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J Dent Res 86(1):35-40, 2007
© 2007 International and American Associations for Dental Research

RESEARCH REPORT
Biological

Anti-P. gingivalis Response Correlates with Atherosclerosis

P.J. Ford1,*, E. Gemmell1, P. Timms2, A. Chan1, F.M. Preston1, and G.J. Seymour1

1 Oral Biology and Pathology, School of Dentistry, University of Queensland, Brisbane 4072, Australia; and
2 Infectious Diseases Program, Science Research Centre, School of Life Sciences, Queensland University of Technology, Brisbane 4001, Australia

* corresponding author, p.ford{at}uq.edu.au


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Significant associations between atherosclerosis and both Porphyromonas gingivalis, a major periodontopathogen, and the respiratory pathogen, Chlamydia pneumoniae, have been shown. Many individuals with evidence of atherosclerosis demonstrate seropositivity to these pathogens. The aim of the present study was to examine the atherogenic effect of repeated immunizations with either or both of these agents, and to determine if molecular mimicry of bacterial heat-shock protein (HSP), termed GroEL, and host (h) HSP60 was involved. Atherogenesis was examined in apolipoprotein-E-deficient (–/–) mice following intraperitoneal immunizations with P. gingivalis, C. pneumoniae, P. gingivalis, and C. pneumoniae or vehicle. Lesion area in the proximal aorta and levels of serum antibodies to P. gingivalis, C. pneumoniae, and GroEL were measured. The increased pathogen burden of P. gingivalis, but not of C. pneumoniae, enhanced atherosclerosis. hHSP60 was detected in lesions, and in P. gingivalis-immunized mice, lesion development was correlated with anti-GroEL antibody levels, supporting the involvement of molecular mimicry between GroEL and hHSP60.

KEY WORDS: Porphyromonas gingivalis • periodontal disease • antibody response • atherosclerosis • mouse model


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Atherosclerotic cardiovascular disease is the leading cause of death in Western societies. The importance of the role of infection and inflammation in atherosclerosis is now widely accepted. Chronic inflammatory periodontal diseases are among the most common infections, with Porphyromonas gingivalis a major periodontopathogen (Consensus Report for Periodontal Diseases, 1996). Studies have repeatedly shown associations between periodontal disease and cardiovascular disease (DeStefano et al., 1993; Grau et al., 1997; Valtonen, 1999). We have identified P. gingivalis in atherosclerotic plaques and have shown that the prevalence of periodontopathic bacteria in these tissues was higher than that of other bacteria examined (Ford et al., 2005). Chlamydia pneumoniae is a common pathogen of the upper respiratory tract, and an association between infection with this organism and cardiovascular disease has been shown to be strong (Yamashita et al., 1998). Until recently, studies have concentrated on the effect of infection with a single pathogen. It has been postulated (Epstein, 2002) that multiple pathogens are involved, and that "pathogen burden", or the aggregate pathogen load, is a more significant risk factor than any single infection. One mechanism by which infection may enhance atherosclerosis can be explained in terms of the immune response to the bacterial heat-shock protein (HSP), GroEL. HSPs are expressed by cells on exposure to stress. Due to structural similarity or "molecular mimicry" of host (h) HSP60 and GroEL, an immune response generated by the host directed at pathogenic HSP may result in an autoimmune response.

