS injection for 6 weeks all successfully induced peri-implantitis in mice. Mice have the advantages of mature gene editing technology, low cost, and short time to induce peri-implantitis. It has applications in the study of pathogenesis, non-surgical treatments, and interactions with other diseases. However, compared with large animals, mice also have a number of disadvantages that limit their application.

Human β-Defensin 3 Inhibition of P. gingivalis LPS-Induced IL-1β Production by BV-2 Microglia through Suppression of Cathepsins B and L.

Cathepsin B (CatB) is thought to be essential for the induction of Porphyromonas gingivalis lipopolysaccharide (Pg LPS)-induced Alzheimer's disease-like pathologies in mice, including interleukin-1β (IL-1β) production and cognitive decline. However, little is known about the role of CatB in Pg virulence factor-induced IL-1β production by microglia. We first subjected IL-1β-luciferase reporter BV-2 microglia to inhibitors of Toll-like receptors (TLRs), IκB kinase, and the NLRP3 inflammasome following stimulation with Pg LPS and outer membrane vesicles (OMVs). To clarify the involvement of CatB, we used several known CatB inhibitors, including CA-074Me, ZRLR, and human β-defensin 3 (hBD3). IL-1β production in BV-2 microglia induced by Pg LPS and OMVs was significantly inhibited by the TLR2 inhibitor C29 and the IκB kinase inhibitor wedelolactonne, but not by the NLRPs inhibitor MCC950. Both hBD3 and CA-074Me significantly inhibited Pg LPS-induced IL-1β production in BV-2 microglia. Although CA-074Me also suppressed OMV-induced IL-1β production, hBD3 did not inhibit it. Furthermore, both hBD3 and CA-074Me significantly blocked Pg LPS-induced nuclear NF-κB p65 translocation and IκBα degradation. In contrast, hBD3 and CA-074Me did not block OMV-induced nuclear NF-κB p65 translocation or IκBα degradation. Furthermore, neither ZRLR, a specific CatB inhibitor, nor shRNA-mediated knockdown of CatB expression had any effect on Pg virulence factor-induced IL-1β production. Interestingly, phagocytosis of OMVs by BV-2 microglia induced IL-1β production. Finally, the structural models generated by AlphaFold indicated that hBD3 can bind to the substrate-binding pocket of CatB, and possibly CatL as well. These results suggest that Pg LPS induces CatB/CatL-dependent synthesis and processing of pro-IL-1β without activation of the NLRP3 inflammasome. In contrast, OMVs promote the synthesis and processing of pro-IL-1β through CatB/CatL-independent phagocytic mechanisms. Thus, hBD3 can improve the IL-1β-associated vicious inflammatory cycle induced by microglia through inhibition of CatB/CatL.

Modulation of myeloid cell functions by long noncoding RNAs RN7SK and HCG11

Abstract Objective Expressional dynamics of long noncoding RNAs (lncRNA)” regulates a gamut of biological processes including immunity. Role of lncRNA in response to oral pathogens and their regulatory impact on the innate immune functions of myeloid cells remain poorly explored. Therefore, we hypothesize that periodontal pathogens may cause the alteration of lncRNA profiles in myeloid cells and modulate their innate immune functions. Method CD14+ monocytes sorted from human PBMCs were differentiated into macrophages (MΦ) or dendritic cells (DC), and challenged with periopathic bacteria (A. actinomycetemcomitans [Aa] and P. gingivalis [Pg] for 4, 12 or 24 h. lncRNA expression was done at these time points by RT-qPCR array. Differentially expressed lncRNAs were assessed for: 1) cell migration , 2) phagocytosis, and 3) antigen uptake/processing in . Cell surface markers were analysed by flow-cytometry. Result Challenge of DCs with Pg, Pg LPS, Aa, and Aa LPS separately result in the differential expression of 21 lncRNAs (19 up, 2 down). Knockdown of LncRNA, SNHG11, NUTM2A-AS1, MCM3AP-AS1, JPX, and HCG11 (upregulated) and RN7SK (downregulated) were used to evaluate myleoid cell functions. HCG11 knockdown revealed attenuation of cell migration, while RN7SK significantly enhanced antigen uptake/processing in APCs . Importantly, RN7SK knockdown results in downregulation of M2 Mϕ surface markers (CD163, CD206 or Dectin) and concomitant increase in M1 Mϕ markers (MHC II or CD32) suggesting its critical role in macrophage polarization. Conclusion Our results show that periodontal pathogens alter lncRNA profiles and impair innate immune functions in myeloid cells, thereby suggesting critical roles of lncRNAs in periodontopathogenesis. Supported by NIH/NIDCR R03 DE027147, R01DE027980, and R21DE026259 to AN.

