The Long Noncoding RNA IFNG-AS1 Promotes T Helper Type 1 Cells Response in Patients with Hashimoto’s Thyroiditis

The long noncoding (lnc) RNA-Ifng-AS1 plays an essential role in the transcription of the gene encoding IFN-γ by Th1 cells, and its human ortholog, IFNG-AS1, is expressed in human Th1 cells. However, IFNG-AS1 contributing to Th1 cells’ response in Hashimoto’s thyroiditis (HT) patients has not been reported. Twenty-eight HT patients and 20 healthy controls were enrolled in the study. The proportion of circulating Th1 cells and the level of T-bet, IFNG mRNA were increased in HT patients, the expression of IFNG-AS1 was upregulated and positively correlated with the proportion of circulating Th1 cells or T-bet, and IFNG expression, or serum level of anti-thyroglobulin antibody/thyroperoxidase antibody in HT patients. IFNG-AS1 regulated the expression of IFNG at both transcriptional and translational level in human CD4+ T cells. Furthermore, strong positive correlations between the increased transcript level of IFNG-AS1 and the increased transcript level of T-bet or IFNG were revealed in thyroid tissues from HT patients. Our results indicate that enhanced expression of lncRNA-IFNG-AS1 contributes to Th1 cell response in HT patients and may be involved in the pathogenesis of HT.

presence of interleukin 12 12,13 . T-bet is the key transcription factor, contributing to the transcription of IFNG in Th1 cells 14 . Recent studies have shown that Th1 cells might be involved in the development of HT 7,15,16 . However, our understanding of increased Th1 cells in HT patients remains largely unknown.
Long noncoding RNAs (lncRNAs) are increasingly appreciated as key regulators of genome expression, and only some of their functions have been characterized 17 . In some well-studied cases, lncR-NAs play critical roles in various biological processes and diseases [18][19][20] . However, only a few lncRNAs, such as lnc-DC, NRON, Gas5, have been described in the regulation of the immune system by regulating particular genes [21][22][23] . Ifng-AS1 (Ifng antisense RNA 1), also named NeST (nettoie Salmonella pas Theiler's), or Tmevpg1 (Theiler's murine encephalomyelitis virus persistence candidate gene 1), a lncRNA was initially identified as a candidate gene for the control of Theiler's virus persistence 24 . Ifng-AS1 and its human ortholog are located adjacent to IFN-γ -encoding gene in both mouse and human. Recent studies have described that Tmevpg1 (Ifng-AS1, NeST) is recognized to be a key checkpoint that requires T-bet for active transcription and contributes to Ifng expression in Th1 cells 25,26 . However, it is not yet known whether IFNG-AS1 regulates Th1 cells response in HT patients. Therefore, we explore the role of IFNG-AS1 in the pathogenesis of HT.
In this study, we investigated whether the expression of IFNG-AS1 is dysregulated in HT patients. We found that the expression of IFNG-AS1 was upregulated, and positively correlated with the proportion of circulating Th1 cells in HT patients. These findings provide new insights in understanding the role of IFNG-AS1 in the pathogenesis of HT.

