Evaluation of mRNA expression of the transcription factors of Th1 and Th2 subsets (T-bet and GATA-3) in periodontal health and disease - A pilot study in south Indian population.

BACKGROUND: Based on their respective pro- or anti-inflammatory cytokine profiles, the Th1/Th2 paradigm explains pathogenic mechanisms involved in periodontal disease. Establishment of Th1 and Th2 subsets from a naive T-cell precursor depends on transcriptional regulation. The aim of this study was to compare the expression of master transcription factor regulators T-bet and GATA-3, respectively, to indicate the predominance of Th1 and Th2 subsets in the presence and absence of periodontal disease.
MATERIALS AND METHODS: A gingival tissue biopsy sample was obtained from each of 10 severe periodontitis patients (>5 mm attachment loss) and 10 periodontally healthy patients (no attachment loss). Biopsies were immediately processed by real-time reverse transcriptase polymerase chain reaction and the difference in mRNA expression of T-bet and GATA-3 was assessed for each group.
RESULTS: The mRNA expression of T-bet was marginally increased about 1.31-fold in disease, while the GATA-3 levels showed a significant decrease of 4.39-fold in disease.
CONCLUSION: The advanced periodontal lesions lack Th2 cells, which produce anti-inflammatory cytokines. The biopsies were therefore dominated by Th1 cells, which activate macrophages and osteoclasts.

INTRODUCTION

Although periodontitis is microbial in origin, destructive processes are mediated through an exaggerated host response resulting in increased pro-inflammatory cytokine release. The imbalance between the invading microorganisms and the host response results in periodontal disease.[1] It has been postulated that “appropriate” cytokine production results in protective immunity, while “inappropriate” cytokine production leads to tissue destruction and disease progression.[2]

The central role played by T-cells in the etiopathogenesis of periodontal disease was recognized by following the seminal work of Ivanyi and Lehner.[3] However, with a greater understanding of their biological functions, T-cells are now believed to be involved in the homeostasis of periodontal tissues,[4] modulation of the inflammatory/immune responses,[5] and mediation of the bone loss observed in periodontal disease.[6] T-cells are classified based on their function into various categories such as helper T-cells, cytotoxic T-cells, and regulatory T-cells. Originally, the presence of two different subsets of Th cells-Th1 and Th2 was described by Parish and Liew.[7] Later, Mossman and Coffman delineated two distinct cytokine profiles associated with the Th1 and Th2 cells. Th1 cells play an important role in phagocytosis, complement fixation, and opsonization,[8] while Th2 cells are involved in immunoglobulin (Ig) class switching in B-cells. Naive T-Helper cells can be differentiated into either Th1 cells or Th2 cells, depending on their cytokine nature and transcriptional factors.[910] Glimcher in 2007 stated that the Th1 cell expresses the interleukin (IL-12) receptor subunit IL-12Rβ2, which further commits a cell to proceed differentiation program. The commitment is dependent on the activation of transcription factors, signal transducers and activators of transcription (STAT-4), and T-bet.[1112]

Conversely, newly activated Th cells exposed to IL-4 differentiate into a Th2 phenotype which secretes IL-4, IL-5, and IL-13.[13] Expression of these cytokines is transcriptionally co-regulated by STAT-6, GATA-3, and c-maf.[1415] Th1 and Th2 development is mutually antagonistic and self-reinforcing, in part by the interferon-gamma (IFN-γ) produced by Th1 cells and IL-4 by the Th2 cells.[16] Although a number of studies have documented the preponderance of either the Th1/Th2 signature cytokines in gingival tissues, saliva, and gingival crevicular fluid, however, there are fewer reports on the transcriptional activity within the gingival tissues. Hence, the aim of this study was to evaluate the gene expression of the master transcription factors of the Th1/Th2 subsets namely, T-bet and GATA-3 in the gingival tissues of healthy and chronic periodontitis patients.

MATERIALS AND METHODS

Ten gingival tissue biopsy samples from each group (periodontal health group and advanced chronic periodontitis group) of age and gender matched subjects were recruited from the outpatient Department of Ragas Dental College and Hospital, Chennai. An informed written consent by the patient to participate in this pilot study was obtained. The study has been approved by the Institutional Review Board, Ragas Dental College and Hospital. The following groups of patients and individuals were recruited for the study.

Group A were healthy subjects who had probing depth (PD) <3 mm with no clinical attachment loss and bleeding on probing (BOP) <10% and Group B comprised of patients with advanced chronic periodontitis who had PD ≥8 mm, clinical attachment loss ≥5 mm, and BOP in more than at least one or more teeth indicated for extraction.

The crown lengthening patients were referred from the Department of Endodontics for crown exposure to perform postendodontic restorations and crown preparation. The periodontal status was assessed and found to be periodontally healthy. Diseased tissue samples were obtained from patients indicated for extraction in advanced/severe periodontitis. The dimension of the tissue sample was about 6 mm.

The following patients were excluded from the study: 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, pregnant, lactating mothers, and smokers.

