Endogenous retrovirus promotes the aberrant T cell differentiation in systemic lupus erythematosus via RIG-I pathway

Endogenous retrovirus promotes the aberrant T cell differentiation in systemic lupus erythematosus via RIG-I pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Endogenous retrovirus promotes the aberrant T cell differentiation in systemic lupus erythematosus via RIG-I pathway Ming Zhao, Xiaoli Min, Yaqin Yu, Zhi Hu, Lianlian Ouyang, Yueqi Qiu, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3939567/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The dysregulated differentiation of T lymphocyte play an important role in systemic lupus erythematosus (SLE). However, the underlying mechanism remains unclear. Here, we showed that many transcripts derived from human endogenous retroviruses (HERVs) were highly expressed in CD4 + T cells from SLE patients due to DNA hypomethylation, some of which were characterized by double strand RNAs (dsRNAs). Excessive dsRNAs promoted Th1/Th17 differentiation and inhibited Treg cell differentiation via the activation of dsRNA sensor retinoic acid-inducible gene I (RIG-I). And T cell-specific ablation of RIG-I alleviated disease progression in experimental autoimmune encephalomyelitis (EAE) mice model and lupus-like mice model. Importantly, we demonstrated that dsRNA-activated RIG-I protein bind lactate dehydrogenase A (LDHA) and regulate histone lysine 18 lactylation (H3K18Lac) and acetylation (H3K18Ac) modifications in T cell differentiation via changing lactate level. Collectively, our findings uncover a novel role and mechanism of HERVs and RIG-I in regulating the aberrant differentiation of T cells in SLE patients. Health sciences/Diseases/Immunological disorders/Autoimmune diseases/Systemic lupus erythematosus Health sciences/Pathogenesis/Immunopathogenesis/Adaptive immunity/Cellular immunity/Lymphocyte differentiation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Systemic lupus erythematosus (SLE) is a potentially fatal, chronic, multisystem autoimmune disorder 1 , characterized by overproduction of numerous inflammatory cytokines and aberrant differentiation of pathogenic T lymphocyte 2 . In SLE disease, abnormal activation and proliferation of pathogenic CD4 + T cells (including the Th1, Th2, Th17 and Tfh cell subsets) contribute to the secretion of a variety of inflammatory factors (such as IFN-γ, IL-4, IL-17A and IL-21, etc.) and assist B cells in production of high levels of auto-antibodies, whereas inflammation-suppressing Treg cells show a quantitative and/or qualitative deficiencies associated with uncontrolled autoinflammation 3 – 5 . Previous studies showed that genetic factors and epigenetic changes such as DNA hypomethylation contributed to T cells over-activation and SLE pathogenesis 6 – 8 . However, the precise mechanism causing T cell abnormalities in SLE remains unclear. Human endogenous retroviruses (HERVs) are genetic remnants of retroviruses that were integrated into the human genome millions of years ago, and make up as much as 8% (over 500,000 individual elements) of the human genome 9 , 10 . Recombination has resulted in the majority of HERV elements existing as solitary long terminal repeats (LTRs) 11 , and only a few HERVs retain the ability of encoding virus proteins. Previous findings suggested that HERVs encoded proteins were involved in the pathogenesis of SLE 12 , 13 . For example, HERV-E clone 4 − 1 and ERV envelope glycoprotein, gp70, were implicated as an autoantigen to result in lupus 13 , 14 . In addition, recent research revealed that DNA hypomethylation driven transcriptional activation of HERV LTRs can drive proinflammatory response via the formation of double-stranded RNA (dsRNA) and activation of the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) recognition pathway in some tumors 15 , 16 , which play an anti-tumor role. However, the role of HERVs in human genome is still poor understand in autoimmune diseases up to now. Whether there exists the transcription activation of HERVs in SLE CD4 + T cells, which has been shown to be hypomethylation in SLE patients, and whether HERVs lead to RLR pathway activation and aberrant T cells differentiation in SLE patients need to be investigated. RIG-I, one member of the RLRs, is a key sensor of viral dsRNA that recruits mitochondrial antiviral signaling protein (MAVS) and relays the signal to the kinases TBK1, which mediates transcriptional induction of type I interferons (I-IFN) and interferon stimulation genes (ISGs) via interferon-regulatory factor-3 (IRF-3) and IRF-7 17,18 , play an important role in antiviral innate immune response. Evidence supports the idea that the I-IFN pathway activation contributes to SLE susceptibility 19 , 20 . Beside of innate immune cells, RIG-I signal genes and many interferon stimulation genes were overexpression in various of cells, especially CD4 + T cells in SLE patient 21 , 22 . However, the roles of dsRNA sensors RIG-I in adaptive immune cells remain unclear. In this study, we screened and identified many overexpressed HERVs in CD4 + T cells from SLE patients and found some HERVs formed dsRNAs to induced activation of RIG-I signaling pathway. And we revealed the role of HERVs derived dsRNAs and RIG-I in regulating effector T-cell differentiation and disease progression of lupus-like mice. Mechanistically, we also demonstrated the effect of activated RIG-I on histone lactylation and acetylation via regulating lactate dehydrogenase A (LDHA) and lactate level during T cells differentiation. Our findings uncovered a novel mechanism underlying the aberrant T cells differentiation in SLE. Results Screening and identification of the differentially expressed HERVs in lupus CD4 + T cells To screen the highly expressed HERVs transcripts in CD4 + T cells of SLE patients, we performed RNA sequencing (RNA-seq) and whole transcriptome sequencing and identified the LTRs derived from HERVs with Repeatmasker online software ( https://www.repeatmasker.org ). 34,992 HERVs based on RNA-seq and 38,006 HERVs based on whole transcriptome sequencing were discovered in CD4 + T cells obtained from peripheral blood of SLE patients and HCs (Fig. 1 a). 204 up-regulated and 38 down-regulated HERVs ( P < 0.05) were identified by RNA-seq (Fig. 1 b), as well as 1674 up-regulated and 542 down-regulated HERVs ( P < 0.05) were identified by whole transcriptome sequencing in SLE patients compared to HCs (Fig. 1 c). Based on the expression levels and change folds of HERVs, 17 HERVs in RNA-seq (log2 fold change (FC) ≥ 1, FPKM in SLE ≥ 20, and exclusion of HERVs overlapped with other genes’ exon) (Fig. 1 e) and 18 HERVs in the whole transcriptome sequencing (log2 fold change (FC) ≥ 1, P < 0.05, FPKM in SLE ≥ 7, and exclusion of HERVs overlapped with other genes’ exon) were screened (Fig. 1 f). Combined the two sequencing results, 18 HERVs with high expression abundance were chosen according to the two sequencing data to verify by RT-qPCR, except for HERVs size below 100bp and with poor primer specificity. The results showed that the expression levels of MER21C, ERV3-16A3_I-int, LTR16A, THE1B-367, LTR40C, MLT1F1, LTR56, MER4A1, MLT1A0-130, THE1C-288, THE1C, MLT1B, MLT2B3, THE1B-362, MER65A and MLT2A2 were significantly higher in CD4 + T cells of SLE patients than those in HCs (Fig. 1 g). Previous sthdies showed that these ancient retroviruses have been integrated into the human genome, some of which were reactivated transcriptionally under specific conditions 23 . To clarify the derivation of these highly expressed HERVs in SLE CD4 + T cells, we analyzed their genomic locations by IGV_Win_2.6.0. We found that the genomic locations of these highly expressed HERVs were predominantly distributed on chromosomes 1, 4, 13, and 21, and the locations of HERVs on the same chromosome were contiguous (supp Fig. 1 a). Interestingly, these HERVs were localized in the intronic region or intergenic region of interferon-stimulated genes (ISGs), including OAS2 , OAS3 , DDX60L , DDX60 , LY6E , MX1 , MX2 , IFI44L , IFI44 and EPSTI1 (supp Fig. 1 b and 1 c), which were also highly expressed in CD4 + T cells of SLE patients and correlated positively with the expression levels of HERVs (supp Fig. 1 d, e). HERVs are mainly localized in heterochromatin and repressed by epigenetic silencing at steady state 24 , 25 . Previous studies have shown that DNA hypomethylation, leading to ISGs overexpression in SLE patients, contributed to SLE pathogenesis 26 . Here, we detected the DNA methylation status of these highly expressed HERVs including ERV3-16A3_I-int, LTR40C, LTR16A, THE1B, MER21C, and MLT1F1 in CD4 + T cells of SLE patients. The results showed that the mean CpG methylation levels of these HERVs were significantly decreased in SLE CD4 + T cells compared with HCs (Fig. 2 a-c). Moreover, we also found that 5-aza-2’-deoxycytidine (5-AZA-CdR), a methyltransferase-specific inhibitor, induced DNA hypomethylation and overexpression of these HERVs in CD4 + T cells (Fig. 2 d, e). Those results suggested that transcriptional activation of HERVs might be due to DNA hypomethylation in CD4 + T cells of SLE patients. HERVs formed dsRNAs and activated RIG-I pathway in SLE CD4 + T cells HERVs transcription leads to dsRNA production, which has been indicated in tumor 15 . To investigate whether these highly expressed HERVs formed dsRNAs in CD4 + T cells of SLE, we performed dsRNA enrichment assay with RNase A digestion, which cleaves single-stranded RNA (ssRNA) and preserves dsRNA under high salt conditions, and dsRNA-specific antibody J2 pulldown assays 27 . Both two assays indicated that ERV3-16A3_I-int, MER65A, MLT2A2 and MLT1B expressed as dsRNAs in SLE CD4 + T cells because of more than 100 times enrichments in RNA digested with RNase A or J2 antibody (Fig. 3 a, b). To investigate whether the RIG-I signal pathway could be triggered by HERVs derived dsRNAs to activate type I interferons pathway, we obtained ERV3-16A3_I-int RNA by T7 RNA in vitro transcription kit and annealed to form dsRNA (supp Fig. 2 ). ERV3-16A3_I-int dsRNA and poly(I:C), a synthetic dsRNA analog as positive control, were electrotransfected into human naive CD4 + T cells respectively. We observed that upon dsRNA treatment, naive CD4 + T cells initiated the strong activation of the RIG-I signal pathway. The mRNA and protein expression of RIG-I-like receptors (RLRs) pathway genes (such as DDX58 and IFR7) and phosphorylation level of IRF3 protein were remarkably elevated in poly(I:C) or ERV3-16A3_I-int-treated CD4 + T cells (Fig. 3 c-e). In addition, our results also showed that the expression levels of type I IFN (IFNα and IFNβ) and the IFN-stimulated gene 15 (ISG15) were significantly induced after Poly(I:C) or ERV3-16A3_I-int treatment (Fig. 3 c, d). Importantly, we observed that ERV3-16A3_I-int dsRNA can be bond strongly by RIG-I proteins (Fig. 3 f). In addition, the ubiquitination level of RIG-I, which is a hallmark of dsRNA-stimulated RIG-I activation, was increased in dsRNA treated CD4 + T cells (Fig. 3 g). These results indicated that HERVs-derived dsRNA could trigger RIG-I-mediated innate immune response in T cells. Then, we sought to determine the levels of dsRNA and RIG-I signal pathway genes in lupus CD4 + T cells. J2 immunofluorescence staining showed a significant enrichment of dsRNAs in SLE CD4 + T cells (Fig. 3 h). We observed that the expression levels of DDX58, TRIM25, IRF7 and ISG15 were significantly increased in CD4 + T cells of SLE patients compared with healthy controls (Fig. 3 i). Furthermore, we found that not only the protein level but also the level of ubiquitination of RIG-I were markedly increased in lupus CD4 + T cells (Fig. 3 j). We next characterized RIG-I expression in naive CD4 + T cells and memory/effector CD4 + T cells of SLE patients and healthy controls. Our data showed a significantly high RIG-I expression in CD4 + CD45RA + CD45RO − naive CD4 + T cells as well as in CD4 + CD45RA − CD45RO + memory/effector CD4 + T cells of SLE patients (Fig. 3 k). These data indicated that HERVs derived dsRNA induced the activation of RIG-I signal pathway, which may play an role in regulating T cells differentiation in CD4 + T cells of SLE. HERV-derived dsRNA regulated T cell differentiation via activation of RIG-I pathway Abnormal differentiation of pathogenic T cells plays an important role in the pathogenesis of SLE 28 . We observed that the expression of ERV3-16A3_I-int was significantly increased in Th1 and Th17 cells, as well as the protein levels of RIG-I and IRF7 were markedly increased in Th1, Th17 and Treg cells and the phosphorylation levels of TBK1 and IRF3 were increased in Th1 and Th17, compared to Th0 cells (supp Fig. 3 ). To investigate the role of dsRNA/RIG-I pathway in CD4 + T cell differentiation, RNA-seq was performed in CD3 and CD28 antibodies activated naïve CD4 + T cells transfected with poly(I:C). The expression profiles of genes related to the effect T cell differentiation were shown in Fig. 4 a. The expression levels of IFNG (Th1), TBX21 (Th1) and RORC (Th17) were up-regulated and GATA3 (Th2) and FOXP3 (Treg) expression were down-regulated after poly(I:C) treatment, which were also validated by RT-qPCR (Fig. 4 b). Then, naïve CD4 + T cells were cultured in vitro under polarization conditions of different T-cell subclasses for 3 days, and transfected with Poly(I:C) or ERV3-16A3_I-int dsRNA to activate the RIG-I signaling pathway. FCM analysis showed that Th1 and Th17 cell differentiation was increased and Treg cell differentiation was significantly inhibited in the poly(I:C) and ERV3-16A3_I-int transfected group compared with mock controls (Fig. 4 c, d) (supp Fig. 4 b, d). The mRNA levels of IFNG, TBX21, IL17A and RORC1 in T cells with poly(I:C) or ERV3-16A3_I-int stimulation were significantly increased, and the mRNA levels of FOXP3 and TGFB1 in Treg cells with poly(I:C) or ERV3-16A3_I-int stimulation were significantly decreased (Fig. 4 e-h) (supp Fig. 4 b, d). The cytokine production of IFN-γ (in the supernatants of Th1) was increased (Fig. 4 e, f), and the level of TGFβ (in the supernatants of Treg) was decreased in the poly(I:C) and ERV3-16A3_I-int transfected group (Fig. 4 g, h). No difference was observed in Th2 cell differentiation after poly(I:C) and ERV3-16A3_I-int treatment (supp Fig. 4 a, c). In contrast, we specifically inhibited expression of ERV3-16A3-Int via transfecting ERV3-16A3-Int smart silence in naïve CD4 + T cells under polarization conditions of different T cell subclasses (supp Fig. 4 e). We observed that ERV3-16A3-Int knockdown decreased Th1 and Th17 cell differentiation and increased Treg cell differentiation by FCM and RT-qPCR compared with negative controls (Fig. 4 i, j) (supp Fig. 4 g), but no significant effect on Th2 cell differentiation (supp Fig. 4 f). These results suggested that HERV-derived dsRNAs play an important role in regulating T cell differentiation. Next, we transfected healthy naive CD4 + T cells with siRNA targeting DDX58 (si-RIG-I) and stimulated them to differentiate into T cell subsets in vitro. As expected, cells transfected with si-RIG-I showed decreased the expression of RIG-I in CD4 + T cells (Fig. 5 a). FCM and RT-qPCR analysis showed that the percentages of Th1 cells and Th17 cells were decreased and the percentage of Treg cells was increased in T cells with si-RIG-I transfection compared with negative control (Fig. 5 b, d and e). Furthermore, we generated Ddx58 -knockout ( Ddx58 KO) mice. The mRNA and protein expression of RIG-I was significantly reduced in splenic CD4 + T cells of Ddx58 KO mice compared with WT mice (Fig. 5 f). Naïve CD4 + T cells from KO and WT mice spleen were cultured in vitro under T cell-polarizing conditions for 3 days. Similar changes in Th1, Th17 and Treg cell differentiation were also observed in Ddx58 KO mice. (Fig. 5 g). Activation of the RIG-I signal pathway induced by dsRNA elevated I-IFN expression. Previously research demonstrated that STAT1 was a key transcription factor downstream of I-IFN 29 , and regulated transcription of TBX21 gene in Th1 cell differentiation 30 , 31 . Here, we observed that the protein level of T-Bet and the protein phosphorylation level of STAT1 was increased in poly(I:C)-treated Th1 cells (supp Fig. 5 a, b). STAT1 inhibitor fludarabine reduced STAT1 phosphorylation and T-bet protein expression in Th1 cells with poly(I:C) stimulation (supp Fig. 5 c), and inhibited Th1 cell differentiation (supp Fig. 5 d, e). On the other hand, RLR-activated p-IRF3 represses Treg cell differentiation by preventing both bindings of Smad3 with TGFbR and Smad transcriptional complex formation 32 . Our data showed that reducing IRF3 expression partially alleviated the inhibitory effect of poly(I:C) on Treg cell differentiation (supp Fig. 6 ). Taken together, the above results indicated that HERVs dsRNA regulated T cells differentiation via activation of RIG-I pathway. RIG-I deficiency