Epidemiological studies have shown an increased risk of atherosclerotic disease with periodontal infection, although this association is not strong (Khader et al., 2004). An inherent problem with human studies is the presence of confounding variables, since periodontitis and cardiovascular disease share common risk factors, such as diet, lifestyle, infection history, and genetic background. Studies with apolipoprotein-E-deficient (apoE) mice have provided support for this association, demonstrating that inoculation with P. gingivalis resulted in enhanced atherosclerosis (Li et al., 2002; Lalla et al., 2003). Additionally, elevated antibody levels to P. gingivalis have been associated with coronary heart disease (Pussinen et al., 2003) and carotid artery stenosis (Taniguchi et al., 2003). In this light, the current study examined the humoral immune response to P. gingivalis in relation to atherosclerosis development in the apoE mouse model. P. gingivalis and C. pneumoniae infections, although located at different sites, contribute to the total pathogen burden. The atherogenic effect of the immune response to multiple pathogens has not previously been examined. The aim of the present study was to determine the effect of immunization with P. gingivalis and C. pneumoniae on antibody responses and the development of atherosclerosis in apoE–/– mice.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacteria
A clinical isolate of C. pneumoniae (strain AO3) was grown in Hep2 cells as previously described (Polkinghorne et al., 2006). Cells were harvested, re-suspended in sucrose phosphate glutamic acid (SPG) medium, and frozen at –70°C until use. P. gingivalis ATCC 33277 was cultured anaerobically as described previously (Bird and Seymour, 1987). Bacteria were harvested from Wilkins-Chalgren anaerobe broth (Oxoid, Basingstoke, Hampshire), re-suspended in phosphate-buffered saline (PBS), and transported in an anaerobic state for injection.

Mice
This project was approved by the University of Queensland animal ethics review committee. Six-week-old male apoE (–/–) mice were obtained from the Animal Resources Centre (Canning Vale, Australia) and fed regular chow.

Immunization Procedure
Ten groups of mice (6/group) each received a different intraperitoneal immunization regime (TableGo). Injections were weekly and consisted of viable P. gingivalis (108 organisms/100 µL/mouse) or C. pneumoniae (108 IFU/100 µL/mouse). As controls, groups of mice received injections of vehicle (sterile PBS or uninfected Hep2 cells diluted in SPG [100 µL/mouse], respectively). Mice were killed at 18 wks of age (after 11 injections) or at 34 wks of age (after 22 injections). Mice were anesthetized with ketamine (600 µg/mouse) and xylazine (3 mg/mouse) delivered intraperitoneally. Blood was collected from the left ventricle, then the heart was perfused with PBS. The contents of the chest cavity were removed and immersed in 2% neutral buffered formalin for 16 hrs at 4°C, then in 30% sucrose in PBS at 4°C for 2 days. The tissue was embedded in Tissue Tek® OCT (Optimal Cutting Temperature) embedding medium (Sakura Finetek U.S.A., Inc., Torrance, CA, USA), quenched, and stored in liquid nitrogen.


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  		Table. Intraperitoneal Immunization Regimes
 
Assessment of Atherosclerotic Lesion Area
Frozen sections of the proximal aorta were prepared (Paigen et al., 1987). Briefly, 5 10-µm-thick sections per mouse, each separated by 100 µm, were stained with Oil red O (Sigma-Aldrich, St. Louis, MO, USA) and counterstained with hematoxylin. Intermediate sections were retained for immunohistological analysis. Sections were evaluated quantitatively for lesion area by means of an image analysis program (Spot, Diagnostic Instruments, Sterling Heights, MI, USA). Total lesion area per section was determined, and mean lesion area per section per mouse calculated.

Serum Antibody Levels
Serum levels of anti-P. gingivalis, anti-C. pneumoniae, and anti-GroEL antibodies were measured by an ELISA technique (Gemmell et al., 2000, 2002a). Briefly, P. gingivalis ATCC 33277 (5 µg/mL), C. pneumoniae AO3 (5 µg/mL), or recombinant (r) GroEL (purified from Escherichia coli, Stressgen Biotechnologies Corporation, Victoria, Canada) (2 µg/mL) was coated onto 96-well plates (Maxisorb Immunoplates, Nunc, Roskilde, Denmark). Diluted serum samples were added, followed by peroxidase-conjugated sheep anti-mouse IgG (The Binding Site, Birmingham, UK). Substrate containing 0.0075% H2O2 and 2.5 mM O’tolidine (Eastman Kodak, Rochester, NY, USA) was added, the reaction stopped after 10 min with 3 M HCl, and the optical density of the wells read at an absorbance of 450 and 655 nm. Antibody levels were determined from a standard curve of dilutions of normal mouse IgG (Caltag Laboratories, Burlingame, CA, USA) coated onto plates. Wells with PBS in place of serum samples were used to determine background values.

