Evaluation of transcription factor that regulates T helper 17 and regulatory T cells function in periodontal health and disease.

BACKGROUND: The differentiation of naοve T helper (Th) cells towards Th17 and regulatory T cells (Treg) is regulated by the transcription factors retinoic acid related orphan receptor gamma transcription (RORYt) and Forkhead box p3 (Foxp3), respectively. An imbalance in the activity of these transcription factors could result in the dysregulation of Th17/Treg response.
MATERIALS AND METHODS: Total RNA was isolated from gingival tissue obtained from 10 patients, each from periodontally healthy and diseased groups. The gene expression of RORYt and Foxp3 was measured by real-time reverse transcription polymerization chain reaction using total RNA isolates from gingival tissues group when compared to the healthy group, while Foxp3 demonstrated a 6.68 ± 0.03 fold decrease of expression in diseased group when compared to healthy group.
CONCLUSION: Our results indicate a functional imbalance in the Th17/Treg response in periodontal disease group when compared to the periodontally healthy group.

Periodontitis is an inflammatory disease of the supporting tissues of the teeth, initiated by a specific group of microorganisms, and characterized by destruction of the tooth supporting structures.[1] This destruction is mediated by the host response. As periodontal disease is a T helper (Th) cell dominant lesion, its role in the etiopathogenesis of periodontal disease has classically been described based on the Th1/Th2 paradigm.[2] This model suggested that the stable early periodontal lesion is characterized by protective Th1 cells while the late progressive lesion is dominated by the destructive Th2 cells.[3]

Studies in recent years have reported discrepancies with this model. The simplistic Th1/Th2 model did not adequately explain many findings with respect to Th cell-mediated immune responses.[4] The identification of Th17 cells helped explain some of the deficiencies in the classic Th1/Th2 model.[5] The Th17 cells may play an important role in the pathogenesis of periodontal disease.

T helper 17 cells and T regulatory cells play opposite roles in the immune response.[6] The Th17 cells expressing the retinoic acid-related orphan receptor gamma transcription (RORYt) factor have a predominantly pro-inflammatory role by its ability to recruit Polymorphonuclear cells (PMN), macrophages and amplify the inflammatory response by producing IL-17, TNFa. These cells are involved in the pathogenesis of inflammatory and autoimmune disease like Rheumatoid Arthritis, Systemic Lupus erythematosis, etc. While the regulatory T cells (Treg) cells expression the Forkhead box p3 (Foxp3) have a regulatory function and suppress effector T cell function and thus have an anti-inflammatory role.[7] The imbalance between this effector and regulatory mechanism may have a role to play in inducing an inappropriate immune-mediated response that would be destructive.[8]

The activity and differentiation of Th17 and Treg cells are regulated by master transcription factors like RORYt and Foxp3 (Forkhead box p3) respectively.[7] Recent evidence suggests that an imbalance between the Th17 and Treg cells activity may be important in the development of chronic systemic inflammatory lesions.[6]

The aim of this study was to evaluate the activity of Th17 and Treg cells in periodontal health and disease at the transcription level.

Materials and Methods

The age and gender matched subjects were recruited from the outpatient Department of Periodontics, Ragas Dental College and Hospital, Chennai. A written consent by the patient to participate in the study was obtained. The study has been approved by Ragas Dental College Institutional Review Board. The age of subjects in both the healthy and diseased groups was ranging from 35 to 55 years. The subjects will be divided into two groups:

Group A: Healthy subjects (control group) who had probing depth <3 mm with no clinical attachment loss, bleeding on probing <10% and no radiographic bone loss. Group B: Advanced chronic periodontitis patient (test group) who had probing depth ≥7 mm, clinical attachment loss ≥5 mm with bleeding on probing, and evidence of radiographic bone loss >2/3rd and involving more than 4 sites/teeth indicated for extraction.

Exclusion criteria

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

Smokers gingival sample

Healthy tissue samples were obtained from patients who had reported to the department for crown lengthening or during impacted/IIIrd molar removal

Diseased tissue samples from patients indicated for extraction in advanced/severe periodontitis

The dimension of the gingival tissue sample would be 3 mm × 2 mm

Tissue samples were completely minced and homogenized in a homogenizer using TRY reagent. The obtained tissue samples were thoroughly washed with phosphate buffered saline (PBS) and stored in RNA later (RNAase free solution) and stored in −80°C until further RNA extraction is carried out. Total RNA was isolated from health and diseased cells by using total RNA isolation reagent (Trisol, Medox) kit.