relieved EAE mice model and lupus-like mice models through mediating the aberrant T cell differentiation To further determine the role of RIG-I in autoimmune diseases, we isolated naive CD4 + T cells from WT and Ddx58 KO mice and injected them into the tail veins of Rag2 -/- mice respectively. After 5 days of T cell transfer, Rag 2 -/- mice were induced by immunization with myelin oligodendrocyte glycoprotein (MOG35-55). On the 9th day after immunization, we performed a second T cell-transfer. Mice were sacrificed for analysis after 16 days of EAE immunization (Fig. 6 a). Ddx58 KO- Rag2 -/- mice developed more mild signs of EAE with disease onset advanced as quantified by clinical score or weight loss (Fig. 6 b, c) and had decreased demyelination compared to WT- Rag2 -/- mice (Fig. 6 d). In addition, FCM showed that Th1 cell proportion in the spleen and Th17 cell proportions in the spleen and draining lymph nodes (dLNs) were decreased, and Treg cell proportion in dLNs was increased in Ddx58 -KO- Rag2 -/- mice (Fig. 6 e-g). Then, we investigated whether RIG-I deficiency in CD4 + T cells affects the progression of lupus. We generated mice with a Ddx58 allele flanked by loxP sites ( Ddx58 f/f ). Mice with confirmed germline transmission were crossed with CD4-Cre transgenic mice to generate a conditional knockout mouse model with RIG-I expression deficiency specifically in CD4 + T cells ( Ddx58 f/f CD4-Cre mice, Ddx58 CKO) (supp Fig. 7 b-d). We treated the 7-week-old Ddx58 CKO and Ddx58 f/f mice with imiquimod (IMQ) three times a week to induce lupus-like mice model. After 8 weeks of IMQ stimulation, there was a significant decrease of spleen weight in Ddx58 CKO without body weight difference compared to Ddx58 f/f mice (Fig. 7 a) (supp Fig. 7 e). In addition, the Ddx58 CKO mice showed higher survival rate and a lower ratio of urine protein/creatine than Ddx58 f/f mice (supp Fig. 7 f) (Fig. 7 b). Furthermore, the serum levels of anti-dsDNA antibody and anti-nuclear antibody (ANA) were decreased in Ddx58 CKO mice compared with Ddx58 f/f mice (Fig. 7 c, d). Morphological examination by H&E and PAS staining showed the relieved kidney damage in Ddx58 CKO mice (Fig. 7 e). Consistently, immunofluorescence staining showed that renal C3 and IgG immune complex depositions were also decreased in Ddx58 CKO mice (Fig. 7 f). We next measured the proportions of effect T cell subsets in the spleen and dLNs. We observed significant decreases in the proportions of Th1 and Th17 cells in the spleen and the proportions of Th1, GCB cells and plasma cells in dLNs of Ddx58 CKO mice compared to Ddx58 f/f mice (Fig. 7 g). In contrast, RIG-I deficiency elevated the frequency of Treg cells in the spleen and dLNs of Ddx58 CKO mice (Fig. 7 g). No significant differences were observed in the proportion of Th2 cells (Fig. 7 g). We found that the production of IFN-γ, IL-17A, TNFα, IL-10 and IL-6 was decreased in the serum of Ddx58 CKO mice compared to Ddx58 f/f mice without significant changes in IL-4 and IL-2 protein levels (Fig. 7 h) (supp Fig. 7 g). In addition, similar relieves of lupus-like phenotypes and dysregulated T cell differentiation were also observed in the other lupus-like mice model, chronic graft-versus-host disease (cGVHD) mice model (supp Fig. 8 ). Collectively, these data indicate that RIG-I deficiency in CD4 + T cells alleviates disease progression in lupus mice. RIG-I regulates histone lactylation and acetylation in T cells by modulating LDHA activity To demonstrate the mechanism of RIG-I in regulating T cell differentiation, we first enriched proteins by RIG-I antibody in co-IP experiment and identified RIG-I binding proteins in CD4 + T cells under Th1 polarization condition by mass spectrometry (MS) (supp Fig. 9 a). The result showed lactate dehydrogenase A (LDHA) was enriched by RIG-I antibody in Th1 cells (supp Fig. 9 b). Co-IP and western blot results verified that RIG-I bind LDHA protein in Th1 cells with and without poly(I:C) stimulation, but no difference in enrichment levels of LDHA protein between two groups. (Fig. 8 a). In addition, we observed no marked change in the expression of LDHA in Th1 cells with poly(I:C) stimulation (Fig. 8 a), suggesting RIG-I activation has no effect on LDHA protein level in Th1 cells. Next, we determine whether LDHA activity was regulated by RIG-I. Our results showed that the LDHA activity and lactate level were significantly increased in Th1 cells by stimulation with RIG-I agonist poly(I:C) (Fig. 8 b), whereas the opposite results were observed in Th1 cells after transfection with siRIG-I (Fig. 8 c). We further explored the function of LDHA in Th1 cell differentiation. The results showed that LDHA knockdown in naive CD4 + T cells under Th1 polarization condition down-regulated LDHA expression, which inhibited the differentiation of Th1 cells (supp Fig. 10a, b). In addition, we also found that silencing LDHA expression could partially block the effect of poly(I:C) on Th1 differentiation (supp Fig. 10c, d), indicating LDHA may contribute to dsRNAs-induced Th1 differentiation. It has been reported that LDHA regulated lactate production to influence histone lysine lactylation and acetylation 33 – 35 . Here, we observed that the levels of lactylated histone H3K18 (H3K18Lac) and acetylated H3K18 (H3K18Ac) were increased in Th1 cells differentiation with poly(I:C) stimulation compared with mock control (Fig. 8 d). In contrast, we also observed that the histone H3K18Lac and H3K18Ac levels was decreased in Th1 cells after transfection with siRIG-I (Fig. 8 e). Moreover, we found that the enrichment levels of H3K18Lac and H3K18Ac in gene loci were increased in poly(I:C)-treated Th1 cells by CUT&TAG (Fig. 8 f). We validated that the levels of both H3K18Lac and H3K18Ac in IFNG and TBX21 gene promoter regions were increased in poly(I:C)-treated Th1 cells compared to mock control by ChIP-qPCR (Fig. 8 g). In contrast to Th1 cell, we observed that the LDHA activity and lactate level were significantly decreased in Treg cells with poly(I:C) treatment (supp Fig. 11a). The expression of LDHA as well as the levels of H3K18Lac and H3K18Ac were obviously decreased in Treg cells with poly(I:C) stimulation (supp Fig. 11b). No significant increase in levels of H3K18Lac and H3K18Ac of Th2 and Th17 cells with poly(I:C) stimulation (supp Fig. 11c). Collectively, these data suggested that dsRNA activated RIG-I activation may regulate Th1 and Treg cell differentiation via LDHA-mediated histone lactylation and acetylation. Discussion In this study, we explored the expression, roles, and potential mechanism of HERVs in CD4 + T cells of SLE. We identified a lot of transcripts derived from HERVs loci in CD4 + T cells by both RNA-seq and transcriptome-seq (identifying HERVs with or without ploy A tail), most of which were higher expression in SLE due to DNA hypomethylation. Some of the up-regulated HERVs were characterized by dsRNAs and associated with RLR signaling pathway activation. Activation of RIG-I signal pathway triggered by dsRNAs promoted pathogenic T cell differentiation via regulating histone lysine lactylation and acetylation and STAT1 phosphorylation, and blocking the pathway decreased proportion of pathogenic T cell and alleviated the progression of autoimmune diseases. This study highlights that the important role of HERVs and dsRNA mediated RIG-I activation in T cell differentiation. Human endogenous retroviruses (HERVs), members of the long terminal repeat (LTR) retrotransposons repetitive element class, make up at least 8–10% of the human genome 10 . Most HERV sequences have acquired numerous mutations over time and therefore do not have protein-coding potential or the potential to generate infectious viral particles 36 , 37 . Epigenetic modifications are required in maintaining the transcriptional silencing of HERVs 38 . Alteration of epigenetic modification contributed to transcriptional activation of HERVs in diseases (such as tumor, aging and autoimmune diseases) 39 – 41 . A lot of evidences have shown the DNA hypomethylation and aberrant histone modifications in CD4 + T cells of SLE, which contribute to SLE pathogenesis 42 , 43 . In this study, we found that DNA hypomethylation favored transcriptional activation of HERVs and hyperexpression of the RIG-I signal pathway (supp Fig. 1 2), which was closely related to exacerbating the progress in SLE disease. Furthermore, our results identified many up-regulated HERV transcripts with LTR, most of which located in introns or intergenic region of interferon stimulated genes in genome and were positively associated with interferon gene signatures expression in CD4 + T cells of SLE (supp Fig. 1 ). DNA hypomethylation in these HERVs loci according to our results, as well as most interferon genes hypomethylation identified by previous studies 26 , account for the transcriptional activation of HERVs in SLE CD4 + T cells. Besides of transcriptional activation, the decreased expression of DICER and AGO2, dsRNA cleaving enzymes for intercellular dsRNA degradation 27 , was identified in SLE CD4 + T cells (supp Fig. 1 3), which may result in the accumulation of dsRNA in SLE CD4 + T cells. Although RLR pathway-mediated dsRNA sensing in innate immune cells has been well studied to play anti-virus role 44 , the sense and function of dsRNA in adaptive immune is poorly understood. Here we found that dsRNA derived from HERVs could be sensed and activated RLR signal pathway, including up-regulating RIG-I, MDA5 and IRF7 expression and phosphorylation of TBK1 and IRF3, leading to type I IFN gene activation in SLE CD4 + T cells. Previous study showed that the RLRs drive distinct immune gene activation and response polarization to mediate an M1/inflammatory signature while suppressing the M2/wound healing phenotype 45 . In this study, we first found that HERV dsRNAs activated RIG-I had the opposite regulation on effector T cells differentiation, including promoting Th1 and Th17 cells differentiation and inhibiting Treg cell differentiation. RIG-I deficiency improved the imbalance of Th1/Th17 and Treg cells lupus-like mice and alleviated the autoimmune diseases. Those findings indicated that RIG-I sensing dsRNAs in T cells not only induced IFN-I gene expression, but also promoted the dysregulated T cells differentiation, contributing to SLE pathogenesis. The activated RIG-I interacts with the mitochondrial antiviral signaling proteins (MAVS), which forms a multilayered protein complex and then catalyzes the activation of the serine/threonine-protein kinase 1 (TBK1) 46 , 47 . TBK1 phosphorylates the transcription factors IRF3 and IRF7, which then activate the expression of I-IFN (IFNα and IFNβ), leading to a massive inflammatory response 48 , 49 . Recent studies discovered that RIG-I might be critical for the differentiation of pro-inflammatory T cells, which promoted the expression of IFNG and TBX21 by reducing CpG methylation in the TBX21 promoter in CD8 + T cells 50 . Tumor cell-intrinsic RIG-I signaling mediates the release of immunogenic extracellular vesicles (EVs), which increase proportions of CD8 + IFNγ + T cells and potent cytotoxic antitumor immunity 51 . In this study, we revealed RIG-I activation in T cells induced Th1 differentiation via I-IFN inducing STAT1 phosphorylation to up-regulate TBX21 expression. And inhibition of STAT1 phosphorylation decreased the expression of TBX21, which completely inhibited the effect of activated RIG-I on Th1 cell differentiation. Importantly, we also found a novel mechanism under RIG-I regulated T cell differentiation through identifying LDHA bind to RIG-I protein in Th1 cells. We found that RIG-I activation influenced the activity and expression of LDHA during T cells differentiation, which indicated that the activated RIG-I also was involved in glycolysis process in T cells. Aerobic glycolysis is a metabolic hallmark of activated T cells and has been implicated in augmenting effector T cell responses 52 . LDHA is an important glycolytic enzyme to support aerobic glycolysis by the conversion of pyruvate to lactate, which has been proven to maintain high levels of acetyl-CoA to enhance histone acetylation and transcription of IFNG 35 . Ablation of LDHA in T cells protects mice from immunopathology triggered by excessive IFN-γ expression or deficiency of regulatory T cells 53 . Recently, LDHA was reported to induce histone lactylation, promoting gene transcription like histone acetylation 54 , 55 . H3K18Lac promotes reparative gene expression during M1 macrophage polarization to promote immune homeostasis 56 , and regulates early remote activation of the reparative transcriptional response in monocytes 57 . However, there is no report about histone lactylation in T cells differentiation. Here, our data showed that activity of LDHA and intracellular lactate were increased by dsRNAs-activated RIG-I and both the levels of H3K18Lac and H3K18Ac and their enrichments in IFNG and TBX21 promoters were upregulated in poly(I:C)-treated Th1 cells. An inverse histone lactylation and acetylation modification changes also observed in Treg cells. These findings suggest LDHA mediated histone lactylation and acetylation modifications and on T cell differentiation genes may be a noncanonical downstream pathway of activated RIG-I in regulating CD4 + T cell differentiation. However, the distinct effect and mechanism of RIG-I activation on LDHA and histone lactylation in different effect T cells still need to be investigated. In summary, our study provided a model that DNA hypomethylation induced HERV dsRNA accumulation in SLE CD4 + T cells activates RIG-I and I-IFN pathway and promotes LDHA mediated histone lactylation and acetylation, leading to pathogenic T cell differentiation in SLE. This finding uncovers the roles of HERVs and RIG-I pathway in adaptive immune response and SLE pathogenesis, and suggests that maintaining the silencing of HERVs by epigenetic interference or targeting RIG-I pathway may be a good way for SLE therapy. Methods Patients and controls. We collected blood samples of SLE patients and healthy controls (HC) in Second Xiangya Hospital of Central South University. Diagnosis of SLE disease was based on the following classification criteria: the 1982 revised criteria for the classification of SLE 58 . Healthy controls matched with age and gender were recruited. Written informed consent was provided by All subjects. This study was approved by the Ethics Committee of the Second Xiangya Hospital. The basic characteristics of all subjects were listed in Supplementary Table 1 and Supplementary Table 2. Mice. Rag2 − / − mice were purchased from the Shanghai Research Center For Model Organisms. B6D2F1 mice were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. The Ddx58 knockout (KO) mice were generated by Shanghai Model Organisms Center, Inc. For the Ddx58 CKO mice generation, the loxP- Ddx58 -loxP mice were constructed by Cyagen Bioscience Inc.