Human periodontal ligament cells exhibit no endotoxin tolerance upon stimulation with Porphyromonas gingivalis lipopolysaccharide

Background/Objectives

Endotoxin tolerance is characterized by a state of hyporesponsiveness after confrontation with endotoxins such as lipopolysaccharides (LPS) at low concentrations. The aim of this study was to investigate, whether pretreatment with Porphyromonas gingivalis leads to endotoxin tolerance induction and possible alterations in toll‐like receptor (TLR) 2‐ and 4‐induced response in human periodontal ligament cells (hPDLCs).

Material and Methods

Primary hPDLCs were pretreated with P. gingivalis (0.1 or 0.3 μg/mL) LPS for 24 hours and afterwards treated with one of the following stimuli: P. gingivalis LPS (1 μg/mL); TLR4 agonist Escherichia coli LPS (0.1 μg/mL; 1 μg/mL); TLR2 agonist Pam3CSK4 (0.1 μg/mL; 1 μg/mL). The protein expression of interleukin (IL)‐6, IL‐8 and monocyte chemotactic protein‐1 was analyzed with quantitative polymerase chain reaction and enzyme‐linked immunosorbent assay. Gene expression levels of TLR2 and TLR4 were determined by quantitative polymerase chain reaction.

Results

Pretreatment of cells with low concentrations of P. gingivalis LPS did not result in lower production of IL‐6, IL‐8 and monocyte chemotactic protein‐1 compared to control group. In some cases, pretreated cells exhibited lower gene expression levels of TLR2 and TLR4 compared to non‐pretreated cells.

Conclusion

The results of this study implicate that hPDLCs do not develop endotoxin tolerance. Furthermore, the amplitude of the inflammatory response shows no significant dependency on TLR2 and TLR4 expression levels.     

Interleukin‐1 receptor‐associated kinase‐M in gingival epithelial cells attenuates the inflammatory response elicited by Porphyromonas gingivalis

Takahashi N, Honda T, Domon H, Nakajima T, Tabeta K, Yamazaki K. Interleukin‐1 receptor‐associated kinase‐M in gingival epithelial cells attenuates the inflammatory response elicited by Porphyromonas gingivalis. J Periodont Res 2010; 45: 512–519. © 2010 John Wiley & Sons A/S Background and Objective: Recent studies have revealed that negative regulatory molecules, including interleukin‐1 receptor‐associated kinase‐M (IRAK‐M), control the overactivation of Toll‐like receptor (TLR) signaling. The role of IRAK‐M in human gingival epithelial cells (HGECs), which express TLRs, remains unclear. The present study examined the role of IRAK‐M on interleukin‐8 and macrophage chemoattractant protein‐1 (MCP‐1) expression in HGECs stimulated with Porphyromonas gingivalis and TLR ligands. Material and Methods: Primary HGECs and an SV40 T‐antigen‐immortalized HGEC line (epi 4) were stimulated with live or heat‐killed P. gingivalis, P. gingivalis lipopolysaccharide or the synthetic lipopeptide PAM₃CSK₄, and subsequent expression of IRAK‐M, interleukin‐8 and MCP‐1 was evaluated at the mRNA and protein levels. The effects of IRAK‐M on interleukin‐8 and MCP‐1 expressions were evaluated by IRAK‐M‐specific RNA interference (RNAi)‐based loss‐of‐function assay. Results: All tested stimulants up‐regulated the expression of IRAK‐M in HGECs. The P. gingivalis lipopolysaccharide or PAM₃CSK₄ increased MCP‐1 expression, whereas live P. gingivalis down‐regulated the MCP‐1 expression in HGECs. However, IRAK‐M RNAi increased the expression of MCP‐1 irrespective of up‐ or down‐regulation mediated by the respective stimulants. Interleukin‐8 gene expression, up‐regulated by all tested stimulants, was further enhanced by IRAK‐M RNAi. In contrast, IRAK‐M RNAi had no effect on the interleukin‐8 protein levels, irrespective of the stimulant, indicating that post‐translational modification, not IRAK‐M, controls interleukin‐8 protein expression. Conclusion: Interleukin‐1 receptor‐associated kinase‐M appeared to have distinct regulatory roles on the interleukin‐8 and MCP‐1 produced by HGECs, further suggesting an important role for interleukin‐8 in the immune reponse to periodontopathic bacteria.     

