Skip to main content
Download PDF

Increased HERV-E clone 4–1 expression contributes to DNA hypomethylation and IL-17 release from CD4+ T cells via miR-302d/MBD2 in systemic lupus erythematosus

Cell Communication and Signaling volume 17, Article number: 94 (2019) Cite this article

Abstract

Background

Increased human endogenous retroviruses E clone 4–1 (HERV-E clone 4–1) mRNA expression is observed in systemic lupus erythematosus (SLE) patients and associates with the disease activity. In this study, we want to further investigate the mechanism of HERV-E clone 4–1 mRNA upregulation and its roles in SLE progression.

Methods

CD4+ T cells were isolated from venous blood of SLE patients or healthy controls and qRT-PCR was used to detect HERV-E clone 4–1 mRNA expression. We then investigated the regulation of Nuclear factor of activated T cells 1 (NFAT1) and Estrogen receptor-α (ER-α) on HERV-E clone 4–1 transcription and the functions of HERV-E clone 4–1 3′ long terminal repeat (LTR) on DNA hypomethylation and IL-17 release.

Results

We found HERV-E clone 4–1 mRNA expression was upregulated in CD4+ T cells from SLE patients and positively correlated with SLE disease activity. This is associated with the activation of Ca2+/calcineurin (CaN)/NFAT1 and E2/ER-α signaling pathway and DNA hypomethylation of HERV-E clone 4–1 5’LTR. HERV-E clone 4–1 also takes part in disease pathogenesis of SLE through miR-302d/Methyl-CpG binding domain protein 2 (MBD2)/DNA hypomethylation and IL-17 signaling via its 3’LTR.

Conclusions

HERV-E clone 4–1 mRNA upregulation is due to the abnormal inflammation/immune/methylation status of SLE and it could act as a potential biomarker for diagnosis of SLE. HERV-E clone 4–1 also takes part in disease pathogenesis of SLE via its 3’LTR and the signaling pathways it involved in may be potential therapeutic targets of SLE.

Background

Systemic lupus erythematosus (SLE) is an autoimmune disease in which autoreactive CD4+ T cells play an important role [1]. Genetic interactions with environmental factors, particularly ultraviolet light exposure, infection and hormonal factors, might initiate the disease, resulting in immune dysregulation at the level of cytokines, T cells, B cells and macrophages [2].

Human endogenous retroviruses (HERV) are descendants of occasional germline invasion by exogenous retroviruses which occupy as much as 8% of the human genome [3]. HERV-E clone 4–1 is inserted in the short arm of chromosome 19 at position 19p12 upstream of the ZNF66 gene locus and in the antisense orientation. This full-length HERV-E clone 4–1 is considered to be an LTR2C prototype containing 5′ and 3′ LTR elements that are 95.5% identical and encompass gag, pol and env genes (GenBank: M10976, Additional file 1: Figure S1) [4]. Enhanced expression of mRNA from HERV-E clone 4–1 was reported in SLE than healthy controls (HCs) [5, 6], and our former study demonstrated that HERV-E clone 4–1 mRNA expression was increased in SLE patients, and the expression level of HERV-E clone 4–1 was associated with SLE disease activity index (SLEDAI) [7]. HERV-E clone 4–1 5’LTR/LTR2C was hypomethylated in CD4+ T cells from SLE patients [7,8,9] which might have close relationship with its expression.

In this study, we sought to further investigate the mechanism of HERV-E clone 4–1 mRNA upregulation and its roles in SLE progression, and to estimate the potential value of HERV-E clone 4–1 in acting as a biomarker and therapeutic target for SLE.

Methods

Ethics and selection of patients

This research was approved by the Institutional Research Ethics Committee of Shanghai General Hospital and abided by the ethical guidelines of the Declaration of Helsinki. All the patients involved in this study were adult and written informed consents were obtained from all the patients. All patients with SLE were diagnosed in accordance with the 1997 ACR revised criteria for classification of SLE. Disease activity was assessed using the SLE disease activity index (SLEDAI), and active disease was defined as an SLEDAI score ≥ 5. Age- and sex-matched healthy controls were recruited from the medical staff at Shanghai General Hospital.

Isolation, culture and treatment of CD4+ T cells

Peripheral blood mononuclear cells (PBMC) were isolated from venous blood of SLE patients or healthy controls using Ficoll-paque density gradient centrifugation. Purified CD4+ T cells were negatively isolated from PBMCs by CD4+ T-cell isolation kits (STEMCELL Technologies, Vancouver, Canada) according to the manufacturer’s protocol. CD4+ T cell purity was routinely > 90% as verified through flow cytometry. The cells were then cultured in Xvivo 15 medium (Lonza, Walkersville, MD, USA) supplemented with 10% human AB serum (Valley Biomedical, Winchester, VA, USA) at 37 °C with 5% CO2. The treatments of the cells were: TNF-α (HY-P7058, MedChemExpress, NJ, USA), 10 ng/ml, 24 h; IL-6 (HY-P7044, MedChemExpress), 10 ng/ml, 24 h; 17β-estradiol (estradiol/E2) (HY-B0141, MedChemExpress), 100 nmol/L, 24 h; Lipopolysaccharides (LPS) (L8880, Solarbio, Beijing, China), 100 ng/ml, 24 h; ultraviolet B (UVB), 50 mJ/cm2 [10]; hydroxychloroquine sulfate (HCQ sulfate) (HY-B1370, MedChemExpress), 6 μg/ml, 24 h; 5-Azacytidine (5-aza C) (HY-10586, MedChemExpress), 1 mM, 24 h; prednisolone (HY-17463, MedChemExpress), 10 ng/ml, 24 h; AZD9496 (HY-12870, MedChemExpress), 5 nM, 24 h.