Identification of Infiltrating Cells
CD4+ and CD8+ T-cell subsets, Mac-3+ macrophages, and CD19+ B-cells were labeled according to an immunoperoxidase method. Following antigen retrieval (CD4, 1 mM EDTA, pH 8–9; CD8, CD19, 10 mM citrate buffer, pH 6 for 15 min, followed by heating at 85–90 C for 15 min), sections were incubated with the following primary monoclonal antibodies: FITC-conjugated rat anti-mouse CD4 and CD8 and rat anti-mouse Mac-3 and CD19 (1:50) (Pharmingen, San Diego, CA, USA). This was followed by application of the Super PicTure Polymer Detection Kit (Zymed, Invitrogen, Carlsbad, CA, USA). Nuclei were counterstained with methyl green. Thymus within the sections was used as positive controls. PBS in place of the primary antibody acted as negative controls.

Identification of HSP60
HSP60 present in aortic sections was labeled by an avidin-biotin immunoperoxidase method (Gemmell et al., 2002b), with mouse anti-mammalian HSP60 (1:50) (LK-1 clone, Stressgen Biotechnologies Corp., Victoria, Canada). Sections were incubated with the primary antibody, biotinylated goat anti-mouse immunoglobulins (Zymed), and then streptavidin peroxidase (Zymed). The peroxidase was developed in a liquid DAB substrate-chromagen system (Zymed). Nuclei were counterstained with methyl green.

Statistical Analysis
Multivariate analysis of variance with the general linear model was used to test for differences in the lesion areas and serum antibody levels of the different groups. Pairs of groups were tested for significance by the Student’s t test. The association between levels of serum antibodies and lesion area was measured by the Pearson correlation coefficient, and the statistical significance of the correlation was determined from exact tables (Snedecor and Cochran, 1967). We used the Minitab statistical package (Minitab Inc., State College, PA, USA) to perform the analyses.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Assessment of Atherosclerotic Lesion Area
After 11 weekly injections, mice immunized with C. pneumoniae demonstrated increased atherogenesis compared with those immunized with P. gingivalis or vehicle (p < 0.001). P. gingivalis-immunized mice showed no significant lesions at this stage. After 22 weekly immunizations, however, atherosclerotic lesions were greater in mice immunized with P. gingivalis alone, compared with those mice immunized with P. gingivalis followed by C. pneumoniae (p < 0.043), with those immunized with either bacterium followed by vehicle (p < 0.036), or with controls (p < 0.001). All immunized mice developed lesions greater than those of the controls (p < 0.05) (Figs 1Go, 2Go).


Figure 1
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  		Figure 1. Mice immunized with P. gingivalis (A) and C. pneumoniae (B) demonstrated marked atherosclerotic lesions in the proximal aorta, as observed with Oil red O staining, compared with control mice (C) after 26 wks of immunizations. Dark-staining areas in the wall of the aorta are lipid-filled atherosclerotic lesions. Cross-sections of the proximal aorta show atherosclerotic lesions dominated by Mac-3 staining cells (D). Endothelial cells and some lesional cells stain positively for HSP60 (E). Original magnification 40X. Scale shown represents 100 µm.

 

Figure 2
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  		Figure 2. Mean cross-sectional atherosclerotic lesion area of the proximal aorta of mice immunized with P. gingivalis (Pg), C. pneumoniae (Cp), or vehicle (v). Results are mean ± SEM of 6 mice per group. *P < 0.001; **P < 0.036; ***P < 0.043.

 
Serum Antibody Levels
Elevated anti-P. gingivalis IgG serum antibody levels were demonstrated in all P. gingivalis-immunized mice compared with control mice (p < 0.001). Mice that had received 22 immunizations of P. gingivalis had significantly higher levels of specific antibody than did all other groups (p < 0.001). Mice receiving both bacteria, however, showed a reduced antibody response compared with those immunized with P. gingivalis alone (p < 0.001). This response was further reduced if P. gingivalis was followed by C. pneumoniae, compared with C. pneumoniae followed by P. gingivalis (p < 0.008) (Fig. 3AGo).


