Semaphorin3A released from human dental pulp cells inhibits the increase in interleukin-6 and CXC chemokine ligand 10 production induced by tumor necrosis factor-α through suppression of nuclear factor-κB activation.

Human dental pulp cells (HDPCs) play an important role in pulpitis. Semaphorin3A (Sema3A), which is an axon guidance molecule, is a member of the secretory semaphorin family. Recently, Sema3A has been reported to be an osteoprotective factor and to be involved in the immune response. However, the role of Sema3A in dental pulp inflammation remains unknown. The aim of this study was to reveal the existence of Sema3A in human dental pulp tissue and the effect of Sema3A which is released from tumor necrosis factor (TNF)-α-stimulated HDPCs on production of proinflammatory cytokines, such as interleukin (IL)-6 and CXC chemokine ligand 10 (CXCL10), from HDPCs stimulated with TNF-α. Sema3A was detected in inflamed pulp as compared to normal pulp. HDPCs expressed Neuropilin-1(Nrp1) which is Sema3A receptor. TNF-α increased the levels of IL-6 and CXCL10 in HDPCs in time-dependent manner. Sema3A inhibited production of these two cytokines from TNF-α-stimulated HDPCs. TNF-α induced soluble Sema3A production from HDPCs. Moreover, antibody-based neutralization of Sema3A further promoted production of IL-6 and CXCL10 from TNF-α-stimulated HDPCs. Sema3A inhibited nuclear factor (NF)-κB P65 phosphorylation and inhibitor κBα degradation in TNF-α-stimulated HDPCs. These results indicated that Sema3A is induced in human dental pulp, and TNF-α acts on HDPCs to produce Sema3A, which partially inhibits the increase in IL-6 and CXCL10 production induced by TNF-α, and that the inhibition leads to suppression of NF-κB activation. Therefore, it is suggested that Sema3A may regulate inflammation in dental pulp and be novel antiinflammatory target molecule for pulpitis.

IL-8 as a Potential Therapeutic Target for Periodontitis and Its Inhibition by Caffeic Acid Phenethyl Ester In Vitro.

Salivary levels of interleukin-8 (IL-8) are elevated in patients with periodontitis. Caffeic acid phenethyl ester (CAPE) improves the periodontal status in subjects. However, whether CAPE can reduce IL-8 expression is unclear. We collected saliva to determine proinflammatory cytokine levels and used subgingival calculus and surrounding tissues from patients with periodontitis for oral microbiota analysis via 16s ribosomal RNA gene sequencing. THP-1 cells were stimulated with sterile-filtered saliva from patients, and target gene/protein expression was assessed. IL-8 mRNA expression was analyzed in saliva-stimulated THP-1 cells treated with CAPE and the heme oxygenase-1 (HO-1) inhibitor tin-protoporphyrin (SnPP). In 72 symptomatic individuals, IL-8 was correlated with periodontal inflammation (bleeding on probing, r = 0.45; p < 0.001) and disease severity (bleeding on probing, r = 0.45; p < 0.001) but not with the four oral microbiota species tested. Reduced salivary IL-8 secretion was correlated with effective periodontitis treatment (r = 0.37, p = 0.0013). In THP-1 cells, saliva treatment induced high IL-8 expression and IKK2 and nuclear factor-κB (NF-κB) phosphorylation. However, the IKK inhibitor BMS-345541, NF-κB inhibitor BAY 11-7082, and CAPE attenuated saliva-induced IL-8 expression. CAPE induced HO-1 expression and inhibited IKK2, IκBα, and NF-κB phosphorylation. Blocking HO-1 decreased the anti-inflammatory activity of CAPE. The targeted suppression of IL-8 production using CAPE reduces inflammation and periodontitis.

Environmental lead effects on gene expression in oral epithelial cells.