Quantitative reverse transcription-PCR (qRT-PCR)

Total RNAs of cells were extracted using Trizol (Invitrogen) according to the instructions provided by the manufacturer. Reverse transcription was performed using the Primescript RT Master Mix (Takara, Otsu, Japan), and cDNA was amplified using SYBR-Green Premix (Takara). The expression of HERV-E clone 4–1 gag was normalized to the expressions of GAPDH. The data were analyzed by delta Ct method. Primers of HERV-E clone 4–1 gag used in this study were imported from other published articles [5,6,7] and the primers were, F: 5′-CACATGGTGGAGAGTCGTGTTT-3′ and R: 5′-GCTTGCGGCTTTTCAGTATAGG-3′; GAPDH, F: 5′-GGAGTCCACTGGCGTCTTC-3′ and R: 5′-GCTGATGATCTTGAGGCTGTTG-3′. Primers for HERV-E clone 4–1 3’LTR were, F: 5′-TCGCCACTTCTCCTGTTGTC-3′ and R: 5′-TATTCGGCCGGGATCATTGG-3′.

Oligonucleotide, plasmids and transfection

SiRNA, miR-302d mimics and corresponding negative controls were transfected by Hiperfect transfection reagent (Qiagen, Valencia, CA, USA) and plasmids were transfected by Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) into cells. Nuclear factor of activated T cells 1 (NFAT1) siRNA and Estrogen receptor-α (ER-α) siRNA were obtained from Santa Cruz Biotechnology (sc-36,055, sc-29,305, Santa Cruz, CA, USA). The 3’LTR of HERV-E clone 4–1 were cloned into pcDNA 3.1 plasmid and the recombinant plasmid was transfected into cells to obtain the 3’LTR mRNA overexpression.

Western blot analysis

Cells were lysed using radioimmunoprecipitation (RIPA) lysis buffer (Beyotime, Shanghai, China). Protein concentrations were detected using bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA). Total proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). Antibodies used in the assays were NFAT1 antibody (ab2722, Abcam, Cambridge, UK), ER-α antibody (#8644, Cell Signaling Technology) and GAPDH antibody (#5174, Cell Signaling Technology), IRF9 antibody (#76684, Cell Signaling Technology), Methyl-CpG binding domain protein 2 (MBD2) antibody (ab38646, Abcam) and IL-17 antibody (ab77171, Abcam).

Luciferase assay

An NFAT luciferase reporter plasmid (pNFAT-Luc) containing NFAT1 binding promoter elements was used to detect the NFAT1 transcriptional activity. CD4+ T cells were co-transfected with a mixture of 300 ng pNFAT-Luc reporter and 5 ng pRL-TK Renilla luciferase reporter. After different treatment, the luciferase activities were measured using the Dual Luciferase Reporter assay (Promega, Madison, WI, USA). pRL-TK Renilla luciferase reporter was used to normalize the transfection efficiency.

Full-length sequences of HERV-E clone 4–1 5′ LTR containing wild-type of NFAT1 or ER-α predicted binding site was inserted into PGL3-Basic luciferase reporter vector (Promega). Mutant reporter plasmids were prepared using Mutagenesis Kit (Stratagene, La Jolla, CA, USA). Cells were co-transfected with a mixture of 300 ng firefly luciferase reporter, 5 ng pRL-TK Renilla luciferase reporter, and NFAT1 or ER-α plasmids. After 48 h of incubation, the luciferase activities were quantified using the Dual Luciferase Assay System (Promega). The sequences of 3’LTR of HERV-E clone 4–1 mRNA or MBD2 3’UTR containing potential wild-type or mutant binding sites of miR-302d were constructed into pmirGLO vectors (Promega). The luciferase vectors and miR-302d mimics were transfected into CD4+ cells along with pRL-TK vector. The dual-luciferase Reporter assay system (Promega) was used to detect luciferase activity. pRL-TK Renilla luciferase reporter was used to normalize the transfection efficiency.

Chromatin immunoprecipitation (ChIP)

ChIP assay was conducted using EZ ChIP kit (Millipore, Billerica, MA, USA) and NFAT1 antibody (ab2722, Abcam) or ER-α antibody (#8644, Cell Signaling Technology) according to the instruction of the manufacturer. The primers specific to HERV-E clone 4–1 5′ LTR were: 5′-CTCCCCAACCTCCCCTTTTC-3′ and 5′-TGAGAAACATGACTGGGGGC-3′. Normal rabbit IgG (A7016, Beyotime, Shanghai, China) was used to control the nonspecific immunoprecipitation.

DNA extraction and global methylation analysis

Assays of DNA extraction and global methylation analysis was described in our previous study [11].

Enzyme-linked immunosorbent assay

The concentration of IL-17 in culture supernatants were measured by Human IL-17 ELISA Kit (ab119535, Abcam) according to the manufacturer’s instructions. Optical density values were read at 450 nm using ELx800 Absorbance Microplate Reader (BioTek, VT, USA).

Statistical analysis

Statistical analysis was performed using the SPSS program (version 18.0; SPSS, Chicago, IL, USA). The statistical significance of differences between two groups was tested using Student’s t test. Spearman’s analysis was used to test correlation. P < 0.05 was considered as statistically significant.

Results

HERV-E clone 4–1 mRNA expression was upregulated in CD4+ T cells from SLE patients

In our former study, we found that HERV-E clone 4–1 mRNA expression was higher in lupus CD4+ T cells than in cells from healthy controls and the HERV-E clone 4–1 mRNA expression level was positively correlated with SLE disease activity [7]. To continue our study, first, we used new samples (Additional file 1: Table S1) to prove HERV-E clone 4–1 mRNA expression was higher in SLE CD4+ T cells than in cells from healthy controls using Quantitative reverse transcription-PCR (qRT-PCR) (Fig. 1a). We also found that HERV-E clone 4–1 mRNA expression level was higher in active patients than that of inactive patients (Fig. 1b) and positively correlated with SLE disease activity (Fig. 1c). We also followed-up some patients who got oral prednisolone and hydroxychloroquine treatment and the activity of SLE changed from active to inactive. We found that the HERV-E clone 4–1 mRNA expressions decreased as the SLEDAI decreased (Fig. 1d and e). But the HERV-E clone 4–1 mRNA expressions of the inactive patients were also higher than that of HCs (Fig. 1f). What’s more, to assess the diagnostic value of HERV-E clone 4–1 mRNA for SLE, we performed Receiver Operating Characteristic (ROC) curve analysis to differentiate SLE from HC with the relative HERV-E clone 4–1 mRNA expressions of the SLE patients and healthy controls (Fig. 1f). The Area Under Curve (AUC) was 0.760, the 95% confidence interval (95% CI) was 0.622 to 0.897, and the best Youden’s index is 0.5. This indicated that HERV-E clone 4–1 mRNA might have good diagnostic value for SLE and could act as a potential diagnostic biomarker for SLE.

