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.     

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.     

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.     

PI3K/Akt mediates expression of TNF‐α mRNA and activation of NF‐κB in calyculin A‐treated primary osteoblasts

Objective: The effect of calyculin A (CA), a serine/threonine protein phosphatase inhibitor, on tumor necrosis factor‐α (TNF‐α) in primary osteoblasts was investigated to determine whether protein phosphatases could affect primary osteoblasts and if so which signaling pathways would be involved. Materials and methods: Primary osteoblasts were prepared from newborn rat calvaria. Cells were treated with 1 nM CA for different time periods. The expressions of TNF‐α and GAPDH mRNA were determined by RT‐PCR. Cell extracts were subjected to SDS‐PAGE and the activation of Akt and NF‐κB were analyzed by western blotting. Results: Calyculin A‐treatment markedly increased the expression of TNF‐α mRNA and enhanced the phosphorylation level of Akt (Ser473) in these cells. Pretreatment with the PI3K inhibitor LY294002 suppressed the increase in TNF‐α mRNA expression and the phosphorylation of Akt in response to CA. Western blot analysis showed that CA stimulated the phosphorylation and nuclear translocation of NF‐κB in primary osteoblasts, and these responses were blocked by pretreatment with LY294002. Conclusion: Calyculin A elicits activation of PI3K/Akt pathway which leads to expression of TNF‐α mRNA and activation of NF‐κB. This NF‐κB activation involves both phosphorylation and nuclear translocation of NF‐κB.     

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.     

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.     

Experience with anti‐TNF‐α therapy for orofacial granulomatosis

J Oral Pathol Med (2011) 40 : 14–19 Background: Orofacial granulomatosis (OFG) can be challenging to treat and experience with anti‐TNF‐α therapy is limited. We report our experience with infliximab (IFX) and adalimumab (ADA) for OFG in 14 patients, the largest reported series to date. Methods: A review of patients receiving induction and maintenance IFX for OFG +/? Crohn’s disease (CD) for active oral disease failing other therapies was performed. Clinical response defined by global physician assessment, aided by oral disease activity scores, was assessed at 2 months, 1 and 2 years. ADA was considered for patients failing IFX. Adverse events were recorded. Predictors of need for anti‐TNF‐α therapy were determined by comparison with OFG patients not requiring anti‐TNF‐α from our overall OFG database (n = 207). Results: Fourteen patients (9 men) were treated with IFX [OFG only (n = 7), OFG with CD (n = 7)]. Nine patients received concomitant immunosuppression. Median duration of treatment was 18 months. Short‐term response was achieved in 10/14 (71%) patients. Eight of 14 (57%) and 4/12 (33%) patients remained responsive at 1 and 2 years, respectively. Two patients who failed IFX responded to ADA. Factors predicting need for anti‐TNF‐α therapy were oral sulcal involvement, intestinal CD and a raised C‐reactive protein (CRP). Oral sulcal involvement predicted response at 1 and 2 years. Intestinal CD did not predict response. The only significant adverse event was an IFX infusion reaction. Conclusion: IFX provided good short‐term response for most OFG patients; however, a significant proportion lost response long term. Adverse events were uncommon. Patients failing IFX may respond to ADA.     

Antigen-specific T-cell receptor Vβ expression in Porphyromonas gingivalis–specific T-cell lines

FACS analysis was used to determine the expression of 15 T-cell receptor Vβ families on CD4 and CD8 cells in Porphyromonas gingivalis—specific T-cell lines established from eight P. gingivalis—positive. adult periodontitis and seven P. gingivalis—positive healthy or gingivitis subjects. All 15 T-cell receptor Vβ families were expressed by the T-cell lines, although a significantly higher proportion of the CD4 cells expressed the 5.2-3 Vβ region compared with the other 14 families, including the 5.3 region, suggesting that it is the 5.2 family which is overexpressed. This was also true for the CD8 cells, with the exception of the 3.1 region in adult periodontitis T-cell lines and the 3.1, 13.1/13.3 and 21.3 regions in healthy or gingivitis lines. Between the two clinical groups, a significantly lower percentage of 13.1/13.3-positive CD8 cells was noted in the adult periodontitis lines compared with the healthy or gingivitis lines. There was a significant reduction in DNA synthesis by the lines in the presence of P. gingivalis outer membrane antigens and fixed irradiated lymphoblastoid cell lines compared with cultures containing untreated irradiated lymphoblastoid cell lines and in cultures containing anti-class II major histocompatibility complex antibody in comparison with all other cultures. The results of this study have shown that P. gingivalis preferentially induces the T-cell receptor Vβ5.2 family on CD4 and CD8 cells in P. gingivalis-specific T-cell lines and that activation of T cells by P. gingivalis outer membrane antigens may be by antigen-specific rather than superantigen activity.     

Lower antibody response to Porphyromonas gingivalis associated with immunoglobulin G Fcgamma receptor IIB polymorphism

Background and Objective: Human FcγRIIB is one of the receptors for immunoglobulin G (IgG) and suppresses the activation of B lymphocytes through cross‐linking with the B cell receptor via immune complexes. This function of FcγRIIB is essential for the negative regulation of antibody production. Our previous study has demonstrated the gene polymorphism FcγRIIB‐I232T to be associated with periodontitis. The polymorphism FcγRIIB‐232T has been reported to inhibit B‐cell antigen receptor signaling more effectively compared to FcγRIIB‐232I, while other groups concluded that FcγRIIB‐232T had no ability to inhibit activatory receptors. In this study, we examined whether FcγRIIB‐I232T polymorphism would change the IgG antibody response to the periodontopathic bacteria Porphyromonas gingivalis. Material and Methods: Forty‐seven patients with periodontitis were genotyped with the direct sequencing of genome DNA. Serum IgG and specific IgG subclass levels for the sonicate of P. gingivalis and the recombinant 40 kDa outer membrane protein (OMP) were determined. Results: No significant difference in the total IgG level and IgG response to P. gingivalis sonicate were observed between sera from FcγRIIB‐232T carriers and non‐carriers. The FcγRIIB‐232T carriers revealed a significantly lower IgG₂ response to P. gingivalis 40 kDa OMP compared to non‐carriers (p = 0.04, Mann–Whitney U‐test). Lower responses of FcγRIIB‐232T carriers were also observed in specific IgG and IgG₁ levels. The FcγRIIB‐232T carriers revealed a low level of IgG₂ response to P. gingivalis 40 kDa OMP, even with a high average probing pocket depth. Conclusion: These results suggest that association of the FcγRIIB‐232T allele with periodontitis might be related to the lower levels of antibody response to P. gingivalis.     

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.