Porphyromonas gingivalis inhibits M2 activation of macrophages by suppressing α‐ketoglutarate production in mice

Reprograming of metabolic pathways is critical in governing the polarization of macrophages into classical proinflammatory M1 or alternative anti‐inflammatory M2 phenotypes in metabolic diseases, such as diabetes. Porphyromonas gingivalis, a keystone pathogen of periodontitis, causes an imbalance in M1/M2 activation, resulting in a hyperinflammatory environment that promotes the pathogenesis of periodontitis. However, whether P. gingivalis infection modulates metabolic pathways to alter macrophage polarization remains unclear. Bone‐marrow‐derived macrophages (BMDMs) were collected from 6‐week‐old female C57BL/6 mice and stimulated with P. gingivalis, P. gingivalis‐derived LPS or IL‐4. Relative gene expression and protein production were measured by quantitative real‐time PCR, RNA sequencing and western blotting. Colorimetric assays were also performed to assess the amounts of α‐ketoglutarate (α‐KG) and succinate. P. gingivalis or P. gingivalis‐derived LPS‐induced inflammatory responses enhanced M1 macrophages and suppressed M2 macrophages, even in the presence of IL‐4. P. gingivalis inhibited Idh1/2 and Gpt1/2 mRNA expression, and increased Akgdh mRNA expression, thus decreasing the ratio of α‐KG/succinate. Supplementation of cell‐permeable dimethyl‐α‐KG dramatically restored M2 activation during P. gingivalis infection. Our study suggests that P. gingivalis maintains a hyperinflammatory state by suppressing the production of α‐KG by M2 macrophages.     

E‐selectin expression induced by Porphyromonas gingivalis in human endothelial cells via nucleotide‐binding oligomerization domain‐like receptors and Toll‐like receptors

Porphyromonas gingivalis, an important periodontal pathogen, has been proved to actively invade cells, induce endothelial cell activation, and promote development of atherosclerosis. Innate immune surveillance, which includes the activity of nucleotide‐binding oligomerization domain (NOD)‐like receptors (NLRs) and Toll‐like receptors (TLRs), are essential for the control of microbial infections; however, the roles of receptor families in P. gingivalis infections remain unclear. Here, we examined the roles of NLRs and TLRs in endothelial cell activation caused by P. gingivalis. Live P. gingivalis and whole cell sonicates were used to stimulate endothelial cells, and both showed upregulation of E‐selectin as well as NOD1, NOD2, and TLR2. In addition, silencing of these genes in endothelial cells infected with P. gingivalis led to a reduction in E‐selectin expression. Porphyromonas gingivalis also induced nuclear factor‐κB (NF‐κB) and P38 mitogen‐activated protein kinase (MAPK) activity in endothelial cells, whereas small interfering RNA targeting NOD1 significantly reduced these signals. Moreover, inhibition of either NOD2 or TLR2 inhibited NF‐κB significantly, but had only a weak inhibitory effect on P38 MAPK signaling. Direct inhibition of NF‐κB and P38 MAPK significantly attenuated E‐selectin expression induced by P. gingivalis in endothelial cells. Taken together, these findings suggest that NOD1, NOD2, and TLR2 play important, non‐redundant roles in endothelial cell activation following P. gingivalis infection.     

