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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgment
  9. References

T helper 17(Th17) cell is a new subset of CD4+ T cells that produce a proinflammatory cytokine interleukin-17 (IL-17). Th17 cells have recently been shown to play a critical role in many autoimmune diseases that had previously been thought to be Th1 dominant. Although Hashimoto’s thyroiditis (HT) was thought to be a Th1-type disease, the contributions of Th17 cells to the pathogenesis remain unclear. In this study, we investigated the expression levels of Th1/Th17 cell-associated factors in peripheral blood mononuclear cells (PBMC) and plasma from patients with HT by quantitative real-time polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). Our results showed that the expression levels of Th1 cells-related T-bet and interferon-γ (IFN-γ) mRNA in PBMC from HT significantly decreased. However, the mRNA of Th17 coherent retinoic acid-related orphan nuclear receptor gamma t (RORγt) and IL-17 in patients with HT increased. In addition, a negative correlation between T-bet and RORγt mRNA expression was found in patients with HT, and the similar phenomena also appeared on the levels of mRNA and plasma concentration between IFN-γ and IL-17. It suggested that Th17 cells rather than Th1 cells predominated among patients suffering from HT, and Th17 cells might be involved in the pathogenesis of HT.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgment
  9. References

Th1 cells can induce cell-mediated inflammatory responses that are responsible for the induction of some autoimmune diseases [1]. Interferon (IFN)-γ is produced mainly by Th1 cells, and it has a critical role in activating macrophages to increase their microbicidal responses. T-bet is the key transcription factor of Th1 [2]. Recently, the emergence of Th17 cells and its produced proinflammatory cytokine IL-17 has raised a great deal of new questions and hot research topic [3, 4]. RORγt is identified as the specific transcription factor of Th17 cell [5]. IL-17 produced by Th17 can induce many inflammatory cytokines secretion and chemokine expression [6, 7], which involved in immune response against extracellular antigen and the induction of many organ-specific autoimmune diseases, such as, rheumatoid arthritis, multiple sclerosis, experimental autoimmune encephalomyelitis, inflammatory bowel disease and psoriasis [6–9].

Hashimoto’s thyroiditis (HT) is an autoimmune disease characterized by the destruction of thyroid cells by lymphocytes and antibody-mediated immune processes [10]. It is one of the most important causes of hypothyroidism in iodine-sufficient areas of the world. But the initiating event of the process is not well understood. The previous views that Th1/Th2 imbalance led to inflammatory factor and infiltrated Th1 cells destroy the thyroid gland have been well documented in the pathogenesis of HT [11].

However, little investigation has been focus on the role of Th17 in HT pathogenesis. In this study, we attempted to discover the relationship between Th1 and Th17 in HT disease by detecting the mRNA of Th1, Th17-associated cytokines, transcription factors in PBMC and the plasma concentrations of IFN-γ and IL-17.

Patients and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgment
  9. References

Patients.  Twenty-four patients diagnosed newly with HT were included in this study from January 2009 to September 2009 from the Affiliated People’s Hospital of Jiangsu University. Among these patients, 21 women and three men ranged from 23 to 60 years old (the average age, 37.24 ± 6.21). Before the blood samples were collected, all patients were untreated. The diagnosis of HT was based on commonly accepted clinical and laboratory criteria: positive for antithyroglobulin and/or antiperoxidase (TgAb and TPOAb, respectively) at high titres; negativity for anti-TSH receptor (TRAb); thyroid ultrasound imaging suggestive of a chronic thyroiditis; the existence of a firm goitre, thyroid dysfunction [12]. Twenty-two volunteers were studied as control, namely 18 women and four men ranging from 28 to 41 years old (the average age, 32 ± 5.36). This project was approved by the ethical committee of the Affiliated People’s Hospital of Jiangsu University, and all the individuals were consent informed.

Blood samples.  Peripheral blood samples were collected from volunteers and patients. The collection tubes contained 0.2 ml sodium heparin. The blood samples were centrifuged at 200 g/min 4 °C for 5 min, and then the supernatant was collected and stored at −70 °C for cytokines assay, and the sediment was separated for preparing PBMC by standard Ficoll-Hypaque density centrifugation. The PBMC were stored at −70 °C with Trizol for extracting total RNA.