BACKGROUND AND OBJECTIVE: Host responses in periodontitis span a range of local and emigrating cell types and biomolecules. Accumulating evidence regarding the expression of this disease across the population suggests some component of genetic variation that controls onset and severity of disease, in concert with the qualitative and quantitative parameters of the oral microbiome at sites of disease. However, there remains little information regarding the capacity of accruing environmental stressors or modifiers over a lifespan at both the host genetic and microbial ecology levels to understand fully the population variation in disease. This study evaluated the impact of environmental lead exposure on the responses of oral epithelial cells to challenge with a model pathogenic oral biofilm. METHODS AND RESULTS: Using NanoString technology to quantify gene expression profiles of an array of 511 host response-associated genes in the epithelial cells, we identified an interesting primary panel of basal responses of the cells with numerous genes not previously considered as major response markers for epithelial cells, eg, interleukin (IL)-32, CTNNB1, CD59, MIF, CD44 and CD99. Even high levels of environment lead had little effect on these constitutive responses. Challenge of the cells with the biofilms (Streptococcus gordonii/Fusobacterium nucleatum/Porphyromonas gingivalis) resulted in significant increases in an array of host immune-related genes (134 of 511). The greatest magnitude in differential expression was observed with many genes not previously described as major response genes in epithelial cells, including IL-32, CD44, NFKBIA, CTSC, TNFAIP3, IL-1A, IL-1B, IL-8 and CCL20. The effects of environmental lead on responses to the biofilms were mixed, although levels of IL-8, CCL20 and CD70 were significantly decreased at lead concentrations of 1 and/or 5 µmol/L. CONCLUSION: The results provided new information on a portfolio of genes expressed by oral epithelial cells, targeted substantial increases in an array of immune-related genes post-biofilm challenge, and a focused impact of environmental lead on these induced responses.

Human ß-Defensin 3 Inhibition of P. gingivalis LPS-Induced IL-1ß 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-1ß (IL-1ß) production and cognitive decline. However, little is known about the role of CatB in Pg virulence factor-induced IL-1ß production by microglia. We first subjected IL-1ß-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 ß-defensin 3 (hBD3). IL-1ß 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-1ß production in BV-2 microglia. Although CA-074Me also suppressed OMV-induced IL-1ß 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-1ß production. Interestingly, phagocytosis of OMVs by BV-2 microglia induced IL-1ß 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-1ß without activation of the NLRP3 inflammasome. In contrast, OMVs promote the synthesis and processing of pro-IL-1ß through CatB/CatL-independent phagocytic mechanisms. Thus, hBD3 can improve the IL-1ß-associated vicious inflammatory cycle induced by microglia through inhibition of CatB/CatL.

Knockdown of TRPM2 promotes osteogenic differentiation of human periodontal ligament stem cells by modulating NF-κB /NLRP3 pathway.

Periodontitis is characterized by periodontal destruction triggered by chronic inflammation. The optimal treatment for periodontitis is to improve the periodontal microenvironment, reduce inflammation and achieve periodontal regeneration. Recently, the role of TRPM2 in inflammatory diseases has been reported. However, the function of TRPM2 in periodontal disease and the biological mechanism remain elusive. Therefore, this study aimed to identify the role and explore the underlying mechanisms of TRPM2 in periodontal disease. Here, we first identified the characterization of human periodontal ligament stem cells (PDLSCs). Oil Red O Staining and Alizarin Red mineralized matrix were used to evaluate the multi-differentiation capacity of cells. Flow cytometry was employed to detect MSC-specific surface markers of hPDLSCs. hPDLSCs were treated with 0, 5, 10 or 40 µg/mL of TNF-α for 72 h. Western blot assay were performed to examine the expression of Transient receptor potential cation channel, subfamily M, member 2 (TRPM2) in hPDLSCs. CCK8 and colony formation assays were used to detect the cell viability and proliferation of hPDLSCs, which revealed that TRPM2 knockdown promoted hPDLSCs proliferation. Then, ALP activity in hPDLSCs was detected by ALP activity detection kit. Next, the expression of ALP and Runx2 in hPDLSCs was detected by immunofluorescence staining. The result showed that TRPM2 knockdown promoted osteogenic differentiation and affected the genes expression of osteogenic. Finally, the expressions of p-p65, p65, p-IκBα, IκBα and NLRP3 in hPDLSCs were detected by western blot assay. Together, these results suggested that knockdown of TRPM2 accelerated osteogenic differentiation of hPDLSCs through mediating NF-κB /NLRP3 pathway.

The neutralising and stimulatory effects of antimicrobial peptide LL-37 in human gingival fibroblasts.

