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Infection and Immunity, December 2005, p. 8050-8059, Vol. 73, No. 12
0019-9567/05/$08.00+0 doi:10.1128/IAI.73.12.8050-8059.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Department of Medicine, Section of Infectious Diseases,1 Department of Microbiology, Boston University School of Medicine,6 Department of Periodontology and Oral Biology, Goldman School of Dental Medicine, Boston University, Boston, Massachusetts 02118,5 Department of Conservative Dentistry, Tokushima University School of Dentistry, Tokushima 770-8504,2 Department of Oral Microbiology, Kanagawa Dental College, Yokosuka 238-9580, Japan,4 School of Dentistry, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan3
Received 22 April 2005/ Returned for modification 8 August 2005/ Accepted 21 September 2005
| ABSTRACT |
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| INTRODUCTION |
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The association between human periodontal disease, a chronic bacterial infection of the tissue that supports the teeth, and atherosclerosis has been suggested on the basis of epidemiological studies (1, 2, 16, 19, 26, 29, 30). Porphyromonas gingivalis, the primary infectious agent of adult periodontal disease, possesses a broad array of virulence factors, including LPS, hemagglutinins, proteases, capsular polysaccharide, and two types of fimbriae (41-kDa major fimbria and 67-kDa minor fimbria). In recent animal experimental studies, other groups have demonstrated that infection of heterozygous apolipoprotein E (ApoE) knockout mice with P. gingivalis increased the mean area and extent of atherosclerotic lesions histologically relative to those in uninfected animals (7) and accelerated the progression of atherosclerosis (27). We also demonstrated that ApoE knockout (ApoE/) mice challenged with wild-type (WT) P. gingivalis presented with increased atherosclerotic plaque and expressed TLR2 and -4 in aortic tissue (15). Despite early detection of an invasion-impaired P. gingivalis fimbria-deficient mutant (FimA) in the blood and in aortic arch tissue, ApoE/ mice challenged with the FimA mutant did not present with up-regulation of TLR expression or accelerated atherosclerosis (15).
The endothelium, a continuous cellular monolayer lining the blood cells, has an enormous range of important homeostatic roles (42). When this homeostatic balance is disturbed, endothelial dysfunction develops and may contribute to the pathogenesis of atherosclerosis (42). We and others have previously demonstrated that P. gingivalis can actively invade aortic, heart, and vein endothelial cells and coronary artery smooth muscle cells (8-10) and that fimbriae are required for this invasion process. However, it is not clear how P. gingivalis infection of endothelial cells modulates the inflammatory response of these cells. In this study, we examined the expression of TLRs and chemokines in endothelial cells in response to P. gingivalis infection and determined the functional activity of TLRs on human aortic endothelial cells (HAEC). Our results indicate that invasive P. gingivalis bacteria, but not purified outer cell membrane components, up-regulate TLR expression on HAEC and stimulate HAEC to respond to TLR2- and -4-specific ligands.
| MATERIALS AND METHODS |
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Bacterial strains and growth conditions. P. gingivalis WT strains 381 and 33277 were maintained on anaerobic blood agar plates (BBL media; Becton Dickinson Co., Cockeysville, Md.). The P. gingivalis fimA (major 41-kDa fimbria) mutant (DPG3) (31) and a 67-kDa minor fimbrial mutant (MF1) (Y. Takahashi et al., unpublished data) were constructed in WT strain 381. P. gingivalis fimbrial mutants (DPG3 and MF1) were cultured in the presence of erythromycin (10 µg/ml) and tetracycline (1 µg/ml) as required, respectively. All bacteria were grown at 37°C in an anaerobic environment containing 85% N2, 5% H2, and 10% CO2 for 3 to 5 days. At 24 h prior to infection assays, P. gingivalis was transferred from plates into brain heart infusion broth (Difco, Detroit, Mich.) containing 0.5% yeast extract (Difco), 10 µg/ml hemin, 1 µg/ml vitamin K, and antibiotics as necessary and grown until the optical density at 660 nm reached 1.0.
