|Year : 2010 | Volume
| Issue : 1 | Page : 24-29
Aggregatibacter actinomycetemcomitans modulates toll-like receptors 2 and 4 in gingival epithelial cells in experimental periodontitis
Mythily Srinivasan1, Krithika N Kodumudi1, Dominique M Galli2
1 Department of Oral Pathology, Medicine and Radiology, Indiana University-School of Dentistry, Indianapolis, Indiana, USA
2 Department of Oral Biology, Indiana University-School of Dentistry, Indianapolis, Indiana, USA
|Date of Web Publication||18-Nov-2011|
Department of Oral Pathology, Medicine and Radiology, Indiana University School of Dentistry, 1121 West Michigan Street, Indianapolis, IN 46202
Source of Support: Indiana University School of Dentistry, Conflict of Interest: None
| Abstract|| |
Background: Periodontitis is a common bacterial infection precipitated by exaggerated host responses to the oral microorganisms, As the first cells to encounter the oral pathogens the gingival epithelial cells (GEC) respond via toll-like receptors (TLR) that recognize conserved microbial patterns. Here we investigated the expression of TLR-2 and TLR-4 in GEC of naοve mice in response to infection with the periodontal pathogen Aggregatibacter actinomycetemcomitans (Aa). Methods: 7-9 weeks old mice were induced experimental periodontitis by inoculating the palatal gingival with 1x109 colony forming units of Aa. Mice were sacrificed 50 days later and intact maxilla harvested. The degree of alveolar bone loss was determined by micro-CT. Single suspensions of the epithelial cells isolated from palatal gingival tissues were assessed for the expressions of TLR-2 and TLR-4 proteins by flow cytometry and for TLR-2 and TLR-4 mRNA by reverse transcriptase PCR. Differences between the control and the disease groups were determined by students't- test. Results: Both TLR-2 and TLR-4 proteins were significantly elevated in the GEC of mice infected with A. actinomycetemcomitans when compared to the control group. The ratio of TLR-4 to TLR-2 mRNA was upregulated in chornic periodontitis as compared with the control group. Conclusion: Our data suggest that TLR-2 and TLR-4 are regulated differentially in A. actinomycetemcomitans-induced periodontitis.
Keywords: Aggregatibacter actinomyctemcomitans , Periodontitis, TLR-2, TLR-4
|How to cite this article:|
Srinivasan M, Kodumudi KN, Galli DM. Aggregatibacter actinomycetemcomitans modulates toll-like receptors 2 and 4 in gingival epithelial cells in experimental periodontitis. J Int Clin Dent Res Organ 2010;2:24-9
|How to cite this URL:|
Srinivasan M, Kodumudi KN, Galli DM. Aggregatibacter actinomycetemcomitans modulates toll-like receptors 2 and 4 in gingival epithelial cells in experimental periodontitis. J Int Clin Dent Res Organ [serial online] 2010 [cited 2019 Dec 6];2:24-9. Available from: http://www.jicdro.org/text.asp?2010/2/1/24/89992
| Introduction|| |
Periodontitis is a prevalent infectious disease characterized by the destruction of soft and hard tissues supporting the teeth. It affects 50% of the adult population worldwide.  The primary etiological agents of periodontitis are Gram-negative bacteria. The gingival epithelial cells (GEC) are the first cells to encounter these oral pathogens. The host-microbe interplay is facilitated by toll-like receptors (TLR) expressed by GEC that interact with highly conserved pathogen-associated molecular patterns (PAMP). ,, TLR-2 recognizes bacterial products such as peptidoglycan, lipoproteins, and lipoteichoic acids, while TLR-4 binds lipopolysaccharide, a major component of the Gram-negative bacterial cell wall. , CD14 is a co-receptor that facilitates the recognition of peptidoglycan and lipopolysaccharide by TLR-2 and TLR-4. Binding of these cell wall components to TLR-2 and TLR-4 stimulates the production of proinflammatory cytokines. Considerable evidence suggests a critical role for TLR-2 and TLR-4 in periodontal disease pathology. , Elevated expression of TLR-2 and TLR-4 proteins has been observed in inflamed gingiva in human periodontitis.  In addition, polymorphisms in TLR-4 and CD14 have been associated with susceptibility to periodontitis. , Two of the most common and best characterized periodontal pathogens are the Gram-negative Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis. Exvivo assays showed that P. gingivalis upregulates TLR-2 expression and signaling in GEC inducing cytokine secretion. ,,, Invivo studies confirmed that TLR-2 deficient mice were resistant to P. gingivalis-induced periodontitis.  In contrast, data obtained ex vivo suggested that A.actinomycetemcomitans interacts with both TLR-2 and TLR-4. , This observation has not been confirmed invivo. In this study, we investigated the relationship between A. actinomycetemcomitans infection, TLR expression and periodontal pathology in a mouse model of experimental periodontitis. We report that invivo infection with live A. actinomycetemcomitans modulates the expression of both TLR-2 and TLR-4 but not CD14 in GEC.
