|Year : 2012 | Volume
| Issue : 3 | Page : 156-162
Comparison of systemic levels of regulatory T cells in periodontal health and disease
Pinnamaneni Indumathy1, KV Arun2, T. S. S. Kumar2, A Raja3, P Sai Prashanth4
1 Department of Periodontics, Panineeya Mahavidyalaya Institute of Dental Sciences and Research Centre, Road No 5, Kamala Nagar, Hyderabad, AP, India
2 Department of Periodontics, Ragas Dental College and Hospitals, Chennai, Tamil Nadu, India
3 Department of Animal Biotechnology, Tamilnadu Veterinary and Animal Sciences University, Chennai, Tamil Nadu, India
4 Consultant Endodontist, Denty's Dental Care, Hyderabad, AP, India
|Date of Web Publication||15-Oct-2012|
Senior Lecturer, Department of Periodontics, Panineeya Maha Vidyalaya Institute of Dental Sciences, Road No 5, Kamala Nagar, Dilsukh Nagar, Hyderabad, A.P.
Source of Support: None, Conflict of Interest: None
Aim: Treg cells have been identified to play an important role in regulating T-cell responses and thereby affecting the Th1/Th2 cytokine balance. However, their role in periodontal disease is yet to be identified. Alterations in the expression of regulatory T cells in periodontal tissues could result in a change in their systemic levels. The aim of the study was to identify the regulatory T-cell subsets in systemic circulation and to compare their levels in patients with periodontal health and disease.
Materials and Methods: Thirty-five peripheral blood samples were collected from each of two groups of patients (periodontal health: Group A, and periodontal disease: Group B). The samples were processed for flow cytometric analysis to detect and compare the expression of Treg cells (regulatory T cells). Statistical analysis was done using the Student's t test.
Results: The mean Treg cells for the health group (Group A) was 311,724 cells/ml with a standard deviation of 250,411. The mean Treg cells for the disease group (Group B) was 260,809 cells/ml with a standard deviation of 187,900. P value was 0.508, which is statistically not significant.
Conclusion: There is no statistically significant difference between Treg cell levels in periodontal health and disease, with respect to systemic circulation, although there is a slight decrease in levels in periodontitis compared to health. Apart from Th1/Th2 cells, the regulatory T cells may also contribute to the overall T-cell phenotype. Further studies are needed to confirm this hypothesis.
Keywords: Flow cytometry, periodontitis, peripheral blood, treg cells
|How to cite this article:|
Indumathy P, Arun K V, Kumar T, Raja A, Prashanth P S. Comparison of systemic levels of regulatory T cells in periodontal health and disease. J NTR Univ Health Sci 2012;1:156-62
|How to cite this URL:|
Indumathy P, Arun K V, Kumar T, Raja A, Prashanth P S. Comparison of systemic levels of regulatory T cells in periodontal health and disease. J NTR Univ Health Sci [serial online] 2012 [cited 2020 Mar 31];1:156-62. Available from: http://www.jdrntruhs.org/text.asp?2012/1/3/156/102440
| Introduction|| |
It is now recognized that although periodontitis is microbial in origin, destructive processes are mediated through an exaggerated host response resulting in increased proinflammatory cytokine release. The imbalance between the invading micro-organisms and the host response results in periodontal disease. 
Regulatory cells were described for the first time in the early 1970s by Gershon et al. and were called suppressor cells.  Regulatory cells act via immunosuppression rather than activation. They inhibit the proliferation and activation of other T cells, including helper T cells, thereby modulating their response. They are induced in an antigen-specific manner and show immunosuppressive effects through a cell-cell contact-dependent manner or through the production of inhibitory cytokines. 
Several studies have reported an increased infiltration of Treg cells in gingival tissues affected by periodontal disease. [4-8] This increased infiltration has been attributed to the prevention of excessive destructive activity against self-antigens such as Hsp60 ,, and collagen type I antibody. 
The inflammatory/immune responses generated in the periodontium have been reported to exhibit systemic effects. The systemic spill-over of proinflammatory cytokines such as IL-1, IL-6, and TNF-α has been well documented., This systemic spill-over has been thought to be responsible for the ability of periodontal disease to modulate the course of several diseases such as cardiovascular diseases and diabetes mellitus. ,
In addition to the inflammatory cytokines, periodontal disease has also been reported to affect the immune cells in systemic circulation. Increase in white blood cell count , B-cell responses , have been determined in systemic circulation in patients affected with moderate-to-severe periodontal disease. A hyper-inflammatory response has been elicited from the mononuclear/phagocyte cells isolated from the peripheral blood in patients with periodontitis. 