BET protein inhibitor apabetalone represses Porphyromonas gingivalis LPS-induced macrophage M1 polarization via regulating miR-130a/STAT3 axis

Periodontitis is a frequent chronic inflammatory disorder destroying periodontium. Recent studies have revealed the role of bromodomain and extraterminal domain inhibitor (BETi) and microRNA (miR)-130a in regulating macrophage polarization and pro-inflammatory response. However, little is known about whether apabetalone (a novel BETi) and miR-130a are correlated with chronic inflammatory state in periodontitis by regulating macrophage polarization. Here murine RAW264.7 macrophages were applied as an in vitro inflammatory model. After treatment with Porphyromonas gingivalis-derived lipopolysaccharide (Pg LPS) and apabetalone, the expression of macrophage M1 polarization markers and inflammatory cytokines was assessed using real-time PCR, western blot, and enzyme-linked immuno sorbent assay (ELISA). MiR-130a level was assessed using real-time PCR, and the target gene was identified using dual luciferase reporter assay. We demonstrated that apabetalone repressed Pg LPS-induced macrophage M1 polarization in a dose-dependent manner, as evidenced by decreased expression of inducible nitric oxide synthase (iNOS), CD86, and pro-inflammatory cytokines, and increased expression of Arg-1 and CD206. Mechanistically, Pg LPS increased miR-130a expression in macrophages, whereas apabetalone treatment repressed the effect. Functionally, forced expression of miR-130a promoted macrophage M1 polarization, and signal transducer and activator of transcription (STAT)-3 was the direct target gene of miR-130a in the process. Taken together, apabetalone decreases Pg LPS-induced macrophage M1 polarization via regulating miR-130a-3p/STAT3 axis, and may be a promising target for the clinical management of periodontitis.

Gender differences in immunological response of African-American juveniles with Grade C molar incisor pattern periodontitis.

Prevalence of Grade C molar incisor periodontitis (C/MIP) in females (F) and males (M) is controversial, although some studies suggest higher prevalence in females. The objective of this study was to evaluate differences in clinical parameters, and levels of cyto/chemokines in gingival crevicular fluid (GCF) and peripheral blood response. GCF and blood were collected from 79 C/MIP African-American participants (53F and 26M) and healthy controls (58F and 38M), aged 5 to 23. Blood was stimulated with ultrapure LPS from Escherichia coli (Ec) and Porphyromonas gingivalis (Pg) and we quantified levels of 14 cyto/chemokines. Clinical parameters were collected before and 12 months following treatment RESULTS: No clinical parameters or age differences were found between males and females, although age was negatively correlated with response to treatment. GCF levels of TNFα, IFNγ, MIP1α, and MCP1 from diseased and sites and healthy sites IFNγ levels were higher in M (P<0.05). C/MIP females presented higher Pg and Ec LPS induced levels of Eotaxin, IFNγ, and GMCSF (P<0.05), whereas healthy males presented higher Ec LPS induced levels of Eotaxin and IFNγ (P<0.05). Inflammatory profiles were also different among genders in disease (P=0.004). Although males seemed to present few elevated inflammatory markers in the GCF in disease and in health, females presented an elevated systemic inflammatory response to LPS in disease, which indicates a possible differential susceptibility to inflammation. Future studies need to determine if sex hormones have a role in the peripheral host response and in the pathogenesis of C/MIP.