The tissue samples were immersed in an RNAase inhibitor containing solution (RNA later, Invitrogen, Bangalore, India) and homogenized immediately. Total RNA was obtained from the homogenates using Trizol total RNA extraction kit (Medox, Chennai, Tamil Nadu, India). The first step in real time reverse transcription polymerase chain reaction (q-PCR) was the conversion of the total RNA to a complementary cDNA which was then carried out using a high capacity cDNA reverse transcription kit (Applied Biosystems, Invitrogen BioServices, Bangalore, India). The quantitative real-time amplification was done using a SYBR Green containing master mix kit (Qiagen, Germany) to evaluate the mRNA expression of T-bet and GATA-3 in periodontal health and disease.

Reaction was performed using 5.0 μl of 2X reaction buffer, 1 μl of 100 nM of both forward and reverse primers of gene of interest, 1 μl of forward and reverse primers of 18S RNA (internal control gene), and 3.0 μl of diluted cDNA was made up to a 10 μl reaction. The thermal cycling protocol was as follows: 95°C for 5 min, followed by 40 cycles of PCR at 95°C for 30 s and 58°C for 30 s. All reactions were performed in triplicate along with no template control. Melt curve analysis was performed using the thermal cycling programed at 50–95°C for each sample to determine the presence of multiple amplicons, nonspecific products, and contaminants. The relative amount of mRNA was calculated by using the comparative Ct method.

The primer sequences of the genes are as follows:

T-bet - Forward primers: 5’-TCCAAGTTTAATCAGCACCAGA-3’

Reverse primers: 5’-TGACAGGAATGGGAACATCC-3’

GATA-3 - Forward primers: 5’-CTCATTAAGCCCAAGCGAAG-3’

Reverse primers: 5’-TCTGACAGTTCGCxACAGGAC-3’

18 s RNA - Forward primers: 5’-CGC TTC CTT ACC TGG TTG AT-3’

Reverse primers: 5’-GAG CGA CCA AAG GAA CCA TA-3’.

Data interpretation

The values obtained in the qRT-PCR was interpreted using the formula of fold change, 2−ΔΔct = (ct of gene of interest − ct of internal control) of disease sample – (ct of gene of interest − ct of internal control) of health sample.[1718]

This form of equation was used to compare the gene expression in two different samples (health and disease) or (treated and untreated). Each sample is related to an internal control gene.

RESULTS

The mean delta Ct value for the T-bet gene in the health group was 12.35 and 11.23 in the diseased group, respectively. There was a 1.12-fold increase in the mRNA expression of T-bet in disease when compared to health, which was not significant [Figure 1].

Figure 1

Bar graph representing the fold change of mRNA expression of T-bet in periodontal health and disease

The mean delta Ct value for the GATA-3 gene in the health group was 10.18 and 14.58 in the diseased group, respectively. There was a −4.39-fold decrease in the mRNA expression of GATA-3 in disease when compared to health, which was significant [Figure 2].

Figure 2

Bar graph representing the fold change of mRNA expression of GATA-3 in periodontal health and disease

Figures 3 and 4 depict the amplification plots and dissociation curve of T-bet, GATA-3, respectively.

Figure 3

Amplification plots of T-bet and GATA-3

Figure 4

Dissociation curve of T-bet and GATA-3

T-bet expression was similar in health and disease, whereas GATA-3 expression was reduced 4-fold [Table 1]. Thus, the diseased and healthy biopsies had similar levels of Th1 cells, but the diseased biopsies were significantly reduced in Th2 cells.

Table 1

Expression of T-bet and GATA-3 in periodontal health and disease

DISCUSSION

We found a significant and profound decrease in GATA-3 levels in periodontally diseased tissues compared with health, and a minor change in T-bet levels. The decrease in GATA-3 indicates a down shifted Th2 cell activation. The periodontally diseased biopsies were therefore dominated by T-bet activated Th1 cells, which secrete high levels of IFN-γ responsible for stimulating macrophage collagenase production, osteoclasts development, and classical features of advanced periodontitis. The lack of Th2 activity suppressed IL-4 and IL-10 expression, which would have dampened the IFN-γ mediated inflammatory response and exerts the suppressive effect on osteoclastogenesis.[19]

Therefore, we have confirmed the importance of the cytokine milieu for both the differentiation and functioning of the Th profile in advanced periodontitis,[20] Th1 cytokines were associated with early stage of lesions, while the destructive Th2 cytokines in the advanced lesion.[21]

The results of our study are in agreement with that of Fujihashi et al.[2223] In addition, Zhao et al.[24] explained how Th1 and Th2 cell cytokine expression might influence each other. Other studies indicated that Th1and Th2 subsets have counter balancing and regulatory effects at the cytokine and transcriptional levels.[24] We, therefore, suggested that the decrease in Th2 IL-4 and IL-10 activities resulted from the over-activation of Th1 cell activity in advanced periodontitis lesions. These results must, however, be interpreted with caution. The diseased samples in our study were the end stage of periodontal disease with active inflammation, the levels of the anti-inflammatory IL-4 levels were likely to be decreased. This decreased IL-4 production could have directly influenced in lowered GATA-3 expression observed in our samples.

CONCLUSION

The Th1 response predominates over Th2 in advanced periodontitis because GATA-3 production by Th2 cells is profoundly decreased. How this is brought about is not clear and a better understanding of how cytokine production interacts with the transcriptional regulation of Th2 cells in advanced periodontitis could lead to the development of new agents that may enhance the therapy of advanced periodontal lesions. However, further investigations on the protein levels are required to evaluate GATA-3 and T-bet levels in periodontal tissues.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.