Porphyromonas gingivalis induces autophagy in THP‐1‐derived macrophages

Autophagy provides a mechanism for the turnover of cellular organelles and proteins through a lysosome‐dependent degradation pathway and is a possible mechanism in inflammatory disease. Periodontitis is an inflammatory disease caused by periodontal pathogens. Porphyromonas gingivalis, an important periodontal pathogen, activates cellular autophagy to provide a replicative niche while suppressing apoptosis in endothelial cells. However, the molecular basis for a causal relationship between P. gingivalis and autophagy is unclear. This research examines the involvement of P. gingivalis in autophagy through light chain 3 (LC3) and autophagic proteins, and the role of P. gingivalis‐induced autophagy in the clearance of P. gingivalis and inflammation. To investigate the molecular mechanism of autophagy induced by P. gingivalis, PMA‐differentiated THP‐1‐derived macrophages were infected with live P. gingivalis. The P. gingivalis increased the formation of autophagosomes in a multiplicity of infection‐dependent manner, as well as autophagolysosomes. Porphyromonas gingivalis activated LC3‐I/LC3‐II conversion and increased the conjugation of autophagy‐related 5 (ATG5) –ATG12 and the expression of Beclin1. The expressions of Beclin1, ATG5–ATG12 conjugate, and LC3‐II were significantly inhibited by the presence of 3‐methyladenine, an autophagy inhibitor. Interestingly, 3‐methyladenine increased the survival of P. gingivalis and proinflammatory cytokine interleukin‐1β production. The data indicate that P. gingivalis induces autophagy in PMA‐differentiated THP‐1‐derived macrophages and in turn, macrophages eliminate P. gingivalis through an autophagic response, which can lead to the restriction of an excessive inflammatory response by downregulating interleukin‐1β production. The induction of autophagy by P. gingivalis may play an important role in the periodontal inflammatory process and serve as a target for the development of new therapies.     

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.     

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.     

Mechanism and implications of CXCR4‐mediated integrin activation by Porphyromonas gingivalis

In monocytes and macrophages, the interaction of Porphyromonas gingivalis with Toll‐like receptor 2 (TLR2) leads to the activation of a MyD88‐dependent antimicrobial pathway and a phosphatidylinositol‐3 kinase (PI3K) ‐dependent pro‐adhesive pathway, which activates the β₂‐integrin complement receptor 3 (CR3). By means of its fimbriae, P. gingivalis binds CXC‐chemokine receptor 4 (CXCR4) and induces crosstalk with TLR2 that inhibits the MyD88‐dependent antimicrobial pathway. In this paper, we investigated the impact of the P. gingivalis‐CXCR4 interaction on the pro‐adhesive pathway. Using human monocytes, mouse macrophages, or receptor‐transfected cell lines, we showed that the binding of P. gingivalis fimbriae to CXCR4 induces CR3 activation via PI3K, albeit in a TLR2‐independent manner. An isogenic strain of P. gingivalis expressing mutant fimbriae that do not interact with CXCR4 failed to efficiently activate CR3, leading to enhanced susceptibility to killing in vivo compared with the wild‐type organism. This in vivo observation is consistent with previous findings that activated CR3 mediates safe entry of P. gingivalis into macrophages. Taken together with our previous work, these results indicate that the interaction of P. gingivalis with CXCR4 leads to inhibition of antimicrobial responses and enhancement of pro‐adhesive responses, thereby maximizing its adaptive fitness in the mammalian host.     

Effects of interleukin‐1β and tumor necrosis factor‐α on macrophage inflammatory protein‐3α production in synovial fibroblast‐like cells from human temporomandibular joints

Background

Interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α) are key mediators of the intracapsular pathological conditions of the temporomandibular joint (TMJ). Therefore, the gene expression profiles in synovial fibroblast‐like cells (SFCs) from patients with internal derangement of the TMJ were examined after they were stimulated with IL‐1β or TNF‐α to determine which genes were altered.