(Guangzhou, China) and CD4 cre mice (stock no. 022071) were purchased from Shanghai Model Organisms Center, Inc. The Ddx58 -floxed mice were bred with CD4 cre transgenic mice to generate Ddx58 f/f CD4 cre ( Ddx58 CKO) mice. This study was approved by the Ethics Committee of the Chinese Academy of Medical Sciences and Peking Union Medical College. Animal models Chronic graft-versus-host disease (cGVHD) model. A total of 5×10 7 CD8 + T cells-depleted lymphocytes of Ddx58 CKO and Ddx58 f/f female mice were injected into B2D6F1 female mice via tail veins. Urine was collected weekly and the serum samples were collected at the the end of the observation period. By the end of 12 weeks, mice were sacrificed for further experiments. IMQ-induced lupus model. Epicutaneous Application of imiquimod leads to systemic autoimmunity is a recognized new lupus model 59 , 60 . To induce lupus-like disease, Ddx58 CKO and Ddx58 f/f mice were treated topically with 5% IMQ cream (Sichuan Med-shine Pharmaceutical, H20030128). Mice were treated with IMQ cream applied to the ear skin three times a week for 8 weeks. Urine samples were collected weekly and serum samples fortnightly. By the end of 8 weeks, mice were sacrificed for further experiments. The urine protein test kit for urine protein, and the serum anti-double-stranded DNA (dsDNA) IgG and antinuclear antibody (ANA) IgG levels were detected by ELISA kits (CUSABIO, China). Flow cytometry was used to analyze immune cells in the draining lymph nodes (dLNs) and spleens of the models. Analysis of C3 and IgG deposits in kidneys using multi-IHC stains. EAE disease model. EAE was induced by complete Freund’s adjuvant (CFA)- MOG35-55 peptide immunization (Hooklabs) and scored daily. Briefly, 2×10 6 naive CD4 + T cells of Ddx58 KO and WT female mice were injected into Rag2-/- female mice via tail veins. Mice were then injected subcutaneously into the neck with 200µl containing 200µg MOG35-55 peptide (Hooklabs) emulsified in complete Freund’s adjuvant (Sigma-Aldrich). Mice were also injected intraperitoneally with 500ng of pertussis toxin (Listlabs) on days 0 and 2 after immunization. Mice were monitored daily for morbidity and scored according to the following scoring criteria: 0, no symptoms, active and mobile; 0.5, partial weakness of the tail; 1, completely paralyzed tails, less active; 2, hind limb weakness, hobbling gait; 2.5, partial paralysis of hind limbs and complete paralysis of a single hind limb; 3, complete hind limb paralysis; 3.5, complete paralysis of both hind limbs, partial paralysis of forelimbs; 4, completely paralyzed of all limbs, losing mobility; 5, moribund or death. Cell isolation. Density gradient centrifugation (GE Healthcare) was used to isolate the peripheral blood mononuclear cells (PBMCs) from the peripheral blood of healthy controls and SLE patients. Then CD4 + T cells were isolated from the PBMCs by Miltenyi beads according to the manufacturer’s instructions (Miltenyi Biotec). In vitro human T cell differentiation. Naive CD4 + T cells were purified from PBMCs using the human Naive CD4 + T Cell Isolation Kit (Miltenyi Biotec), and then cells were stimulated with plate-bound anti-CD3 (5µg/ml, Calbiochem, catalog 217570) and anti-CD28 (2µg/ml, Calbiochem, catalog 217669) under the Supplemental Table 3 polarizing conditions. We performed cell culture in 24-well plates with a total volume of 1 ml/well of culture medium with 1×10 6 naive CD4 + T cells. The medium was refreshed on day 3. In vitro mouse T cell differentiation. Naive CD4 + T cells from mouse spleen were purified using mouse naive CD4 + T cell isolation kit II (Miltenyi Biotec), and the purity of the enriched subset was validated by flow cytometry and was generally higher than 95%. Purified naive CD4 + T cells were stimulated with plate-bound anti-CD3 (5µg/ml, eBioscience, catalog 16-0031-85) and anti-CD28 (2µg/ml, eBioscience, catalog 16-0281-85) for 3 days under different polarizing conditions (Supplemental Table 4). We performed cell culture in 24-well plates with a total volume of 1 ml/well of culture medium with 1×10 6 naive CD4 + T cells. Transfection of siRNA, poly(I:C), and ERV3-16A3_I-int RNA. The Human T Cell Nucleofector Kit and Amaxa Nucleofector System (Lonza) for T cell transfection. Briefly, naive CD4 + T cells were induced differentiation into different T cells for 3 days. The cells were collected and resuspended in 100 µL transfection reagents, and 10µL siRNA (20µM), or 1µL Smart Silence (20µM), or 500ng poly(I:C), or 10µg ERV3-16A3_I-int RNA was added and transfected into the cells by electroporation using the nucleofector program V-024 in the Amaxa Nucleofector apparatus (Lonza). After being cultured under RPMI 1640 complete medium (Gibco) for 6 hours, the cells were transferred to fresh complete medium under different polarizing conditions for 48 to 72 hours and then harvested for subsequent experiments. Flow cytometry. Briefly, T cells were incubated with fluorescein-labeled surface-labeled antibodies at 4°C for 30 minutes protected from light. For cytokines, cells were stimulated with Leukocyte Activation Cocktail, with BD GolgiPlug™ at 37°C and 5% CO2 for 6 hours. For intracellular staining, cells were fixed and permeabilized using the Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences) or transcription factor buffer set (BD Pharmingen), and then stained with fluorescent antibodies for 30 minutes at 4°C in the dark. Information on antibodies is shown in Supplemental Extended Table 1. The expression of cytokines, surface markers, and transcriptional factors was determined by flow cytometry using FACS Canto II (BD Biosciences) or Cytek® Northern Lights™-CLC (CYTEK Biosciences), and the data were analyzed by the Flowjo software. Chromatin immunoprecipitation (ChIP) qPCR. Th1 cells transfected with the poly(I:C) were isolated. ChIP with anti-H3K18Ac, anti-H3K18Lac were used to detect IFNG and TBX21 enrichment, which was performed by a ChIP kit (Millipore). The detailed protocols were previously described 61 . Immunoprecipitated DNA and input DNA were assessed using real-time PCR. The resulting DNA fragments were purified and subjected to PCR with the use of primers encompassing the D-box region of the TBX21 and IFNG gene promoters. The primers used in the present study were shown in Supplemental Table 5. CUT&Tag. CUT&Tag was performed according to the Hyperactive Universal CUT&Tag Assay Kit for Illumina(Vazyme, TD903) for Th1 cells. In brief, Th1 cells were collected and counted, of which the cell viability was > 85%. Cells were separated into 100,000 cell aliquots in each sample and incubated on ice for 10 min with 100ul of pre-cooled NE buffer for obtaining cell nuclei. Cells were centrifuged at 600×g for 5 min at room temperature and then resuspended in 100µl wash buffer in each sample. ConA Beads were activated in the binding buffer. Transfer 100µl of nuclei to an 8-strip tube containing 10µl activated ConA Beads, invert to mix and incubate at room temperature for 10 min. Beads were separated with a magnetic and supernatant was removed. 1µl of the primary antibody was diluted 1:50 in antibody buffer. The primary antibodies H3K18la (PTM-Bio, PTM1406RM) and H3K18ac (PTM-Bio, PTM-114RM) were used in this study. Cells were incubated overnight at 4°C. The primary antibody was replaced with the secondary antibody diluted to 1:100 in the dig-wash buffer. Samples were incubated for 45min at room temperature on the nutator. The secondary antibody was removed, and samples were washed 3 times in the dig-wash buffer. 2µl pA/G-Tnp was added in 98µl dig-300 buffer for per sample. Samples were incubated for 1h at room temperature on the nutator. Samples were washed 3 times with dig-300 buffer and then resuspended in 50 µl tagmentation buffer. Samples were incubated at 37°C for 1h. DNA was extracted with DNA Extract Beads. Fragmented DNA after purification was amplified by PCR. PCR conditions were set to 72°C for 3min, 95°C for 3min, 11 cycles of 98°C for 10sec, 60°C for 5sec, and 72°C for 1min. VAHTS DNA Clean Beads were used to purify the PCR product. Libraries were indexed using Nextera Indexes, and 150-bp paired-end sequencing was performed on Illumina Novaseq instruments. RNA extraction and Quantitative reverse transcription PCR (RT-qPCR). Total RNA was extracted from T cells using TRIzol (Invitrogen). RNA quality control was conducted with a NanoDrop spectrophotometer and an Agilent 2100 Bioanalyzer (Thermo Fisher Scientific). 1µg of total RNA was reverse-transcribed using PrimeScript RT reagent Kits With gDNA Eraser (Takara). RT-qPCR was performed on a Fast Real-time PCR system (Roche) with iTaq Universal SYBR Green (BioRad). The relative expression levels of genes were calculated by the 2 −ΔCt method, which normalized to the reference gene β-actin. The primers are listed in Supplemental Extended Table 1. Western Blot. Total protein was isolated from T cells by IP lysis buffer (Beyotime) supplemented with protease inhibitors (Roche) and phosphatase inhibitor (Beyotime). The proteins were quantified by Pierce BCA Protein Assay Kit (Thermo). The primary antibodies and secondary antibodies were used in this study as Supplemental Extended Table 2. The quantification of proteins was normalized to GAPDH or β-actin by densitometry using ImageJ software. Co-IP. Co-IP assays were performed with the Dynabeads Protein G (Life Technologies) for immunoprecipitation. First, we extracted protein from fresh cells using IP lysis buffer supplemented with protease and phosphatase inhibitors. Rabbit anti-RIG-I Ab (Abcam, ab180675) or Rabbit anti-LDHA Ab (Abcam, ab52488) was added to the lysates, forming a new antibodies-bait-target complex. Then, the antibodies-bait-protein complexes were eluted from the beads and dissociated by boiling in protein loading buffer. Finally, the presence of the target protein was evaluated by Western blot. Bisulfite sequencing polymerase chain reaction (BSP). Genomic DNA was isolated from CD4 + T cells or Jurkat cells using the QIAamp DNA Mini Kit (Qiagen). DNA was bisulfifite converted using EZ DNA Methylation-Lightning™ Kit (Zymo Research). The bisulfite-treated DNA was amplified via nested PCR amplification reactions with specific primers, which were designed using the online MethPrimer software( http://www.urogene.org/methprimer/ ). The primer information used to amplify the target fragment are shown in Supplementary Table 6. For each PCR, 0.25mM dNTP mix (Promega), 0.2µM forward and reverse primers, 2.5 U of Taq DNA Polymerase (Promega), and 5×Green GoTaq® Reaction Buffer (Promega) were used in a 20µl total reaction volume. Here 100ng of bisulfite-treated DNA was used as the template of the first PCR, whereas 4µl of PCR1 product was used as the template for the second PCR. Thermal cycling conditions consisted of one cycle of 2 min at 96°C, followed by 40 cycles of 10s at 96°C, 30s at 55°C and 1 min at 72°C, and a final extension at 72°C for 10min. The PCR products were purified by gel extraction (Promega) from a 1.5% agarose gel and ligated into the pMD™18-T Vector (Takara). The ligation products were used to transform competent Escherichia coli cells (strain DH5a) using standard procedures, and blue/white screening was used to select ten independent clones from each specimen were sequenced (Sangon). The final sequence results were analyzed by online QUMA software ( http://quma.cdb.riken.jp/ ). DsRNA analysis by RNase digestion. According to previous research for dsRNA analysis by RNase A digestion 27 , 5µg total RNA of CD4 + T cells was dissolved in 32µL H 2 O and mixed well with 17.5µL NaCl (1 M stock). Then, 0.5µL RNase A (10mg/ml stock, Thermo Fisher Scientific) or H 2 O was mixed to a total volume of 50µL and incubated at room temperature for 10 min. 1mL TRIzol was added to the mixture. The levels of HERVs were detected by RT-qPCR with ACTB as an internal control. The ratios of (HERV/ACTB)RNaseA/(HERV/ACTB)H 2 O were calculated as enrichment folds. DsRNA analysis by J2 pulldown. Purified total RNA from CD4 + T cells was used for the J2 pulldown assay. Purified total RNA from CD4 + T cells was used for the J2 pulldown assay. J2 antibody (Scicons) and mouse IgG control (Merck) (1µg per pulldown) were incubated with Protein G dynabeads (Merck), respectively, for 30min at room temperature. 30µg RNA was mixed with 500µl immunoprecipitation (IP) buffer. Then, the whole mixture was added with washed beads and rotated at 4°C for 2h. Afterward, the beads were incubated in 50µl Proteinase K digestion solution. 1 ml Trizol was directly added to the eluate for RNA purification and RT-qPCR analysis as described above. RNA-FISH. We used the RNA-FISH to study the subcellular distribution of ERV3-16A3_I-int. Fluoresce-conjugated ERV3-16A3_I-int probes labelled with Cy3 and FISH kits were generated from RiboBio (China). Briefly, 4% paraformaldehyde (supplementing 5% TritonX-100) was used to fix CD4 + T cells of SLE and HCs (30 min). The fixed cells were incubated with ERV3-16A3_I-int probes in hybridization buffer at 37◦C overnight. Nuclei were stained with DAPI. Immunofluorescent staining. CD4 + T cells were fixed with cold methanol at -20℃ for 15 min. Then the cells were incubated with 0.2% Triton (Sigma) at 4℃ for 5 min. The samples were incubated with primary antibody (Scicons) at 4℃ for overnight and then secondary antibody (Abcam) for 1h at room temperature. Mouse kidney were fixed in formalin and embedded in paraffin. Hematoxylin and eosin (H&E) stains and Periodic Acid-Schiff (PAS) stains were used to assess lymphocytic infiltration and glycogen deposition in the kidney. To assess the immune complex deposition in the kidney, we stained paraffin-embedded renal sections with rabbit anti-C3 antibody (Abcam) and HRP-conjugated anti-mouse IgG. The opal 7-Color Manual IHC Kit (Perkin Elmer) was used for fluorescence labeling. Information on antibodies was provided in Supplemental Extended Table 2. RNA-Sequencing. RNA-seq dataset was taken from CD4 + T cells of 4 healthy female controls and 4 female SLE patients. The controls ranged in age from 22–51 years of age average age 31.6, and the patients ranged in age from 19–44 years old with an average age of 34.2 years old. The patients without drug therapy had a SLE disease activity index (SLEDAI) ranging from 7 to 23 with an average score of 14.8. RNA library preparation was performed as described in previous researches 62 . The HERVs were obtained from RepeatMasker for expression analyses by stringtie 63 , 64 . The differential analysis was performed using DESeq 65 . Whole transcriptome sequencing. Whole transcription sequencing data included 5 healthy female controls and 5 female SLE patients. The controls ranged in age from 21–32 years of age average age 24.2, and the patients ranged in age from 15–32 years old with an average age of 22.2 years old. The patients without drug treatment had SLEDAI ranging from 6 to 13 with an average score of 9.4. After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform and 150bp paired-end reads were generated. The raw data were processed through Fastp 66 . The data were then mapped to the human reference genome hg38 with Hisat2 v2.0.5 62 . The differential analysis was performed using DESeq 65 . Statistical Analysis. SPSS 22.0 was used for statistical analysis and calculation. Comparisons of the means between experimental variables were made via unpaired two-sided Student’s t-test for normally distributed variables or Mann-Whitney for non-normally distributed variables. P < 0.05 was regarded as significant. Declarations Ethics approval. The study was approved by the Ethics Committee of the Second Xiangya Hospital of Central South University and the Ethics Committee of Chinese Academy of Medical Sciences and Peking Union Medical College. All participants provided written informed consent. Funding This work was supported by the National Natural Science Foundation of China (No. 82030097 and No. 32141004), the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (2022-RC310-04), and the National Key R&D Program of China (2022YFC3601803). Author Contributions X.M. conceptualized the studies, analyzed the data, and wrote the manuscript. X.M, Y.Y., Z.H., L.YO., Y.Q., J.W., C.Z. and S.Y. performed the experiments. H.Z., J.W., M.Zheng. and Q.L. collected samples of SLE and handled the clinical information of patients. Q.L. and S.J. contributed to conception and design of the study. D.Y. provided suggestions for the studies. M.Z. conceptualized the studies, supervised the experiments, analyzed results and wrote the manuscript. Declaration of Competing Interest The authors declare no competing financial interests. References Lazar S, Kahlenberg JM (2023) Systemic Lupus Erythematosus: New Diagnostic and Therapeutic Approaches. 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Supplementary Files SupplementalTable.docx SupplementaryExtendedTable1.docx SupplementaryExtendedTable2.docx SupplementaryFig1.pdf SupplementaryFig2.pdf SupplementaryFig3.pdf SupplementaryFig4.pdf SupplementaryFig5.pdf SupplementaryFig6.pdf SupplementaryFig7.pdf SupplementaryFig8.pdf SupplementaryFig9.pdf SupplementaryFig10.pdf SupplementaryFig11.pdf SupplementaryFig12.pdf SupplementaryFig13.