OBJECTIVES: To investigate the effects of LL-37, a broad spectrum antimicrobial peptide expressed in periodontal tissues, on human gingival fibroblast responsiveness to microbial challenge and to explore the direct effects of LL-37 on human gingival fibroblasts. DESIGN: The effect of LL-37 on bacterial lipopolysaccharide-induced expression of Interleukin (IL-6) and chemokine C-X-C motif ligand (CXCL) 8 was determined by enzyme linked immunosorbent assay (ELISA). LL-37's influence on bacterial lipopolysaccharide-induced IκBα degradation was investigated by western blot. DNA microarray analysis initially determined the direct effects of LL-37 on gene expression, these findings were subsequently confirmed by quantitative polymerase chain reaction and ELISA analysis of selected genes. RESULTS: Bacterial lipopolysaccharide-induced IL-6 and CXCL8 production by human gingival fibroblasts was significantly reduced in the presence of LL-37 at concentrations in the range of 1-10 µg/ml. LL-37 led to a reduction in lipopolysaccharide-induced IκBα degradation by Escherichia coli lipopolysaccharide and Porphyromonas gingivalis lipopolysaccharide (10 µg/ml). LL-37 (50 µg/ml) significantly altered the gene expression of 367 genes in human gingival fibroblasts by at least 2-fold. CXCL1, CXCL2, CXCL3, Interleukin-24 (IL-24), CXCL8, Chemokine (C-C motif) Ligand 2, and Suppressor of Cytokine Signalling 3 mRNA were significantly upregulated by LL-37. LL-37 also significantly stimulated expression of CXCL8, hepatocyte growth factor and CXCL1 at the protein level. CONCLUSION: LL-37 plays an important regulatory role in the immunomodulatory activity of gingival fibroblasts by inhibiting lipopolysaccharide -induced expression of inflammatory cytokines and directly stimulating the expression of an array of bioactive molecules involved in inflammation and repair.

Effects of nuclear factor-κB signaling pathway on periodontal ligament stem cells under lipopolysaccharide-induced inflammation.

Lipopolysaccharide (LPS) induces inflammatory stress and apoptosis. This study focused on the effect of nuclear factor kappa B (NF-κB) signaling pathway on proliferation and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) after LPS induction and its mechanism. We first isolated hPDLSCs from human tooth root samples in vitro. Then, flow cytometry detected positive expression of cell surface antigens CD146 and STRO-1 and negative expression of CD45, suggesting the hPDLSCs were successfully isolated. LPS significantly induced increased apoptosis and diminished proliferation of hPDLSCs. The NF-κB pathway agonist phorbol 12-myristate 13-acetate (PMA) or p65 overexpression inhibited the proliferation of LPS-treated hPDLSCs and promoted apoptosis. PMA also promoted LPS-induced up-regulation of the expression of inflammatory factors TNF-α and IL-6 and down-regulation of the expression of anti-inflammatory factor IL-10. Additionally, LPS was confirmed to lead to a reduction of alkaline phosphatase (ALP) activity, calcium nodules, and expression of osteogenic markers Runt-related transcription factor 2 (Runx2) and osteopontin. This reduction could be promoted by PMA. Western blotting further indicated that PMA could promote LPS-induced decrease of expression of p65 (cytoplasm), and total cellular proteins IKKα and IKKß in hPDLSCs, while protein expression of p-IκBα (cytoplasm) and p65 (nucleus), and p-IκBα/IκBα ratio was elevated. By contrast, inhibition of the NF-κB pathway (PDTC) or small-interfering RNA targeting NF-κB/p65 (p65 siRNA) showed the opposite results. In conclusion, activation of NF-κB signaling in LPS-induced inflammatory environment can inhibit the proliferation and osteogenic differentiation of hPDLSCs. This study provides a theory foundation for the clinical treatment of periodontitis.

NF-κB inhibition promotes apoptosis in androgen-independent prostate cancer cells by the photothermal effect via the IκBα/AR signaling pathway.