Design of primers and response conditions.  According to Genbank sequences, the primers were designed by Primer Premier 5.0 software and synthesized by Shanghai Sangon Biological Engineering Technology & Service Company. All sequences of primers are shown in Table 1.

Table 1.   The primer sequences and amplicons’ length used in this study.
GeneSequence(5′-3′)Accession numberLength (bp)
T-bet5′-GTTCCCATTCCTGTCATTTACT-3′ 5′-TCTCCGTCGTTCACCTCAA-3′NM 013351.1339
IFN-γ5′-TATTCGGTAACTGACTTG-3′ 5′-AATCACATAGCCTTGC-3′NM 000619.2378
RORγt5′-CCTGGGCTCCTCGCCTGACC-3′ 5′-TCTCTCTGCCCTCAGCCTTGCC-3′NM 001001523.1171
IL-175′-CAAGACTGAACACCGACTAAG-3′ 5′-TCTCCAAAGGAAGCCTGA-3′NM 002190.2231
IL-23P195′-AACCCATTAGGACTTGTGC-3′ 5′-CTGAGCCACCCAGGAAAG-3′NM 016584.2213
IL-65′-CAAGACTGAACACCGACTAAG-3′ 5′-CTGGCTCTGAAACAAAGGAT-3′NM 000600.2331
β-actin5′-CACGAAACTACCTTCAACTCC-3′ 5′-CATACTCCTGCTTGCTGATC-3′NM 001101.3265

RNA extraction and cDNA synthesis.  Following the manufacturer’s instructions, total RNA from PBMC was extracted with Trizol (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized with reverse transcription reagent kits (Toyobo, Tokyo, Japan). All RNA samples were heated at 65 °C for 10 min to denature the secondary structure with the template and cooled immediately on ice for 5 min. Total RNA (1 μg) was reversely transcribed in a total volume of 20 μl, containing oligo (dT) 1 μl, dNTP (10 mm) 2 μl, 5 × RT buffer 4 μl, ReverTraAce (100 U/μl) 1 μl, RNAse Inhibitor 1 μl, DEPC H2O added up to 20 μl, response conditions: 42 °C 20 min; 99 °C 5 min and 4 °C 5 min. The cDNA was stored at −20 °C.

Construction of T-bet, IFN-γ, RORγt, IL-17, IL-23p19, IL-6 and β-actin recombinant plasmids calibrator.  PCR was performed in the PCR thermal cycler (Eppendorf, Hauppauge, NY, USA). The program consisted of an initial denaturation step for 5 min at 94 °C followed by 30 cycles, with each cycle consisting of 30-s denaturing at 94 °C, 30-s annealing at 56 °C and 30-s extension at 72 °C. The reaction was completed by a final 5-min extension at 72 °C.

Purified T-bet, IFN-γ, RORγt, IL-17, IL-23p19, IL-6 and β-actin PCR amplicons were transformed to PMD18-T vector (Invitrogen, Carlsbad, CA, USA) to establish recombinant plasmids PMD18-T-bet, IFN-γ, RORγt, IL-17, IL-23p19, IL-6 and β-actin. All these recombinant plasmids were transformed into competent E. coli DH5α and inoculated into 1.5% agar Amp-resistant plate and then cultured at 37 °C for 12–14 h. Positive clones were initially identified by sequencing. Part of positive clones were further amplified and accurately quantified with a nucleic acid-protein ultraviolet instrument. Tenfold serial dilution of the recombinant plasmid DNAs was used as calibrator and stored at −20 °C to use.

RT-qPCR detects objective genes expression.  The expression of objective genes (T-bet, IFN-γ, RORγt, IL-17, IL-23p19 and IL-6) was detected by quantitative real-time polymerase chain reaction (RT-qPCR). All PCR were performed using the Corbett Rotor Gene 6000 system (Promega, Madison, WI, USA) in a total volume of 20 μl, containing 1 μl cDNA, 10 μl 2 × SYBR Green1 Premix (Takara, Dallian, China), 0.3 μl 10 μm each primer and 8.4 μl water. The specificity of the amplicons was controlled by a melting curve analysis. The copy numbers of T-bet, IFN-γ, RORγt, IL-17, IL-23p19, IL-6 and β-actin transcripts in samples were calculated with the Corbett software according to corresponding standard curves. The copy numbers of gene/%β-actin^103 represent the ratio of the gene. A no-template negative control was also included in each experiment, and all samples were measured in triplicate.