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Carcinogenesis, Cancer Research Institute and School of Basic Medicine, Central South University","correspondingAuthor":false,"prefix":"","firstName":"Lianlian","middleName":"","lastName":"Ouyang","suffix":""},{"id":272474672,"identity":"9eea2873-ce5a-4d78-91c7-aa9a5ed6482c","order_by":5,"name":"Yueqi Qiu","email":"","orcid":"","institution":"Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Yueqi","middleName":"","lastName":"Qiu","suffix":""},{"id":272474673,"identity":"3a0e8d14-f28a-4eb4-9935-f50405059086","order_by":6,"name":"Hongjun zhao","email":"","orcid":"","institution":"Department of Rheumatology, Xiangya Hospital, Central South University","correspondingAuthor":false,"prefix":"","firstName":"Hongjun","middleName":"","lastName":"zhao","suffix":""},{"id":272474674,"identity":"cb1ecf65-7f91-413f-b664-c227439efb4f","order_by":7,"name":"Jiali Wu","email":"","orcid":"","institution":"Department of Dermatology, Hunan Key Laboratory of Medical Epigenomics, Second Xiangya Hospital, Central South University","correspondingAuthor":false,"prefix":"","firstName":"Jiali","middleName":"","lastName":"Wu","suffix":""},{"id":272474675,"identity":"48c2146f-c558-49eb-a718-8db4014bcc6c","order_by":8,"name":"Chun Zou","email":"","orcid":"","institution":"Department of Dermatology, Hunan Key Laboratory of Medical Epigenomics, Second Xiangya Hospital, Central South University","correspondingAuthor":false,"prefix":"","firstName":"Chun","middleName":"","lastName":"Zou","suffix":""},{"id":272474676,"identity":"e4c9f7fe-a7b2-463d-9581-5453d5ef9bae","order_by":9,"name":"Meiling Zheng","email":"","orcid":"","institution":"Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Meiling","middleName":"","lastName":"Zheng","suffix":""},{"id":272474677,"identity":"c6dcd4a2-dd65-4e5d-a50d-b8b4f9ebffd4","order_by":10,"name":"Shuang Yang","email":"","orcid":"","institution":"Department of Dermatology, Hunan Key Laboratory of Medical Epigenomics, Second Xiangya Hospital, Central South University","correspondingAuthor":false,"prefix":"","firstName":"Shuang","middleName":"","lastName":"Yang","suffix":""},{"id":272474678,"identity":"1f3ce7e3-fdaa-4a69-8860-a878827ccd0d","order_by":11,"name":"Jia Sujie","email":"","orcid":"","institution":"Department of Pharmacy, The Third Xiangya Hospital, Central South University","correspondingAuthor":false,"prefix":"","firstName":"Jia","middleName":"","lastName":"Sujie","suffix":""},{"id":272474679,"identity":"2b9b0765-b05d-49ab-8162-b64c3728ea61","order_by":12,"name":"Di Yu","email":"","orcid":"https://orcid.org/0000-0003-1721-8922","institution":"The University of Queensland","correspondingAuthor":false,"prefix":"","firstName":"Di","middleName":"","lastName":"Yu","suffix":""},{"id":272474680,"identity":"99bd17ea-1a2e-435a-b98c-586c777a1191","order_by":13,"name":"Qianjin Lu","email":"","orcid":"https://orcid.org/0000-0002-1504-4896","institution":"Institute of Dermatoloy, Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Qianjin","middleName":"","lastName":"Lu","suffix":""}],"badges":[],"createdAt":"2024-02-08 10:11:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3939567/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3939567/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51078344,"identity":"b7ce6029-ea2f-42a6-8ca9-1269af7795d1","added_by":"auto","created_at":"2024-02-13 18:53:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":538307,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScreening and identification of the highly expressed HERVs in CD4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+ \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eT Cells of SLE.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e The number of identified HERVs by RNA-seq and whole transcriptome sequencing. \u003cstrong\u003eb, c\u003c/strong\u003e The number of differential expression of HERVs in RNA-seq and the whole transcriptome sequencing. \u003cstrong\u003ed\u003c/strong\u003e Schematic diagram depicting the screening of HERVs\u003cstrong\u003e \u003c/strong\u003ein CD4\u003csup\u003e+ \u003c/sup\u003eT Cells of SLE. FPKM, fragments per kilobase of transcript per million mapped reads. \u003cstrong\u003ee, f\u003c/strong\u003e The differential HERVs were identified with significantly higher expression in RNA-seq and the whole transcriptome sequencing according to stringent thresholds. \u003cstrong\u003eg\u003c/strong\u003e RT-qPCR analysis of the identified HERVs expression in CD4\u003csup\u003e+ \u003c/sup\u003eT Cells of SLE (n= 19) and HCs (n= 16). (unpaired 2-tailed Student’s t test and Mann-Whitney test for \u003cstrong\u003eg\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-3939567/v1/bcc35de5126882edfd6f42ce.png"},{"id":51078336,"identity":"612afdb3-4e09-4bf4-8c36-6f860160da3a","added_by":"auto","created_at":"2024-02-13 18:53:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":632200,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetection of DNA methylation status in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eHERV\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e loci in CD4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+ \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eT Cells of SLE. a \u003c/strong\u003eCpG islands in \u003cem\u003eHERV\u003c/em\u003e loci (Blue bars represented upstream or downstream areas of genes; red bars represented gene body areas; yellow box represented detected CpG region).\u003cstrong\u003e b, c\u003c/strong\u003e DNA methylation levels of HERVs in CD4\u003csup\u003e+ \u003c/sup\u003eT cells of HCs and SLE (n=5). \u003cstrong\u003ed\u003c/strong\u003e DNA methylation changes in HERVs in Jurkat cells treated with 5-AZA-CdR (n=3). \u003cstrong\u003ee\u003c/strong\u003e Expression of HERVs in Jurkat cells treated with 5-AZA-CdR (n=3). (unpaired 2-tailed Student’s t test for \u003cstrong\u003ec-e\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-3939567/v1/4f1b92a2946418e54aee5c11.png"},{"id":51078347,"identity":"0bc5b946-876e-4b48-9de2-66120d074579","added_by":"auto","created_at":"2024-02-13 18:53:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1095590,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHERV-derived dsRNAs triggered RLRs pathway activation in SLE CD4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+ \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eT cells. a, b \u003c/strong\u003eThe dsRNA enrichments of HERVs were assessed by RNase A digestion and J2-dsRNA pulldown (n=3). \u003cstrong\u003ec-e \u003c/strong\u003eqPCR and Western blot analysis of RIG-I signal pathway genes in CD4\u003csup\u003e+ \u003c/sup\u003eT cells with poly(I:C) or ERV3-16A3_I-int treatment. \u003cstrong\u003ef\u003c/strong\u003e RIP assay assessed the binding capacity of ERV3-16A3_I-int to RIG-I. \u003cstrong\u003eg \u003c/strong\u003eIP and Western blot detected the ubiquitination level of RIG-I protein in CD4\u003csup\u003e+ \u003c/sup\u003eT cells treated with poly(I:C).\u003cstrong\u003e h\u003c/strong\u003e Expression and distribution of ERV3-16A3_I-int and dsRNA in CD4\u003csup\u003e+ \u003c/sup\u003eT cells of SLE and HCs detected by FISH and immunofluorescence of J2 antibody staining. \u003cstrong\u003ei\u003c/strong\u003e qPCR analysis of RIG-I signal pathway genes in CD4\u003csup\u003e+ \u003c/sup\u003eT cells of SLE (n=31) and HCs (n=13). \u003cstrong\u003ej\u003c/strong\u003e IP and Western blot detected the ubiquitination level of RIG-I protein and expression level of RIG-I in CD4\u003csup\u003e+ \u003c/sup\u003eT cells from healthy donors (n=20) and patients with SLE (n=27). \u003cstrong\u003ek\u003c/strong\u003e Representative flow cytometry and quantification of RIG-I in CD45RA\u003csup\u003e+\u003c/sup\u003eCD45RO\u003csup\u003e- \u003c/sup\u003enaive CD4\u003csup\u003e+ \u003c/sup\u003eT cells and CD45RA\u003csup\u003e-\u003c/sup\u003eCD45RO\u003csup\u003e+\u003c/sup\u003e memory/effector CD4\u003csup\u003e+ \u003c/sup\u003eT cells from healthy donors or patients with SLE (n=22 for healthy donors, n=18 for patients with SLE). (unpaired 2-tailed Student’s t test for \u003cstrong\u003ec, d, i, j and k\u003c/strong\u003e)\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-3939567/v1/93d73bd3b62960d99d26ebd7.png"},{"id":51078355,"identity":"2d820a39-0112-48c5-bca6-8b50e06c5b6b","added_by":"auto","created_at":"2024-02-13 18:53:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":935099,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDsRNAs regulated T cell differentiation in vitro. a \u003c/strong\u003eRNA-seq analysis of expression of genes associated with T cell differentiation in naive CD4\u003csup\u003e+ \u003c/sup\u003eT cells with poly(I:C) treatment. \u003cstrong\u003eb\u003c/strong\u003e RT-qPCR analysis of genes associated with T cell differentiation in naive CD4\u003csup\u003e+ \u003c/sup\u003eT cells with poly(I:C) treatment. \u003cstrong\u003ec, d, i\u003c/strong\u003e Representative flow cytometry of Th1 and Treg cells transfected with poly(I:C), ERV3-16A3_I-int RNA or smart silence ERV3-16A3_I-int treatment (n=4). \u003cstrong\u003ee-h, j\u003c/strong\u003e The mRNA expression of genes associated with T cell differentiation in CD4\u003csup\u003e+ \u003c/sup\u003eT cells transfected with poly(I:C), ERV3-16A3_I-int RNA or smart silence ERV3-16A3_I-int and the protein levels of IFN-γ and TGFβ in cultured supernatants from normal human CD4\u003csup\u003e+ \u003c/sup\u003eT cells transfected with poly(I:C), ERV3-16A3_I-int RNA or smart silence ERV3-16A3_I-int (n=4) (unpaired 2-tailed Student’s t test for\u003cstrong\u003e b-j\u003c/strong\u003e)\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-3939567/v1/c329a92443218805e911af26.png"},{"id":51078335,"identity":"f9096aed-300a-4488-bc6f-44d3945a39a8","added_by":"auto","created_at":"2024-02-13 18:53:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":549218,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLoss of RIG-I in CD4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+ \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eT cells affected T cell differentiation in vitro. a \u003c/strong\u003eWestern blot of\u003cstrong\u003e \u003c/strong\u003ethe expression of RLRs signalling pathway in CD4\u003csup\u003e+ \u003c/sup\u003eT cells transfected with siNC or siRIG-I. \u003cstrong\u003eb-e\u003c/strong\u003e The percentage of Th1, Th2, Th17, and Treg cells transfected with siNC or siRIG-I was detected by flow cytometry (n=3). \u003cstrong\u003ef\u003c/strong\u003e Western blot and qPCR (n=3) of RIG-I in CD4\u003csup\u003e+ \u003c/sup\u003eT cell of WT and \u003cem\u003eDdx58\u003c/em\u003e KO mice. \u003cstrong\u003eg\u003c/strong\u003e Naive CD4\u003csup\u003e+ \u003c/sup\u003eT cells of WT and \u003cem\u003eDdx58\u003c/em\u003e KO mice were cultured under Th1/Th2/Th17/Treg cell-polarized conditions for 3 days. Representative flow cytometry of Th1, Th2, Th17 and Treg cells (n=3). (unpaired 2-tailed Student’s t test for \u003cstrong\u003eb-g\u003c/strong\u003e)\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-3939567/v1/a57be56265507a520622f338.png"},{"id":51078350,"identity":"0b62cadc-777a-455b-984e-dffd94d34580","added_by":"auto","created_at":"2024-02-13 18:53:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":712559,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRIG-I deficiency improved EAE mice model via regulating Th1/Th17 and Treg cell differntiation in vivo. a \u003c/strong\u003eScheme of adoptive transfer of naïve CD4\u003csup\u003e+ \u003c/sup\u003eT cells from WT or \u003cem\u003eDdx58\u003c/em\u003e KO mice into \u003cem\u003eRag2-/-\u003c/em\u003e mice followed by EAE immunization for 16 days. \u003cstrong\u003eb, c\u003c/strong\u003e Weight loss and clinical scores of Rag2-/- mice after the induction of EAE were assessed every day (n=4). \u003cstrong\u003ed\u003c/strong\u003e The degree of demyelination of spinal cords from WT-\u003cem\u003eRag2-/- \u003c/em\u003eand \u003cem\u003eDdx58\u003c/em\u003e KO-\u003cem\u003eRag2-/-\u003c/em\u003e mice with EAE was analyzed by luxol fast blue staining. \u003cstrong\u003ee-g\u003c/strong\u003e Representative flow cytometry of CD4\u003csup\u003e+\u003c/sup\u003eIFNγ\u003csup\u003e+ \u003c/sup\u003eTh1 cells (e), CD4\u003csup\u003e+\u003c/sup\u003eIL17A\u003csup\u003e+ \u003c/sup\u003eTh17 cells (f), CD4\u003csup\u003e+\u003c/sup\u003eFOXP3\u003csup\u003e+ \u003c/sup\u003eTreg cells (g) (n=4). (unpaired 2-tailed Student’s t test for \u003cstrong\u003eb,c, e-h\u003c/strong\u003e)\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-3939567/v1/01f994ae910a17abbc3e02de.png"},{"id":51079473,"identity":"860efaa5-e348-4113-9967-b27591132b09","added_by":"auto","created_at":"2024-02-13 19:01:00","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1879923,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRIG-I deficiency inhibited pathogenic T cell differentiation and relieved IMQ-iduced lupus-like model mice. a \u003c/strong\u003eThe spleen weight of IMQ-iduced lupus-like model mice. \u003cstrong\u003eb\u003c/strong\u003e The ratio of urine protein/creatine in IMQ-iduced lupus-like model mice. \u003cstrong\u003ec, d\u003c/strong\u003e Serum levels of anti-dsDNA IgG and anti-nuclear antibody (IgG) in \u003cem\u003eDdx58\u003c/em\u003e\u003csup\u003ef/f\u003c/sup\u003e (n=5) and \u003cem\u003eDdx58\u003c/em\u003e CKO mice (n=11). \u003cstrong\u003ee\u003c/strong\u003e Representative morphology (by H\u0026amp;E and PAS staining) and histological scoring of kidneys. \u003cstrong\u003ef\u003c/strong\u003e C3 and IgG deposition in the kidney sections were assessed by immunofluorescence staining. Scale bar: 50μm. \u003cstrong\u003eg\u003c/strong\u003e Representative flow cytometry of CD4\u003csup\u003e+\u003c/sup\u003eIFNγ\u003csup\u003e+ \u003c/sup\u003eTh1 cells, CD4\u003csup\u003e+\u003c/sup\u003eIL4\u003csup\u003e+ \u003c/sup\u003eTh2 cells, CD4\u003csup\u003e+\u003c/sup\u003eIL17A\u003csup\u003e+ \u003c/sup\u003eTh17 cells, CD4\u003csup\u003e+\u003c/sup\u003eFOXP3\u003csup\u003e+ \u003c/sup\u003eTreg cells, CD4\u003csup\u003e-\u003c/sup\u003eB220\u003csup\u003e-\u003c/sup\u003eCD138\u003csup\u003e+ \u003c/sup\u003eplasma cells and CD4\u003csup\u003e-\u003c/sup\u003eB220\u003csup\u003e+\u003c/sup\u003eFAS\u003csup\u003e+\u003c/sup\u003eGL7\u003csup\u003e+ \u003c/sup\u003eGCB cells. \u003cstrong\u003eh\u003c/strong\u003e Cytokine levels in serum of IMQ-iduced lupus-like model mice (f/f n=5, CKO n=11). \u003cem\u003eDdx58\u003c/em\u003e\u003csup\u003ef/f\u003c/sup\u003e mice (n=5) and \u003cem\u003eDdx58\u003c/em\u003e CKO mice (n=11), (unpaired 2-tailed Student’s t test and Mann-Whitney test for \u003cstrong\u003ea-d\u003c/strong\u003e, and \u003cstrong\u003eg, h\u003c/strong\u003e)\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-3939567/v1/94f87f08f8aec8dd02723e22.png"},{"id":51078354,"identity":"48a2b47a-d0a0-464b-99ff-dde914d0b201","added_by":"auto","created_at":"2024-02-13 18:53:02","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":990897,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003edsRNA/RIG-I axis regulated histone modification in Th1 cells by modulating LDH activity. a \u003c/strong\u003eCo-IP and western blot detected the interaction between RIG-I and LDHA protein. \u003cstrong\u003eb, c\u003c/strong\u003e LDHA activity and Lactate level in Th1 cells with dsRNA stimulation or siRIG-I treatment. \u003cstrong\u003ed, e\u003c/strong\u003e The expression levels of RIG-I and LDHA, histone H3K18Lac and H3K18Ac in Th1 cells with poly(I:C) stimulation or siRIG-I treatment.\u003cstrong\u003e f \u003c/strong\u003eHeatmap of the enrichments of\u003cstrong\u003e \u003c/strong\u003eH3K18Lac and H3K18Ac in gene loci in Th1 cells with poly(I:C) stimulation.\u003cstrong\u003e g \u003c/strong\u003eThe levels of\u003cstrong\u003e \u003c/strong\u003eH3K18Lac and H3K18Ac in gene promoters of \u003cem\u003eIFNG\u003c/em\u003e and \u003cem\u003eTBX21 \u003c/em\u003ewere detected by ChIP-qPCR in Th1 cells with poly(I:C) stimulation. (unpaired 2-tailed Student’s t test for \u003cstrong\u003eb, c, \u003c/strong\u003eand \u003cstrong\u003eg\u003c/strong\u003e)\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-3939567/v1/195e31f0a6573d59b740841b.png"},{"id":51078357,"identity":"e48ab366-cf3d-474f-a555-21b66cdd4053","added_by":"auto","created_at":"2024-02-13 18:53:02","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":266090,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe model of HERVs regulating RIG-I pathway and T cell differentiation in SLE CD4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e T cells. \u003c/strong\u003eMultiple HERVs were highly expressed due to DNA hypomethylation in CD4\u003csup\u003e+ \u003c/sup\u003eT cells from SLE patients, which induced activation of RIG-I signaling via forming dsRNA. Mechanistically, RIG-I increased LDHA activity upon dsRNA stimulation, which promoted histone acetylation and lactylation in Th1 cells, resulting in increased Th1 cell differentiation. In addition, dsRNA stimulation induced overexpression of type I IFN (IFNα and IFNβ), which regulated T-Bet by modulating STAT1 phosphorylation. Overall, our data show that dsRNA-induced RIG-I signal favors pathogenic T-cell differentiation and secretion of inflammatory factors, providing strong evidence for the important role of HERVs in lupus pathogenesis.