Fig. 1
figure 1

HERV-E clone 4–1 mRNA expression was upregulated in CD4+ T cells from SLE patients. a and b) Relative expression of HERV-E clone 4–1 mRNA in CD4+ cells from SLE patients (N = 27) and healthy controls (HCs) (N = 21) or active SLE patients (N = 24) and inactive SLE patients (N = 3) compared using the unpaired Student’s t test. c Correlation between expression of HERV-E clone 4–1 mRNA and SLEDAI score analyzed with Spearman’s analysis. d and e SLEDAI score and HERV-E clone 4–1 mRNA expression in patients who got oral prednisolone and hydroxychloroquine treatment (N = 9); data were compared using the paired Student’s t test. f Relative expression of HERV-E clone 4–1 mRNA in CD4+ cells from inactive SLE patients (N = 11) and HCs (N = 21) compared using the unpaired Student’s t test. g ROC curve of relative HERV-E clone 4–1 mRNA expression for differentiating SLE patients from HCs. Data were represented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Full size image

NFAT1 activity was increased in SLE and associated with increased HERV-E clone 4–1 mRNA

To explain why HERV-E clone 4–1 mRNA was upregulated in CD4+ T cells from SLE patients, we wondered if some transcription factors could promote the transcription of HERV-E clone 4–1 mRNA. Since the 5′ LTR contained the transcription factor binding sites [12], this region was used to predict the potential transcription factors. Using TransFac and JASPAR database, we found some transcription factors that might regulate the expression of HERV-E clone 4–1 mRNA. NFAT1, which was proved to play critical roles in SLE [13] caught our attention. First, full length fragment of the human Human endogenous retroviral DNA (4–1) 5′ LTR with wild type (wt) or mutant (mut) predicted NFAT1 binding site was inserted into the luciferase reporter plasmid (Fig. 2a). Then, we use NFAT1 overexpression plasmids to overexpress NFAT1 and NFAT1 siRNA to knockdown NFAT1 (Fig. 2b-e). Luciferase reporter analysis showed that overexpression of NFAT1 led to an increase in luciferase activity of the wt HERV-E clone 4–1 5’LTR plasmid in CD4+ T cells, while mut NFAT1 binding site attenuated the increase of luciferase activity (Fig. 2f). In addition, Chromatin immunoprecipitation (ChIP) assay clearly showed that the predicted NFAT1-binding site in HERV-E clone 4–1 5′ LTR presented the ability to bind to NFAT1 protein (Fig. 2g). Moreover, qRT-PCR analysis showed that overexpression of NFAT1 could increase the expression of HERV-E clone 4–1 mRNA and knockdown of NFAT1 with siRNA could decrease the expression of HERV-E clone 4–1 mRNA (Fig. 2h and i). Then, we collected CD4+ T cells of SLE patients and HCs to detected NFAT1 activity using NFAT luciferase reporter assay and HERV-E clone 4–1 mRNA expression. We found NFAT1 activity was upregulated in CD4+ T cells from SLE patients (Fig. 2j) and higher in active patients than that of inactive patients (Fig. 1k). What’s more, the relative NFAT1 activity had strong correlation with HERV-E clone 4–1 mRNA expression (Fig. 2l). So, these results all together suggested that NFAT1 could induce HERV-E clone 4–1 mRNA expression via binding to its 5′ LTR. We also detected the influence of some factors on NFAT1 activity and HERV-E clone 4–1 mRNA expression (Fig. 2m and n).

Fig. 2
figure 2

NFAT1 activity was upregulated in CD4+ T cells from SLE patients and closely associated with increased HERV-E clone 4–1 mRNA expression. a Predicted wild-type (wt) binding sites and corresponding mutant (mut) sites of NFAT1 on HERV-E clone 4–1 5’LTR. b-e qRT-PCR and western-blot assays showing relative NFAT1 mRNA and protein expression in CD4+ cells from SLE patient with NFAT1 overexpression or knockdown. f Luciferase assays were performed in CD4+ T cells from SLE patient transfected with wt or mut luciferase reporter. Each luciferase activity was normalized to the value obtained in the cells transfected with vector (N = 3). g ChIP assay was used to assess NFAT1 binding site at HERV-E clone 4–1 5’LTR. h and i) Relative HERV-E clone 4–1 mRNA expression in CD4+ cells from SLE patient with NFAT1 overexpression or knockdown (N = 3). j and k Relative NFAT1 activity in CD4+ cells from SLE patients (N = 15) and HCs (N = 13) or active SLE patients (N = 12) and inactive SLE patients (N = 3) compared using the unpaired Student’s t test. l Correlation between relative NFAT1 activity and relative HERV-E clone 4–1 mRNA expression analyzed with Spearman’s analysis (N = 15). m and n CD4+ T cells from SLE patient were treated with TNF-α, IL-6, E2, LPS, UVB, HCQ or prednisolone in vitro. Relative NFAT1 activity (L) and relative HERV-E clone 4–1 mRNA expression (m) were detected. Data were represented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Full size image

E2 could upregulate HERV-E clone 4–1 mRNA expression via ER-α in CD4+ T cells from SLE patients