Visfatin regulates Pg LPS-induced proinflammatory/prodegradative effects in healthy and inflammatory periodontal cells partially via NF-κB pathway

Periodontitis is a widespread chronic infectious-inflammatory disease associated with multiple systemic diseases. Visfatin is an adipokine-enzyme that can be locally produced by human periodontal ligament cells (hPDLCs) and human gingival fibroblasts (hGFs). It can upregulate proinflammatory cytokines and matrix metalloproteinases (MMPs) in various types of cells. However, the effects of visfatin on healthy and inflammatory human periodontal cells as well as the underlying molecular mechanisms remain unclear. This study firstly demonstrated visfatin expression was highly elevated in inflamed human gingiva and Pg LPS-treated hPDLCs. Moreover, recombinant visfatin significantly upregulated the expression of proinflammatory cytokines (TNF-α, IL-1β and IL-6) and prodegradative factors (EMPPRIN, MMP1, MMP3 and MMP13) in hPDLCs. Next, we found the levels of proinflammatory and prodegradative cytokines were significantly increased in visfatin-overexpressing hPDLCs, and decreased in visfatin-silencing inflammatory hGFs (iGFs) when treated with Pg LPS. In the absence of Pg LPS, visfatin silencing failed to affect the expression of these factors in iGFs, and overexpression of visfatin upregulated MMPs but no other factors in hPDLCs. Furthermore, marked NF-κB pathway activation with increased phosphorylation of p65 was observed in visfatin-overexpressing hPDLCs. BAY11-7082, a specific inhibitor of NF-κB, partially reversed the upregulation proinflammatory and prodegradative factors induced by visfatin overexpression. Taken together, this study showed that visfatin critically regulates Pg LPS-induced proinflammatory/prodegradative effects in healthy and inflammatory periodontal cells partially via NF-κB pathway. The findings suggest that visfatin is closely involved in the development of periodontitis, and may serve as a promising novel biomarker and therapeutic target for periodontitis management.

Intergenerational effects of pre-pregnancy chronic lipopolysaccharide from Porphyromonas gingivalis on the learning, memory and seizure susceptibility of offspring

ObjectiveThe aim of this study was to investigate the effects of pre-pregnancy chronic exposure to Porphyromonas gingivalis LPS (Pg LPS) on the learning, memory, and seizure susceptibility of the offspring. DesignTo achieve periodontitis, Pg LPS (5 μg/kg) was injected into the gingival of five female rats every 48 h for three weeks. Five control female rats received saline (0.9 %) and five female were kept intact. The concentrations of TNF-α and IL-6 were measured in the blood samples. One week after the final injection, females were mated with intact males. Following birth and weaning, two male and two female offspring were randomly selected from each mother, and new groups of male and female offspring were defined for behavioral assessments. Morris water maze was used to evaluate spatial memory, shuttle box was used to investigate avoidance memory and a pentylenetetrazole-induced seizure was used to evaluate seizure susceptibility in the offspring. ResultsSpatial learning and avoidance memory significantly decreased in both male and female offspring of Pg LPS-exposed female rats, compared to the control offspring. Latency to reach seizure stages 1 and 2 significantly increased in the male offspring, but not the female offspring of Pg LPS-exposed female, compared to the control offspring. However, no significant difference was found in latency to reach stages 3–5. ConclusionPre-pregnancy exposure to Pg LPS could affect some behavioral functions in both male and female offspring intergenerationally.

Lipoxin A4 promotes autophagy and inhibits overactivation of macrophage inflammasome activity induced by Pg LPS.