\u003c/p\u003e","description":"","filename":"Fig9.png","url":"https://assets-eu.researchsquare.com/files/rs-3939567/v1/8559b5fc6ac452b0433bff1f.png"},{"id":75875322,"identity":"bda91a07-3d7c-428d-b7f5-d267843b42d5","added_by":"auto","created_at":"2025-02-10 07:43:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8890513,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3939567/v1/5780b795-fc1c-4009-b437-d50956c9e26d.pdf"},{"id":51078349,"identity":"ebd8ad32-faa0-484a-ba06-fc26ed2bc051","added_by":"auto","created_at":"2024-02-13 18:53:01","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":29554,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTable.docx","url":"https://assets-eu.researchsquare.com/files/rs-3939567/v1/59f4cd2f7f299329957e9eb6.docx"},{"id":51078342,"identity":"49cd3fd9-b1a1-4dd1-8489-8c2a1db4734e","added_by":"auto","created_at":"2024-02-13 18:53:00","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":15637,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryExtendedTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-3939567/v1/a9c030a6c796b8a4b4d815f3.docx"},{"id":51079476,"identity":"dbf9e3af-7228-4975-91a1-dcb7c8fe888f","added_by":"auto","created_at":"2024-02-13 19:01:00","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":25933,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryExtendedTable2.docx","url":"https://assets-eu.researchsquare.com/files/rs-3939567/v1/0e8ec5e56b6c9136d4785de2.docx"},{"id":51078346,"identity":"5b946289-cfd2-4e7e-9b7a-5a1aec252637","added_by":"auto","created_at":"2024-02-13 18:53:01","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1047407,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3939567/v1/46b0ce01decd96e5b6f065d3.pdf"},{"id":51078353,"identity":"a0fa5d44-4230-4fd2-9e6b-728372478fe9","added_by":"auto","created_at":"2024-02-13 18:53:02","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":853276,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3939567/v1/3b0fbac4e099539afb7a6b4f.pdf"},{"id":51080216,"identity":"f23f52cf-b164-4789-9cac-05491ecd332d","added_by":"auto","created_at":"2024-02-13 19:09:02","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":1380758,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3939567/v1/fdf32f588efbb8a0867f8095.pdf"},{"id":51079478,"identity":"85f43811-088b-445f-9ed5-2a9ab5ac8baa","added_by":"auto","created_at":"2024-02-13 19:01:01","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":610639,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3939567/v1/719d6e3e2b941630f61db647.pdf"},{"id":51079479,"identity":"09686a57-2f54-45bb-b99b-7c6824bdb986","added_by":"auto","created_at":"2024-02-13 19:01:01","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":912207,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3939567/v1/1eb58b4f29563ff47360d4e8.pdf"},{"id":51079480,"identity":"57397baa-6499-40ef-a4fd-e5a0c88f9639","added_by":"auto","created_at":"2024-02-13 19:01:02","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":714294,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig6.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3939567/v1/810a69d9f497696d417b3480.pdf"},{"id":51078341,"identity":"509b3697-e8ee-4f06-bc3d-32da3e700423","added_by":"auto","created_at":"2024-02-13 18:53:00","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":1432270,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig7.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3939567/v1/12235e8468d57c0b86e88733.pdf"},{"id":51078360,"identity":"e8d4bc7a-60e1-4001-9a04-bf97940f6960","added_by":"auto","created_at":"2024-02-13 18:53:02","extension":"pdf","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":16183659,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig8.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3939567/v1/ffc94044ea75dd5c0b791b71.pdf"},{"id":51078340,"identity":"47327cc7-ff2d-4997-9f08-d66abf11f8ff","added_by":"auto","created_at":"2024-02-13 18:53:00","extension":"pdf","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":738580,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig9.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3939567/v1/9cb2675b18f0cceef9b868bb.pdf"},{"id":51078352,"identity":"1b7cad3e-b403-4450-9c68-64e859e7728b","added_by":"auto","created_at":"2024-02-13 18:53:01","extension":"pdf","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":821798,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig10.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3939567/v1/5fbc36eada0aeef2a8ff5b81.pdf"},{"id":51080214,"identity":"2c400096-1ffa-4fe7-9d03-7ec3bbf1ac67","added_by":"auto","created_at":"2024-02-13 19:09:00","extension":"pdf","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":833401,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig11.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3939567/v1/5bc28098c4d1ded4aa161cff.pdf"},{"id":51078351,"identity":"78b73e21-6386-4950-9d4b-671f711092b4","added_by":"auto","created_at":"2024-02-13 18:53:01","extension":"pdf","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":1102984,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig12.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3939567/v1/258fb9a74ca7ce718e842a6d.pdf"},{"id":51078359,"identity":"79345958-1228-4199-b7b1-3eeebd640c08","added_by":"auto","created_at":"2024-02-13 18:53:02","extension":"pdf","order_by":16,"title":"","display":"","copyAsset":false,"role":"supplement","size":258715,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig13.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3939567/v1/cea3492ba3fae7c9fc5ba7e2.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Endogenous retrovirus promotes the aberrant T cell differentiation in systemic lupus erythematosus via RIG-I pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSystemic lupus erythematosus (SLE) is a potentially fatal, chronic, multisystem autoimmune disorder\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, characterized by overproduction of numerous inflammatory cytokines and aberrant differentiation of pathogenic T lymphocyte\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. In SLE disease, abnormal activation and proliferation of pathogenic CD4\u003csup\u003e+\u003c/sup\u003e T cells (including the Th1, Th2, Th17 and Tfh cell subsets) contribute to the secretion of a variety of inflammatory factors (such as IFN-γ, IL-4, IL-17A and IL-21, etc.) and assist B cells in production of high levels of auto-antibodies, whereas inflammation-suppressing Treg cells show a quantitative and/or qualitative deficiencies associated with uncontrolled autoinflammation\u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Previous studies showed that genetic factors and epigenetic changes such as DNA hypomethylation contributed to T cells over-activation and SLE pathogenesis\u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. However, the precise mechanism causing T cell abnormalities in SLE remains unclear.\u003c/p\u003e \u003cp\u003eHuman endogenous retroviruses (HERVs) are genetic remnants of retroviruses that were integrated into the human genome millions of years ago, and make up as much as 8% (over 500,000 individual elements) of the human genome\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Recombination has resulted in the majority of HERV elements existing as solitary long terminal repeats (LTRs)\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, and only a few HERVs retain the ability of encoding virus proteins. Previous findings suggested that HERVs encoded proteins were involved in the pathogenesis of SLE\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. For example, HERV-E clone 4\u0026thinsp;\u0026minus;\u0026thinsp;1 and ERV envelope glycoprotein, gp70, were implicated as an autoantigen to result in lupus\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. In addition, recent research revealed that DNA hypomethylation driven transcriptional activation of HERV LTRs can drive proinflammatory response \u003cem\u003evia\u003c/em\u003e the formation of double-stranded RNA (dsRNA) and activation of the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) recognition pathway in some tumors\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, which play an anti-tumor role. However, the role of HERVs in human genome is still poor understand in autoimmune diseases up to now. Whether there exists the transcription activation of HERVs in SLE CD4\u003csup\u003e+\u003c/sup\u003e T cells, which has been shown to be hypomethylation in SLE patients, and whether HERVs lead to RLR pathway activation and aberrant T cells differentiation in SLE patients need to be investigated.\u003c/p\u003e \u003cp\u003eRIG-I, one member of the RLRs, is a key sensor of viral dsRNA that recruits mitochondrial antiviral signaling protein (MAVS) and relays the signal to the kinases TBK1, which mediates transcriptional induction of type I interferons (I-IFN) and interferon stimulation genes (ISGs) via interferon-regulatory factor-3 (IRF-3) and IRF-7\u003csup\u003e17,18\u003c/sup\u003e, play an important role in antiviral innate immune response. Evidence supports the idea that the I-IFN pathway activation contributes to SLE susceptibility\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Beside of innate immune cells, RIG-I signal genes and many interferon stimulation genes were overexpression in various of cells, especially CD4\u003csup\u003e+\u003c/sup\u003e T cells in SLE patient\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. However, the roles of dsRNA sensors RIG-I in adaptive immune cells remain unclear.\u003c/p\u003e \u003cp\u003eIn this study, we screened and identified many overexpressed HERVs in CD4\u003csup\u003e+\u003c/sup\u003e T cells from SLE patients and found some HERVs formed dsRNAs to induced activation of RIG-I signaling pathway. And we revealed the role of HERVs derived dsRNAs and RIG-I in regulating effector T-cell differentiation and disease progression of lupus-like mice. Mechanistically, we also demonstrated the effect of activated RIG-I on histone lactylation and acetylation via regulating lactate dehydrogenase A (LDHA) and lactate level during T cells differentiation. Our findings uncovered a novel mechanism underlying the aberrant T cells differentiation in SLE.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eScreening and identification of the differentially expressed HERVs in lupus CD4\u003csup\u003e+\u003c/sup\u003e T cells\u003c/h2\u003e \u003cp\u003eTo screen the highly expressed HERVs transcripts in CD4\u003csup\u003e+\u003c/sup\u003e T cells of SLE patients, we performed RNA sequencing (RNA-seq) and whole transcriptome sequencing and identified the LTRs derived from HERVs with Repeatmasker online software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.repeatmasker.org\u003c/span\u003e\u003cspan address=\"https://www.repeatmasker.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). 34,992 HERVs based on RNA-seq and 38,006 HERVs based on whole transcriptome sequencing were discovered in CD4\u003csup\u003e+\u003c/sup\u003e T cells obtained from peripheral blood of SLE patients and HCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). 204 up-regulated and 38 down-regulated HERVs (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were identified by RNA-seq (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), as well as 1674 up-regulated and 542 down-regulated HERVs (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were identified by whole transcriptome sequencing in SLE patients compared to HCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Based on the expression levels and change folds of HERVs, 17 HERVs in RNA-seq (log2 fold change (FC)\u0026thinsp;\u0026ge;\u0026thinsp;1, FPKM in SLE\u0026thinsp;\u0026ge;\u0026thinsp;20, and exclusion of HERVs overlapped with other genes\u0026rsquo; exon) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee) and 18 HERVs in the whole transcriptome sequencing (log2 fold change (FC)\u0026thinsp;\u0026ge;\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, FPKM in SLE\u0026thinsp;\u0026ge;\u0026thinsp;7, and exclusion of HERVs overlapped with other genes\u0026rsquo; exon) were screened (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Combined the two sequencing results, 18 HERVs with high expression abundance were chosen according to the two sequencing data to verify by RT-qPCR, except for HERVs size below 100bp and with poor primer specificity. The results showed that the expression levels of MER21C, ERV3-16A3_I-int, LTR16A, THE1B-367, LTR40C, MLT1F1, LTR56, MER4A1, MLT1A0-130, THE1C-288, THE1C, MLT1B, MLT2B3, THE1B-362, MER65A and MLT2A2 were significantly higher in CD4\u003csup\u003e+\u003c/sup\u003e T cells of SLE patients than those in HCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePrevious sthdies showed that these ancient retroviruses have been integrated into the human genome, some of which were reactivated transcriptionally under specific conditions\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. To clarify the derivation of these highly expressed HERVs in SLE CD4\u003csup\u003e+\u003c/sup\u003e T cells, we analyzed their genomic locations by IGV_Win_2.6.0. We found that the genomic locations of these highly expressed HERVs were predominantly distributed on chromosomes 1, 4, 13, and 21, and the locations of HERVs on the same chromosome were contiguous (supp Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Interestingly, these HERVs were localized in the intronic region or intergenic region of interferon-stimulated genes (ISGs), including \u003cem\u003eOAS2\u003c/em\u003e, \u003cem\u003eOAS3\u003c/em\u003e, \u003cem\u003eDDX60L\u003c/em\u003e, \u003cem\u003eDDX60\u003c/em\u003e, \u003cem\u003eLY6E\u003c/em\u003e, \u003cem\u003eMX1\u003c/em\u003e, \u003cem\u003eMX2\u003c/em\u003e, \u003cem\u003eIFI44L\u003c/em\u003e, \u003cem\u003eIFI44\u003c/em\u003e and \u003cem\u003eEPSTI1\u003c/em\u003e (supp Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), which were also highly expressed in CD4\u003csup\u003e+\u003c/sup\u003e T cells of SLE patients and correlated positively with the expression levels of HERVs (supp Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, e).\u003c/p\u003e \u003cp\u003eHERVs are mainly localized in heterochromatin and repressed by epigenetic silencing at steady state\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Previous studies have shown that DNA hypomethylation, leading to ISGs overexpression in SLE patients, contributed to SLE pathogenesis\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Here, we detected the DNA methylation status of these highly expressed HERVs including ERV3-16A3_I-int, LTR40C, LTR16A, THE1B, MER21C, and MLT1F1 in CD4\u003csup\u003e+\u003c/sup\u003e T cells of SLE patients. The results showed that the mean CpG methylation levels of these HERVs were significantly decreased in SLE CD4\u003csup\u003e+\u003c/sup\u003e T cells compared with HCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-c). Moreover, we also found that 5-aza-2\u0026rsquo;-deoxycytidine (5-AZA-CdR), a methyltransferase-specific inhibitor, induced DNA hypomethylation and overexpression of these HERVs in CD4\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, e). Those results suggested that transcriptional activation of HERVs might be due to DNA hypomethylation in CD4\u003csup\u003e+\u003c/sup\u003e T cells of SLE patients.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eHERVs formed dsRNAs and activated RIG-I pathway in SLE CD4\u003csup\u003e+\u003c/sup\u003e T cells\u003c/h2\u003e \u003cp\u003eHERVs transcription leads to dsRNA production, which has been indicated in tumor\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. To investigate whether these highly expressed HERVs formed dsRNAs in CD4\u003csup\u003e+\u003c/sup\u003e T cells of SLE, we performed dsRNA enrichment assay with RNase A digestion, which cleaves single-stranded RNA (ssRNA) and preserves dsRNA under high salt conditions, and dsRNA-specific antibody J2 pulldown assays\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Both two assays indicated that ERV3-16A3_I-int, MER65A, MLT2A2 and MLT1B expressed as dsRNAs in SLE CD4\u003csup\u003e+\u003c/sup\u003e T cells because of more than 100 times enrichments in RNA digested with RNase A or J2 antibody (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate whether the RIG-I signal pathway could be triggered by HERVs derived dsRNAs to activate type I interferons pathway, we obtained ERV3-16A3_I-int RNA by T7 RNA in vitro transcription kit and annealed to form dsRNA (supp Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). ERV3-16A3_I-int dsRNA and poly(I:C), a synthetic dsRNA analog as positive control, were electrotransfected into human naive CD4\u003csup\u003e+\u003c/sup\u003e T cells respectively. We observed that upon dsRNA treatment, naive CD4\u003csup\u003e+\u003c/sup\u003e T cells initiated the strong activation of the RIG-I signal pathway. The mRNA and protein expression of RIG-I-like receptors (RLRs) pathway genes (such as DDX58 and IFR7) and phosphorylation level of IRF3 protein were remarkably elevated in poly(I:C) or ERV3-16A3_I-int-treated CD4\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec-e). In addition, our results also showed that the expression levels of type I IFN (IFNα and IFNβ) and the IFN-stimulated gene 15 (ISG15) were significantly induced after Poly(I:C) or ERV3-16A3_I-int treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, d). Importantly, we observed that ERV3-16A3_I-int dsRNA can be bond strongly by RIG-I proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). In addition, the ubiquitination level of RIG-I, which is a hallmark of dsRNA-stimulated RIG-I activation, was increased in dsRNA treated CD4\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). These results indicated that HERVs-derived dsRNA could trigger RIG-I-mediated innate immune response in T cells.