Quantification of RNA quantitative real time-polymerization chain reaction analysis of retinoic acid-related orphan receptor gamma transcription and Forkhead box p3 reagents

Trisol kit has the following components:

Phenol, guanidium isothiocyanate, urea, detergents, buffering agents and stabilizers

Chloroform (Molecular biology grade)

Isopropanol (Molecular biology grade)

75% ethanol (Analytical grade) - To 7.5 ml of absolute ethanol 2.5 ml of autoclaved deionized water was added.

All the reactions described above were carried out in triplicate.

Forkhead box p3

Forward: ACC TAC GCC ACG CTC ATC

Reverse: TCA TTG AGT GTC CGC TGC T.

Related orphan receptor gamma

Forward: AGA AGG ACA GGG AGC CAA G

Reverse: CAA GGG ATC ACT TCA ATT TGT G.

18s rRNA

Forward: CGC TTC CTT ACC TGG TTG AT

Reverse: GAG CGA CCA AAG GAA CCA TA.

Once the amplification was generated at the end of the reaction, the corresponding cycle threshold (ct) values were obtained.

Data interpretation

The values obtained in the real-time reverse transcription polymerization chain reaction (RT-PCR) was interpreted by using the formula of fold change is 2−ΔΔct(17) this form of equation may be used to compare the gene expression in two different samples (health and disease). Each sample is related to an internal control gene. 2−ΔΔct = (ct of gene of interest−ct internal control) of disease sample−(ct of gene of interest−ct internal control) of health sample.

Results

Gingival samples were obtained from 10 patients from each of periodontally healthy and diseased groups and total RNA isolated and extracted. The mRNA expression for RORYt and Foxp3 was evaluated by using real-time RT-PCR. The results of this study showed are summarized in Table 1. Retinoic acid-related orphan receptor gamma transcription. The results reveal that the mean value of the health group was 25.39 ± 0.01 and the disease group was 19.71 ± 0.01. The delta CT value derived from the formula. 2−ΔΔct = (ct of gene of interest−ct internal control) of disease sample−(ct of gene of interest−ct internal control) of health sample and showed 6.80 ± 0.01 for health group and 5.49 ± 0.01 for diseased group. This shows that the amplification has occurred in fewer cycles in disease than in health. This is represented as fold change from health to disease.

Table 1

Fold change of RORYt and Foxp3 expression in periodontal diseased tissue when compared to healthy tissue

Mean ± SD − Internal control = Delta ct.

2−ΔΔct = 19.71 − 14.22 = 5.49 (disease group).

=25.4 − 18.59 = 6.80 (health group).

Fold change = 6.68 − 5.49 = 1.31.

An increase of 1.31 ± 0.01 fold change was observed in disease when compared to health as shown in Graph 1. It's Suggesting an indirect proportion in the fold change by greater amplification of expression of rory, with low levels of cycles in the diseased group than the health with more number of cycles with lesser expression of rory.

Graph 1

Bar graph representing Forkhead box p3 in periodontal diseased tissue when compared to healthy tissue

Similarly in FOXP3 results reveals that the mean value of health group is 24.32 ± 0.02 and diseased groups 26.64 ± 0.03. The delta CT values derived from the formula. 2−ΔΔct = (ct of gene of interest−ct internal control) of disease sample−(ct of gene of interest−ct internal control) of health sample, it showed 5.73 ± 0.02 for healthy group and 12.42 ± 0.03 for diseased group.

Mean ± SD – internal control = Delta ct.

2−ΔΔct = 26.64 − 14.22 = 12.42 (disease group).

24.32 − 18.59 = 5 − 73 (health group).

Fold change = health group – disease group.

Fold change = 5.73 − 12.42= −6.68.

This shows that the amplification has not occurred with less or no expression in disease group than in health group. This is represented as fold change from health to disease. A decrease of −6.68 ± 0.0 was observed in disease group when compared to health as shown in Graph 2. It also suggests that an indirect proportion with delta CT value with less or no expression of FOXP3 in disease condition when compared to health.

Graph 2

Bar graph representing Forkhead box p3 in periodontal diseased tissue when compared to healthy tissue

Discussion

Periodontal disease is a chronic inflammatory disease involving various immune cells particularly T lymphocytes. The debate as to which Th cell has a major role in the etiopathogenesis of periodontal disease has been till date in conclusion.[1] Mossmans studies resulted in the Th1/Th2 paradigm and its role in periodontal disease was proposed, but this model did not adequately explain the various discrepancies with regard to the predominant Th cell observed in diseased tissue.[4]