ObjectiveTo explore the role of lipoxin A4 (LXA4) on inflammasome and inflammatory activity in macrophages activated by Porphyromonas gingivalis lipopolysaccharide (PgLPS) one of the major causative agents of chronic periodontitis.MethodsThe mouse macrophage cell line RAW264.7 was used to produce an activated inflammation model. Markers of inflammasome and inflammatory activity and autophagy were assessed by ELISA, reverse transcription polymerase chain reaction (RT-PCR), and Western blot assay.ResultsMarkers of inflammasome activity, inflammation and autophagy increased with Pg LPS concentration. They also increased with increasing exposure to Pg LPS up to 12h but decreased at 24h. However, markers of autophagy increased. Phosphorylated NF-κBp65 decreased with LXA4, which was similar to results obtained with the autophagy inducer, rapamycin.ConclusionsLXA4 promoted autophagy and inhibited activation of inflammasomes and inflammation markers in macrophage inflammation induced by PgLPS and this action was linked to the phosphorylation of NF-κB.

Lipopolysaccharides of Fusobacterium nucleatum and Porphyromonas gingivalis increase RANKL-expressing neutrophils in air pouches of mice

Increases of neutrophils and osteoclasts are pathological changes of periodontitis. RANKL is an osteoclast differentiation factor. The effect of periodontopathogen LPS on RANKL-expressing neutrophils has not been clarified yet. We evaluated numerical changes of RANKL-expressing neutrophils in air pouches of mice injected with LPSs of Fusobacterium nucleatum and Porphyromonas gingivalis. Mice with air pouches were assigned into saline (C)-, E. coli LPS- (Ec LPS)-, F. nucleatum LPS (Fn LPS)-, P. gingivalis LPS (Pg LPS)-, and Fn LPS and Pg LPS (Fn + Pg LPS)-injected groups. CD11b+Ly6G+ neutrophils and CD11b+Ly6G+RANKL+ neutrophils in blood and air pouch exudates were determined by flow cytometry. In blood, compared to the C group, the Fn LPS group showed increases of CD11b+Ly6G+ neutrophils and CD11b+Ly6G+RANKL+ neutrophils whereas the Pg LPS group showed no significant differences. These increases in the Fn LPS group were not different to those in the Ec LPS group. In exudates, Fn LPS and Pg LPS groups showed increases of CD11b+Ly6G+ neutrophils and CD11b+Ly6G+RANKL+ neutrophils compared to the C group. Increased levels in the Fn LPS group were not different to those in the Ec LPS group, but Pg LPS group was lower than those in the Ec LPS group. In blood and exudates, the Fn + Pg LPS group showed no difference in levels of these neutrophils compared to the Ec LPS group. LPSs of F. nucleatum and P. gingivalis increased RANKL-expressing neutrophils although the degrees of increases were different. These suggest that periodontopathogen LPS can act as a stimulant to increase RANKL-expressing neutrophils.

LncRNAs modulate immune response in myeloid cells challenged with periodontal pathogens

Abstract Objective Long noncoding RNAs (lncRNA) are an emerging class of regulatory RNAs. LncRNAs have been implicated in differentiation and regulation of various immune cells. However, the role of lncRNA in myeloid cell regulation, in the context of periodontal inflammation remains poorly studied. Here we examined lncRNA-mediated regulation of myeloid cells upon challenge with periopathic bacteria. Method CD14+ monocytes were purified from PBMCs and were differentiated into macrophage (Mϕ) or dendritic cells (DC). Monocytes were differentiated to M1, M2 Mϕ, or DC in presence of GM-CSF, M-CSF or GM-CSF+IL-4 respectively for 7 days. Mϕ and DC were challenged A. actinomycetemcomitans (Aa), P. gingivalis (Pg) or treated with LPS from Aa and Pg. Total RNA isolated from periopathogen treated cells was used for qPCR. LncRNA knockdown was achieved by siRNA transfection using Lipofectamine 2000 and cell surface markers, cytokine analysis, antigen uptake/processing and phagocytosis assays were performed. Result DC challenged with Pg LPS exhibit differential expression of various lncRNAs (19 were up, 2 were down). Expression of selected lncRNAs in periopathogen challenged Mϕ/DC show that lncRNA are sensitive to periopathogen challenge. Cells transfected with lncRNA targeting siRNA showed skewing of M1 or M2 Mϕ polarization markers and altered secretion of pro- or anti-inflammatory cytokines upon periopathogen challenge. Finally, lncRNA knockdown resulted in functional impairment of Mϕ and DC function as reflected by decreased antigen presentation and phagocytosis. Conclusion We have functionally characterized lncRNAs in myeloid cells challenged with periopathogens and demonstrated their regulatory role in shaping immune response.