\u003c/p\u003e \u003cp\u003eThen, we sought to determine the levels of dsRNA and RIG-I signal pathway genes in lupus CD4\u003csup\u003e+\u003c/sup\u003e T cells. J2 immunofluorescence staining showed a significant enrichment of dsRNAs in SLE CD4\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh). We observed that the expression levels of DDX58, TRIM25, IRF7 and ISG15 were significantly increased in CD4\u003csup\u003e+\u003c/sup\u003e T cells of SLE patients compared with healthy controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei). Furthermore, we found that not only the protein level but also the level of ubiquitination of RIG-I were markedly increased in lupus CD4\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej). We next characterized RIG-I expression in naive CD4\u003csup\u003e+\u003c/sup\u003e T cells and memory/effector CD4\u003csup\u003e+\u003c/sup\u003e T cells of SLE patients and healthy controls. Our data showed a significantly high RIG-I expression in CD4\u003csup\u003e+\u003c/sup\u003eCD45RA\u003csup\u003e+\u003c/sup\u003eCD45RO\u003csup\u003e\u0026minus;\u003c/sup\u003e naive CD4\u003csup\u003e+\u003c/sup\u003e T cells as well as in CD4\u003csup\u003e+\u003c/sup\u003eCD45RA\u003csup\u003e\u0026minus;\u003c/sup\u003eCD45RO\u003csup\u003e+\u003c/sup\u003e memory/effector CD4\u003csup\u003e+\u003c/sup\u003e T cells of SLE patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek). These data indicated that HERVs derived dsRNA induced the activation of RIG-I signal pathway, which may play an role in regulating T cells differentiation in CD4\u003csup\u003e+\u003c/sup\u003e T cells of SLE.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eHERV-derived dsRNA regulated T cell differentiation via activation of RIG-I pathway\u003c/h2\u003e \u003cp\u003eAbnormal differentiation of pathogenic T cells plays an important role in the pathogenesis of SLE\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. We observed that the expression of ERV3-16A3_I-int was significantly increased in Th1 and Th17 cells, as well as the protein levels of RIG-I and IRF7 were markedly increased in Th1, Th17 and Treg cells and the phosphorylation levels of TBK1 and IRF3 were increased in Th1 and Th17, compared to Th0 cells (supp Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). To investigate the role of dsRNA/RIG-I pathway in CD4\u003csup\u003e+\u003c/sup\u003e T cell differentiation, RNA-seq was performed in CD3 and CD28 antibodies activated na\u0026iuml;ve CD4\u003csup\u003e+\u003c/sup\u003e T cells transfected with poly(I:C). The expression profiles of genes related to the effect T cell differentiation were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea. The expression levels of IFNG (Th1), TBX21 (Th1) and RORC (Th17) were up-regulated and GATA3 (Th2) and FOXP3 (Treg) expression were down-regulated after poly(I:C) treatment, which were also validated by RT-qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThen, na\u0026iuml;ve CD4\u003csup\u003e+\u003c/sup\u003e T cells were cultured in vitro under polarization conditions of different T-cell subclasses for 3 days, and transfected with Poly(I:C) or ERV3-16A3_I-int dsRNA to activate the RIG-I signaling pathway. FCM analysis showed that Th1 and Th17 cell differentiation was increased and Treg cell differentiation was significantly inhibited in the poly(I:C) and ERV3-16A3_I-int transfected group compared with mock controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, d) (supp Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, d). The mRNA levels of IFNG, TBX21, IL17A and RORC1 in T cells with poly(I:C) or ERV3-16A3_I-int stimulation were significantly increased, and the mRNA levels of FOXP3 and TGFB1 in Treg cells with poly(I:C) or ERV3-16A3_I-int stimulation were significantly decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee-h) (supp Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, d). The cytokine production of IFN-γ (in the supernatants of Th1) was increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee, f), and the level of TGFβ (in the supernatants of Treg) was decreased in the poly(I:C) and ERV3-16A3_I-int transfected group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg, h). No difference was observed in Th2 cell differentiation after poly(I:C) and ERV3-16A3_I-int treatment (supp Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, c).\u003c/p\u003e \u003cp\u003eIn contrast, we specifically inhibited expression of ERV3-16A3-Int \u003cem\u003evia\u003c/em\u003e transfecting ERV3-16A3-Int smart silence in na\u0026iuml;ve CD4\u003csup\u003e+\u003c/sup\u003e T cells under polarization conditions of different T cell subclasses (supp Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). We observed that ERV3-16A3-Int knockdown decreased Th1 and Th17 cell differentiation and increased Treg cell differentiation by FCM and RT-qPCR compared with negative controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei, j) (supp Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg), but no significant effect on Th2 cell differentiation (supp Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). These results suggested that HERV-derived dsRNAs play an important role in regulating T cell differentiation.\u003c/p\u003e \u003cp\u003eNext, we transfected healthy naive CD4\u003csup\u003e+\u003c/sup\u003e T cells with siRNA targeting \u003cem\u003eDDX58\u003c/em\u003e (si-RIG-I) and stimulated them to differentiate into T cell subsets in vitro. As expected, cells transfected with si-RIG-I showed decreased the expression of RIG-I in CD4\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). FCM and RT-qPCR analysis showed that the percentages of Th1 cells and Th17 cells were decreased and the percentage of Treg cells was increased in T cells with si-RIG-I transfection compared with negative control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, d and e). Furthermore, we generated \u003cem\u003eDdx58\u003c/em\u003e-knockout (\u003cem\u003eDdx58\u003c/em\u003e KO) mice. The mRNA and protein expression of RIG-I was significantly reduced in splenic CD4\u003csup\u003e+\u003c/sup\u003e T cells of \u003cem\u003eDdx58\u003c/em\u003e KO mice compared with WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). Na\u0026iuml;ve CD4\u003csup\u003e+\u003c/sup\u003e T cells from KO and WT mice spleen were cultured in vitro under T cell-polarizing conditions for 3 days. Similar changes in Th1, Th17 and Treg cell differentiation were also observed in \u003cem\u003eDdx58\u003c/em\u003e KO mice. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eActivation of the RIG-I signal pathway induced by dsRNA elevated I-IFN expression. Previously research demonstrated that STAT1 was a key transcription factor downstream of I-IFN\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, and regulated transcription of \u003cem\u003eTBX21\u003c/em\u003e gene in Th1 cell differentiation\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Here, we observed that the protein level of T-Bet and the protein phosphorylation level of STAT1 was increased in poly(I:C)-treated Th1 cells (supp Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b). STAT1 inhibitor fludarabine reduced STAT1 phosphorylation and T-bet protein expression in Th1 cells with poly(I:C) stimulation (supp Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec), and inhibited Th1 cell differentiation (supp Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed, e). On the other hand, RLR-activated p-IRF3 represses Treg cell differentiation by preventing both bindings of Smad3 with TGFbR and Smad transcriptional complex formation\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Our data showed that reducing IRF3 expression partially alleviated the inhibitory effect of poly(I:C) on Treg cell differentiation (supp Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Taken together, the above results indicated that HERVs dsRNA regulated T cells differentiation via activation of RIG-I pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eRIG-I deficiency relieved EAE mice model and lupus-like mice models through mediating the aberrant T cell differentiation\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further determine the role of RIG-I in autoimmune diseases, we isolated naive CD4\u003csup\u003e+\u003c/sup\u003e T cells from WT and \u003cem\u003eDdx58\u003c/em\u003e KO mice and injected them into the tail veins of \u003cem\u003eRag2\u003c/em\u003e-/- mice respectively. After 5 days of T cell transfer, Rag\u003cem\u003e2\u003c/em\u003e-/- mice were induced by immunization with myelin oligodendrocyte glycoprotein (MOG35-55). On the 9th day after immunization, we performed a second T cell-transfer. Mice were sacrificed for analysis after 16 days of EAE immunization (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). \u003cem\u003eDdx58\u003c/em\u003e KO-\u003cem\u003eRag2\u003c/em\u003e-/- mice developed more mild signs of EAE with disease onset advanced as quantified by clinical score or weight loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, c) and had decreased demyelination compared to WT-\u003cem\u003eRag2\u003c/em\u003e-/- mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). In addition, FCM showed that Th1 cell proportion in the spleen and Th17 cell proportions in the spleen and draining lymph nodes (dLNs) were decreased, and Treg cell proportion in dLNs was increased in \u003cem\u003eDdx58\u003c/em\u003e-KO-\u003cem\u003eRag2\u003c/em\u003e-/- mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee-g).\u003c/p\u003e \u003cp\u003eThen, we investigated whether RIG-I deficiency in CD4\u003csup\u003e+\u003c/sup\u003e T cells affects the progression of lupus. We generated mice with a \u003cem\u003eDdx58\u003c/em\u003e allele flanked by loxP sites (\u003cem\u003eDdx58\u003c/em\u003e\u003csup\u003ef/f\u003c/sup\u003e). Mice with confirmed germline transmission were crossed with \u003cem\u003eCD4-Cre\u003c/em\u003e transgenic mice to generate a conditional knockout mouse model with RIG-I expression deficiency specifically in CD4\u003csup\u003e+\u003c/sup\u003eT cells (\u003cem\u003eDdx58\u003c/em\u003e\u003csup\u003ef/f\u003c/sup\u003e\u003cem\u003eCD4-Cre\u003c/em\u003e mice, \u003cem\u003eDdx58\u003c/em\u003e CKO) (supp Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb-d). We treated the 7-week-old \u003cem\u003eDdx58\u003c/em\u003e CKO and \u003cem\u003eDdx58\u003c/em\u003e\u003csup\u003ef/f\u003c/sup\u003e mice with imiquimod (IMQ) three times a week to induce lupus-like mice model. After 8 weeks of IMQ stimulation, there was a significant decrease of spleen weight in \u003cem\u003eDdx58\u003c/em\u003e CKO without body weight difference compared to \u003cem\u003eDdx58\u003c/em\u003e\u003csup\u003ef/f\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea) (supp Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee). In addition, the \u003cem\u003eDdx58\u003c/em\u003e CKO mice showed higher survival rate and a lower ratio of urine protein/creatine than \u003cem\u003eDdx58\u003c/em\u003e\u003csup\u003ef/f\u003c/sup\u003e mice (supp Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). Furthermore, the serum levels of anti-dsDNA antibody and anti-nuclear antibody (ANA) were decreased in \u003cem\u003eDdx58\u003c/em\u003e CKO mice compared with \u003cem\u003eDdx58\u003c/em\u003e\u003csup\u003ef/f\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec, d). Morphological examination by H\u0026amp;E and PAS staining showed the relieved kidney damage in \u003cem\u003eDdx58\u003c/em\u003e CKO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee). Consistently, immunofluorescence staining showed that renal C3 and IgG immune complex depositions were also decreased in \u003cem\u003eDdx58\u003c/em\u003e CKO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next measured the proportions of effect T cell subsets in the spleen and dLNs. We observed significant decreases in the proportions of Th1 and Th17 cells in the spleen and the proportions of Th1, GCB cells and plasma cells in dLNs of \u003cem\u003eDdx58\u003c/em\u003e CKO mice compared to \u003cem\u003eDdx58\u003c/em\u003e\u003csup\u003ef/f\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg). In contrast, RIG-I deficiency elevated the frequency of Treg cells in the spleen and dLNs of \u003cem\u003eDdx58\u003c/em\u003e CKO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg). No significant differences were observed in the proportion of Th2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg). We found that the production of IFN-γ, IL-17A, TNFα, IL-10 and IL-6 was decreased in the serum of \u003cem\u003eDdx58\u003c/em\u003e CKO mice compared to \u003cem\u003eDdx58\u003c/em\u003e\u003csup\u003ef/f\u003c/sup\u003e mice without significant changes in IL-4 and IL-2 protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eh) (supp Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg). In addition, similar relieves of lupus-like phenotypes and dysregulated T cell differentiation were also observed in the other lupus-like mice model, chronic graft-versus-host disease (cGVHD) mice model (supp Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Collectively, these data indicate that RIG-I deficiency in CD4\u003csup\u003e+\u003c/sup\u003e T cells alleviates disease progression in lupus mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eRIG-I regulates histone lactylation and acetylation in T cells by modulating LDHA activity\u003c/h2\u003e \u003cp\u003eTo demonstrate the mechanism of RIG-I in regulating T cell differentiation, we first enriched proteins by RIG-I antibody in co-IP experiment and identified RIG-I binding proteins in CD4\u003csup\u003e+\u003c/sup\u003e T cells under Th1 polarization condition by mass spectrometry (MS) (supp Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea). The result showed lactate dehydrogenase A (LDHA) was enriched by RIG-I antibody in Th1 cells (supp Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eb). Co-IP and western blot results verified that RIG-I bind LDHA protein in Th1 cells with and without poly(I:C) stimulation, but no difference in enrichment levels of LDHA protein between two groups. (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). In addition, we observed no marked change in the expression of LDHA in Th1 cells with poly(I:C) stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea), suggesting RIG-I activation has no effect on LDHA protein level in Th1 cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we determine whether LDHA activity was regulated by RIG-I. Our results showed that the LDHA activity and lactate level were significantly increased in Th1 cells by stimulation with RIG-I agonist poly(I:C) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb), whereas the opposite results were observed in Th1 cells after transfection with siRIG-I (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec). We further explored the function of LDHA in Th1 cell differentiation. The results showed that LDHA knockdown in naive CD4\u003csup\u003e+\u003c/sup\u003e T cells under Th1 polarization condition down-regulated LDHA expression, which inhibited the differentiation of Th1 cells (supp Fig.\u0026nbsp;10a, b). In addition, we also found that silencing LDHA expression could partially block the effect of poly(I:C) on Th1 differentiation (supp Fig.\u0026nbsp;10c, d), indicating LDHA may contribute to dsRNAs-induced Th1 differentiation.\u003c/p\u003e \u003cp\u003eIt has been reported that LDHA regulated lactate production to influence histone lysine lactylation and acetylation\u003csup\u003e\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Here, we observed that the levels of lactylated histone H3K18 (H3K18Lac) and acetylated H3K18 (H3K18Ac) were increased in Th1 cells differentiation with poly(I:C) stimulation compared with mock control (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed). In contrast, we also observed that the histone H3K18Lac and H3K18Ac levels was decreased in Th1 cells after transfection with siRIG-I (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ee). Moreover, we found that the enrichment levels of H3K18Lac and H3K18Ac in gene loci were increased in poly(I:C)-treated Th1 cells by CUT\u0026amp;TAG (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ef). We validated that the levels of both H3K18Lac and H3K18Ac in \u003cem\u003eIFNG\u003c/em\u003e and \u003cem\u003eTBX21\u003c/em\u003e gene promoter regions were increased in poly(I:C)-treated Th1 cells compared to mock control by ChIP-qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003eIn contrast to Th1 cell, we observed that the LDHA activity and lactate level were significantly decreased in Treg cells with poly(I:C) treatment (supp Fig.\u0026nbsp;11a). The expression of LDHA as well as the levels of H3K18Lac and H3K18Ac were obviously decreased in Treg cells with poly(I:C) stimulation (supp Fig.