A Sialidase‐Deficient Porphyromonas gingivalis Mutant Strain Induces Less Interleukin‐1β and Tumor Necrosis Factor‐α in Epi4 Cells Than W83 Strain Through Regulation of c‐Jun N‐Terminal Kinase Pathway

Background: Porphyromonas gingivalis is one of the major periodontal pathogens. In a previous study, a mouse abscess model showed that sialidase deficiency of P. gingivalis weakened its virulence, but the mechanism behind this observation remains unknown. Methods: A sialidase‐deficient mutant strain (△PG0352) and a complemented strain (com△PG0352) were constructed. Epi4 cells were stimulated by wild‐type strain P. gingivalis W83, △PG0352, or com△PG0352. Real‐time polymerase chain reaction was carried out to detect expression of virulent genes in P. gingivalis and interleukin (IL)‐1β, IL‐6, IL‐8, and tumor necrosis factor (TNF)‐α in epi4 cells. Activities of sialidase, gingipains, and lipopolysaccharide (LPS) were compared among the different P. gingivalis strains. Levels of IL‐1β and TNF‐α in the epi4 cells supernatant were detected by enzyme‐linked immunosorbent assay and levels of p38, extracellular signal‐regulated kinase, c‐Jun N‐terminal kinase (JNK), and phospho‐c‐Jun were detected by western blotting. Results: Compared with P. gingivalis W83 and com△PG0352, activities of Kgp and Rgp gingipains and amount of LPS decreased in △PG0352, whereas there were no differences in LPS activity among these three strains. Level of phospho‐JNK was lower in epi4 cells stimulated by △PG0352. △pG0352 induced less IL‐1β and TNF‐α and more IL‐8 in epi4 cells; differences in IL‐1β and TNF‐α could not be detected after JNK blocking. Conclusion: A sialidase‐deficient P. gingivalis mutant strain induces less IL‐1β and TNF‐α in epi4 cells than W83 strain through regulation of JNK pathway.     

Toll‐Like Receptor 2 Knockdown Modulates Interleukin (IL)‐6 and IL‐8 but not Stromal Derived Factor‐1 (SDF‐1/CXCL12) in Human Periodontal Ligament and Gingival Fibroblasts

Background: Fibroblasts are now seen as active components of the immune response because these cells express Toll‐like receptors (TLRs), recognize pathogen‐associated molecular patterns, and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual inasmuch as different studies have reported that it can be an agonist for Toll‐like receptor 2 (TLR2) and an antagonist or agonist for Toll‐like receptor 4 (TLR4). This study investigates and compares whether signaling through TLR2 or TLR4 could affect the secretion of interleukin (IL)‐6, IL‐8, and stromal derived factor‐1 (SDF‐1/CXCL12) in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). Methods: After small interfering RNA‐mediated silencing of TLR2 and TLR4, HGF and HPDLF from the same donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL‐6, IL‐8, and CXCL12 mRNA expression and protein secretion were evaluated by quantitative polymerase chain reaction and enzyme‐linked immunosorbent assay, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPDLF by all the stimuli applied. Knockdown of TLR2 decreased IL‐6 and IL‐8 in response to P. gingivalis LPS, or Pam2CSK4 and Pam3CSK4, in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. Conclusion: These results suggest that signaling through TLR2 by gingival and periodontal ligament fibroblasts can control the secretion of IL‐6 and IL‐8, which contribute to periodontal pathogenesis, but do not interfere with CXCL12 levels, an important chemokine in the repair process.     

Tumor Necrosis Factor‐α and Porphyromonas gingivalis Lipopolysaccharides Decrease Periostin in Human Periodontal Ligament Fibroblasts