When selecting the potential transcript factors that might regulate the expression of HERV-E clone 4–1 mRNA, ER-α, which was the receptor of E2 drew our attention. This is because SLE has a predilection for females of child-bearing age who have relatively high estrogen level and estrogen is also a risk factor for SLE [14] and HERV-E was upregulated in breast cancer and ovarian cancer [15, 16]. Then, we further explored the role of E2 and ER-α in SLE. Accordingly, full length fragment of the human endogenous retroviral DNA (4–1) 5′ LTR with wild type (wt) or mutant (mut) predicted ER-α binding site was inserted into the luciferase reporter plasmid (Fig. 3a). Then, we use ER-α overexpression plasmids to overexpress ER-α and ER-α siRNA to knockdown ER-α (Fig. 3b-e). Luciferase reporter analysis showed that overexpression of ER-α led to an increase in luciferase activity of the wt HERV-E clone 4–1 5’LTR plasmid in CD4+ T cells, while mut ER-α binding site attenuated the increase of luciferase activity (Fig. 3f). In addition, ChIP assay clearly showed that the predicted ER-α-binding site in HERV-E clone 4–1 5′ LTR presented the ability to bind to ER-α protein (Fig. 3g). Moreover, qRT-PCR analysis showed that ER-α plasmids could increase the expression of HERV-E clone 4–1 mRNA while ER-α antagonist AZD9496 maleate and ER-α siRNA could decrease the expression of HERV-E clone 4–1 mRNA (Fig. 3h-j). In addition, AZD9496 and ER-α siRNA could reverse the upregulated HERV-E clone 4–1 mRNA expression induced by E2 (Fig. 3k and l). So, these results all together suggested that E2 could also upregulate HERV-E clone 4–1 mRNA expression via ER-α in CD4+ T cells from SLE patients.

Fig. 3
figure 3

E2 could upregulate HERV-E clone 4–1 mRNA expression via ER-α in CD4+ T cells from SLE patients. a Predicted wild-type (wt) binding sites and corresponding mutant (mut) sites of ER-α on HERV-E clone 4–1 5’LTR. b-e qRT-PCR and western-blot assays showing relative ER-α mRNA and protein expression in CD4+ cells from SLE patient with ER-α overexpression or knockdown. f Luciferase assays were performed in CD4+ T cells from SLE patient transfected with wt or mut luciferase reporter. Each luciferase activity was normalized to the value obtained in the cells transfected with vector. g ChIP assay was used to assess ER-α binding site at HERV-E clone 4–1 5’LTR. h Relative HERV-E clone 4–1 mRNA expression in CD4+ cells from SLE patient with ER-α overexpression compared using the paired Student’s t test. i and j Relative HERV-E clone 4–1 mRNA expression in CD4+ cells from SLE patient with ER-α inhibition using siRNA or AZD9496 compared using the paired Student’s t test. k and l Relative HERV-E clone 4–1 mRNA expression in CD4+ cells from SLE patient when ER-α siRNA or AZD9496 was used after E2 treatment compared using the paired Student’s t test. Data were represented as mean ± SD, N = 3. *P < 0.05, **P < 0.01, ***P < 0.001

Full size image

DNA hypomethylation of HERV-E clone 4–1 5’LTR contributed to the increase of HERV-E clone 4–1 mRNA

In our former study, we found the HERV-E clone 4–1 5’LTR was hypomethylated in CD4+ T cells from SLE patients and its methylation could be inhibited by 5-aza C [7]. Here, we investigated whether this DNA hypomethylation was involved in the NFAT1 or ER-α induced HERV-E clone 4–1 mRNA upregulation. We found that HERV-E clone 4–1 mRNA expressions were upregulated when NFAT1 or ER-α was overexpressed or 5-aza C was used in CD4+ T cells from SLE patients and HCs (Fig. 4a-d). In CD4+ T cells from SLE patients and HCs, HERV-E clone 4–1 mRNA expressions were higher when both NFAT1 was overexpressed and 5-aza C was used than that when NFAT1 was overexpressed or 5-aza C was used (Fig. 4a and b); accordingly, HERV-E clone 4–1 mRNA expressions were higher when both ER-α was overexpressed and 5-aza C was used than that when ER-α was overexpressed or 5-aza C was used (Fig. 4c and d). Besides, the times of HERV-E clone 4–1 mRNA upregulation were higher in CD4+ T cells from SLE patients than that of HCs when NFAT1 or ER-α was overexpressed (Fig. 4e and f), and the times of HERV-E clone 4–1 mRNA upregulation were higher in CD4+ T cells from HCs than that of SLE patients when 5-aza C was used (Fig. 4g). These results together suggested that DNA hypomethylation contributed to the upregulation of HERV-E clone 4–1 mRNA induced by NFAT1 and ER-α.

Fig. 4
figure 4

DNA hypomethylation of HERV-E clone 4–1 5’LTR contributed to upregulation of HERV-E clone 4–1 mRNA induced by NFAT1 and ER-α. CD4+ T cells from SLE patient or HC were treated with NFAT1 plasmids, ER-α plasmids, 5-aza C alone or in combination with 5-aza C in vitro. Relative HERV-E clone 4–1 mRNA expression (a-d) were detected. e and f Times of HERV-E clone 4–1 mRNA upregulation were compared by the Student’s t test in CD4+ T cells from SLE patient and HC when NFAT1 or ER-α was overexpressed. g Times of HERV-E clone 4–1 mRNA upregulation were compared by the Student’s t test in CD4+ T cells from SLE patient and HC when 5-aza C was used. Data were represented as mean ± SD, N = 3. *P < 0.05, **P < 0.01, ***P < 0.001

Full size image

HERV-E clone 4–1 3’LTR induced DNA hypomethylation and IL-17 release via miR-302d/MBD2