Although Tregs have been characterized in gingival tissues, there is paucity in the literature regarding the regulatory T cells in systemic circulation following periodontal disease. It is thus not yet clear if these cells exert any effect on the systemic inflammatory status following periodontal disease.
| Aim|| |
The aim of the present study was to identify and compare the levels of regulatory T cells in the systemic circulation of patients in periodontal health and disease, using monoclonal anti-human GITR Fluorescein to detect Treg cells through flow cytometric analysis.
| Materials and Methods|| |
35 patients who attended the outpatient Department of Ragas Dental College and Hospitals, Chennai, were enrolled in the study. Patients were divided into two groups based on their periodontal health status. Informed consent was obtained from all the patients. The study was undertaken following approval from the institutional review board.
Group A: healthy gingiva - Patients exhibiting no signs of periodontal disease, determined by the absence of clinical attachment loss, absence of bleeding on probing. PPD <3 mm.
Group B: periodontitis - Patients with teeth exhibiting PPD ≥5 mm and CAL ≥3 mm and radiographic evidence of bone loss in atleast six teeth. 
- Patients with history of periodontal therapy or antibiotic therapy in the past 6 months.
- Patients with history of systemic diseases that may affect the periodontal status.
- Pregnancy and lactation.
2 ml of peripheral blood drawn from patients using venepuncture from the antecubital fossa to be used for flow cytometry analysis as well as for total and differential white-blood-cell-count estimations.
Peripheral blood was drawn prior to the onset of Phase I periodontal therapy in periodontitis patients. All patients underwent complete hematological investigation to rule out systemic diseases and conditions. Samples collected into vaccutainers containing anticoagulant (EDTA).
Processing for Flow Cytometry Analysis
2 ml of peripheral blood was collected by venipuncture and dispensed in 3 ml vaccutainers containing EDTA. To a 15 ml conical centrifuge tube, 3.0 ml HISTOPAQUE® -1077 was added and brought to room temperature. To 1 ml of peripheral blood, 2 ml of freshly prepared phosphate-buffered saline (PBS) was added to make up to 3 ml. 3 ml whole blood was carefully layered onto the HISTOPAQUE® -1077 and centrifuged at 2700 rpm for exactly 30 min at room temperature. After centrifugation, the upper layer was carefully aspirated to within 0.5 cm of the opaque interface containing mononuclear cells. This upper layer was discarded. The opaque interface was carefully transferred into a clean conical centrifuge tube.
10 ml isotonic PBS was added to this tube and mixed by gentle aspiration and centrifuged at 2000 rpm for 10 min. Supernatant was then aspirated and discarded. The cell pellet was resuspended in 5 ml isotonic PBS and mixed by gentle aspiration and centrifuged at 2000 rpm for 10 min. The above step was repeated again, supernatant discarded, and cell pellet resuspended in 0.5 ml PBS. Cell count was performed using hemocytometer and count adjusted to 10 6 /ml. The resulting suspension was divided into control and test samples each containing equal volumes. Both test and control samples were centrifuged at 2000 rpm for 5 min, and the supernatant was aspirated and discarded. To control sample, 400 μl of 4% paraformaldehyde was added, and the sample was stored at 4°C for flow cytometry analysis. Cells were Fc blocked by adding 25 μl of human IgG to test samples and maintaining for 15 min at room temperature.
10 μl of fluroscein-conjugated anti-human GITR antibody was added and kept for incubation for 30-45 min at 2-8°C. Unreacted antibody was removed by washing cells twice in 4 ml PBS, by centrifuging at 3000 rpm for 5 min. Supernatant was aspirated and discarded. 400 μl of 4% paraformaldehyde was added, and test sample stored at 4°C for flow cytometry analysis[Figure 1].
Flow Cytometry Analysis
Samples were transferred to flow tubes and cytometric analysis was performed with an excitation wavelength of 488 nm and an emission fluorescence of 530 nm. The fluorescence from the analyzed cells was gated in the F1 region. The fluorochrome used was carboxyfluoroscein. Results were measured as the number of events emitting fluorescence per ten thousand cells run through the flow cytometer. These profiles of cells were displayed as dot plots or histograms. The x axis shows the intensity of the detected signal and y axis measures the number of events (cells counted)[Figure 2].