Background: Periostin is a matricellular protein essential for tissue integrity and maturation and is believed to have a key function as a modulator of periodontal ligament (PDL) homeostasis. The aim of this study is to evaluate whether periodontal disease‐associated pathogen‐related virulence factors (endotoxins/lipopolysaccharides [LPS]) and proinflammatory cytokines alter the expression of periostin in PDL cells. Methods: Human PDL cultures were exposed to inflammatory mediators (tumor necrosis factor‐α [TNF‐α]), bacterial virulence factors (Porphyromonas gingivalis LPS) or a combination in a biomechanically challenged environment. Culture conditions were applied for 24 hours, 4 days, and 7 days. Periostin and TGF‐β inducible gene clone H3 (βIGH3) mRNA expression from cell lysates were analyzed. Periostin and βIGH3 proteins were also detected and semiquantified in both cell lysates and cell culture supernatants by Western blot. In addition, periostin localization by immunofluorescence was performed. Analysis of variance and Fisher tests were used to define the statistical differences among groups (P <0.05). Results: In a mechanically challenged environment, periostin protein was more efficiently incorporated into the matrix compared to the non‐loaded controls (higher levels of periostin in the supernatant in the non‐loaded group). Interestingly, chronic exposure to proinflammatory cytokines and/or microbial virulence factors significantly decreased periostin protein levels in the loaded cultures. There was greater variability on βIGH3 levels, and no particular pattern was clearly evident. Conclusions: Inflammatory mediators (TNF‐α) and bacterial virulence factors (P. gingivalis LPS) decrease periostin expression in human PDL fibroblasts. These results support a potential mechanism by which periostin alterations could act as a contributing factor during periodontal disease progression.     

Resveratrol attenuates the pathogenic and inflammatory properties of Porphyromonas gingivalis

Porphyromonas gingivalis has been strongly associated with chronic periodontitis, which affects tooth‐supporting tissues. This Gram‐negative anaerobic bacterium produces a repertoire of virulence factors that modulate tissue destruction directly or indirectly by the induction of inflammatory processes. The aim of this study was to investigate the effects of resveratrol, a major polyphenol found in grapes and wine, on the growth and virulence properties of P. gingivalis as well as on gingival keratinocyte tight junction integrity and the host inflammatory response. Resveratrol exhibited antibacterial activity that may result from damage to the bacterial cell membrane. Resveratrol also killed a pre‐formed P. gingivalis biofilm and reduced bacterial adherence to matrix proteins. In addition, resveratrol had a protective effect on the integrity of the keratinocyte tight junctions by inhibiting its breakdown by P. gingivalis. This may be related to the ability of resveratrol to inhibit the protease activities of P. gingivalis. Lastly, resveratrol reduced P. gingivalis‐mediated activation of the NF‐κB signaling pathway and attenuated TREM‐1 gene expression as well as soluble TREM‐1 secretion in monocytes. The effect on NF‐κB activation likely results from the ability of resveratrol to act as a PPAR‐γ agonist. In summary, the antibacterial, anti‐adherence, and antiprotease properties of resveratrol, as well as its ability to protect the gingival keratinocyte barrier and attenuate the inflammatory response in monocytes suggest that it may be a promising novel therapeutic agent for treating periodontal disease.     

l‐arginine‐dependent nitric oxide production of a murine macrophage‐like RAW 264.7 cell line stimulated with Porphyromonas gingivalis lipopolysaccharide

The aim of this study was to determine nitric oxide (NO) production of a murine macrophage cell line (RAW 264.7 cells) when stimulated with Porphyromonas gingivalis lipopolysaccharides (Pg‐LPS). RAW264.7 cells were incubated with i) various concentrations of Pg‐LPS or Salmonella typhosa LPS (St‐LPS), ii) Pg‐LPS with or without l ‐arginine and/or N^G‐monomethyl‐l ‐arginine (NMMA), an arginine analog or iii) Pg‐LPS and interferon‐γ (IFN‐γ) with or without anti‐IFN‐γ antibodies or interleukin‐10 (IL‐10). Tissue culture supernatants were assayed for NO levels after 24 h in culture. NO was not observed in tissue culture supernatants of RAW 264.7 cells following stimulation with Pg‐LPS, but was observed after stimulation with St‐LPS. Exogenous l ‐arginine restored the ability of Pg‐LPS to induce NO production; however, the increase in NO levels of cells stimulated with Pg‐LPS with exogenous l ‐arginine was abolished by NMMA. IFN‐γ induced independent NO production by Pg‐LPS‐stimulated macrophages and this stimulatory effect of IFN‐γ could be completely suppressed by anti‐IFN‐γ antibodies and IL‐10. These results suggest that Pg‐LPS is able to stimulate NO production in the RAW264.7 macrophage cell model in an l ‐arginine‐dependent mechanism which is itself independent of the action of IFN‐γ.     