Since 3’UTRs of mRNAs were reported to act as natural miRNA sponges and could serve as competitive endogenous RNAs (ceRNAs) of other genes through sharing the common miRNAs [17,18,19,20]. We want to explore whether the 3’LTR of HERV-E clone 4–1 mRNA could act as a miRNA sponge and act ceRNAs of other genes. Through programs based on microRNA.org and Targetscan, we found that there was a potential binding site of miR-302d in the 3’LTR of HERV-E clone 4–1 mRNA (Fig. 5a). Then, we performed luciferase reporter assays to determine this interaction. Luciferase assay showed that miR-302d mimics could decrease the luciferase activity of reporter containing wt 3’LTR of HERV-E clone 4–1 while mut binding site attenuated the increase of luciferase activity (Fig. 5b). This suggested that 3’LTR of HERV-E clone 4–1 could bind to miR-302d and act as a sponge for miR-302d. We also found MBD2 was another potential target of miR-302d (Fig. 5a) and verified the interaction between MBD2 3’UTR and miR-302d using luciferase assay (Fig. 5c). Then, we found that overexpression of 3’LTR of HERV-E clone 4–1 (Fig. 5d) increased the protein levels of MBD2 and miR-302d mimics could rescue the increase of MBD2 protein by the 3’LTR (Fig. 5e). These results suggested that HERV-E clone 4–1 acts as a ceRNA of MBD2 to positively regulate MBD2 expression in 3’LTR and miR-302d dependent manners. We also detected the expression of IRF-9 which was a proved target of miR-302d in SLE [21] and found that overexpression of 3’LTR of HERV-E clone 4–1 increased the protein levels of IRF9 and miR-302d mimics could rescue the increase of IRF9 protein by the 3’LTR (Fig. 5e).

Fig. 5
figure 5

HERV-E clone 4–1 3’LTR induced DNA hypomethylation and IL-17 release via miR-302d/MBD2 in CD4+ T cells of SLE. a Predicted binding sites of miR-302d in the 3’LTR of HERV-E clone 4–1 mRNA and MBD2 3’UTR. b and c Effects of miR-302d on the expression of luciferase reporter genes containing HERV-E clone 4–1 3’LTR or MBD2 3’UTR. Luciferase activity was normalized to the value obtained in cells transfected with NC oligonucleotides. d Relative expression of HERV-E clone 4–1 3’LTR mRNA when CD4+ T cells were transfected with HERV-E clone 4–1 3’LTR expression plasmids. e Western blot analysis of IRF9 and MBD2 proteins in CD4+ T cells from SLE patients transfected with HERV-E clone 4–1 3’LTR expression plasmids and/or miR-302d mimics and the corresponding negative controls. Relative global DNA methylation level (f), intracellular IL-17 level (g-i) and IL-17 level in culture supernatants (j) in CD4+ T cells from SLE patient transfected with HERV-E clone 4–1 3’LTR expression plasmids, miR-302d mimics or MBD2 expression plasmids and the corresponding negative controls

Full size image

The mRNA levels of MBD2 in was increased in CD4+ T cells of SLE patients and inversely correlated with global DNA methylation and positively correlated with and SLEDAI score [22, 23]. What’s more, MBD2 was found to stimulates Th17 cell differentiation and IL-17 release in other autoimmune diseases [24,25,26] and IL-17 play critical functions in the pathophysiology of SLE [27, 28] So, MBD2 might play important roles in SLE progression. Then, we intended to further study the role of HERV-E clone 4–1, miR-302d and MBD2 in global DNA methylation and IL-17 expression in CD4+ T cells of SLE patients. CD4+ T cells were transfected with HERV-E clone 4–1 3’LTR expression plasmids, miR-302d mimics or MBD2 expression plasmids. Global DNA methylation levels, intracellular IL-17 level and IL-17 level in culture supernatants were subsequently measured. The results showed that global DNA methylation level decreased when CD4+ T cells of SLE were transfected with 3’LTR expression plasmids or MBD2 expression plasmids and increased when transfected with miR-302d mimics (Fig. 5f). Intracellular IL-17 level and IL-17 level in culture supernatants increased when CD4+ T cells of SLE were transfected with 3’LTR expression plasmids or MBD2 expression plasmids and decreased when transfected with miR-302d mimics (Fig. 5g-j). All together, these results suggested that HERV-E clone 4–1 3’LTR induce DNA hypomethylation and IL-17 release via miR-302d/MBD2 in CD4+ T cells of SLE.

Discussion

Some studies had proved that HERV-E clone 4–1 mRNA expression was increased in SLE patients, and the expression level of HERV-E clone 4–1 was associated with SLE disease activity [5,6,7], however, they didn’t thoroughly investigate the function and mechanism of HERV-E clone 4–1 in SLE. In this study, we investigated the mechanism of HERV-E clone 4–1 mRNA upregulation in CD4+ T cells from SLE patients and its roles in SLE progression. First, we found NFAT1 could induce HERV-E clone 4–1 mRNA expression by binding to its 5′ LTR. NFAT1, which is a key factor of Ca2+/ calcineurin (CaN)/NFAT signaling pathways, was verified to be activated in SLE [13]. We also demonstrated that NFAT1 activity was upregulated in SLE and positively correlated with HERV-E clone 4–1 mRNA expression. NFAT1 are phosphorylated and reside in the cytoplasm in resting cells; upon stimulation, they are dephosphorylated by calcineurin, translocate to the nucleus, and become transcriptionally active [29,30,31]. Then the activated NFAT1 can regulate transcription of some inflammatory cytokines such as IL-6, IL-8, TNF-α and interferon-γ (IFN-γ) [32,33,34,35]. Furthermore, we found TNF-α, IL-6, E2, LPS, UVB could upregulate NFAT1 activity and HERV-E clone 4–1 mRNA expression and these factors play critical roles in SLE [14, 36,37,38]. These results together may explain the roles of NFAT1 in HERV-E clone 4–1 mRNA expression in SLE.