Methods

Ribonucleic acid was isolated from SFCs after IL‐1β or TNF‐α treatment. Gene expression profiling was performed using oligonucleotide microarray analysis. On the basis of the results of this assay, we investigated the kinetics of macrophage inflammatory protein‐3α (MIP‐3α) gene expression using PCR, and protein production in TMJ SFCs stimulated by IL‐1β or TNF‐α using an ELISA. Inhibition experiments were performed with MAPK and NFκB inhibitors. SFCs were stimulated with IL‐1β or TNF‐α after treatment with inhibitors. The MIP‐3α levels were measured using an ELISA.

Results

Macrophage inflammatory protein‐3α was the gene most upregulated by IL‐1β‐ or TNF‐α stimulation. The mRNA and protein levels of MIP‐3α increased in response to IL‐1β in a time‐dependent manner. In contrast, during TNF‐α stimulation, the MIP‐3α mRNA levels peaked at 4 h, and the protein levels peaked at 8 h. In addition, the IL‐1β‐ and TNF‐α‐stimulated MIP‐3α production was potently reduced by the MAPK and NFκB signaling pathway inhibitors.

Conclusion

Interleukin‐1β and TNF‐α increased the MIP‐3α production in SFCs via the MAPK and NFκB pathways. These results suggest that the production of MIP‐3α from stimulation with IL‐1β or TNF‐α is one factor associated with the inflammatory progression of the internal derangement of the TMJ.     

Immunoprotective effects of a hemin‐binding peptide derived from hemagglutinin‐2 against infection with Porphyromonas gingivalis

The principal etiologic agent in periodontal disease, Porphyromonas gingivalis, generates cysteine proteases that bind heme with domains such as hemagglutinin‐2 (HA2). High‐affinity HA2–hemin binding supplies the porphyrin and ferric iron needed for growth and virulence. The DHYAVMISK peptide, recently identified at the hemin‐binding site of HA2, inhibits hemin binding. We now evaluate the protective effect of vaccination with DGFPGDHYAVMISK (termed DK) against P. gingivalis using a rat infection model. Rats immunized with DK generated anti‐peptide serum IgGs and salivary sIgAs (as measured by ELISA). In a subcutaneous abscess model, the protective effect of immunization was then investigated by measuring abscess size following subcutaneous injection with P. gingivalis. In an oral infection model, a ligature inoculated with P. gingivalis was used to induce periodontitis. The degree of bone erosion, ordinarily provoked by infection, was then evaluated by micro‐computed tomography. We found that anti‐peptide antibody titers of serum IgGs and salivary sIgAs for rats immunized with DK and adjuvant were significantly higher than for sham‐immunized rats (injected with adjuvant/PBS alone; P < .05). In the subcutaneous abscess model, the DK + adjuvant‐vaccinated rats recovered faster than sham‐vaccinated animals, with their abscess sizes significantly smaller (P < .05). Further, in the experimental periodontitis model, bone loss at the molar palatal side for DK + adjuvant‐vaccinated rats was significantly lower than for sham‐vaccinated animals (P < .05). Collectively, these data demonstrate the potential of (DK) peptide immunization in terms of eliciting an immunoprotective effect against infection with P. gingivalis.     

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.     

Immunization by Arg‐gingipain A DNA vaccine protects mice against an invasive Porphyromonas gingivalis infection through regulation of interferon‐γ production

We previously demonstrated that a Porphyromonas gingivalis rgpA DNA vaccine induced protective immune responses against P. gingivalis infection in mice (Yonezawa et al. Infect Immun 2001: 69: 2858–2864). In the present study, reduction in lethality against infection by lethal doses of P. gingivalis was observed in the rgpA DNA vaccine‐immunized mice. Cytokine levels in the mouse model with nonlethal doses of infection by P. gingivalis were evaluated to analyze the mechanism of protection by immunization with the rgpA DNA vaccine. After nonlethal challenge with invasive P. gingivalis W50, production of interleukin (IL)‐2, IL‐4, IL‐5 and IL‐12 was elevated; however, interferon (IFN)‐γ was lower in the serum of the DNA vaccine‐immunized mice than in the serum of nonimmunized mice. The regulation of IFN‐γ production elicited by immunization with the rgpA DNA vaccine may play a significant role in protection against P. gingivalis infection in mice.     

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.