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ORIGINAL ARTICLE
Year : 2015  |  Volume : 19  |  Issue : 6  |  Page : 624-627  

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


1 Department of Periodontology and Implantology, G. Pulla Reddy Dental College, Kurnool, Andhra Pradesh, India
2 Department of Periodontology and Implantology, Ragas Dental College, Chennai, Tamil Nadu, India
3 Department of Periodontology and Implantology, Meghana Dental College, Nizamabad, Andhra Pradesh, India

Date of Web Publication28-Dec-2015

Correspondence Address:
Nichenametla Rajesh
Department of Periodontology and Implantology, G. Pulla Reddy Dental College, Kurnool, Andhra Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-124X.164748

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   Abstract 

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.

Keywords: Th1 cells, Th2 cells, real-time reverse transcriptase polymerase chain reaction, periodontitis


How to cite this article:
Rajesh N, Arun KV, Subbu Kumar TS, Reddy KK, Alamelu S, Reddy BR. 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. J Indian Soc Periodontol 2015;19:624-7

How to cite this URL:
Rajesh N, Arun KV, Subbu Kumar TS, Reddy KK, Alamelu S, Reddy BR. 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. J Indian Soc Periodontol [serial online] 2015 [cited 2019 Oct 23];19:624-7. Available from: http://www.jisponline.com/text.asp?2015/19/6/624/164748


   Introduction Top


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.[9],[10] 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.[11],[12]

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.[14],[15] 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 Top


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.[17],[18]

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 Top


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

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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

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[Figure 3] and [Figure 4] depict the amplification plots and dissociation curve of T-bet, GATA-3, respectively.
Figure 3: Amplification plots of T-bet and GATA-3

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Figure 4: Dissociation curve of T-bet and GATA-3

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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

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   Discussion Top


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.[22],[23] 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.[2],[4] 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 Top


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.

 
   References Top

1.
Seymour GJ, Gemmell E, Reinhardt RA, Eastcott J, Taubman MA. Immunopathogenesis of chronic inflammatory periodontal disease: Cellular and molecular mechanisms. J Periodontal Res 1993;28:478-86.  Back to cited text no. 1
    
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Gemmell E, Seymour GJ. Immunoregulatory control of Th1/Th2 cytokine profiles in periodontal disease. Periodontol 2000 2004;35:21-41.  Back to cited text no. 2
    
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Ivanyi L, Lehner T. Stimulation of lymphocyte transformation by bacterial antigens in patients with periodontal disease. Arch Oral Biol 1970;15:1089-96.  Back to cited text no. 3
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Gemmell E, Yamazaki K, Seymour GJ. The role of T cells in periodontal disease: Homeostasis and autoimmunity. Periodontol 2000 2007;43:14-40.  Back to cited text no. 4
    
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Teng YT, Nguyen H, Gao X, Kong YY, Gorczynski RM, Singh B, et al. Functional human T-cell immunity and osteoprotegerin ligand control alveolar bone destruction in periodontal infection. J Clin Invest 2000;106:R59-67.  Back to cited text no. 6
    
7.
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Romagnani S. Lymphokine production by human T cells in disease states. Annu Rev Immunol 1994;12:227-57.  Back to cited text no. 8
    
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Mosmann TR, Cherwinski H, Bond MW, Giedlin MA, Coffman RL. Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol 1986;136:2348-57.  Back to cited text no. 9
    
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Mosmann TR, Coffman RL. TH1 and TH2 cells: Different patterns of lymphokine secretion lead to different functional properties. Annu Rev Immunol 1989;7:145-73.  Back to cited text no. 10
    
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Glimcher LH. Trawling for treasure: Tales of T-bet. Nat Immunol 2007;8:448-50.  Back to cited text no. 11
    
12.
Szabo SJ, Kim ST, Costa GL, Zhang X, Fathman CG, Glimcher LH. A novel transcription factor, T-bet, directs Th1 lineage commitment. Cell 2000;100:655-69.  Back to cited text no. 12
    
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Zhu J, Min B, Hu-Li J, Watson CJ, Grinberg A, Wang Q, et al. Conditional deletion of Gata3 shows its essential function in T(H)1-T(H)2 responses. Nat Immunol 2004;5:1157-65.  Back to cited text no. 13
    
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Takeda K, Tanaka T, Shi W, Matsumoto M, Minami M, Kashiwamura S, et al. Essential role of Stat6 in IL-4 signalling. Nature 1996;380:627-30.  Back to cited text no. 14
    
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Yamashita M, Onodera A, Nakayama T. Immune mechanisms of allergic airway disease: Regulation by transcription factors. Crit Rev Immunol 2007;27:539-46.  Back to cited text no. 15
    
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Yamazaki K, Nakajima T, Hara K. Immunohistological analysis of T cell functional subsets in chronic inflammatory periodontal disease. Clin Exp Immunol 1995;99:384-91.  Back to cited text no. 16
    
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Winer J, Jung CK, Shackel I, Williams PM. Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. Anal Biochem 1999;270:41-9.  Back to cited text no. 17
    
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Schmittgen TD, Zakrajsek BA. Effect of experimental treatment on housekeeping gene expression: Validation by real-time, quantitative RT-PCR. J Biochem Biophys Methods 2000;46:69-81.  Back to cited text no. 18
    
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Moore KW, de Waal Malefyt R, Coffman RL, O'Garra A. Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol 2001;19:683-765.  Back to cited text no. 19
    
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Baker PJ, Howe L, Garneau J, Roopenian DC. T cell knockout mice have diminished alveolar bone loss after oral infection with Porphyromonas gingivalis. FEMS Immunol Med Microbiol 2002;34:45-50.  Back to cited text no. 20
    
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Seymour GJ, Gemmell E. Cytokines in periodontal disease: Where to from here? Acta Odontol Scand 2001;59:167-73.  Back to cited text no. 21
    
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Fujihashi K, Yamamoto M, Hiroi T, Bamberg TV, McGhee JR, Kiyono H. Selected Th1 and Th2 cytokine mRNA expression by CD4(+) T cells isolated from inflamed human gingival tissues. Clin Exp Immunol 1996;103:422-8.  Back to cited text no. 22
    
23.
Fujihashi K, Yamamoto M, McGhee JR, Kiyono H. Type 1/type 2 cytokine production by CD4+T cells in adult periodontitis. J Dent Res 1994;73:204.  Back to cited text no. 23
    
24.
Zhao L, Zhou Y, Xu Y, Sun Y, Li L, Chen W. Effect of non-surgical periodontal therapy on the levels of Th17/Th1/Th2 cytokines and their transcription factors in Chinese chronic periodontitis patients. J Clin Periodontol 2011;38:509-16.  Back to cited text no. 24
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1]



 

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