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

Figures

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  Fig. 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

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  Fig. 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…

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  Fig. 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…

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  Fig. 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

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  Fig. 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

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  Fig. 6. Network diagram hypothesis showing relationship between HERV-E clone 4–1 and SLE