| Materials and Methods|| |
C57BL/6 male mice, 7-9 weeks old, were obtained from the Jackson laboratory (Ben Harbor, MA) and maintained under specific pathogen-free conditions at the Indiana University School of Dentistry bioresearch facility. Periodontal infection was induced in naοve mice after obtaining approval from the Institutional Animal Care and Use Committee. Briefly, A. actinomycetemcomitans (ATCC 29522) was grown to mid-exponential phase in TSBYE (Tryptic Soy Broth, 0.6% yeast) in the presence of 10% CO 2 . The palatal gingiva of mice (n = 6) were injected with 1×10 9 colony-forming unit (CFU) of bacteria suspended in 20 μl of sterile PBS. Additionally, 1×10 9 bacteria in 50μl of PBS containing 2% carboxymethylcellulose were placed in the oral cavity at time points 0 h, 48 h, and 96 h. , The control group included sham-infected mice (n = 6) that received PBS with carboxymethylcellulose only. Colonization and effectiveness of infection was confirmed by the detection of A. actinomycetemcomitans in gingival tissues by polymerase chain reaction. 
Micro-computerized tomography (micro-CT)
Mice were sacrificed 50 days after infection. Maxilla with intact teeth were fixed in neutral buffered formaldehyde, hemi-sected and scanned by micro-computed tomography (CT) in three spatial planes at a resolution of 10 μm.  Alveolar bone loss was determined by measuring the distance from the cemento-enamel junction (CEJ) to the alveolar bone crest (ABC) in two dimensional micro-CT images.
Isolation of leucocytes and epithelial cells
The palatal gingival tissue of the upper molars was dissected, cut into pieces, rinsed in Ca 2+ - and Mg 2+ -free Hanks' balanced salt solution (CMF-HBSS) and digested in RPMI 1640 containing 0.15 mg/mL type II collagenase and 0.1 mg/mL DNase for 90-120 minutes at 37°C. Purified leucocytes were isolated using a discontinuous gradient of 44% and 70% Percoll, washed and counted using a hemocytometer. , For the isolation of epithelial cells digested pieces of gingival tissue were initially incubated at 37°C in serum-free RPMI supplemented with 0.145 mg/mL DTT and 0.37 mg/mL EDTA to dissociate epithelial cells followed by disaggregation with 3 mg/ml dispase and 1 mg/ml DNase. After passing through a 50% Percoll density gradient, purified epithelial cells were collected from the interface, washed and counted.  The purity of the epithelial cell preparation was assessed by intracellular cytokeratin staining with anti-mouse pan-cytokeratin antibody (C11: sc-8018, Santa Cruz laboratories, CA) followed by phycoerythrin (PE)-conjugated rabbit anti-mouse IgG and analyzed by flow cytometry. 
Single cell suspensions of GEC were sequentially stained with biotinylated anti-mouse CD14 mAb, anti-mouse TLR-2 mAb or an isotype-matched control antibody followed by PE-conjugated streptavidin or FITC-conjugated goat anti-mouse IgG respectively for 30 min at 4°C.  To stain TLR-4 cells they were incubated with FITC conjugated anti-mouse TLR-4. All antibodies were obtained from RandD Systems, Minneapolis, MN. Labeled cells were resuspended in PBS containing 1% paraformaldehyde and analyzed using a FACS Calibur flow cytometer (BD Biosciences, CA). Three independent experiments each counting 10,000 events were performed.