A Follicle-Stimulating Hormone Exacerbates the Progression of Periapical Inflammation Through Modulating the Cytokine Release in Periodontal Tissue.

Menopause is directly related to a systemically low grade of inflammation, indicating that postmenopausal women might be more prone to the development of inflammation. The high levels of circulating follicle-stimulating hormone (FSH) may cause hypogonadal bone loss during postmenopausal osteoporosis independent of estrogen. Previous research revealed that FSH could aggravate alveolar bone loss during experimental periapical lesions in ovariectomized rats; however, the mechanisms for these effects remain unclear. In this study, we showed that FSH enhanced the expression and secretion of the proinflammatory cytokines interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha to a significant degree in human periodontal ligament (hPDL) cells. Furthermore, FSH upregulated Porphyromonas gingivalis lipopolysaccharide (pg LPS)-induced proinflammatory cytokine production. Furthermore, in vivo studies demonstrated that FSH increased the levels of the aforementioned cytokines in the serum and enhanced the expression of Toll-like receptor 4 at both the messenger RNA and protein levels in hPDL cells and periodontal tissues. Our research suggests that high FSH levels may regulate the immune status of periodontal tissues during the postmenopausal period and, to a certain extent, suggested that postmenopausal women might be more prone to the development of inflammation of the periapical periodontitis and more obvious bone loss.

Histone Lys demethylase KDM3C demonstrates anti-inflammatory effects by suppressing NF-κB signaling and osteoclastogenesis.

Histone Lys-specific demethylases (KDMs) play a key role in many biological processes through epigenetic mechanisms. However, the role of KDMs in inflammatory responses to oral bacterial infection is poorly understood. Here, we show a novel regulatory role of KDM3C in inflammatory responses to oral bacterial infection. KDM3C expression is transiently suppressed in human and mouse macrophages exposed to LPS from Porphyromonas gingivalis (Pg LPS). Loss of KDM3C in both human and mouse macrophages led to notable induction of proinflammatory cytokines in response to Pg LPS stimulation. Also, KDM3C depletion led to strong induction of p65 phosphorylation and accelerated nuclear translocation in cells exposed to Pg LPS. Kdm3C knockout (KO) in mice led to increased alveolar bone destruction upon induction of experimental periodontitis or pulp exposure compared with those of the wild-type (WT) littermates. The Kdm3C KO mice also revealed an increased number of osteoclasts juxtaposed to the bony lesions. We also confirmed enhanced osteoclastogenesis by bone marrow-derived macrophages isolated from the Kdm3C KO compared with the WT controls. These findings suggest an anti-inflammatory function of KDM3C in regulating the inflammatory responses against oral bacterial infection through suppression of NF-κB signaling and osteoclastogenesis.-Lee, J. Y., Mehrazarin, S., Alshaikh, A., Kim, S., Chen, W., Lux, R., Gwack, Y., Kim, R. H., Kang, M. K. Histone Lys demethylase KDM3C demonstrates anti-inflammatory effects by suppressing NF-κB signaling and osteoclastogenesis.