Materials and Methods
Subjects and samples. Twenty-eight patients with HT, including twenty-three females and five males were enrolled into the study. The main clinical characteristics of these patients are summarized in Table 1. All patients were diagnosed by clinical manifestation and auxiliary examination, including B-ultrasonic and laboratory criteria. The serum concentration of free triiodothyronine (FT3), free thyroxine (FT4), thyroid stimulating hormone (TSH), TgAb, and TPOAb were measured by LDX-800 (BECKMAN COULTER, California, USA), according to the manufacturer's instructions. Ten HT patients with hypothyroidism had a high level of TSH and low level of FT4, other patients with euthyroid had a normal level of both TSH and FT4. All patients had a positive test for TgAb and TPOAb. Twenty age-and sex-matched healthy subjects were included as controls. All healthy subjects were free of thyroid-specific autoantibodies and had no history of thyroid disease or other autoimmune diseases. The number of peripheral leukocytes was within normal range. Peripheral blood samples were obtained from all patients and healthy controls.
Fresh tissue samples from the thyroid gland of ten HT patients were collected from thyroidectomy and stored at − 80 °C. Lymphocytic infiltration was detected in thyroid samples. Thyroid tissues from five patients with simple goiter were used as control thyroid samples.
The study conformed to the principles outlined in the Declaration of Helsinki and in accordance with the approved guidelines. Written informed consent was obtained from each participant prior to blood samples collection. All samples were taken in accordance with the regulations and approval of the Affiliated People's Hospital of Jiangsu University.
Cell isolation and purification. Human peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation over Ficoll-Hypaque solution (Haoyang Biological Technology Co., Tianjin, China) and stored at − 80 °C until use for quantitative real-time PCR (qRT-PCR). Human CD4 + T cells were purified from PBMCs by magnetic beads using CD4 + T cell Isolation Kit (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) according to the manufacturer's instructions. Human CD4 + T cells were cultured in RPMI-1640 medium (Gibco, California, USA) supplemented with 10% fetal bovine serum (Gibco, California, USA) for transfection. Thyroid mononuclear cells (TMCs) were obtained from thyroid specimens, which were minced and digested with collagenase II (Sigma-Aldrich, St. Louis, MO) for 1-2 h at 37 °C and then isolated by density-gradient centrifugation over Ficoll-Hypaque solution. Cell viability was found to be more than 95%.
Flow cytometric analysis. Separated PBMCs were resuspended at 1 × 10 6 /ml in RPMI-1640 medium containing 10% fetal bovine serum and stimulated with 50 ng/ml of phorbol myristate acetate (PMA; Sigma-Aldrich, California, USA) and 1 μ g/ml of ionomycin (Sigma-Aldrich, California, USA) for 2 hours and then incubated for an additional 4 hours in the presence of 1 μ g/ml of brefeldin-A (eBioscience, San Diego, USA) at 37 °C and 5% CO 2 . After the incubation, the suspended cells were stained with phycoerythrin-cyanin 5 (PE-Cy5) -conjugated anti-human CD3 mAb and fluorescein isothiocyanate (FITC) -conjugated anti-human CD8 mAb (eBioscience, San Diego, USA) against cell surface antigens for thirty minutes at 4 °C in the dark. Cells were then fixed and permeabilized using an intracellular staining kit (Invitrogen, Carlsbad, USA), followed by incubation for 45 minutes at 4 °C in the dark with PE-conjugated anti-human IFN-γ mAb (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The stained cells were analyzed with Accuri C6 (Becton Dickinson, San Jose, USA). To analyze the proportion of Th1 cells, the population of CD3 + CD8 -IFN-γ + cells was defined as Th1 cells.
Small interfering RNA knockdown. Small interfering RNA (siRNA) (Ribobio, Guangzhou, China) was designed against the sequence of IFNG-AS1. Nonspecific scramble siRNA was used as negative control (NC). The purified human CD4 + T cells were transfected with the IFNG-AS1 siRNA or NC at 100 nM dose using the Entranster-R (Engreen Biosystem, Co Ltd, Beijing, China) according to the manufacturers' instructions for 48 hours in the presence of 0.5 μ g/ml functional anti-human CD3 mAb plus 2 μ g/ml functional anti-human CD28 mAb (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). IFN-γ + cells were measured by flow cytometric analysis.
Statistical analysis. Student's unpaired t test was applied for two comparisons in accordance with the standard t test. Mann-Whitney U test was used to analyze the difference between the two groups. Correlation between variables was determined by Pearson's correlation coefficient. p value < 0.05 was considerate as significant (*p < 0.05, **p < 0.01, ***p < 0.001). Data were analyzed with GrapPadPrism version 5 software (GraphPad Software, Inc., San Diego, USA).