Reverse transcriptase polymerase chain reaction (RT-PCR)
Total cellular RNA was isolated from naοve mouse intestinal epithelial cells and the GEC 50 days post-infection using a Qiagen kit (Invitrogen, Carlsbad, CA) and reverse transcribed with the iScript cDNA synthesis kit (Bio-Rad, CA). Equal amounts of cDNA were used to amplify the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH), CD14, TLR-2 and TLR-4 with Platinum PCR Supermix (Invitrogen, Austin, TX). Primers used: 5'GCCAAACGGGTCATC ATCTC-3' and 5' GACACATTGGGGGTAGGAAC-3' for GAPDH, 5'-CAAACTGGAGACTCTGGAAG-3' and 5'- CTGTAGGAAACAAAGGCATC-3' for TLR-2, 5'-CAGTCGGTCAGCAAACGCCTTCTTC-3' and 5'-CAAGGCAGGCTAGCAGGAAAGGGTG-3' for TLR-4, and 5' TGCGAGCTAGACGAGGAAAGTTGT-3' and 5'-ACCGT AAGCCGCTTTAAGGACAGA-3' for CD14. Number of amplification cycles and annealing temperatures were 35 cycles at 55°C for 30s. The PCR products were visualized by gel electrophoresis. The images acquired with the KODAK GEL Logic system were used for densitometric analysis of the amplified bands by the Kodak 1D image analysis software (Eastman Kodak Company, Rochester, MN).
Differences in lymphocyte count, degree of bone loss, protein and mRNA levels between sham- and A. actinomycetemcomitans-infected mice were determined by Student's paired t-test. A value of P < 0.05 was considered significant.
| Results|| |
Alveolar bone loss and tissue inflammation are the hallmarks of periodontitis.  Unlike the control group A. actinomycetemcomitans-infected mice exhibited alveolar bone loss as demonstrated by micro-CT analysis [Figure 1]A-D. In addition, the distance between the CEJ and ABC measured 50 days after infection was significantly increased in this latter group [Figure 1]E. Histological examination showed proliferation of the junctional epithelium and inflammatory cell infiltration in the gingiva of A. actinomycetemcomitans-infected mice only (data not shown). The number of leukocytes isolated from the palatal gingiva at day 50 post-infection was significantly increased in A. actinomycetemcomitans-infected mice when compared to control mice (P < 0.001) [Figure 1]F. Taken together these observations confirmed that infection with A. actinomycetemcomitans induces periodontitis in mice. ,
|Figure 1: Alveolar bone loss in C57Bl/6 mice infected with A. actinomycetemcomitans (Aa). Alveolar bone loss in Aa- and sham-infected mice (n = 6/group) was assessed by micro-CT. Representative two-dimensional images and three-dimensional reconstructed images (from the buccal side) are shown (A, B: sham-infected; C, D: Aa-infected). (E) Distance in mm between cementoenamel junction (CEJ) and the alveolar bone crest (ABC). (F) Number of leukocytes isolated from the palatal gingiva of Aaand sham-infected mice on day 50 post-infection. Data presented are mean ± SE. *P < 0.05|
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The relationship between A. actinomycetemcomitans infection and TLR activation was evaluated by assessing surface protein expression of CD14, TLR-2 and TLR-4 in the GEC by flow cytometry. Purity of the epithelial cell preparation was confirmed by examining cellular morphology (data not shown) and intracellular cytokeratin expression [Figure 2]A and E. Both TLR-2 and TLR-4 proteins were significantly elevated in the GEC of mice infected with A. actinomycetemcomitans when compared to the control group [Figure 2]C-E. No significant difference was observed in the expression of CD14 between the two groups [Figure 2]B and E.
