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
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.
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.
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.
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.
KeywordsHERV-E clone 4–1 Systemic lupus erythematosus Transcription factors DNA hypomethylation miR-302d MBD2
- 5-aza C
Area Under Curve
Human endogenous retroviruses
Long terminal repeats
Methyl-CpG binding domain protein 2
Nuclear factor of activated T cells
Open Reading Frames
Peripheral blood mononuclear cells
Quantitative reverse transcription-PCR
Systemic lupus erythematosus
SLE disease activity index
Tumor necrosis factor-a
Systemic lupus erythematosus (SLE) is an autoimmune disease in which autoreactive CD4+ T cells play an important role . 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 .
Human endogenous retroviruses (HERV) are descendants of occasional germline invasion by exogenous retroviruses which occupy as much as 8% of the human genome . 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) . 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) . 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.
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 ; 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).
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 .
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 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.
HERV-E clone 4–1 mRNA expression was upregulated in CD4+ T cells from SLE patients
NFAT1 activity was increased in SLE and associated with increased HERV-E clone 4–1 mRNA
E2 could upregulate HERV-E clone 4–1 mRNA expression via ER-α in CD4+ T cells from SLE patients
DNA hypomethylation of HERV-E clone 4–1 5’LTR contributed to the increase of HERV-E clone 4–1 mRNA
HERV-E clone 4–1 3’LTR induced DNA hypomethylation and IL-17 release via miR-302d/MBD2
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.
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 . 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 . 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 . Hydroxychloroquine, which could block Ca2+/CaN/NFAT signaling pathway through inhibiting the sustained Ca2+ storage release from the endoplasmic reticulum , 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 . 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.
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.
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.
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.
This work was supported by grants from the National Natural Science Foundation of China (Grant No. 81573031 and 81773310).
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
The authors declare that they have no competing interests.
- 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.CrossRefGoogle 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
- 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.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
Open AccessThis 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.