Different effects of P. gingivalis LPS and E. coli LPS on the expression of interleukin-6 in human gingival fibroblasts

Objective. Gingival fibroblasts (GFs) produce pro-inflammatory cytokines in response to stimulation with lipopolysaccharide (LPS) of Porphyromonas gingivalis, which is thought to be mediated by activation of toll-like receptors (TLR)2 and TLR4. The present study investigated the expression of interleukin (IL)-6, TLR2, and TLR4 in GFs of seven different donors upon stimulation with P. gingivalis LPS. The effects of P. gingivalis LPS were compared with those of TLR4 agonist Escherichia coli LPS and TLR2 agonist Pam3CSK4. Materials and methods. GFs were stimulated with P. gingivalis LPS, E. coli LPS or Pam3CSK4 and the expression of IL-6, TLR2 and TLR4 was measured by qPCR. The surface expression of TLR2 and TLR4 was measured by flow cytometry. Results. In GFs from three donors, P. gingivalis LPS and Pam3CSK4 induced a markedly lower increase in IL-6 expression than E. coli LPS. This was accompanied by significant down-regulation of the TLR2 and TLR4 expression. In GFs from another four donors, an increase in IL-6 expression upon stimulation with P. gingivalis LPS and Pam3CSK4 was similar or even higher than that induced by E. coli LPS. In GFs of these donors, all stimuli induced an up-regulation of both mRNA and protein expression of TLR2 and did not influence that of TLR4. Conclusions. This study suggests that P. gingivalis LPS and E. coli LPS differently regulate cytokine production in human gingival fibroblasts. Regulation of the expression level of TLR2 and TLR4 by periodontal pathogens might be an important factor controlling the inflammatory response in GFs.     

Moesin‐induced signaling in response to lipopolysaccharide in macrophages

Zawawi KH, Kantarci A, Schulze‐Späte U, Fujita T, Batista EL Jr, Amar S, Van Dyke TE. Moesin‐induced signaling in response to lipopolysaccharide in macrophages. J Periodont Res 2010; 45: 589–601.©2010 John Wiley & Sons A/S Background and Objective: Many physiological and pathophysiological conditions are attributable in part to cytoskeletal regulation of cellular responses to signals. Moesin (membrane‐organizing extension spike protein), an ERM (ezrin, radixin and moesin) family member, is involved in lipopolysaccharide (LPS)‐mediated events in mononuclear phagocytes; however, its role in signaling is not fully understood. The aim of this study was to investigate the LPS‐induced moesin signaling pathways in macrophages. Material and Methods: Macrophages were stimulated with 500 ng/mL LPS in macrophage serum‐free medium. For blocking experiments, cells were pre‐incubated with anti‐moesin antibody. Moesin total protein and phosphorylation were studied with western blotting. Moesin mRNA was assessed using quantitative real‐time PCR. To explore binding of moesin to LPS, native polyacrylamide gel electrophoresis (PAGE) gel shift assay was performed. Moesin immunoprecipitation with CD14, MD‐2 and Toll‐like receptor 4 (TLR4) and co‐immunoprecipitation of MyD88–interleukin‐1 receptor‐associated kinase (IRAK) and IRAK–tumor necrosis factor receptor‐activated factor 6 (TRAF6) were analyzed. Phosphorylation of IRAK and activities of MAPK, nuclear factor κB (NF‐κB) and IκBα were studied. Tumor necrosis factor α, interleukin‐1β and interferon β were measured by ELISA. Results: Moesin was identified as part of a protein cluster that facilitates LPS recognition and results in the expression of proinflammatory cytokines. Lipopolysaccharide stimulates moesin expression and phosphorylation by binding directly to the moesin carboxyl‐terminus. Moesin is temporally associated with TLR4 and MD‐2 after LPS stimulation, while CD14 is continuously bound to moesin. Lipopolysaccharide‐induced signaling is transferred downstream to p38, p44/42 MAPK and NF‐κB activation. Blockage of moesin function interrupts the LPS response through an inhibition of MyD88, IRAK and TRAF6, negatively affecting subsequent activation of the MAP kinases (p38 and ERK), NF‐κB activation and translocation to the nucleus. Conclusion: These results suggest an important role for moesin in the innate immune response and TLR4‐mediated pattern recognition in periodontal disease.     