Figure 3
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  		Figure 3. Serum antibody levels of immunized mice. (A) Anti-P. gingivalis; (B) anti-C. pneumoniae; and (C) anti-GroEL serum IgG antibody levels of mice immunized with P. gingivalis (Pg), C. pneumoniae (Cp), or vehicle (v). Results are mean ± SEM of 6 mice per group.

 
Generally, production of anti-C. pneumoniae IgG antibodies was less than for anti-P. gingivalis IgG antibodies. These antibodies were elevated in all mice immunized with C. pneumoniae only (p < 0.001), as well as in mice immunized with both bacteria (p < 0.036), compared with controls. However, reduced levels of specific antibody were demonstrated if C. pneumoniae was followed by P. gingivalis compared with C. pneumoniae only (p < 0.001). No significant reduction occurred if C. pneumoniae followed P. gingivalis (Fig. 3BGo).

Production of anti-GroEL IgG antibodies was lower than for antibodies to the whole organisms. In general, higher levels of anti-GroEL antibodies were observed in mice immunized with P. gingivalis compared with C. pneumoniae. After 11 wks of immunizations, immunized mice demonstrated anti-GroEL antibody levels higher than those of control mice (p < 0.007), and for P. gingivalis-immunized mice, these levels were greater than for those immunized with C. pneumoniae (p < 0.049). After 22 injections of P. gingivalis, levels of anti-GroEL antibodies were increased compared with those in mice immunized 11 times (p < 0.001). This was not so for C. pneumoniae. Co-immunization inhibited levels of anti-GroEL antibodies compared with P. gingivalis alone, but only if P. gingivalis was given first (p < 0.044) (Fig. 3CGo).

Association of Serum Antibody Levels and Lesion Size
The association of the antibody response of mice killed after 22 injections with lesion surface area was measured. This was found to be significant for mice immunized with P. gingivalis alone (Groups 2, 5, and 7 analyzed), but not for mice immunized with C. pneumoniae alone (Groups 3, 6, and 7 analyzed). For P. gingivalis-immunized groups, levels of anti-P. gingivalis antibodies as well as levels of anti-GroEL antibodies were correlated with lesion surface area (r = 0.96, p < 0.05; r = 0.97, p < 0.05, respectively).

Immunohistology
The atherosclerotic lesions were macrophage/foam-cell-dominated, with most lipid deposits being intracellular. There was an inflammatory cell infiltrate composed almost entirely of macrophages, with very few CD4-, CD8-, or CD19-positive cells observed (Fig. 1DGo). HSP60 was expressed by endothelial cells and some inflammatory cells (Fig. 1EGo). This was observed in both immunized and control mice.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of the present study show that increasing the pathogen burden of P. gingivalis enhanced atherosclerosis. This was not shown for C. pneumoniae. It was also demonstrated that atherosclerotic lesions developed later with P. gingivalis immunization compared with C. pneumoniae. After 22 weekly injections of either bacterium, however, lesions were of comparable size. Interestingly, when P. gingivalis immunization was followed by C. pneumoniae, there was reduced enhancement of disease compared with immunization with P. gingivalis only. This showed that a pre-existing exposure to P. gingivalis altered the atherogenic effect of C. pneumoniae. Therefore, as well as pathogen burden, the net response to both pathogens determined the progression of atherosclerotic disease.

The response of co-immunized mice was inhibited compared with that of mice immunized with P. gingivalis only. These mice also demonstrated reduced atherosclerotic lesion development, suggesting a role for the anti-P. gingivalis immune response in atherogenesis. Increased total burden of P. gingivalis, therefore, enhanced specific antibody production and atherosclerosis. When a C. pneumoniae immune response was superimposed on an existing P. gingivalis response, however, anti-P. gingivalis antibody production and atherosclerosis were inhibited. The antibody response has previously been shown to be modulated by co-immunization with P. gingivalis and F. nucleatum in mice (Gemmell et al., 2004). Exposure to P. gingivalis in the presence of an anti-C. pneumoniae response caused an inhibition of anti-C. pneumoniae antibody production, but this was not associated with reduced atherosclerosis. These results show that the immune response to multiple pathogens appears to be much more complex than simply the sum of the responses to the separate pathogens.