Primary HAEC cultures. Primary HAEC (Cascade Biologics, Portland, Oreg.) were maintained and grown in M200 supplemented with low-serum growth supplement (Cascade Biologics) at 20 µl/ml at 37°C in 5% CO2 in tissue culture flasks. Confluent second- to fourth-passage cells were used in all experiments. For infection studies, HAEC were plated at concentrations of 6 x 105 to 8 x 105 and 1.25 x 105 to 1.66 x 105 cells/well in 6- and 24-well flat-bottom plates, respectively.
Preparation of heat-killed P. gingivalis and P. gingivalis fimbriae and LPS. Heat-killed P. gingivalis was prepared by heating a bacterial suspension for 10 min at 60°C. Purification of the major fimbrial protein of P. gingivalis strain MF1, the minor fimbrial protein of P. gingivalis strain DPG3, and whole native fimbrial protein (major and minor fimbriae) of P. gingivalis strain 33277 was done by a modification of procedures described previously (41). The fimbrial preparations were analyzed for LPS contamination by electrophoresis by loading polyacrylamide gels stained with silver nitrate, and contamination of LPS was not detected in these preparations. P. gingivalis LPS was prepared by the hot phenol-water technique (11, 44). LPS preparations were analyzed for protein contamination by a bicinchoninic acid protein assay kit (Pierce, Rockford, Ill.) and by electrophoresis by overloading polyacrylamide gels and staining them with Coomassie blue or silver nitrate.
Invasion of HAEC by P. gingivalis. P. gingivalis invasion of HAEC monolayers was quantified by determining the number of CFU recovered following metronidazole treatment as described previously (36). To examine the effects of invasion on TLR expression in response to live invasive P. gingivalis, we preincubated HAEC with cytochalasin D (1 µg/ml in dimethyl sulfoxide [DMSO]), an inhibitor of actin polymerization, for 1 h as described previously (8). In preliminary experiments, cytochalasin D was assessed for toxicity for both P. gingivalis and HAEC viability and found to have no adverse effects at concentrations of up to 5 µg/ml. The multiplicity of infection (MOI) was calculated based on the number of endothelial cells per well in six-well flat-bottom plates at confluence.
Fluorescence-activated cell sorter analysis. Confluent endothelial cell monolayers incubated with P. gingivalis cells, purified fimbriae, or purified LPS were incubated with anti-human TLR2, -3, -4, -6, and -9 mouse monoclonal antibodies or isotype-matched immunoglobulin G (IgG; 5 µg/ml) in accordance with the manufacturer's instructions and labeled with fluorescein isothiocyanate-labeled goat anti-mouse IgG (1:100 dilution), and 10,000 events were analyzed by flow cytometry with a FACScan flow cytometer (Becton Dickinson). The viability and integrity of HAEC after infection were confirmed by the trypan blue exclusion method and microscopic morphological observation, as well as side and forward scatter signal determination by FACScan (data not shown).
TLR functional assay. P. gingivalis were added to confluent HAEC monolayers in 24-well culture plates at an MOI of 100 and incubated for 5 h. Nonadherent bacteria were removed by washing, and HAEC infected with P. gingivalis were stimulated with 10 ng/ml or 10 µg/ml of E. coli O111:B4 LPS (TLR4 ligand) or 100 ng/ml of SLTA (TLR2 ligand) for 24 h. For blocking of TLR4 function or LPS activity, HAEC infected with P. gingivalis were also treated with 10 µg/ml of mouse anti-human TLR4 monoclonal antibody, an isotype-matched control IgG, or 10 µg/ml of polymyxin B for 1 h before LPS stimulation; LPS was then added to HAEC in the presence of TLR4 monoclonal antibody, isotype-matched control antibody, or polymyxin B, and HAEC were incubated for 24 h. The culture supernatant fluids were collected and stored at 20°C until enzyme-linked immunosorbent assays (ELISAs) were performed.
Cytokine and chemokine assays.
The concentrations of monocyte chemoattractant protein 1 (MCP-1), interleukin-1ß (IL-1ß), and tumor necrosis factor alpha (TNF-
) in cell culture supernatants were determined with commercially available ELISA kits (BD Biosciences, San Diego, Calif.) in accordance with the manufacturer's instructions.