Hemin binding by Porphyromonas gingivalis strains is dependent on the presence of A‐LPS

Porphyromonas gingivalis is a Gram‐negative black pigmenting anaerobe that is unable to synthesize heme [Fe(II)‐protoporphyrin IX] or hemin [Fe(III)‐protoporphyrin IX‐Cl], which are important growth/virulence factors, and must therefore derive them from the host. Porphyromonas gingivalis expresses several proteinaceous hemin‐binding sites, which are important in the binding/transport of heme/hemin from the host. It also synthesizes several virulence factors, namely cysteine‐proteases Arg‐ and Lys‐gingipains and two lipopolysaccharides (LPS), O‐LPS and A‐LPS. The gingipains are required for the production of the black pigment, μ‐oxo‐bisheme {[Fe(III)PPIX]₂ O}, which is derived from hemoglobin and deposited on the bacterial cell‐surface leading to the characteristic black colonies when grown on blood agar. In this study we investigated the role of LPS in the deposition of μ‐oxo‐bisheme on the cell‐surface. A P. gingivalis mutant defective in the biosynthesis of Arg‐gingipains, namely rgpA/rgpB, produces brown colonies on blood agar and mutants defective in Lys‐gingipain (kgp) and LPS biosynthesis namely porR, waaL, wzy, and pg0129 (α‐1, 3‐mannosyltransferase) produce non‐pigmented colonies. However, only those mutants lacking A‐LPS showed reduced hemin‐binding when cells in suspension were incubated with hemin. Using native, de‐O‐phosphorylated and de‐lipidated LPS from P. gingivalis W50 and porR strains, we demonstrated that hemin‐binding to O‐polysaccharide (PS) and to the lipid A moiety of LPS was reduced compared with hemin‐binding to A‐PS. We conclude that A‐LPS in the outer‐membrane of P. gingivalis serves as a scaffold/anchor for the retention of μ‐oxo‐bisheme on the cell surface and pigmentation is dependent on the presence of A‐LPS.     

DNA from Porphyromonas gingivalis and Tannerella forsythia induce cytokine production in human monocytic cell lines

Toll‐like receptor 9 (TLR9) expression is increased in periodontally diseased tissues compared with healthy sites indicating a possible role of TLR9 and its ligand, bacterial DNA (bDNA), in periodontal disease pathology. Here, we determine the immunostimulatory effects of periodontal bDNA in human monocytic cells (THP‐1). THP‐1 cells were stimulated with DNA of two putative periodontal pathogens: Porphyromonas gingivalis and Tannerella forsythia. The role of TLR9 in periodontal bDNA‐initiated cytokine production was determined either by blocking TLR9 signaling in THP‐1 cells with chloroquine or by measuring IL‐8 production and nuclear factor‐κB (NF‐κB) activation in HEK293 cells stably transfected with human TLR9. Cytokine production (IL‐1β, IL‐6, and TNF‐α) was increased significantly in bDNA‐stimulated cells compared with controls. Chloroquine treatment of THP‐1 cells decreased cytokine production, suggesting that TLR9‐mediated signaling pathways are operant in the recognition of DNA from periodontal pathogens. Compared with native HEK293 cells, TLR9‐transfected cells demonstrated significantly increased IL‐8 production (P < 0.001) and NF‐κB activation in response to bDNA, further confirming the role of TLR9 in periodontal bDNA recognition. The results of PCR arrays demonstrated upregulation of proinflammatory cytokine and NF‐κB genes in response to periodontal bDNA in THP‐1 cells, suggesting that cytokine induction is through NF‐κB activation. Hence, immune responses triggered by periodontal bacterial nucleic acids may contribute to periodontal disease pathology by inducing proinflammatory cytokine production through the TLR9 signaling pathway.     