As expected, antibodies specific for a single antigen, GroEL, were produced at lower levels than those specific for the whole bacteria. After 11 injections, there was a significant anti-GroEL antibody response. An increasing pathogen burden of P. gingivalis, but not of C. pneumoniae, enhanced this response. Similarly to the anti-P. gingivalis antibody response, co-immunization with P. gingivalis given before C. pneumoniae inhibited anti-GroEL antibodies compared with P. gingivalis alone, and this group also exhibited reduced atherosclerotic lesion development. Overall, the anti-GroEL antibody response was stronger in P. gingivalis- than in C. pneumoniae-immunized mice, suggesting that GroEL may be more important in the humoral response to P. gingivalis than to C. pneumoniae.

The results of the present study show that increasing the total burden of the periodontopathogen P. gingivalis caused enhanced atherosclerosis, and that the addition of another pathogen, C. pneumoniae, altered the immune response and net atherosclerotic effect. Further, the increased production of anti-GroEL antibodies with increasing P. gingivalis burden was associated with atherosclerosis severity. These results, along with the expression of hHSP60 by cells of the lesion, support the hypothesis of molecular mimicry as a mechanism involved. Interestingly, the antibody response elicited by P. gingivalis, but not by C. pneumoniae, was correlated with atherosclerosis progression. It is possible that molecular mimicry involving C. pneumoniae GroEL occurs; however, as an obligate intracellular organism, cellular rather than humoral responses could be involved. A comparison of amino acid sequences (GenBank®) reveals very similar homologies between P. gingivalis and C. pneumoniae GroEL with human HSP60 (around 50%). More important than sequence, however, would be determination of the cross-reactive epitopes of the proteins; clearly, further work is required in terms of mapping these.

Molecular mimicry may be one of several mechanisms that could occur in pathogen-induced atherosclerosis. A recent study showed up-regulation of innate immune markers, including Toll-like receptor 2 (TLR2) and TLR4, in aortic tissue soon after oral inoculation of apoE mice with P. gingivalis (Miyamoto et al., 2006). Ligation of TLR by products such as bacterial LPS, fimbriae, and both human and bacterial HSPs initiates signal transduction pathways, leading to enhanced innate inflammatory responses (reviewed in Gibson et al., 2006). TLR4 expression is enhanced by the presence of oxidized low-density lipoprotein (Xu et al., 2001), suggesting that inflammation due to pathogens may act synergistically with hypercholesterolemia to promote atherosclerosis. It has also been reported that direct bacterial invasion of the arterial wall could be a mechanism for atherosclerosis development, since wild-type P. gingivalis, but not a fimbriae-deficient mutant strain, up-regulated aortic TLR2 and TLR4 expression and accelerated atherosclerosis in apoE mice (Gibson et al., 2004).

We have shown that a high pathogen load of P. gingivalis resulted in a marked antibody response, including to GroEL, as well as increased atherosclerosis, compared with a lower pathogen load in this model. Treatment of periodontal disease, which is a means of lowering P. gingivalis load, has been shown to improve endothelial function in humans. albeit without documented cardiovascular disease (Mercanoglu et al., 2004). Establishment of a role for P. gingivalis in human atherosclerosis would provide a basis for targeting individuals with cardiovascular risk for mechanical periodontal treatment and possibly antibiotic therapy. Further mechanistic studies are urgently required.


   ACKNOWLEDGMENTS
 
This work was supported by the Australian Dental Research Foundation.

Received April 27, 2006; Last revision October 4, 2006; Accepted October 4, 2006


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bird PS, Seymour GJ (1987). Production of monoclonal antibodies that recognize specific and cross-reactive antigens of Fusobacterium nucleatum. Infect Immun 55:771–777.[Abstract/Free Full Text]

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Ford PJ, Gemmell E, Hamlet SM, Hasan A, Walker PJ, West MJ, et al. (2005). Cross-reactivity of GroEL antibodies with human heat shock protein 60 and quantification of pathogens in atherosclerosis. Oral Microbiol Immunol 20:296–302.[ISI][Medline]

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