The systemic levels of regulatory T cells in periodontal health and disease were compared by calculating the mean and standard deviation for each group. Student's t test was used for statistical analysis and a P value was calculated. P < 0.05 was considered as significant at 5% level of significance. Statistical analysis was done using SPSS version 10.0.5 for windows.
| Results|| |
The present study assessed the expression of Treg cells in systemic circulation in patients of periodontal health and periodontal disease using the monoclonal anti-human GITR Fluorescein antibody.
35 peripheral blood samples were collected from the two groups: Groups A and B (17 periodontal health and 18 periodontal disease). The patients were assessed for clinical parameters to confirm the periodontal diagnosis [Table 1]. The blood samples were processed for flow cytometric analysis to detect and compare the expression of Treg cells between the two groups.
Fluorescence was measured in terms of M2 which is the number of events (cells) gated per 10,000 cells. Results were obtained by subtracting the number of cells that have shown fluorescence in the test sample (M2 test) from the number of cells that have shown fluorescence in the control sample (M2 control) for each patient.
The number of Treg cells was calculated by correlating with the number of lymphocytes per ml of blood. All 35 samples showed staining for Treg cells (17 health, 18 disease).
T-Test for Independent Samples of Group [Table 2]
This test compared the total WBC count levels in peripheral blood in the two groups. The mean lymphocyte level for health patients (Group A) was 8.1 × 10 6 /ml with a standard deviation of 0.95. For the disease group (Group B), the mean value of WBC count/ml was 8.3 × 10 6 /ml, with a standard deviation of 0.68. P value was 0.432, which was not statistically significant [Figure 3].
T-Test for Independent Samples of Group [Table 3]
This test compared the lymphocyte levels in peripheral blood in the two groups. The mean lymphocyte level for health patients (Group A) was 2.8 × 10 6 /ml with a standard deviation of 0.53. For the disease group (Group B), the mean value of lymphocytes/ml was 3.1 × 10 6 /ml, with a standard deviation of 0.38. The P value was 0.077, which was not statistically significant [Figure 4].
T-Test for Independent Samples of Group [Table 4]
This test compared the Treg cells levels in peripheral blood in the two groups of health and periodontitis. The mean Treg cells for the health group (Group A) was 311,724 cells/ml with a standard deviation of 250,411. The mean Treg cells for the disease group (Group B) was 2,60,809 cells/ml with a standard deviation of 1,87,900. The P value was 0.508, which is statistically not significant [Figure 5].
| Discussion|| |
Periodontal disease has been shown to affect the systemic inflammatory state through various mechanisms. Non-specific inflammatory mediators such as CRP ,, and pro-inflammatory cytokines such as IL-1, TNFα, IL-6 have been reported to be upregulated in systemic circulation following periodontal disease., However, circulatory levels of anti-inflammatory cytokines have not been studied as extensively. As disease occurs as a result of an imbalance between the pro- and anti-inflammatory cytokines, a complete picture of the inflammatory state can be obtained only if both are examined concomitantly, especially considering the redundancy and pleiotropism of cytokines.
Apart from the cytokine levels, periodontal disease is known to affect the cellular response in systemic circulation. An increase in the white blood cell levels  and the presence of hyper-responsive monocytes  has been demonstrated in systemic circulation of patients with periodontal disease. Similarly, Mathur and Michalowicz  demonstrated that T cells exist in peripheral blood that responds to specific periodontal bacteria and their levels are generally higher in patients with periodontitis. These immune cells have been evaluated primarily for their ability to sustain/prolong the pro-inflammatory response.
