Porphyromonas gingivalis and its lipopolysaccharide differentially regulate the expression of cathepsin B in endothelial cells

Porphyromonas gingivalis infection and cathepsins protease upregulation are independently implicated in atherosclerosis worsening. In this study, we evaluated the effects of P. gingivalis infection and P. gingivalis -purified lipopolysaccharide (Pg-LPS) stimulation on the expression of cathepsin B (CATB) in endothelial cells (ECs). Analysis of the enzymatic activity and expression of CATB were investigated at the messenger RNA, protein and protein-phosphorylation levels. Effects of Toll-like receptors 2 and 4 blocking on CATB activity were also analysed. Our results showed that P. gingivalis and Pg-LPS significantly increased the activity of CATB but with different kinetics. The peak of CATB activity was observed 3 h after P. gingivalis infection but it appeared 48 h after Pg-LPS stimulation. The increase of CATB activity was related to its rapid tyrosine-dephosphorylation during P. gingivalis infection, whereas the levels of CATB messenger RNAs and proteins did not vary after P. gingivalis infection or Pg-LPS stimulation. Inhibition of Toll-like-receptors 2 and 4 differentially decreased P. gingivalis and Pg-LPS CATB activations. These results showed for the first time that P. gingivalis infection rapidly affects ECs and modulates CATB activity, whereas Pg-LPS effects appear to be delayed. This study suggests that direct infection of ECs by P. gingivalis may worsen atherosclerotic plaque formation via activation of the CATB pathway.     

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.     

Interleukin‐1α stimulation in monocytes by periodontal bacteria: antagonistic effects of Porphyromonas gingivalis

Periodontal pathogenic bacteria are associated with elevated levels of interleukin‐1α (IL‐1α) but it is unclear if all species can induce cytokine production equally. Porphyromonas gingivalis may be able antagonize IL‐1α induced by other species through the activity of its proteases or lipopolysaccharide (LPS). Monomac‐6 cells and primary human monocytes were treated with culture supernatants from Porphyromonas gingivalis, Fusobacterium nucleatum, Campylobacter rectus, Actinobacillus actinomycetemcomitans, Prevotella intermedius, Veillonella atypical and Prevotella nigrescens. IL‐1α protein levels were measured after 6 h of incubation. In addition, monocytes were co‐stimulated with supernatants from P. gingivalis and other bacteria. The role of P. gingivalis proteases was tested using Arg‐X and Lys‐X mutant strains. The role of LPS was investigated using purified P. gingivalis LPS and polymixin depletion. All species tested induced significant IL‐1α production, but P. gingivalis was the weakest. Co‐stimulation of monocytes with P. gingivalis antagonized the ability of other bacterial species to induce IL‐1α production. This effect was at its greatest with C. rectus (resulting in a 70% reduction). Gingipain mutant strains and chemical inhibition of protease activity did not reduce antagonistic activity. However, 100 ng/ml of P. gingivalis LPS can reproduce the antagonistic activity of P. gingivalis culture supernatants. Periodontitis‐associated bacterial species stimulate IL‐1α production by monocytes. P. gingivalis can antagonize this effect, and its LPS appears to be the crucial component. This study highlights the importance of mixed infections in the pathogenesis of periodontal disease because reduction of pro‐inflammatory cytokine levels may impair the ability of the host to tackle infection.     

Scavenger receptor A is expressed by macrophages in response to Porphyromonas gingivalis,and participates in TNF‐α expression

Introduction: Porphyromonas gingivalis is a periodontopathic bacterium closely associated with generalized aggressive periodontal disease. Pattern recognition receptors (PRRs) participate in host response to this organism. It is likely that PRRs not previously recognized as part of the host response to P. gingivalis also participate in host response to this organism. Methods and Results: Employing qRT‐PCR, we observed increased msr1 gene expression at 2, 6, and 24 h of culture with P. gingivalis strain 381. Flow cytometry revealed increased surface expression of SR‐A protein by the 24 h time point. Macrophages cultured with an attachment impaired P. gingivalis fimA‐ mutant (DPG3) expressed intermediate levels of SR‐A expression. Heat‐killed P. gingivalis stimulated SR‐A expression similar to live bacteria, and purified P. gingivalis capsular polysaccharide stimulated macrophage SR‐A expression, indicating that live whole organisms are not necessary for SR‐A protein expression in macrophage response. As SR‐A is known to play a role in lipid uptake by macrophages, we tested the ability of low‐density lipoprotein (LDL) to influence the SR‐A response of macrophages to P. gingivalis, and observed no effect of LDL on P. gingivalis‐elicited SR‐A expression. Lastly, we observed that SR‐A knockout (SR‐A^?/?) mouse macrophages produced significantly more tumor necrosis factor (TNF)‐α than wild type mouse macrophages cultured with P. gingivalis. Conclusion: These data identify that SR‐A is expressed by macrophages in response to P. gingivalis, and support that this molecule plays a role in TNF‐α production by macrophages to this organism.     