Adreno cortico hormones are an important class of anti-inflammatory/immunosuppressive drugs. They can inhibit the expression of TNF-α and IL-6 and decrease the activity of SLE [39]. Ca2+/CaN/NFAT signaling is an important pathway in the T-cell activation of SLE and some calcineurin inhibitors such as cyclosporine A and tacrolimus have been used in the clinical treatment of SLE [40]. Hydroxychloroquine, which could block Ca2+/CaN/NFAT signaling pathway through inhibiting the sustained Ca2+ storage release from the endoplasmic reticulum [41], was found to repress NFAT1 activity and HERV-E clone 4–1 expression. Prednisolone and hydroxychloroquine are first-line drugs in the treatment of SLE and all the patients followed-up got oral prednisolone and hydroxychloroquine treatment. These reasons interpret it well why HERV-E clone 4–1 mRNA expressions decreased after prednisolone and hydroxychloroquine treatment. So, we hold that the upregulation of HERV-E clone 4–1 mRNA is mainly due to the abnormal inflammation / immune status of SLE which involving many inflammatory cytokines and other risk factors. We also found that E2 could upregulate HERV-E clone 4–1 mRNA expression via ER-α. ER-α is one of the estrogen receptors which can be activated by estrogen and regulate gene transcription in nucleus [42]. Interestingly, HERV-E was upregulated in breast cancer and ovarian cancer [15, 16] and this probably also has close relationship with E2 and ER-α. ER-α antagonist is also a good approach to restrain the expression of HERV-E clone 4–1. Taken together, we think these signaling pathways are good therapeutic targets for HERV-E clone 4–1.

Some studies found the HERV-E clone 4–1 5’LTR was hypomethylated in CD4+ T cells from SLE patients [7,8,9]. We found that DNA hypomethylation contributed to upregulation of HERV-E clone 4–1 mRNA induced by NFAT1 and ER-α. We think DNA hypomethylation of HERV-E clone 4–1 5’LTR is an indispensable factor that account for the upregulation of HERV-E clone 4–1 mRNA for that upregulation of HERV-E clone 4–1 mRNA mainly exists in SLE while not in some other diseases that involving NFAT1 and ER-α activation.

In this study, we found that HERV-E clone 4–1 3’LTR could act as natural miRNA sponges for miR-302d to restrain miR-302d activity. MiR-302d was proved to be downregulated in SLE patient monocytes and could inhibit the type I IFN pathway which was a major contributor to SLE pathogenesis via its target IRF-9 [21]. HERV-E clone 4–1 3’LTR could positively regulate MBD2 expression by acting as a ceRNA of MBD2 via miR-302d and HERV-E clone 4–1 3’LTR could induce DNA hypomethylation and IL-17 release via miR-302d/MBD2 in CD4+T cells of SLE. DNA hypomethylation of immune cells in SLE is associated with immune dysfunction and play important roles in the initiation and development of SLE [43, 44]. IL-17 is a proinflammatory cytokine produced by activated T cells and plays a crucial role in disease pathogenesis and represent an attractive therapeutic target for SLE [27, 28]. Thus, we hold that HERV-E clone 4–1 takes part in disease pathogenesis of SLE through miR-302d/MBD2/DNA hypomethylation and IL-17 signaling via its 3’LTR. So, HERV-E clone 4–1 3’LTR may be a potential therapeutic target of SLE. Taken together, we draw a network diagram hypothesis showing relationship between HERV-E clone 4–1 and SLE which shows the important roles of HERV-E clone 4–1 in SLE pathogenesis (Fig. 6).

Fig. 6
figure 6

Network diagram hypothesis showing relationship between HERV-E clone 4–1 and SLE

Full size image

However, we should admit that we didn’t further investigate the role of HERV-E clone 4–1 proteins and this is a shortcoming of this study. This mainly because there is no specific antibody for these proteins.

Conclusions

In conclusion, we found that HERV-E clone 4–1 mRNA expression was upregulated in CD4+ T cells from SLE patients and could act as a good biomarker for diagnosis of SLE. This is associated with the activation of Ca2+/CaN/NFAT1 and E2/ER-α signaling pathway and DNA hypomethylation of HERV-E clone 4–1 5’LTR. HERV-E clone 4–1 also takes part in disease pathogenesis of SLE through miR-302d/MBD2/DNA hypomethylation and IL-17 signaling via its 3’LTR. These signaling pathways may be potential therapeutic targets of SLE.

Availability of data and materials

Not applicable.

Abbreviations

5-aza C:

5-Azacytidine

AUC:

Area Under Curve

CaN:

Calcineurin

ChIP:

Chromatin immunoprecipitation

E2:

17β-estradiol/estradiol

ER-α:

Estrogen receptor-α

HCQ:

Hydroxychloroquine

HERV:

Human endogenous retroviruses

IFN-γ:

Interferon-γ

LPS:

Lipopolysaccharides

LTRs:

Long terminal repeats

MBD2:

Methyl-CpG binding domain protein 2

NFAT:

Nuclear factor of activated T cells

ORFs:

Open Reading Frames

PBMC:

Peripheral blood mononuclear cells

qRT-PCR:

Quantitative reverse transcription-PCR

ROC:

Operating Characteristic

SLE:

Systemic lupus erythematosus

SLEDAI:

SLE disease activity index

TNF-α:

Tumor necrosis factor-a

UVB:

Ultraviolet B

References

  1. Yin Y, Choi S, Xu Z, Perry D, Seay H, Croker B, et al. Normalization of CD4+ T cell metabolism reverses lupus. Sci Transl Med. 2015;7:274ra218.

    Article  Google Scholar 

  2. Kaul A, Gordon C, Crow M, Touma Z, Urowitz M, van Vollenhoven R, et al. Systemic lupus erythematosus. Nature reviews Disease primers. 2016;2:16039.

    Article  Google Scholar 

  3. Lander E, Linton L, Birren B, Nusbaum C, Zody M, Baldwin J, et al. Initial sequencing and analysis of the human genome. Nature. 2001;409:860–921.

    Article  CAS  Google Scholar 

  4. Le Dantec C, Vallet S, Brooks W, Renaudineau Y. Human endogenous retrovirus group E and its involvement in diseases. Viruses. 2015;7:1238–57.

    Article  Google Scholar 

  5. Ogasawara H, Naito T, Kaneko H, Hishikawa T, Sekigawa I, Hashimoto H, et al. Quantitative analyses of messenger RNA of human endogenous retrovirus in patients with systemic lupus erythematosus. J Rheumatol. 2001;28:533–8.

    CAS  PubMed  Google Scholar 

  6. Piotrowski P, Duriagin S, Jagodzinski P. Expression of human endogenous retrovirus clone 4-1 may correlate with blood plasma concentration of anti-U1 RNP and anti-Sm nuclear antibodies. Clin Rheumatol. 2005;24:620–4.