|Figure 2: Cell surface and transcriptional expression of TLR in GEC of A. actinomycetemcomitans (Aa)- and sham-infected mice isolated day 50 post-infection (n = 6/group). (A-D) Flow cytometric analysis of GEC stained for pan-cytokeratin (A), CD14 (B), TLR-2 (C), and TLR-4 (D). Black and dark grey lines in the representative histograms correspond to staining with the indicated antibodies in GEC from Aa- and sham-infected mice, respectively. The light grey lines represent the isotype-matched control. (E) Mean Ä % staining (% positive - % isotype) from three independent experiments ± SE. (F) cDNA from GEC amplifi ed for GAPDH, CD14, TLR-2 and TLR-4 gene expression by RT-PCR. Target gene expression in murine intestinal epithelial cells (IEC) served as positive control. (G) Densitometric analysis normalized to GAPDH mRNA. Mean values ± SE are shown. *P < 0.05|
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To determine whether the observed changes in the expression of TLR proteins correlated with enhanced transcription of TLR-2 and TLR-4 semi-quantitative RT-PCR was performed. For both animal groups expression levels of CD14, TLR-2 and TLR-4 mRNA were normalized to GAPDH mRNA levels in GEC cells [Figure 2]F. cDNA from mouse intestinal epithelial cells that are known to express CD14, TLR-2 and TLR-4 mRNA served as a positive control. Data obtained revealed differential expression of TLR-4 but not TLR-2 or CD14 transcripts resulting in a significantly higher ratio of TLR-4 to TLR-2 mRNA in the GEC of A. actinomycetemcomitans-infected mice when compared to the control group [Figure 2]G.
| Discussion|| |
A. actinomycetemcomitans is a common periodontal pathogen that has been shown to infect gingival keratinocytes where it modulates transcription of genes involved in stress response and host-pathogen interaction.  TLRs are the major recognition system for such invading bacteria. Hence, activation and regulation of TLRs are pivotal to the initiation of innate inflammatory and immune defense mechanisms to clear microbial infections.  Exvivo functional studies using dead bacteria, bacteria sonicates or cell wall components showed that periodontal pathogens appeared to predominantly use the TLR-2 signaling pathway in human cells except for A. actinomycetemcomitans, which activated TLR-4 to the same extent as TLR-2. ,,,, Recently, Yoshioka et al.  observed that an increase in A. actinomycetemcomitans subgingival plaque numbers correlated positively to elevated TLR-4 activation. The present study is the first to assess epithelial cell expression of TLR-2, TLR-4, and CD14 invivo at the transcriptional and translational level upon stimulation with live A. actinomycetemcomitans. Both TLR-2 and TLR-4 surface protein levels significantly increased in the GEC of A. actinomycetemcomitans-infected mice. Increased protein expression corresponded with an upregulation of gene expression for TLR-4 but not TLR-2, which indicates that the regulation of TLR expression differs. Interestingly, Benakanakere et al.  recently reported that TLR-2 surface expression was inversely regulated at the translational level by miRNA in human oral keratinocytes challenged with P. gingivalis.
It is not clear yet which A. actinomycetemcomitans-specific PAMPs account for the observed TLR activation pattern. Purified A. actinomycetemcomitans LPS and whole cell sonicates were shown to stimulate host cells via TLR-4.  Furthermore, mice harboring a TLR-4 gene mutation were less susceptible to A. actinomycetemcomitans LPS-induced alveolar bone resorption.  Other potential A. actinomycetemcomitans PAMPs that could activate TLRs include outer membrane proteins (Omp) and capsular polysaccharides.  Paul-Stayaseela et al.  recently characterized a highly immunogenic peptidoglycan-associated lipoprotein in an Omp preparation of A. actinomycetemcomitans. Bacterial lipoproteins are known to function as ligands for TLR-2.
Differential TLR-mediated signaling due to differences in subgingival plaque composition could result in distinct cytokine release patterns in the localized periodontal tissue. In return, the type of cytokines produced may influence the T helper (Th) adaptive immune response.  Recently, Kikkert et al.  argued that stimulation of TLR-2 in the absence of TLR-4 activation could result in a Th2-like immune response in periodontitis. Likewise, cytokines produced upon TLR-4 stimulation would favor a Th1 immune response. Indeed, it has been shown that A. actinomycetemcomitans infection induces expression of the Th1 cytokines TNF-a and IFN-g in gingival tissues. ,
In conclusion, our invivo results are consistent with exvivo observations that A. actinomycetemcomitans-induced signaling pathways are mediated through both TLR-2 and TLR-4. This is in contrast to P. gingivalis and most other periodontal pathogens that appear to signal preferentially via TLR-2. ,, Indeed, specific isoforms of P. gingivalis LPS have been shown to antagonize the TLR-4-ediated response and thus may act to reduce inflammatory responses induced by A. actinomycetemcomitans. Accordingly, the relative abundance of a particular periodontal pathogen may influence the nature of the innate and ultimately adaptive immune response in periodontal disease via TLR stimulation.
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