Synergistic effects of lipopolysaccharides from periodontopathic bacteria on pro‐inflammatory cytokine production in an ex vivo whole blood model

Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia have been strongly associated with chronic periodontitis. This disease is characterized by an accumulation of inflammatory cells in periodontal tissue and subgingival sites. The secretion of high levels of inflammatory cytokines by those cells is believed to contribute to periodontal tissue destruction. The aim of this study was to investigate the inflammatory response of whole blood from periodontitis patients following challenges with whole cells of P. gingivalis, T. denticola, and T. forsythia or their lipopolysaccharides (LPS), individually and in combination. Whole blood collected from seven periodontitis patients was stimulated with whole cells or LPS and the production of interleukin (IL)‐1β, IL‐6, IL‐8, and tumor necrosis factor alpha (TNF‐α) were quantified by enzyme‐linked immunosorbent assays. The mono and mixed challenges with whole bacterial cells or LPS induced the secretion of high amounts of IL‐1β, IL‐6, IL‐8, and TNF‐α by the mixed leukocyte population from periodontitis patients. In addition, P. gingivalis LPS, T. denticola LPS, and T. forsythia LPS acted in synergy to induce high levels of IL‐1β and TNF‐α. This study suggests that P. gingivalis, T. denticola, and T. forsythia may contribute to the immunodestructive host response characteristic of periodontitis through synergistic effects of their LPS on the inflammatory response induced by a mixed population of leukocytes.     

Differential profiles of salivary proteins with affinity to Streptococcus mutans lipoteichoic acid in caries‐free and caries‐positive human subjects

Streptococcus mutans is a representative oral pathogen that causes dental caries and pulpal inflammation. Its lipoteichoic acid (Sm.LTA) is known to be an important cell‐wall virulence factor involved in bacterial adhesion and induction of inflammation. Since Sm.LTA‐binding proteins (Sm.LTA‐BPs) might play an important role in pathogenesis and host immunity, we identified the Sm.LTA‐BPs in the saliva of caries‐free and caries‐positive human subjects using Sm.LTA‐conjugated beads and LTQ‐Orbitrap hybrid Fourier transform mass spectrometry. Sm.LTA was conjugated to N‐hydroxysuccinimidyl‐Sepharose^® 4 Fast Flow beads (Sm.LTA‐beads). Sm.LTA retained its biological properties during conjugation, as determined by the expression of nitric oxide and interferon‐γ‐inducible protein 10 in a murine macrophage cell line and activation of Toll‐like receptor 2 (TLR2) in CHO/CD14/TLR2 cells. Sm.LTA‐BPs were isolated from pooled saliva prepared from 10 caries‐free or caries‐positive human subjects each, electrophoresed to see their differential expression in each group, and further identified by high‐resolution mass spectrometry. A total of 8 and 12 Sm.LTA‐BPs were identified with statistical significance in the pooled saliva from the caries‐free and caries‐positive human subjects, respectively. Unique Sm.LTA‐BPs found in caries‐free saliva included histone H4, profilin‐1 and neutrophil defensin‐1, and those in caries‐positive saliva included cystatin‐C, cystatin‐SN, cystatin‐S, cystatin‐D, lysozyme C, calmodulin‐like protein 3 and β‐actin. The Sm.LTA‐BPs found in both groups were hemoglobin subunits α and β, prolactin‐inducible protein, protein S100‐A9, and SPLUNC2. Collectively, we identified Sm.LTA‐BPs in the saliva of caries‐free and caries‐positive subjects, which exhibit differential protein profiles.