Statistical analysis. All statistical analyses were performed by one-way analysis of variance with the Tukey-Kramer multiple-comparison test. Differences in the data were considered significant when the probability was less than 5.0% (P < 0.05).
| RESULTS |
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Blocking P. gingivalis invasion of HAEC with cytoskeleton inhibitors inhibits up-regulation of TLR expression by invasive P. gingivalis infection. To further define the role of P.gingivalis invasion and increased TLR expression, we treated HAEC with cytochalasin D, an inhibitor of actin polymerization and cytoskeleton rearrangements. Treatment of HAEC with cytochalasin D at 1.0 µg/ml inhibited P. gingivalis invasion of HAEC by 96% (Fig. 3A). However, low numbers of P.gingivalis 381 bacteria were capable of invading cytochalasin D-treated HAEC. P. gingivalis-induced expression of TLRs was decreased in cytochalasin D-treated HAEC compared to HAEC without added cytochalasin D (Fig. 3B); however, TLR expression was not completely diminished. These results may be explained by the observation that the invasion efficiency of invasive P. gingivalis 381 with cytochalasin D was still 17-fold higher than that observed with fimA-deficient P. gingivalis strain DPG3 and suggest that very low invasion frequencies are sufficient to alter TLR expression in HAEC.
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in HAEC preinfected with thesame number of bacteria (data not shown). To determine if the TLR2 response was also functionally able to elicit the superinduction of MCP-1 production, similar experiments were performed. HAEC were cultured with SLTA following a P. gingivalis challenge. We also observed that SLTA elicited superinduced MCP-1 production (Fig. 5B). These results demonstrate that P. gingivalis-infected HAEC are primed to respond to defined TLR agonists. Furthermore, these results suggest that the pathway of cellular signal transduction for MCP-1 expression is different from that for IL-1ß and TNF-
expression in HAEC.
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| DISCUSSION |
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Inflammation-dependent induction of TLR2 and -4 expression in intestinal macrophages or epithelial cells from patients with Crohn's disease, ulcerative colitis, or sigmoid diverticulitis has also been recently reported (6, 20). Recent reports have also demonstrated that TLRs are selectively regulated in murine macrophages and human epithelial cells following infection with Mycobacterium avium and nontypeable Haemophilus influenzae, respectively (40, 43). Most interestingly, increased TLR expression has also been associated with inflammatory activation in human atherosclerotic lesions. Xu et al. (45) reported on the preferential expression of TLR4 in lipid-rich and macrophage-infiltrated murine and human atherosclerotic plaques. Recently, two studies have reported that genetic deficiency of TLR4 and myeloid differentiation factor 88 (MyD88), which transduces cell signaling events downstream of the TLRs, is associated with a significant reduction in atherosclerosis through a decrease in macrophage recruitment to the artery wall that was associated with reduced chemokine and cytokine levels in the hypercholesteremic mouse model (5, 33). It has been also reported that TLR4 polymorphism, which attenuates receptor signaling and diminishes the inflammatory response to gram-negative pathogens, is associated with a decreased risk of atherosclerosis (25). Zeuke et al. reported that activation of human coronary artery endothelial cells by LPS, which leads to subsequent production of IL-6, IL-8, and MCP-1, requires TLR4 (46). These studies provide pathophysiologic links among innate immunity, inflammation, and atherosclerosis.
In summary, we demonstrate here that invasive P. gingivalis infection of primary aortic endothelial cells results in increased TLR expression on the cell surface. Furthermore, priming of endothelial cells by invasive P. gingivalis infection leads to increased binding of PAMPs and the induction of TLR-dependent inflammatory responses. Based on these findings, we propose one attractive hypothesis in which P. gingivalis infection of the aortic endothelium can impose a burden on subsequent multiple-pathogen-elicited atherosclerosis. Chronic and episodic stimulation of the endothelium with P. gingivalis may sensitize the endothelium to LPS or other TLR ligands. Our results encourage further studies to determine whether P. gingivalis infection can accelerate atherosclerotic lesion formation via regulation of TLR expression and the innate immune response with TLR signaling molecule-specific knockout mouse models.
| ACKNOWLEDGMENTS |
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| FOOTNOTES |
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