Increased circulating Th1 cells in HT patients. To quantify peripheral Th1 cells in patients with
HT, we first gated on CD3 + CD8cells as CD4 + T cells owing to the downregulated expression of surface membrane CD4 molecule on PBMCs after being stimulated with PMA and ionomycin 27 and then identified IFN-γ + cells to distinguish Th1 cells from activated PBMCs (Fig. 1A). The proportion of peripheral Th1 cells in PBMCs from patients with HT was significantly higher than that in healthy controls (Fig. 1B).
Subsequently, we determined the transcript level of T-bet and IFNG in PBMCs from HT patients and healthy controls by qRT-PCR and found that the transcript level of T-bet and IFNG were substantially greater in HT patients than in healthy controls (Fig. 1C,D). Moreover, a positive correlation between the transcript level of T-bet and the proportion of Th1 cells was found in HT patients (r = 0.4590; p = 0.0140) (Fig. 1E).

Positive correlations between elevated transcript level of IFNG-AS1 and increased circulating Th1 cells in HT patients. IFNG-AS1
is a long noncoding RNA that is comprised of four exons and located at Chromosome 12q15 on the opposing strand to IFNG (Fig. 2A). Ifng-AS1 (Tmevpg1, NeST) expression contributes to driving Th1 cell-dependent Ifng expression, and its human ortholog, IFNG-AS1 (TMEVPG1, NEST), is selectively expressed in Th1 cells 25 . To address the possibility that IFNG-AS1 contributes to increased Th1 cells in HT patients, the transcript level of IFNG-AS1 was determined by qRT-PCR. As shown in Fig. 2B, the transcript level of IFNG-AS1 from PBMCs was increased in HT patients compared with that in healthy controls. Moreover, positive correlations were observed between the transcript level of IFNG-AS1 and the percentage of Th1 cells (r = 0.5010; p = 0.0066) (Fig. 2C) or the transcript level of T-bet (r = 0.6138; p = 0.0005) (Fig. 2D) or the transcript level of IFNG (r = 0.4463; Scientific RepoRts | 5:17702 | DOI: 10.1038/srep17702 p = 0.0173) in HT patients (Fig. 2E). In contrast to the relationship we observed between the transcript level of IFNG-AS1 and the proportion of Th1 cells, there was no correlation between the transcript level of IFNG-AS1 and the proportion of CD8 + IFN-γ + T cells (Supplemental Fig. 1A), which was significantly greater in HT patients than in healthy controls (Supplemental Fig. 1B).

Influence of IFNG-AS1 on the transcription of IFNG in human CD4 + T cells. To determine
whether IFNG-AS1 affects IFNG transcription from CD4 + T cells, human purified CD4 + T cells were transfected with IFNG-AS1-specific siRNA and negative control. Manipulation of IFNG-AS1-specific siRNA resulted in the reduction of the transcript level of IFNG-AS1 and IFNG compared with that of the negative control (Fig. 3A,B). Down-regulated expression of IFNG-AS1 with siRNA resulted in the reduction of the percentage of IFN-γ + cells compared with that of the negative control (Fig. 3C). Moreover, IFNG-AS1-specific siRNA suppressed the percentage of IFN-γ + cells in a dose-dependent manner (Fig. 3D,E). Together, these results indicate that IFNG-AS1 regulates the expression of IFNG in human CD4 + T cells.  index of HT 28 . Our results indicated that there were positive correlations between the transcript level of IFNG-AS1 and the level of TgAb (r = 0.4762, p = 0.0104) or TPOAb (r = 0.3789, p = 0.0468) (Fig. 4A,B).

Upregulated expression of IFNG-AS1, T-bet and IFNG mRNA in thyroid tissues from HT patients.
HT is an organ-specific autoimmune disease characterized by lymphoid infiltration and thyroid structure destruction. To determine whether IFNG-AS1, T-bet and IFNG mRNA were also expressed in local thyroid tissue, qRT-PCR analysis displayed enhanced expression of IFNG-AS1, T-bet and IFNG mRNA in TMCs from patients with HT compared to those from patients with simple goiter (Fig. 5A-C). Strong positive correlations were observed between the transcript level of IFNG-AS1 and T-bet mRNA (r = 0.8652, p = 0.0012) or IFNG mRNA (r = 0.9398, p < 0.0001) in patients with HT (Fig. 5D,E).