The anti-nociceptive effects of Porphyromonas gingivalis lipopolysaccharide

ObjectiveThe objective of this study was to assess the effect of Porphyromonas gingivalis lipopolysaccharide (PG LPS) on acute pain-related behaviour induced in rats and to measure its impact on the levels of pro-inflammatory cytokines (IL-1β, IL-6) and anti-inflammatory (IL-10) cytokines. DesignThe Brennan model was used to induce acute pain like signs in rats' hind paw. Twenty-four hours following the surgery the rats were divided into 5 groups and the affected paws were injected with 0.2 m l of one of three commercialized forms PG LPS doses (high - 1 mg/ml, medium - 0.6 mg/m l and low - 0.2 mg/m l), diclofenac sodium (1 mg/kg) or saline. Tactile allodynia, mechanical hyperalgesia, body temperature and paw swelling were assessed at baseline, 24 h postoperatively and 2 h after the paw injection. The affected and contra-lateral paw tissue was assessed for the mentioned above cytokines levels employing enzyme-linked immunosorbent assay. ResultsThis study may suggest that PG LPS can reduce pain like behaviour via increased levels of anti-inflammatory cytokine IL-10 (5900 ± 748, p < 0.05). The high PG LPS dose and diclofenac reduced the tactile allodynia and mechanical hyperalgesia significantly (42.2 ± 4 and1.6 ± 0.3, p < 0.05). PG LPS high dose increase IL-10 levels while diclofenac reduces IL-1β levels significantly (5900 ± 748 and 1760 ± 271.2). The LPS administration had no effect on paw swelling and did not increase rat’s body temperature. ConclusionThe results demonstrated that PG LPS local application could possess anti- nociceptive properties, which at least in part is mediated by an increase in IL-10 levels.

C/EBPβ and YY1 bind and interact with Smad3 to modulate lipopolysaccharide-induced amelotin gene transcription in mouse gingival epithelial cells.

Junctional epithelium (JE) develops from reduced enamel epithelium during tooth formation and is critical for the maintenance of healthy periodontal tissue through ensuring appropriate immune responses and the rapid turnover of gingival epithelial cells. We have previously shown a relationship between inflammatory cytokines and expression of JE‐specific genes, such as amelotin (AMTN), in gingival epithelial cells. Here, we elucidated the effects of Porphyromonas gingivalis‐derived lipopolysaccharide (Pg LPS) on Amtn gene transcription and the interaction of transcription factors. To determine the molecular basis of transcriptional regulation of the Amtn gene by Pg LPS, we performed real‐time PCR and carried out luciferase assays using a mouse Amtn gene promoter linked to a luciferase reporter gene in mouse gingival epithelial GE1 cells. Gel mobility shift and chromatin immunoprecipitation assays were performed to identify response elements bound to LPS‐induced transcription factors. Next, we analyzed protein levels of the LPS‐induced transcription factors and the interaction of transcription factors by western blotting and immunoprecipitation. LPS increased Amtn mRNA levels and elevated luciferase activities of constructs containing regions between −116 and −238 of the mouse Amtn gene promoter. CCAAT/enhancer‐binding protein (C/EBP) 1–, C/EBP2– and Ying Yang 1 (YY1)–nuclear protein complexes were increased by LPS treatment. Furthermore, we identified LPS‐modulated interactions with C/EBPβ, YY1 and Smad3. These results demonstrate that Pg LPS regulates Amtn gene transcription via binding of C/EBPβ–Smad3 and YY1–Smad3 complexes to C/EBP1, C/EBP2 and YY1 response elements in the mouse Amtn gene promoter.

NFE2L2/NRF2, OGG1, and cytokine responses of human gingival keratinocytes against oxidative insults of various origin.