\u0026nbsp;11b). No significant increase in levels of H3K18Lac and H3K18Ac of Th2 and Th17 cells with poly(I:C) stimulation (supp Fig.\u0026nbsp;11c). Collectively, these data suggested that dsRNA activated RIG-I activation may regulate Th1 and Treg cell differentiation \u003cem\u003evia\u003c/em\u003e LDHA-mediated histone lactylation and acetylation.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we explored the expression, roles, and potential mechanism of HERVs in CD4\u003csup\u003e+\u003c/sup\u003e T cells of SLE. We identified a lot of transcripts derived from HERVs loci in CD4\u003csup\u003e+\u003c/sup\u003e T cells by both RNA-seq and transcriptome-seq (identifying HERVs with or without ploy A tail), most of which were higher expression in SLE due to DNA hypomethylation. Some of the up-regulated HERVs were characterized by dsRNAs and associated with RLR signaling pathway activation. Activation of RIG-I signal pathway triggered by dsRNAs promoted pathogenic T cell differentiation via regulating histone lysine lactylation and acetylation and STAT1 phosphorylation, and blocking the pathway decreased proportion of pathogenic T cell and alleviated the progression of autoimmune diseases. This study highlights that the important role of HERVs and dsRNA mediated RIG-I activation in T cell differentiation.\u003c/p\u003e \u003cp\u003eHuman endogenous retroviruses (HERVs), members of the long terminal repeat (LTR) retrotransposons repetitive element class, make up at least 8\u0026ndash;10% of the human genome\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Most HERV sequences have acquired numerous mutations over time and therefore do not have protein-coding potential or the potential to generate infectious viral particles\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Epigenetic modifications are required in maintaining the transcriptional silencing of HERVs\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Alteration of epigenetic modification contributed to transcriptional activation of HERVs in diseases (such as tumor, aging and autoimmune diseases)\u003csup\u003e\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. A lot of evidences have shown the DNA hypomethylation and aberrant histone modifications in CD4\u003csup\u003e+\u003c/sup\u003e T cells of SLE, which contribute to SLE pathogenesis\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. In this study, we found that DNA hypomethylation favored transcriptional activation of HERVs and hyperexpression of the RIG-I signal pathway (supp Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e2), which was closely related to exacerbating the progress in SLE disease. Furthermore, our results identified many up-regulated HERV transcripts with LTR, most of which located in introns or intergenic region of interferon stimulated genes in genome and were positively associated with interferon gene signatures expression in CD4\u003csup\u003e+\u003c/sup\u003e T cells of SLE (supp Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). DNA hypomethylation in these HERVs loci according to our results, as well as most interferon genes hypomethylation identified by previous studies\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, account for the transcriptional activation of HERVs in SLE CD4\u003csup\u003e+\u003c/sup\u003e T cells. Besides of transcriptional activation, the decreased expression of DICER and AGO2, dsRNA cleaving enzymes for intercellular dsRNA degradation\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, was identified in SLE CD4\u003csup\u003e+\u003c/sup\u003e T cells (supp Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e3), which may result in the accumulation of dsRNA in SLE CD4\u003csup\u003e+\u003c/sup\u003e T cells.\u003c/p\u003e \u003cp\u003eAlthough RLR pathway-mediated dsRNA sensing in innate immune cells has been well studied to play anti-virus role\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, the sense and function of dsRNA in adaptive immune is poorly understood. Here we found that dsRNA derived from HERVs could be sensed and activated RLR signal pathway, including up-regulating RIG-I, MDA5 and IRF7 expression and phosphorylation of TBK1 and IRF3, leading to type I IFN gene activation in SLE CD4\u003csup\u003e+\u003c/sup\u003e T cells. Previous study showed that the RLRs drive distinct immune gene activation and response polarization to mediate an M1/inflammatory signature while suppressing the M2/wound healing phenotype\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. In this study, we first found that HERV dsRNAs activated RIG-I had the opposite regulation on effector T cells differentiation, including promoting Th1 and Th17 cells differentiation and inhibiting Treg cell differentiation. RIG-I deficiency improved the imbalance of Th1/Th17 and Treg cells lupus-like mice and alleviated the autoimmune diseases. Those findings indicated that RIG-I sensing dsRNAs in T cells not only induced IFN-I gene expression, but also promoted the dysregulated T cells differentiation, contributing to SLE pathogenesis.\u003c/p\u003e \u003cp\u003eThe activated RIG-I interacts with the mitochondrial antiviral signaling proteins (MAVS), which forms a multilayered protein complex and then catalyzes the activation of the serine/threonine-protein kinase 1 (TBK1)\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. TBK1 phosphorylates the transcription factors IRF3 and IRF7, which then activate the expression of I-IFN (IFNα and IFNβ), leading to a massive inflammatory response\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Recent studies discovered that RIG-I might be critical for the differentiation of pro-inflammatory T cells, which promoted the expression of IFNG and TBX21 by reducing CpG methylation in the \u003cem\u003eTBX21\u003c/em\u003e promoter in CD8\u003csup\u003e+\u003c/sup\u003eT cells\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Tumor cell-intrinsic RIG-I signaling mediates the release of immunogenic extracellular vesicles (EVs), which increase proportions of CD8\u003csup\u003e+\u003c/sup\u003eIFNγ\u003csup\u003e+\u003c/sup\u003eT cells and potent cytotoxic antitumor immunity\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. In this study, we revealed RIG-I activation in T cells induced Th1 differentiation via I-IFN inducing STAT1 phosphorylation to up-regulate TBX21 expression. And inhibition of STAT1 phosphorylation decreased the expression of TBX21, which completely inhibited the effect of activated RIG-I on Th1 cell differentiation. Importantly, we also found a novel mechanism under RIG-I regulated T cell differentiation through identifying LDHA bind to RIG-I protein in Th1 cells. We found that RIG-I activation influenced the activity and expression of LDHA during T cells differentiation, which indicated that the activated RIG-I also was involved in glycolysis process in T cells.\u003c/p\u003e \u003cp\u003eAerobic glycolysis is a metabolic hallmark of activated T cells and has been implicated in augmenting effector T cell responses\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. LDHA is an important glycolytic enzyme to support aerobic glycolysis by the conversion of pyruvate to lactate, which has been proven to maintain high levels of acetyl-CoA to enhance histone acetylation and transcription of IFNG\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Ablation of LDHA in T cells protects mice from immunopathology triggered by excessive IFN-γ expression or deficiency of regulatory T cells\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Recently, LDHA was reported to induce histone lactylation, promoting gene transcription like histone acetylation\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. H3K18Lac promotes reparative gene expression during M1 macrophage polarization to promote immune homeostasis\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e, and regulates early remote activation of the reparative transcriptional response in monocytes\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. However, there is no report about histone lactylation in T cells differentiation. Here, our data showed that activity of LDHA and intracellular lactate were increased by dsRNAs-activated RIG-I and both the levels of H3K18Lac and H3K18Ac and their enrichments in \u003cem\u003eIFNG\u003c/em\u003e and \u003cem\u003eTBX21\u003c/em\u003e promoters were upregulated in poly(I:C)-treated Th1 cells. An inverse histone lactylation and acetylation modification changes also observed in Treg cells. These findings suggest LDHA mediated histone lactylation and acetylation modifications and on T cell differentiation genes may be a noncanonical downstream pathway of activated RIG-I in regulating CD4\u003csup\u003e+\u003c/sup\u003e T cell differentiation. However, the distinct effect and mechanism of RIG-I activation on LDHA and histone lactylation in different effect T cells still need to be investigated.\u003c/p\u003e \u003cp\u003eIn summary, our study provided a model that DNA hypomethylation induced HERV dsRNA accumulation in SLE CD4\u003csup\u003e+\u003c/sup\u003e T cells activates RIG-I and I-IFN pathway and promotes LDHA mediated histone lactylation and acetylation, leading to pathogenic T cell differentiation in SLE. This finding uncovers the roles of HERVs and RIG-I pathway in adaptive immune response and SLE pathogenesis, and suggests that maintaining the silencing of HERVs by epigenetic interference or targeting RIG-I pathway may be a good way for SLE therapy.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003ePatients and controls.\u003c/b\u003e We collected blood samples of SLE patients and healthy controls (HC) in Second Xiangya Hospital of Central South University. Diagnosis of SLE disease was based on the following classification criteria: the 1982 revised criteria for the classification of SLE\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Healthy controls matched with age and gender were recruited. Written informed consent was provided by All subjects. This study was approved by the Ethics Committee of the Second Xiangya Hospital. The basic characteristics of all subjects were listed in Supplementary Table\u0026nbsp;1 and Supplementary Table\u0026nbsp;2.\u003c/p\u003e \u003cp\u003e\u003cb\u003eMice.\u003c/b\u003e\u003cem\u003eRag2\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice were purchased from the Shanghai Research Center For Model Organisms. B6D2F1 mice were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. The \u003cem\u003eDdx58\u003c/em\u003e knockout (KO) mice were generated by Shanghai Model Organisms Center, Inc. For the \u003cem\u003eDdx58\u003c/em\u003e CKO mice generation, the loxP-\u003cem\u003eDdx58\u003c/em\u003e-loxP mice were constructed by Cyagen Bioscience Inc.(Guangzhou, China) and CD4\u003csup\u003ecre\u003c/sup\u003e mice (stock no. 022071) were purchased from Shanghai Model Organisms Center, Inc. The \u003cem\u003eDdx58\u003c/em\u003e-floxed mice were bred with CD4\u003csup\u003ecre\u003c/sup\u003e transgenic mice to generate \u003cem\u003eDdx58\u003c/em\u003e\u003csup\u003ef/f\u003c/sup\u003e CD4\u003csup\u003ecre\u003c/sup\u003e (\u003cem\u003eDdx58\u003c/em\u003e CKO) mice. This study was approved by the Ethics Committee of the Chinese Academy of Medical Sciences and Peking Union Medical College.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eAnimal models\u003c/h2\u003e \u003cp\u003e \u003cb\u003eChronic graft-versus-host disease (cGVHD) model.\u003c/b\u003e A total of 5\u0026times;10\u003csup\u003e7\u003c/sup\u003e CD8\u003csup\u003e+\u003c/sup\u003eT cells-depleted lymphocytes of \u003cem\u003eDdx58\u003c/em\u003e CKO and \u003cem\u003eDdx58\u003c/em\u003e\u003csup\u003ef/f\u003c/sup\u003e female mice were injected into B2D6F1 female mice via tail veins. Urine was collected weekly and the serum samples were collected at the the end of the observation period. By the end of 12 weeks, mice were sacrificed for further experiments.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIMQ-induced lupus model.\u003c/b\u003e Epicutaneous Application of imiquimod leads to systemic autoimmunity is a recognized new lupus model\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. To induce lupus-like disease, \u003cem\u003eDdx58\u003c/em\u003e CKO and \u003cem\u003eDdx58\u003c/em\u003e\u003csup\u003ef/f\u003c/sup\u003e mice were treated topically with 5% IMQ cream (Sichuan Med-shine Pharmaceutical, H20030128). Mice were treated with IMQ cream applied to the ear skin three times a week for 8 weeks. Urine samples were collected weekly and serum samples fortnightly. By the end of 8 weeks, mice were sacrificed for further experiments.\u003c/p\u003e \u003cp\u003eThe urine protein test kit for urine protein, and the serum anti-double-stranded DNA (dsDNA) IgG and antinuclear antibody (ANA) IgG levels were detected by ELISA kits (CUSABIO, China). Flow cytometry was used to analyze immune cells in the draining lymph nodes (dLNs) and spleens of the models. Analysis of C3 and IgG deposits in kidneys using multi-IHC stains.\u003c/p\u003e \u003cp\u003e\u003cb\u003eEAE disease model.\u003c/b\u003e EAE was induced by complete Freund\u0026rsquo;s adjuvant (CFA)- MOG35-55 peptide immunization (Hooklabs) and scored daily. Briefly, 2\u0026times;10\u003csup\u003e6\u003c/sup\u003e naive CD4\u003csup\u003e+\u003c/sup\u003e T cells of \u003cem\u003eDdx58\u003c/em\u003e KO and WT female mice were injected into \u003cem\u003eRag2-/-\u003c/em\u003e female mice via tail veins. Mice were then injected subcutaneously into the neck with 200\u0026micro;l containing 200\u0026micro;g MOG35-55 peptide (Hooklabs) emulsified in complete Freund\u0026rsquo;s adjuvant (Sigma-Aldrich). Mice were also injected intraperitoneally with 500ng of pertussis toxin (Listlabs) on days 0 and 2 after immunization. Mice were monitored daily for morbidity and scored according to the following scoring criteria: 0, no symptoms, active and mobile; 0.5, partial weakness of the tail; 1, completely paralyzed tails, less active; 2, hind limb weakness, hobbling gait; 2.5, partial paralysis of hind limbs and complete paralysis of a single hind limb; 3, complete hind limb paralysis; 3.5, complete paralysis of both hind limbs, partial paralysis of forelimbs; 4, completely paralyzed of all limbs, losing mobility; 5, moribund or death.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell isolation.\u003c/b\u003e Density gradient centrifugation (GE Healthcare) was used to isolate the peripheral blood mononuclear cells (PBMCs) from the peripheral blood of healthy controls and SLE patients. Then CD4\u003csup\u003e+\u003c/sup\u003e T cells were isolated from the PBMCs by Miltenyi beads according to the manufacturer\u0026rsquo;s instructions (Miltenyi Biotec).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro human T cell differentiation.\u003c/b\u003e Naive CD4\u003csup\u003e+\u003c/sup\u003e T cells were purified from PBMCs using the human Naive CD4\u003csup\u003e+\u003c/sup\u003e T Cell Isolation Kit (Miltenyi Biotec), and then cells were stimulated with plate-bound anti-CD3 (5\u0026micro;g/ml, Calbiochem, catalog 217570) and anti-CD28 (2\u0026micro;g/ml, Calbiochem, catalog 217669) under the Supplemental Table\u0026nbsp;3 polarizing conditions. We performed cell culture in 24-well plates with a total volume of 1 ml/well of culture medium with 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e naive CD4\u003csup\u003e+\u003c/sup\u003e T cells. The medium was refreshed on day 3.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro mouse T cell differentiation.\u003c/b\u003e Naive CD4\u003csup\u003e+\u003c/sup\u003e T cells from mouse spleen were purified using mouse naive CD4\u003csup\u003e+\u003c/sup\u003e T cell isolation kit II (Miltenyi Biotec), and the purity of the enriched subset was validated by flow cytometry and was generally higher than 95%. Purified naive CD4\u003csup\u003e+\u003c/sup\u003e T cells were stimulated with plate-bound anti-CD3 (5\u0026micro;g/ml, eBioscience, catalog 16-0031-85) and anti-CD28 (2\u0026micro;g/ml, eBioscience, catalog 16-0281-85) for 3 days under different polarizing conditions (Supplemental Table\u0026nbsp;4). We performed cell culture in 24-well plates with a total volume of 1 ml/well of culture medium with 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e naive CD4\u003csup\u003e+\u003c/sup\u003e T cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTransfection of siRNA, poly(I:C), and ERV3-16A3_I-int RNA.\u003c/b\u003e The Human T Cell Nucleofector Kit and Amaxa Nucleofector System (Lonza) for T cell transfection. Briefly, naive CD4\u003csup\u003e+\u003c/sup\u003e T cells were induced differentiation into different T cells for 3 days. The cells were collected and resuspended in 100 \u0026micro;L transfection reagents, and 10\u0026micro;L siRNA (20\u0026micro;M), or 1\u0026micro;L Smart Silence (20\u0026micro;M), or 500ng poly(I:C), or 10\u0026micro;g ERV3-16A3_I-int RNA was added and transfected into the cells by electroporation using the nucleofector program V-024 in the Amaxa Nucleofector apparatus (Lonza). After being cultured under RPMI 1640 complete medium (Gibco) for 6 hours, the cells were transferred to fresh complete medium under different polarizing conditions for 48 to 72 hours and then harvested for subsequent experiments.