Enzyme-linked immunosorbent assays (ELISA) for plasma IL-17, IFN-γ, IL-23 and IL-6.  Plasma levels of IL-17, IFN-γ, IL-23 and IL-6 were measured by ELISA, following the manufacturer’s protocols (eBioscience, San Diego, CA, USA). All samples were measured in triplicate.

Statistical analysis.  All statistical analysis was performed using SPSS17.0 (SPSS, Chicago, IL, USA). Data were expressed as the mean ± standard deviation (SD) in text and figures. Comparisons between paired or unpaired groups were made using the appropriate Student’s t-test. For non-parametric data, differences between two groups were analysed by the Mann–Whitney test. Spearman’s correlation was used to test correlation between two continuous variables. < 0.05 was considered to be statistical significance.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgment
  9. References

Electrophoresis identification of PCR amplicons

The lengths of T-bet, IFN-γ, RORγt, IL-17, IL-23p19, IL-6 and β-actin were 339, 378, 171, 231, 213, 331 and 265bp, respectively, and it was consistent with the expected. Identification of positive clone recombinant plasmids was validated by sequencing. These objective gene sequences were in accordance with the Genbank of National Center of Bioinformatics Institute (detailed data not shown).

Levels of T-bet, IFN-γ, RORγt, IL-17, IL-23p19 and IL-6 mRNA in individual patients with HT

The detection range of recombinant plasmid DNAs was from 10 to 108 copies, and the coefficients of variation values ranged from 2.20% to 8.32%; Amplification efficiency ranged from 0.88 to 0.92 and r^2 > 0.99. The expression levels of T-bet, IFN-γ, RORγt, IL-17, IL-23p19 and IL-6 mRNA from patients with HT and controls were measured by RT-qPCR. As shown in Fig. 1(B,D), the mRNAs of Th17-associated cytokine (IL-17) and transcription factor (RORγt) significantly increased in PBMC from patients with HT (< 0.01), and almost every patient with HT had threefold higher than control. However, T-bet and IFN-γ mRNA expression levels obviously decreased in patients with HT(P < 0.01) (Fig. 1A,C).

image

Figure 1.  Expression of each gene by RT-qPCR. A represents T-bet, B represents RORγt, C represents IFN-γ and D represents IL-17.

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The correlations between the mRNA levels of T-bet, RORγt, IFN-γ and IL-17

To assess the relationships of Th1 and Th17 cell-associated factors in PBMC from patients with HT, we examined the correlations between the levels mRNA of T-bet and RORγt, IFN-γ and IL-17 in PBMC of patients with HT. There was a significantly positive correlation between RORγt and IL-17 (Fig. 2A), and a negative correlation between T-bet and RORγt (Fig. 2B). A negative correlation was also found between IL-17 and IFN-γ (Fig. 2C).

image

Figure 2.  The correlation between the mRNA levels of RORγt and IL-17, T-bet and RORγt, IL-17 and IFN-γ, in PBMC with patients with HT (= 24). (A) Interleukin (IL)-17 correlated positively with Th17 cell master regulator gene RORγt. (B) T-bet correlated negatively with RORγt. (C) IFN-γ correlated negatively with IL-17.

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The concentrations of IL-17 and IFN-γ in plasma from patients with HT

Plasma concentrations of IL-17 and IFN-γ measured by ELISA in each group were shown in Table 2. IL-17 level in patients with HT was higher than that in controls, whereas the concentration of IFN-γ in patients with HT was lower than that in volunteers. Those two differences were statistically significant. Additionally, in patients with HT, the concentrations of IL-17 in plasma showed a positive correlation with the mRNA levels of RORγt in PBMC (Fig. 3A), and a similar correlation was found between the IFN-γ plasma concentrations and T-bet mRNA levels (Fig. 3B), but a negative correlation was found between the plasma concentrations of IFN-γ and IL-17 (Fig. 3C).