An acute injection of Porphyromonas gingivalis lipopolysaccharide modulates the OPG/RANKL system and interleukin‐6 in an ovariectomized mouse model

Background/aims: In the present study, we attempted to develop a simulated model to explore the causal effects of periodontal pathogens on skeletal homeostasis in postmenopausal osteoporosis. Methods: Fifty‐three female adult ICR mice were randomly assigned to an experimental group (ovariectomized) or a control group. A single injection of Porphyromonas gingivalis lipopolysaccharide (P. gingivalis‐LPS, ATCC 33277) or Escherichia coli lipopolysaccharide (E. coli‐LPS) was administered intraperitoneally 4 weeks after an ovariectomy. Concentrations of interleukin‐6 (IL‐6), osteoprotegerin (OPG), and the receptor activator of nuclear factor‐κB ligand (RANKL) in serum were subsequently analyzed using an enzyme‐linked immunosorbent assay (ELISA). Results: Under stimulation with P. gingivalis‐LPS or E. coli‐LPS, the concentration of OPG rose in both groups. The serum level of RANKL showed a decreasing trend 24 h after the injection in both groups. After injection of P. gingivalis‐LPS in both the experimental and control animals, the OPG : RANKL ratio increased 24 h after the booster (22.26–620.99, P < 0.05). The serum level of IL‐6 in the experimental group significantly increased 1–6 h after administration of E. coli‐LPS and 1–3 h after administration of P. gingivalis‐LPS (P < 0.05). Conclusions: A single booster injection of P. gingivalis‐LPS induced short‐term changes in OPG, RANKL, and IL‐6 serum levels in this ovariectomized mouse model.     

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.     

Lethal outcome caused by Porphyromonas gingivalis A7436 in a mouse chamber model is associated with elevated titers of host serum interferon‐γ

Background/aims: Septic shock caused by gram‐negative bacteria has been associated with cytokines produced by hosts. Porphyromonas gingivalis A7436, a disseminating strain, caused septic shock‐like symptoms and even animal death in a mouse chamber model. However, P. gingivalis exhibits lower endotoxin activities in its lipopolysaccharide than other typical gram‐negative bacteria. In this study, we examined the effects of P. gingivalis lethal infection on host pro‐inflammatory cytokines production. Methods: Nude and normal BALB/c mice were infected with a lethal dose of P. gingivalis A7436 using a mouse chamber model. Serum levels of tumor necrosis factor, interleukin (IL)‐1β, IL‐12 and interferon‐γ were evaluated. The effects of tumor necrosis factor inhibitor (thalidomide) and anti‐interferon‐γ antibody on infection outcomes were examined. Results: All nude mice survived infectious challenge, whereas 100% of normal mice died with abdominal lesions. Bacterial cultures indicated P. gingivalis dissemination to the circulation. Serum levels of tumor necrosis factor, IL‐1β and IL‐12 showed no significant differences between nude and normal mice. Thalidomide treatment did not protect normal mice from death but decreased remote lesion occurrence, with concurrent reduced bacterial counts recoverable from blood. There was a 3.5‐fold elevation in normal mice serum interferon‐γ titers compared to those of nude mice and anti‐interferon‐γ antibody treatment resulted in 100% protection from lethal outcome. Conclusion: Lethal outcome following P. gingivalis A7436 infection is T‐lymphocyte dependent and involves an increase in systemic interferon‐γ levels. The data further indicate that P. gingivalis transvascular dissemination (bacteremia) alone is not sufficient for lethal outcome.     

Peroxisome proliferator‐activated receptor δ inhibits Porphyromonas gingivalis lipopolysaccharide‐induced activation of matrix metalloproteinase‐2 by downregulating NADPH oxidase 4 in human gingival fibroblasts

We investigated the roles of peroxisome proliferator‐activated receptor δ (PPARδ) in Porphyromonas gingivalis‐derived lipopolysaccharide (Pg‐LPS)‐induced activation of matrix metalloproteinase 2 (MMP‐2). In human gingival fibroblasts (HGFs), activation of PPARδ by GW501516, a specific ligand of PPARδ, inhibited Pg‐LPS‐induced activation of MMP‐2 and generation of reactive oxygen species (ROS), which was associated with reduced expression of NADPH oxidase 4 (Nox4). These effects were significantly smaller in the presence of small interfering RNA targeting PPARδ or the specific PPARδ inhibitor GSK0660, indicating that PPARδ is involved in these events. In addition, modulation of Nox4 expression by small interfering RNA influenced the effect of PPARδ on MMP‐2 activity, suggesting a mechanism in which Nox4‐derived ROS modulates MMP‐2 activity. Furthermore, c‐Jun N‐terminal kinase and p38, but not extracellular signal‐regulated kinase, mediated PPARδ‐dependent inhibition of MMP‐2 activity in HGFs treated with Pg‐LPS. Concomitantly, PPARδ‐mediated inhibition of MMP‐2 activity was associated with the restoration of types I and III collagen to levels approaching those in HGFs not treated with Pg‐LPS. These results indicate that PPARδ‐mediated downregulation of Nox4 modulates cellular redox status, which in turn plays a critical role in extracellular matrix homeostasis through ROS‐dependent regulation of MMP‐2 activity.