Bacterial or tobacco-related insults induce oxidative stress in gingival keratinocytes. The aim of this study was to investigate anti-oxidative and cytokine responses of human gingival keratinocytes (HMK cells) against Porphyromonas gingivalis lipopolysaccharide (Pg LPS), nicotine, and 4-nitroquinoline N-oxide (4-NQO). HMK cells were incubated with Pg LPS (1µl/ml), nicotine (1.54mM), and 4-NQO (1µM) for 24h. Intracellular and extracellular levels of interleukin (IL)-1β, IL-1 receptor antagonist (IL-1Ra), IL-8, monocyte chemoattractant protein (MCP)-1, and vascular endothelial growth factor (VEGF) were measured with the Luminex® xMAP™ technique, and nuclear factor, erythroid 2 like 2 (NFE2L2/NRF2) and 8-oxoguanine DNA glycosylase (OGG1) with Western blots. Data were statistically analyzed by two-way ANOVA with Bonferroni correction. All tested oxidative stress inducers increased intracellular OGG1 levels, whereas only nicotine and 4-NQO induced NFE2L2/NRF2 levels. Nicotine, 4-NQO, and their combinational applications with Pg LPS induced the secretions of IL-1β and IL-1Ra, while that of IL-8 was inhibited by the presence of Pg LPS. MCP-1 secretion was suppressed by nicotine, alone and together with Pg LPS, while 4-NQO activated its secretion. Treatment of HMK cells with Pg LPS, nicotine, 4-NQO, or their combinations did not affect VEGF levels. Pg LPS, nicotine, and 4-NQO induce oxidative stress and regulate anti-oxidative response and cytokine expressions in human gingival keratinocytes differently. These results may indicate that bacterial and tobacco-related insults regulate distinct pathways.

Biological effects of interleukin‐6 on Gingival Fibroblasts: Cytokine regulation in periodontitis

Periodontitis is a bacterial infectious disease, and many inflammatory cytokines regulate periodontitis pathophysiology through a crosstalk between tissue cells and immune cells. Interleukin (IL)-6 is an important cytokine involved in the regulation of host response to bacterial infection. Human Gingival Fibroblasts (HGFs) are the most abundant cells in gingival connective tissues. Various HGF responses to periodontal pathogens or inflammatory cytokines contribute to the development of periodontitis. Lipopolysaccharide derived from Porphyromonas gingivalis (Pg LPS) and IL-1β significantly increase IL-6 production in HGFs. However, IL-6 cannot function in HGFs without the soluble form of the IL-6 receptor (sIL-6R), because HGFs do not express sufficient cell surface IL-6R to bind appreciable levels of IL-6. Importantly, sIL-6R is essential for IL-6 signaling in HGFs, and the sIL-6R is produced by differentiated THP-1 cells treated with IL-6. Calprotectin, a heterodimer of S100A8 and S100A9, is released during inflammation and significantly induces IL-6 production in HGFs via toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) signals. Calprotectin also induces sIL-6R production in differentiated THP-1 cells. IL-6 induces vascular endothelial growth factor (VEGF), matrix-metalloproteinase-1 (MMP-1), and cathepsin L production in HGFs in the presence of sIL-6R. Taken together, calprotectin-induced IL-6 production in HGFs may cause periodontitis progression through a crosstalk of fibroblasts and macrophages. There are many reports that examine how cytokines are released from HGFs to cause beneficial or harmful effects in inflamed periodontal lesions. This review explores the pathophysiology of periodontitis by focusing IL-6-mediated crosstalk of HGFs and macrophages.

Porphyromonas gingivalis lipopolysaccharides act exclusively through TLR4 with a resilience between mouse and human

Porphyromonas gingivalis is a key bacterium in chronic periodontitis, which is associated with several chronic inflammatory diseases. Lipopolysaccharides from P. gingivalis (Pg LPS) can activate multiple cell types via the production of pro-inflammatory cytokines. The receptors for Pg LPS have initially been reported as TLR2, contrasting with the well-studied TLR4 receptor for E. coli LPS; this observation remains controversial since synthetic Pg lipid A activates TLR4 but not TLR2. Despite this observation, the dogma of Pg LPS-mediated TLR2 activation remains the basis of many hypotheses and result interpretations. In the present work, we aimed at determining whether TLR4 or TLR2, or both, mediate Pg LPS pro-inflammatory activity using Pg LPS with different grades of purity, instead of synthetic lipid A from Pg LPS. Here we show that Pg LPS 1) acts exclusively through TLR4, and 2) are differently recognized by mouse and human TLR4 both in vitro and in vivo. Taken together, our results suggest that Pg LPS activity is mediated exclusively through TLR4 and only weakly induces proinflammatory cytokine secretion in mouse models. Caution should be taken when extrapolating data from mouse systems exposed to Pg or Pg LPS to humans.