    Article  Google Scholar 

  7. Wu Z, Mei X, Zhao D, Sun Y, Song J, Pan W, et al. DNA methylation modulates HERV-E expression in CD4+ T cells from systemic lupus erythematosus patients. J Dermatol Sci. 2015;77:110–6.

    Article  CAS  Google Scholar 

  8. Sukapan P, Promnarate P, Avihingsanon Y, Mutirangura A, Hirankarn N. Types of DNA methylation status of the interspersed repetitive sequences for LINE-1, Alu, HERV-E and HERV-K in the neutrophils from systemic lupus erythematosus patients and healthy controls. J Hum Genet. 2014;59:178–88.

    Article  CAS  Google Scholar 

  9. Nakkuntod J, Sukkapan P, Avihingsanon Y, Mutirangura A, Hirankarn N. DNA methylation of human endogenous retrovirus in systemic lupus erythematosus. J Hum Genet. 2013;58:241–9.

    Article  CAS  Google Scholar 

  10. Wu Z, Li X, Qin H, Zhu X, Xu J, Shi W. Ultraviolet B enhances DNA hypomethylation of CD4+ T cells in systemic lupus erythematosus via inhibiting DNMT1 catalytic activity. J Dermatol Sci. 2013;71:167–73.

    Article  CAS  Google Scholar 

  11. Wang X, Zhang C, Wu Z, Chen Y, Shi W. CircIBTK inhibits DNA demethylation and activation of AKT signaling pathway via miR-29b in peripheral blood mononuclear cells in systemic lupus erythematosus. Arthritis Res Ther. 2018;20:118.

    Article  Google Scholar 

  12. Khodosevich K, Lebedev Y, Sverdlov E. Endogenous retroviruses and human evolution. Comparative and functional genomics. 2002;3:494–8.

    Article  CAS  Google Scholar 

  13. Fujii Y, Fujii K, Iwata S, Suzuki K, Azuma T, Saito K, et al. Abnormal intracellular distribution of NFAT1 in T lymphocytes from patients with systemic lupus erythematosus and characteristic clinical features. Clin Immunol. 2006;119:297–306.

    Article  CAS  Google Scholar 

  14. Walker S. Estrogen and autoimmune disease. Clin Rev Allergy Immunol. 2011;40:60–5.

    Article  CAS  Google Scholar 

  15. Frank O, Verbeke C, Schwarz N, Mayer J, Fabarius A, Hehlmann R, et al. Variable transcriptional activity of endogenous retroviruses in human breast cancer. J Virol. 2008:1808–18.

    Article  Google Scholar 

  16. Wang-Johanning F, Liu J, Rycaj K, Huang M, Tsai K, Rosen D, et al. Expression of multiple human endogenous retrovirus surface envelope proteins in ovarian cancer. Int J Cancer. 2007;120:81–90.

    Article  CAS  Google Scholar 

  17. Li J, Tian H, Pan J, et al. Pecanex functions as a competitive endogenous RNA of S-phase kinase associated protein 2 in lung cancer. Cancer Lett. 2017;406:36–46.

    Article  CAS  Google Scholar 

  18. Zheng T, Chou J, Zhang F, et al. CXCR4 3′UTR functions as a ceRNA in promoting metastasis, proliferation and survival of MCF-7 cells by regulating miR-146a activity. Eur J Cell Biol. 2015;94:458–69.

    Article  CAS  Google Scholar 

  19. Gao S, Cheng C, Chen H, et al. IGF1 3′UTR functions as a ceRNA in promoting angiogenesis by sponging miR-29 family in osteosarcoma. J Mol Histol. 2016;47:135–43.

    Article  CAS  Google Scholar 

  20. Zheng L, Li X, Gu Y, et al. The 3′UTR of the pseudogene CYP4Z2P promotes tumor angiogenesis in breast cancer by acting as a ceRNA for CYP4Z1. Breast Cancer Res Treat. 2015;150:105–18.

    Article  CAS  Google Scholar 

  21. Smith S, Fernando T, Wu P, et al. MicroRNA-302d targets IRF9 to regulate the IFN-induced gene expression in SLE. J Autoimmun. 2017;79:105–11.

    Article  CAS  Google Scholar 

  22. Qin H, Zhu X, Liang J, et al. Associations between aberrant DNA methylation and transcript levels of DNMT1 and MBD2 in CD4+T cells from patients with systemic lupus erythematosus. Australas J Dermatol. 2013;54:90–5.

    Article  Google Scholar 

  23. Balada E, Ordi-Ros J, Serrano-Acedo S, et al. Transcript overexpression of the MBD2 and MBD4 genes in CD4+ T cells from systemic lupus erythematosus patients. J Leukoc Biol. 2007;81:1609–16.

    Article  CAS  Google Scholar 

  24. Zhong J, Yu Q, Yang P, et al. MBD2 regulates TH17 differentiation and experimental autoimmune encephalomyelitis by controlling the homeostasis of T-bet/Hlx axis. J Autoimmun. 2014;53:95–104.

    Article  CAS  Google Scholar 

  25. Li X, Sun W, Jia A, et al. MBD2 regulates differentiation and function of Th17 cells in neutrophils- dominant asthma via HIF-1α. J Inflamm. 2018;15:15.

    Article  Google Scholar 

  26. Aijun J, Yueling W, Wenjin S, et al. MBD2 regulates Th17 cell differentiation and experimental severe asthma by affecting IRF4 expression. Mediat Inflamm. 2017;2017:1–10.

    Google Scholar 

  27. Martin JC, Baeten D, Josien R. Emerging role of IL-17 and Th17 cells in systemic lupus erythematosus. Clin Immunol. 2014;154:1–12.

    Article  CAS  Google Scholar 

  28. Koga T, Ichinose K, Kawakami A, Tsokos GC. The role of IL-17 in systemic lupus erythematosus and its potential as a therapeutic target. Expert Rev Clin Immunol. 2019.