Discussion
Lower conservative expression of lncRNAs sequences has prevented most of the sequences from being functionally characterized 29 . Presently, more and more data suggest lncRNAs are mainly divided into   higher transcript level of IFNG-AS1 was found in peripheral blood and thyroid tissues from HT patients. Furthermore, positive correlations were found between the transcript level of IFNG-AS1 and the proportion of Th1 cells, as well as transcript level of T-bet or IFNG in HT patients. Although a study has reported that Tmevpg1 promoted the transcription of Ifng from murine Th1 cells in vitro 25 , the influence of IFNG-AS1 on IFNG transcription in humans is still unknown. To investigate the role of IFNG-AS1 in human CD4 + T cells, we used IFNG-AS1-specific siRNA to knockdown IFNG-AS1 and observed the alteration of IFNG. IFNG-AS1 knockdown resulted in a considerable reduction of IFNG in gene expression and protein expression (IFN-γ ). Interestingly, IFNG-AS1-specific siRNA downregulated the proportion of CD4 + IFN-γ + cells in a dose-dependent manner. In the present study, we provide direct evidence that IFNG-AS1 regulates the transcription of IFNG from human Th1 cells in vitro. However, the epigenetic mechanism of Ifng-AS1 promotes Ifng expression in Th1 cells is poorly understood.
The expression of Tmevpg1 was found in Th1 cells from both mice and human, but was not detected in effector CD8 + T cells under the same culture conditions, which also produced IFN-γ 25 . Our supplemental data also demonstrated that there was no correlation between the transcript level of IFNG-AS1 and increased proportion of CD8 + IFN-γ + T cells in HT patients. One possible interpretation is the different mechanisms of IFN-γ production between Th1 cells and CD8 + T cells. T-bet is the master regulator of Ifng expression in Th1 cells 14 . However, IFN-γ production by effector CD8 + T cells is dependent of Eomesodermin (Eomes), a paralogue of T-bet, which plays key role in the differentiation and function of CD8 + T cells 31 . Another possible interpretation is the difference in regulating IFNG expression by IFNG-AS1 between Th1 cells and CD8 + T cells. Although one model is generally indicated that Ifng-AS1(NeST) was required for Ifng expression in response to infection with Salmonella, and Ifng-AS1(NeST) was found to bind WDR5 component of histone H3 lysine 4 methytransferase complex, and to modify H3K4me3 at the Ifng locus by CD8 + T cells 30 . The differences between these reported findings may be due to different animal models, and the further mechanisms will be investigated in future.
Accumulating studies have demonstrated that HT is a Th1-mediated autoimmune disease because there are abundant Th1 cells infiltrating and thyrocyte destruction in HT patients 14,31,32 . Our results also showed that the proportion of Th1 cells and related genes, such as T-bet and IFNG, were higher in peripheral blood and thyroid gland from HT patients. It is widely accepted that elevated serum concentrations of TgAb and TPOAb are the most common manifestations of HT. These autoantibodies could indicate the development of HT, and IFN-γ production by Th1 cells drives the generation of autoantibodies 28 . We analyzed the correlation between the transcript level of Ifng-AS1 and serum level of autoantibodies. Positive correlations were found between the transcript level of Ifng-AS1 and the level of TgAb or TPOAb. These data suggest that IFNG-AS1 expression could reflect disease severity of HT to some extent.
In summary, our results demonstrate that the lncRNA IFNG-AS1 is significantly increased and may contribute to the pathogenic role of Th1 cells response in HT patients. Further exploration of the mechanism of IFNG-AS1-driven Th1 cells response may lead to better understanding of the pathogenesis of HT.