The photothermal response of nanomaterials provides a basis for many biomedical applications, including diagnosis (e.g., biosensor and photoacoustic imaging) and treatment (e.g., drug delivery and photothermal therapy). The use of nano-materials for cancer phototherapy (solid tumor ablation) can cause cell necrosis and apoptosis. However, photothermal effects using the same material can differ among tumor cell types, and the molecular mechanisms underlying these differences are not clear. We used polydopamine (PDA)-coated branched Au-Ag nanoparticles (Au-Ag@PDA NPs) for the photothermal treatment of two prostate cancer cell lines. The therapeutic effect was evaluated by CCK8, flow cytometry, and expression analyses of related genes by western blotting. Photothermal therapy resulted in oxidative stress in prostate cancer cells and activated the mitochondrial-related apoptosis pathway, increasing the Bax expression. In addition, we observed a greater photothermal treatment effect on the androgen-dependent cells LNCaP than the androgen-independent cells DU145. Pretreatment with an inhibitor of the NF-κB signaling pathway (BAY 11-7082) enhanced the expression of BAX in the DU145 cells and increased the sensitivity of the cells to the heat treatment of Au-Ag@PDA NPs both in vitro and in vivo. Our findings explain the differences in the observed effects of photothermal therapy and provide the direction for further improvements to this strategy.

Biofilm-induced profiles of immune response gene expression by oral epithelial cells.

This study examined the oral epithelial immunotranscriptome response patterns modulated by oral bacterial planktonic or biofilm challenge. We assessed gene expression patterns when epithelial cells were challenged with a multispecies biofilm composed of Streptococcus gordonii, Fusobacterium nucleatum, and Porphyromonas gingivalis representing a type of periodontopathic biofilm compared to challenge with the same species of planktonic bacteria. Of the 579 human immunology genes, a substantial signal of the epithelial cells was observed to 181 genes. Biofilm challenged stimulated significant elevations compared to planktonic bacteria for IL32, IL8, CD44, B2M, TGFBI, NFKBIA, IL1B, CD59, IL1A, CCL20 representing the top 10 signals comprising 55% of the overall signal for the epithelial cell responses. Levels of PLAU, CD9, IFITM1, PLAUR, CD24, TNFSF10, and IL1RN were all elevated by each of the planktonic bacterial challenge vs the biofilm responses. While the biofilms up-regulated 123/579 genes (>2-fold), fewer genes were increased by the planktonic species (36 [S gordonii], 30 [F nucleatum], 44 [P gingivalis]). A wide array of immune genes were regulated by oral bacterial challenge of epithelial cells that would be linked to the local activity of innate and adaptive immune response components in the gingival tissues. Incorporating bacterial species into a structured biofilm dramatically altered the number and level of genes expressed. Additionally, a specific set of genes were significantly decreased with the multispecies biofilms suggesting that some epithelial cell biologic pathways are down-regulated when in contact with this type of pathogenic biofilm.

Salmeterol, a Long-Acting ß2-Adrenergic Receptor Agonist, Inhibits Macrophage Activation by Lipopolysaccharide From Porphyromonas gingivalis.

BACKGROUND: Salmeterol is a long-acting ß2-adrenergic receptor agonist used to treat chronic obstructive pulmonary disease. The authors of the current study previously showed that preincubation of primary microglial-enriched cells with salmeterol could inhibit the inflammatory response induced by Escherichia coli lipopolysaccharide (LPS), a Toll-like receptor (TLR)-4 agonist. In this study, the authors sought to determine if salmeterol had a similar inhibitory effect on the inflammatory response of the murine macrophage cell line RAW264.7 and human monocyte THP-1 to LPS from Porphyromonas gingivalis (PgLPS), an oral microbe implicated in the pathogenesis of periodontal disease. METHODS: RAW264.7 and THP-1 cells were pretreated with salmeterol, followed by PgLPS, and monitored for production of inflammatory mediators by enzyme-linked immunosorbent assay. The nitric oxide concentration and nuclear factor-kappa B (NF-κB) activity were measured by Griess method and secretory alkaline phosphatase reporter activity assay, respectively. Reverse-transcriptase polymerase chain reaction and immunoblot analysis were used to measure messenger RNA and protein levels. Nuclear translocation of NF-κB was detected by immunofluorescence. RESULTS: Pretreatment with salmeterol significantly inhibited production of proinflammatory mediators by RAW264.7 and THP-1 cells. Salmeterol downregulated PgLPS-mediated phosphorylation of the extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinase but not p38 mitogen-activated protein kinases (MAPKs). Salmeterol also attenuated activation of NF-κB via inhibition of nuclear translocation of p65-NFκB, the transcriptional activity of NF-κB and IκBα phosphorylation. CONCLUSION: Salmeterol can significantly inhibit activation of macrophage-mediated inflammation by PgLPS, suggesting that use of salmeterol may be an effective treatment in inhibiting or lessening the inflammatory response mediated through TLR pathway activation.