  29. Luo C, Burgeon E, Rao A. Mechanisms of transactivation by nuclear factor of activated T cells-1. J Exp Med. 1996;184:141–7.

    Article  CAS  Google Scholar 

  30. Rao A, Luo C, Hogan P. Transcription factors of the NFAT family: regulation and function. Annu Rev Immunol. 1997;15:707–47.

    Article  CAS  Google Scholar 

  31. Hogan P, Chen L, Nardone J, Rao A. Transcriptional regulation by calcium, calcineurin, and NFAT. Genes Dev. 2007;17:2205–32.

    Article  Google Scholar 

  32. Sun J, Chen H, Xie Y, Su J, Huang Y, Xu L, et al. Nuclear factor of activated T cells and cytokines gene expression of the T cells in AIDS patients with immune reconstitution inflammatory syndrome during highly active antiretroviral therapy. Mediat Inflamm. 2017;2017:1754741.

    Google Scholar 

  33. Kaunisto A, Henry W, Montaser-Kouhsari L, Jaminet S, Oh E, Zhao L, et al. NFAT1 promotes intratumoral neutrophil infiltration by regulating IL8 expression in breast cancer. Mol Oncol. 2015;9:1140–54.

    Article  CAS  Google Scholar 

  34. Luo C, Burgeon E, Carew J, McCaffrey P, Badalian T, Lane W, et al. Recombinant NFAT1 (NFATp) is regulated by calcineurin in T cells and mediates transcription of several cytokine genes. Mol Cell Biol. 1996;16:3955–66.

    Article  CAS  Google Scholar 

  35. Teixeira L, Fonseca B, Vieira-de-Abreu A, Barboza B, Robbs B, Bozza P, et al. IFN-gamma production by CD8+ T cells depends on NFAT1 transcription factor and regulates Th differentiation. J Immunol. 2005;175:5931–9.

    Article  CAS  Google Scholar 

  36. Umare V, Pradhan V, Nadkar M, Rajadhyaksha A, Patwardhan M, Ghosh K, et al. Effect of proinflammatory cytokines (IL-6, TNF-alpha, and IL-1beta) on clinical manifestations in Indian SLE patients. Mediat Inflamm. 2014;2014:385297.

    Article  Google Scholar 

  37. Chen D, Xie J, Chen H, Yang Y, Zhan Z, Liang L, et al. Infection in southern Chinese patients with systemic lupus erythematosus: Spectrum, drug resistance, outcomes, and risk factors. J Rheumatol. 2016;43:1650–6.

    Article  CAS  Google Scholar 

  38. Deng G, Tsokos G. Pathogenesis and targeted treatment of skin injury in SLE. Nat Rev Rheumatol. 2015;11:663–9.

    Article  CAS  Google Scholar 

  39. Ray A, Sehgal P. Cytokines and their receptors: molecular mechanism of interleukin-6 gene repression by glucocorticoids. J Am Soc Nephrol. 1992;2:S214–21.

    CAS  PubMed  Google Scholar 

  40. Mok C. Calcineurin inhibitors in systemic lupus erythematosus. Best Pract Res Clin Rheumatol. 2017;31:429–38.

    Article  Google Scholar 

  41. Wu S, Chang C, Hsu J, Lu M, Lai N, Li C, et al. Hydroxychloroquine inhibits CD154 expression in CD4(+) T lymphocytes of systemic lupus erythematosus through NFAT, but not STAT5, signaling. Arthritis Res Ther. 2017;19:183.

    Article  CAS  Google Scholar 

  42. Heldring N, Pike A, Andersson S, Matthews J, Cheng G, Hartman J, et al. Estrogen receptors: how do they signal and what are their targets. Physiol Rev. 2007;87:905–31.

    Article  CAS  Google Scholar 

  43. Wu H, Zhao M, Tan L, Lu Q. The key culprit in the pathogenesis of systemic lupus erythematosus: aberrant DNA methylation. Autoimmun Rev. 2016;15:684–9.

    Article  CAS  Google Scholar 

  44. Renaudineau Y, Youinou P. Epigenetics and autoimmunity, with special emphasis on methylation. Keio J Med. 2011;60:10–6.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

Not applicable.

Funding

This work was supported by grants from the National Natural Science Foundation of China (Grant No. 81573031 and 81773310).

Author information

Author notes

  1. Xin Wang, Chaoshuai Zhao and Chengzhong Zhang contributed equally to this work.

Authors and Affiliations

  1. Department of Dermatology, Shanghai General Hospital, Shanghai Jiaotong University School of Medicine, 100 Haining Road, Shanghai, 200080, China

    Xin Wang, Chaoshuai Zhao, Chengzhong Zhang, Xingyu Mei, Jun Song, Yue Sun, Zhouwei Wu & Weimin Shi

Authors
  1. Xin Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chaoshuai Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chengzhong Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xingyu Mei
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jun Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yue Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Zhouwei Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Weimin Shi
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

XW, CZ (Chaoshuai Zhao) and CZ (Chengzhong Zhang) designed and performed the experiments; ZW, XM, JS, YS and WS analyzed and interpreted the data; WS wrote the manuscript. ZW critically revised the manuscript. All authors read and approved the manuscript.

Corresponding authors

Correspondence to Zhouwei Wu or Weimin Shi.

Ethics declarations

Ethics approval and consent to participate

This research was approved by the Institutional Research Ethics Committee of Shanghai General Hospital and abided by the ethical guidelines of the Declaration of Helsinki. Informed consents were obtained from all the patients involved in this study.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Additional file

Additional file 1:

Figure S1. The structure of HERV-E clone 4–1. Table S1. Clinical characteristics of SLE patients and healthy controls. (DOCX 26 kb)

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wang, X., Zhao, C., Zhang, C. et al. Increased HERV-E clone 4–1 expression contributes to DNA hypomethylation and IL-17 release from CD4+ T cells via miR-302d/MBD2 in systemic lupus erythematosus. Cell Commun Signal 17, 94 (2019). https://doi.org/10.1186/s12964-019-0416-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12964-019-0416-5

Keywords

Cell Communication and Signaling

ISSN: 1478-811X