Table 2.   Plasma concentrations of cytokines in patients and controls (mean ± standard deviation).
 Patients with HT (n = 24)Controls (n = 22)P
IL-17 (pg/ml)187.87 ± 35.94 98.39 ± 5.52<0.05
IFN-γ (pg/ml)37.74 ± 3.76106.69 ± 8.47<0.01
IL-23 (pg/ml)155.87 ± 70.16  99.24 ± 12.15<0.05
IL-6 (pg/ml) 71.91 ± 26.83 14.53 ± 6.92<0.01
image

Figure 3.  In patients with HT, the correlation between the mRNA levels of RORγt/T-bet in PBMC and the concentrations of IL-17/IFN-γ in plasma and the concentrations of IL-17 and IFN-γ in plasma. (A) The plasma concentrations of IL-17 correlated positively with the levels of RORγt mRNA in PBMC. (B) The levels of T-bet mRNA in PBMC correlated positively with the plasma concentrations of IFN-γ. (C) The plasma concentrations of IL-17 correlated negatively with IFN-γ.

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The microenvironments of patients with HT contribute to Th17 cells differentiation

To further confirm whether the microenvironments of patients with HT contribute to Th17 cells differentiation or not, we detected the Th17 differentiation-associated factors IL-23p19 and IL-6 by RT-qPCR and ELISA, and we found that IL-23p19 and IL-6 were obviously upregulated and had statistical significance (Fig. 4 and Table 2).

image

Figure 4.  Expression Level of IL-23p19 and IL-6 in patients with HT.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgment
  9. References

HT is an autoimmune disease manifesting as hypothyroidism that is accompanied by massive infiltration of lymphoid cells. A large number of the intrathyroidal lymphocytes in patients are CD4+ T cells, which have been proposed to play important roles in cytokine mediating response [10–17]. Th1 was thought to contribute to a number of autoimmune diseases pathogenesis (including HT) [18–21]. Moreover, Th17 subset has sparked great interest in the role of a broad range of immune-mediated diseases in late 2005 [4, 6]. Th17 cells and their effectors cytokines have being recognized increasingly as important mediators in organ-special autoimmune diseases but relatively unknown about their specific roles in thyroiditis, especially in HT [7, 21–28]. Nictéet al. [29] have suggested an increased differentiation of Th17 lymphocytes and an enhanced synthesis of Th17 cytokines in HT. So the question whether the subsets of Th1 and Th17 are redistributed or not during the development of HT is worth to study.

In our study, the mRNA expression levels of Th1 associated factors were obviously lower and Th17-related factors were significantly higher in patients with HT, which may indicate that Th1/Th17 imbalance existed in PBMC from patients with HT. It may be attributed to some other effectors T cell (T(eff)) cells including Th1 shifting to Th17 or the subsets of CD4+ T cells redistribution under the free triiodothyronine 3 (FT3), free triiodothyronine 4 (FT4), follicle-stimulating hormone, TGAb and TMAb in patients with HT. Therefore, the imbalance of Th1 and Th17 induced or exacerbated the HT. We also found that the microenvironment of patients with HT maybe contribute to Th17 cells differentiation, which was with Figueroa-Vega et al. [30] reports. These results also indicated that Th17 cells may participate in pathogenesis of the autoimmune diseases. But it needs further study to prove whether the measurement of Th17 cell indicate prognostic significance of HT. Our results, together with a previous report demonstrated the importance of Th1 not Th2 immune response for developing thyroiditis using mice deficient for interferon-gamma or IL-4, which clearly indicated that both Th1 and Th17 cells are critical T(eff) subsets for the pathogenesis of spontaneous autoimmune thyroiditis [10, 11, 15, 16, 22]. Although Th17 cells were not the specific source of IL-17 and RORγt, Th17 cells were main source. Our data provided some valuable information to support further research on the precise function of Th17 cells in HT.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgment
  9. References

Our study showed that there was a significant difference between the volunteers and the patients with HT in Th17-related factors expression level, including transcription factors and cytokines in PBMC and plasma, which did indicate that Th17 and its related factors played an important role in HT disease.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgment
  9. References

This work was supported by the National Natural Science Foundation of China (Grant NO. 30871193, 30972748, respectively); Natural Science Foundation of Jiangsu Province Educational Commission (Grant No. 09KJB310001); Innovation Fund for candidate of doctor in Jiangsu Province (Grant NO. CX09B_217Z); Health Department Foundation of Jiangsu Province (Grant NO.H200952).

We thank Dr Valiyeva F and Dr Hu Xu for their editing and proofreading the manuscript.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgment
  9. References