This study was undertaken to determine the effect of periodontitis on cells that are capable of downregulating immune responses in systemic circulation, namely regulatory T cells. Peripheral blood was collected from the antecubital vein, and lymphocytes were separated using Histopaque-1077 following the standard protocol, as reported by other studies. , Regulatory T cells were evaluated in this study using flow cytometry in conformation with the established protocols. ,,
The antibody used to detect regulatory T cells in this study was anti-human gluco-corticoid-inducible tumor necrosis factor-related receptor (GITR). Previous studies support the use of this antibody as a Treg marker. ,,, Although foxp3 is considered to be the master regulator for Treg cells, its expression does not necessarily indicate a functionally active Treg cell. Further, Treg cells have been shown to exhibit plasticity and may thus differentiate to other inflammatory cells such as the Th17 cells.  A weak, transient expression of foxp3 may be overridden by other transcription factors such as ROR-γ under the influence of inflammatory cytokines. GITR, which is an important co-stimulatory molecule involved in the functionality of these cells is thus a more predictable marker of mature regulatory T cells.,,
In this study, a number of Treg cells were detected as a proportion of the total lymphocytes in circulation. There was an increase but no statistically significant difference in the WBC count between health and disease. This result is not in conformity with the previous studies. ,, This discrepancy in our study could either be a result of a variation in population and/or the small sample size used. It has been previously reported that Treg cells constitute 5-10% of the total lymphocytes present in circulation. , The results of this study are in agreement with this proportion, validating the accuracy of our experimental procedure.
There was a decrease in the mean regulatory T cells/ml of peripheral blood (260,809 cells/ml) but no statistically significant difference in this value between periodontal health and disease groups. These results could not be compared with previously established studies in periodontal disease due to lack of available literature as verified by conducting a thorough online search in PubMed (U.S. National Library of Medicine).
The result of this study is in accordance with the study by Alvarado-Sanchez et al. who reported no significant difference in Treg cell levels in the peripheral blood samples of patients with systemic lupus erythematosus when compared with healthy patients. In contradiction to this study, other authors have reported an increase in Treg cell levels in systemic circulation in autoimmune diseases such as rheumatoid arthritis  and in chronic infections such as tuberculosis. 
Our study is more in agreement with some studies that have shown a decrease in Treg cell levels in peripheral blood of patients with various autoimmune diseases. ,, In humans, the circulatory Treg cell levels have been estimated primarily in autoimmune disorders. Although periodontal disease is not an autoimmune disease per se, recent studies have implicated autoimmune destructive processes through self-antigens such as Hsp60 and type I collagen, autoreactive B cells, and T cells. ,,
The role of regulatory T cells in periodontal disease is an area of intense research activity. Several authors have reported an increased presence of regulatory T cells in the gingival tissues in periodontal disease. ,,,,
Okui et al.  have isolated regulatory T cells from the peripheral blood of patient with periodontitis and reported that these cells demonstrated a decreased efficiency in the suppression of T-cell responses.
Previous studies suggest that the Treg cells do not exhibit their normal regulatory function in gingival tissues following periodontal disease. , The results of this study could be interpreted to mean that a similar phenomenon could be observed in systemic circulation, whereby a decrease in the regulatory cells would result in upregulation of the inflammatory state. Evidence for this downregulation of anti-inflammatory regulatory immune responses may be obtained from studies that have shown a decrease in IL-10, IL-4 in periodontal disease. ,
The reason for the decrease in the Treg cells in circulation could be twofold:
- Increased infiltration of Treg cells in the gingival tissues ,,,,, leading to decreased circulatory levels.
- A continuous low-grade, systemic inflammatory state leading to a suppression of the Treg cells or conversion of these cells to inflammatory phenotypes such as Th17.
Some reports have shown a qualitative rather than a quantitative change in Treg cells in peripheral blood in autoimmune diseases. ,,
Host modulatory therapeutic procedures have gained importance in recent years; Treg cells provide a target whereby the proinflammatory effects of other effector T cells could be suppressed by the activation of these cells.
The limitations of this study include:
T-cell phenotype has been shown to be an important risk determinant of periodontal disease. The results from this preliminary study seem to indicate that apart from Th1/Th2 cells, the regulatory T cells may also contribute to the overall T-cell phenotype. Further longitudinal studies that incorporate a large sample that include the effector cytokines such as IL-21,22/IL-10 would help confirm this hypothesis.
- Small sample size as a result of which significance in variation could have perhaps been undetected.
- Concomitant analysis of CTLA4/IL-10 could have given more meaningful information on the functional status of regulatory T cells.
- A longitudinal analysis of these cells following the history of periodontal disease could have provided definitive results.
Understanding the role of Treg cells will have an impact on our understanding and treatment of periodontal disease. The ability to induce certain T-cell subsets with a specific cytokine profile will enable the design of therapies for the prevention and treatment of chronic infections, such as periodontal disease.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4]