With the identification of Th17 cells many of the deficiencies of the Th1/Th2 model could be explained. Though Th17 cells have evolved primarily to provide inflammatory protection against extracellular pathogens, inappropriate activation may have a role in tissue destruction observed in chronic inflammatory lesions through excessive production of cytokines.[6] Treg cells suppress T cell proliferation and activation thus regulating the inflammatory response. Any imbalance between these effector and suppressor mechanism could result in the dysregualtion of the Th cells function, resulting in persistent inflammatory response, which may be associated with tissue destruction.[9]

Both these immune cells have been identified in diseased gingival tissue with the help of either their lineage-specific cytokines IL-17 or by their cell surface markers. The cell surface markers are not well-characterized and do not indicate the differentiation state/effector functions of these cells.[6]

The transcriptional regulation of Th cell differentiation in periodontal disease has gained relevance today due to the ability of Th cell to demonstrate plasticity.[10]

The present study was undertaken to evaluate the expression of Foxp3 and RORYt transcription factors in periodontal health and disease. Healthy gingival tissue samples and diseased gingival samples were obtained from patients according to the exclusion criteria gingival tissues were then homogenized and total RNA isolated real-time reverse transcription polymerization chain reaction analysis was carried out to evaluate the mRNA expression of the transcription factors Foxp3 and RORYt in periodontal health and disease.

This study demonstrated that patients with periodontal disease revealed a marginal insignificant increase in Th17 related transcription factor (RORYt) expression in disease group when compared to periodontally healthy group, while there was a significant decrease in Treg related transcription factor Foxp3 expression in disease group when compared to periodontally healthy group. This result may suggest a downregulation of Treg cells and polarization of Th17 cells in patients with advanced periodontal disease, suggesting a dysregulated immune response.

T helper 17 cells have been described as cells with a proinflammatory role which are regulated by the transcription factor RORYt to secrete primarily IL-17 and to a linear extent IL-6, IL-23.[6] when produced excessively it mediate tissue destruction by regulating IL-6, TNFa, RANKL, MMP.[11] Mean while Treg cells play a crucial role in regulating immune tolerance. Foxp3 is a specific marker expressed as Treg cells and is essential for promoting Treg cells development and function. Treg cells inhibit activated and proliferating of effector T cells by secreting IL-10 and TGFp.[12]

With respect to Foxp3 expression, our study was in agreement with a previous study by (Ernst et al.), but not in agreement with other studies which demonstrated that an increase expression of Foxp3 was found to be associated with chronic periodontal disease than in gingival lesion.[13]

With respect of RORYt expression, our result was in agreement with previous studies[6] where a positive correlate between RORYt expression and bone destruction was established. Recent evidence indicates that both RORYt and Foxp3 can antagonize each other and in addition, Foxp3+ Treg cells can be converted to IL-17 producing cells under suitable conditions such as in chronic inflammation where proinflammatory cytokines may be upregulated. These changes occurring in the microenvironment during chronic inflammation alter the inducing signals that regulate the transcription factors.[14] RORYt production is directly related to the presence of IL-6, IL-21 and TGFp,[6] while Foxp3 production in related to the presence of TGFp and retinoic acid.[1516]

It may be postulated that patients included in the disease group where at an active inflammatory state, the levels of proinflammation cytokines would have been increased, which could have influenced the transcription factor expression.

On the other hand, it may be argued that the changes in the transcription factors expression could be due to these factors being utilized by the differentiating cells and thus resulting in depleted tissue levels. A clear picture could have emerged had the effector cytokine been assessed simultaneously. The absence of these cytokine profiles makes interpretation of our results inconclusive.

In conclusion, our results indicate that a Th17/Treg functional imbalance exists in periodontal disease patients. Based on the results of our study immunotherapy directed at the transcription factor level and the goal of decreasing the inflammation caused by Treg/Th17 imbalance may have a protective effect in patients with periodontal disease.

Conclusion

The T cells play an important role in immune regulation with a balance exists between various Th cell subsets in health. When an imbalance exists, there is a dysregulation of the immune response leading to persistent inflammatory response and destruction of tissue and bone.

This study demonstrated patient with periodontal disease has marginal insignificant increase in Th 17 related transcription factor (RORYt) expression in disease group when compared to periodontally healthy subjects, meanwhile there was a significant decrease in Treg transcription factor FOX P3 expression in diseased group when compared to periodontally healthy group.

In conclusion, our results indicate that a Th17/Treg functional imbalance exists in periodontal disease patients. Due to the limitation of the study design, we could not correlate the Treg/Th17 cell imbalance with severity of disease though it could be speculated that simultaneous Th17 upregulation and Treg cells downregulation may lead to periodontal disease through immune mediated tissue destruction.

Further studies are required to correlate this imbalance with extent of destruction and inducing effector mechanism, which might give further information about the exact role of these cells in disease pathogenesis.