Phosphoglycerol dihydroceramide, a distinctive ceramide produced by Porphyromonas gingivalis, promotes RANKL-induced osteoclastogenesis by acting on non-muscle myosin II-A (Myh9), an osteoclast cell fusion regulatory factor

Among several virulence factors produced by the periodontal pathogen Porphyromonas gingivalis (Pg), a recently identified novel class of dihydroceramide lipids that contains a long acyl-chain has the potential to play a pathogenic role in periodontitis because of its higher level of tissue penetration compared to other lipid classes produced by Pg. However, the possible impact of Pg ceramides on osteoclastogenesis is largely unknown. In the present study, we report that the phosphoglycerol dihydroceramide (PGDHC) isolated from Pg enhanced osteoclastogenesis in vitro and in vivo. Using RAW264.7 cells, in vitro assays indicated that PGDHC can promote RANKL-induced osteoclastogenesis by generating remarkably larger TRAP+ multinuclear osteoclasts compared to Pg LPS in a TLR2/4-independent manner. According to fluorescent confocal microscopy, co-localization of non-muscle myosin II-A (Myh9) and PGDHC was observed in the cytoplasm of osteoclasts, indicating the membrane-permeability of PGDHC. Loss- and gain-of-function assays using RNAi-based Myh9 gene silencing, as well as overexpression of the Myh9 gene, in RAW264.7 cells showed that interaction of PGDHC with Myh9 enhances RANKL-induced osteoclastogenesis. It was also demonstrated that PGDHC can upregulate the expression of dendritic cell-specific transmembrane protein (DC-STAMP), an important osteoclast fusogen, through signaling that involves Rac1, suggesting that interaction of PGDHC with Myh9 can elicit the cell signal that promotes osteoclast cell fusion. Taken together, our data indicated that PGDHC is a Pg-derived, cell-permeable ceramide that possesses a unique property of promoting osteoclastogenesis via interaction with Myh9 which, in turn, activates a Rac1/DC-STAMP pathway for upregulation of osteoclast cell fusion.

Membrane disruptive antimicrobial activities of human β-defensin-3 analogs.

Human beta defensin-3 (HβD-3) is a host-defense protein exhibiting antibacterial activity towards both Gram-negative and Gram-positive bacteria. There is considerable interest in the function of this protein due to its increased salt tolerance and activity against Gram-positive Staphylococcus aureus. In this study, analogs of HβD-3 devoid of N and C terminal regions are investigated to determine the influence of specific structural motif on antimicrobial activity and selectivity between Gram-positive and Gram-negative bacteria. Circular dichroism, fluorescence and solid-state NMR experiments have been used to investigate the conformation and mode of action of HβD3 analogs with various model membranes to mimic bacterial inner and outer membranes and also mammalian membranes. Our studies specifically focused on determining four major characteristics: (i) interaction of HβD3 analogs with phospholipid vesicles composed of zwitterionic PC or anionic PE:PG vesicles and LPS; (ii) conformation of HβD3-peptide analogs in the presence of PC or PE:PG vesicles; (iii) ability of HβD3 analogs to permeate phospholipid vesicles composed of PC or PE:PG; and (iv) activities on bacteria cells and erythrocytes. Our results infer that the linear peptide L25P and its cyclic form C25P are more active than L21P and C21P analogs. However, they are less active than the parent peptide, thus pointing towards the importance of the N terminal domain in its biological activity. The variation in the activities of L21P/C21P and L25P/C25P also suggest the importance of the positively charged residues at the C terminus in providing selectivity particularly to Gram-negative bacteria.