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFlow cytometry.\u003c/b\u003e Briefly, T cells were incubated with fluorescein-labeled surface-labeled antibodies at 4\u0026deg;C for 30 minutes protected from light. For cytokines, cells were stimulated with Leukocyte Activation Cocktail, with BD GolgiPlug\u0026trade; at 37\u0026deg;C and 5% CO2 for 6 hours. For intracellular staining, cells were fixed and permeabilized using the Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences) or transcription factor buffer set (BD Pharmingen), and then stained with fluorescent antibodies for 30 minutes at 4\u0026deg;C in the dark. Information on antibodies is shown in Supplemental Extended Table\u0026nbsp;1. The expression of cytokines, surface markers, and transcriptional factors was determined by flow cytometry using FACS Canto II (BD Biosciences) or Cytek\u0026reg; Northern Lights\u0026trade;-CLC (CYTEK Biosciences), and the data were analyzed by the Flowjo software.\u003c/p\u003e \u003cp\u003e \u003cb\u003eChromatin immunoprecipitation (ChIP) qPCR.\u003c/b\u003e Th1 cells transfected with the poly(I:C) were isolated. ChIP with anti-H3K18Ac, anti-H3K18Lac were used to detect \u003cem\u003eIFNG\u003c/em\u003e and \u003cem\u003eTBX21\u003c/em\u003e enrichment, which was performed by a ChIP kit (Millipore). The detailed protocols were previously described\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Immunoprecipitated DNA and input DNA were assessed using real-time PCR. The resulting DNA fragments were purified and subjected to PCR with the use of primers encompassing the D-box region of the \u003cem\u003eTBX21\u003c/em\u003e and \u003cem\u003eIFNG\u003c/em\u003e gene promoters. The primers used in the present study were shown in Supplemental Table\u0026nbsp;5.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCUT\u0026amp;Tag.\u003c/b\u003e CUT\u0026amp;Tag was performed according to the Hyperactive Universal CUT\u0026amp;Tag Assay Kit for Illumina(Vazyme, TD903) for Th1 cells. In brief, Th1 cells were collected and counted, of which the cell viability was \u0026gt;\u0026thinsp;85%. Cells were separated into 100,000 cell aliquots in each sample and incubated on ice for 10 min with 100ul of pre-cooled NE buffer for obtaining cell nuclei. Cells were centrifuged at 600\u0026times;g for 5 min at room temperature and then resuspended in 100\u0026micro;l wash buffer in each sample. ConA Beads were activated in the binding buffer. Transfer 100\u0026micro;l of nuclei to an 8-strip tube containing 10\u0026micro;l activated ConA Beads, invert to mix and incubate at room temperature for 10 min. Beads were separated with a magnetic and supernatant was removed. 1\u0026micro;l of the primary antibody was diluted 1:50 in antibody buffer. The primary antibodies H3K18la (PTM-Bio, PTM1406RM) and H3K18ac (PTM-Bio, PTM-114RM) were used in this study. Cells were incubated overnight at 4\u0026deg;C. The primary antibody was replaced with the secondary antibody diluted to 1:100 in the dig-wash buffer. Samples were incubated for 45min at room temperature on the nutator. The secondary antibody was removed, and samples were washed 3 times in the dig-wash buffer. 2\u0026micro;l pA/G-Tnp was added in 98\u0026micro;l dig-300 buffer for per sample. Samples were incubated for 1h at room temperature on the nutator. Samples were washed 3 times with dig-300 buffer and then resuspended in 50 \u0026micro;l tagmentation buffer. Samples were incubated at 37\u0026deg;C for 1h. DNA was extracted with DNA Extract Beads. Fragmented DNA after purification was amplified by PCR. PCR conditions were set to 72\u0026deg;C for 3min, 95\u0026deg;C for 3min, 11 cycles of 98\u0026deg;C for 10sec, 60\u0026deg;C for 5sec, and 72\u0026deg;C for 1min. VAHTS DNA Clean Beads were used to purify the PCR product. Libraries were indexed using Nextera Indexes, and 150-bp paired-end sequencing was performed on Illumina Novaseq instruments.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNA extraction and Quantitative reverse transcription PCR (RT-qPCR).\u003c/b\u003e Total RNA was extracted from T cells using TRIzol (Invitrogen). RNA quality control was conducted with a NanoDrop spectrophotometer and an Agilent 2100 Bioanalyzer (Thermo Fisher Scientific). 1\u0026micro;g of total RNA was reverse-transcribed using PrimeScript RT reagent Kits With gDNA Eraser (Takara). RT-qPCR was performed on a Fast Real-time PCR system (Roche) with iTaq Universal SYBR Green (BioRad). The relative expression levels of genes were calculated by the 2\u003csup\u003e\u0026minus;ΔCt\u003c/sup\u003e method, which normalized to the reference gene β-actin. The primers are listed in Supplemental Extended Table\u0026nbsp;1.\u003c/p\u003e \u003cp\u003e \u003cb\u003eWestern Blot.\u003c/b\u003e Total protein was isolated from T cells by IP lysis buffer (Beyotime) supplemented with protease inhibitors (Roche) and phosphatase inhibitor (Beyotime). The proteins were quantified by Pierce BCA Protein Assay Kit (Thermo). The primary antibodies and secondary antibodies were used in this study as Supplemental Extended Table\u0026nbsp;2. The quantification of proteins was normalized to GAPDH or β-actin by densitometry using ImageJ software.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCo-IP.\u003c/b\u003e Co-IP assays were performed with the Dynabeads Protein G (Life Technologies) for immunoprecipitation. First, we extracted protein from fresh cells using IP lysis buffer supplemented with protease and phosphatase inhibitors. Rabbit anti-RIG-I Ab (Abcam, ab180675) or Rabbit anti-LDHA Ab (Abcam, ab52488) was added to the lysates, forming a new antibodies-bait-target complex. Then, the antibodies-bait-protein complexes were eluted from the beads and dissociated by boiling in protein loading buffer. Finally, the presence of the target protein was evaluated by Western blot.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBisulfite sequencing polymerase chain reaction (BSP).\u003c/b\u003e Genomic DNA was isolated from CD4\u003csup\u003e+\u003c/sup\u003e T cells or Jurkat cells using the QIAamp DNA Mini Kit (Qiagen). DNA was bisulfifite converted using EZ DNA Methylation-Lightning\u0026trade; Kit (Zymo Research). The bisulfite-treated DNA was amplified via nested PCR amplification reactions with specific primers, which were designed using the online MethPrimer software(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.urogene.org/methprimer/\u003c/span\u003e\u003cspan address=\"http://www.urogene.org/methprimer/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The primer information used to amplify the target fragment are shown in Supplementary Table\u0026nbsp;6. For each PCR, 0.25mM dNTP mix (Promega), 0.2\u0026micro;M forward and reverse primers, 2.5 U of Taq DNA Polymerase (Promega), and 5\u0026times;Green GoTaq\u0026reg; Reaction Buffer (Promega) were used in a 20\u0026micro;l total reaction volume. Here 100ng of bisulfite-treated DNA was used as the template of the first PCR, whereas 4\u0026micro;l of PCR1 product was used as the template for the second PCR. Thermal cycling conditions consisted of one cycle of 2 min at 96\u0026deg;C, followed by 40 cycles of 10s at 96\u0026deg;C, 30s at 55\u0026deg;C and 1 min at 72\u0026deg;C, and a final extension at 72\u0026deg;C for 10min. The PCR products were purified by gel extraction (Promega) from a 1.5% agarose gel and ligated into the pMD\u0026trade;18-T Vector (Takara). The ligation products were used to transform competent Escherichia coli cells (strain DH5a) using standard procedures, and blue/white screening was used to select ten independent clones from each specimen were sequenced (Sangon). The final sequence results were analyzed by online QUMA software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://quma.cdb.riken.jp/\u003c/span\u003e\u003cspan address=\"http://quma.cdb.riken.jp/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eDsRNA analysis by RNase digestion.\u003c/b\u003e According to previous research for dsRNA analysis by RNase A digestion\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, 5\u0026micro;g total RNA of CD4\u003csup\u003e+\u003c/sup\u003e T cells was dissolved in 32\u0026micro;L H\u003csub\u003e2\u003c/sub\u003eO and mixed well with 17.5\u0026micro;L NaCl (1 M stock). Then, 0.5\u0026micro;L RNase A (10mg/ml stock, Thermo Fisher Scientific) or H\u003csub\u003e2\u003c/sub\u003eO was mixed to a total volume of 50\u0026micro;L and incubated at room temperature for 10 min. 1mL TRIzol was added to the mixture. The levels of HERVs were detected by RT-qPCR with ACTB as an internal control. The ratios of (HERV/ACTB)RNaseA/(HERV/ACTB)H\u003csub\u003e2\u003c/sub\u003eO were calculated as enrichment folds.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDsRNA analysis by J2 pulldown.\u003c/b\u003e Purified total RNA from CD4\u003csup\u003e+\u003c/sup\u003e T cells was used for the J2 pulldown assay. Purified total RNA from CD4\u003csup\u003e+\u003c/sup\u003e T cells was used for the J2 pulldown assay. J2 antibody (Scicons) and mouse IgG control (Merck) (1\u0026micro;g per pulldown) were incubated with Protein G dynabeads (Merck), respectively, for 30min at room temperature. 30\u0026micro;g RNA was mixed with 500\u0026micro;l immunoprecipitation (IP) buffer. Then, the whole mixture was added with washed beads and rotated at 4\u0026deg;C for 2h. Afterward, the beads were incubated in 50\u0026micro;l Proteinase K digestion solution. 1 ml Trizol was directly added to the eluate for RNA purification and RT-qPCR analysis as described above.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNA-FISH.\u003c/b\u003e We used the RNA-FISH to study the subcellular distribution of ERV3-16A3_I-int. Fluoresce-conjugated ERV3-16A3_I-int probes labelled with Cy3 and FISH kits were generated from RiboBio (China). Briefly, 4% paraformaldehyde (supplementing 5% TritonX-100) was used to fix CD4\u003csup\u003e+\u003c/sup\u003e T cells of SLE and HCs (30 min). The fixed cells were incubated with ERV3-16A3_I-int probes in hybridization buffer at 37◦C overnight. Nuclei were stained with DAPI.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunofluorescent staining.\u003c/b\u003e CD4\u003csup\u003e+\u003c/sup\u003e T cells were fixed with cold methanol at -20℃ for 15 min. Then the cells were incubated with 0.2% Triton (Sigma) at 4℃ for 5 min. The samples were incubated with primary antibody (Scicons) at 4℃ for overnight and then secondary antibody (Abcam) for 1h at room temperature. Mouse kidney were fixed in formalin and embedded in paraffin. Hematoxylin and eosin (H\u0026amp;E) stains and Periodic Acid-Schiff (PAS) stains were used to assess lymphocytic infiltration and glycogen deposition in the kidney. To assess the immune complex deposition in the kidney, we stained paraffin-embedded renal sections with rabbit anti-C3 antibody (Abcam) and HRP-conjugated anti-mouse IgG. The opal 7-Color Manual IHC Kit (Perkin Elmer) was used for fluorescence labeling. Information on antibodies was provided in Supplemental Extended Table\u0026nbsp;2.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNA-Sequencing.\u003c/b\u003e RNA-seq dataset was taken from CD4\u003csup\u003e+\u003c/sup\u003e T cells of 4 healthy female controls and 4 female SLE patients. The controls ranged in age from 22\u0026ndash;51 years of age average age 31.6, and the patients ranged in age from 19\u0026ndash;44 years old with an average age of 34.2 years old. The patients without drug therapy had a SLE disease activity index (SLEDAI) ranging from 7 to 23 with an average score of 14.8. RNA library preparation was performed as described in previous researches\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. The HERVs were obtained from RepeatMasker for expression analyses by stringtie\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e,\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. The differential analysis was performed using DESeq\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eWhole transcriptome sequencing.\u003c/b\u003e Whole transcription sequencing data included 5 healthy female controls and 5 female SLE patients. The controls ranged in age from 21\u0026ndash;32 years of age average age 24.2, and the patients ranged in age from 15\u0026ndash;32 years old with an average age of 22.2 years old. The patients without drug treatment had SLEDAI ranging from 6 to 13 with an average score of 9.4. After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform and 150bp paired-end reads were generated. The raw data were processed through Fastp\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. The data were then mapped to the human reference genome hg38 with Hisat2 v2.0.5\u003csup\u003e62\u003c/sup\u003e. The differential analysis was performed using DESeq\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical Analysis.\u003c/b\u003e SPSS 22.0 was used for statistical analysis and calculation. Comparisons of the means between experimental variables were made via unpaired two-sided Student\u0026rsquo;s t-test for normally distributed variables or Mann-Whitney for non-normally distributed variables. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was regarded as significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval.\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Ethics Committee of the Second Xiangya Hospital of Central South University and the Ethics Committee of Chinese Academy of Medical Sciences and Peking Union Medical College. All participants provided written informed consent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (No. 82030097 and No. 32141004), the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (2022-RC310-04), and the National Key R\u0026amp;D Program of China (2022YFC3601803).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eX.M. conceptualized the studies, analyzed the data, and wrote the manuscript. X.M, Y.Y., Z.H., L.YO., Y.Q., J.W., C.Z. and S.Y. performed the experiments. H.Z., J.W., M.Zheng. and Q.L. collected samples of SLE and handled the clinical information of patients. Q.L. and S.J. contributed to conception and design of the study. D.Y. provided suggestions for the studies. M.Z. conceptualized the studies, supervised the experiments, analyzed results and wrote the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLazar S, Kahlenberg JM (2023) Systemic Lupus Erythematosus: New Diagnostic and Therapeutic Approaches. Annu Rev Med 74:339\u0026ndash;352\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen PM, Tsokos GC (2021) T Cell Abnormalities in the Pathogenesis of Systemic Lupus Erythematosus: an Update. Curr Rheumatol Rep 23:12\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao X et al (2022) Iron-dependent epigenetic modulation promotes pathogenic T cell differentiation in lupus. 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Bioinformatics 34:i884\u0026ndash;i890\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3939567/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3939567/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe dysregulated differentiation of T lymphocyte play an important role in systemic lupus erythematosus (SLE). However, the underlying mechanism remains unclear. Here, we showed that many transcripts derived from human endogenous retroviruses (HERVs) were highly expressed in CD4\u003csup\u003e+\u003c/sup\u003e T cells from SLE patients due to DNA hypomethylation, some of which were characterized by double strand RNAs (dsRNAs). Excessive dsRNAs promoted Th1/Th17 differentiation and inhibited Treg cell differentiation via the activation of dsRNA sensor retinoic acid-inducible gene I (RIG-I). And T cell-specific ablation of RIG-I alleviated disease progression in experimental autoimmune encephalomyelitis (EAE) mice model and lupus-like mice model. Importantly, we demonstrated that dsRNA-activated RIG-I protein bind lactate dehydrogenase A (LDHA) and regulate histone lysine 18 lactylation (H3K18Lac) and acetylation (H3K18Ac) modifications in T cell differentiation via changing lactate level. Collectively, our findings uncover a novel role and mechanism of HERVs and RIG-I in regulating the aberrant differentiation of T cells in SLE patients.\u003c/p\u003e","manuscriptTitle":"Endogenous retrovirus promotes the aberrant T cell differentiation in systemic lupus erythematosus via RIG-I pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-13 18:52:53","doi":"10.21203/rs.3.rs-3939567/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1eed71cf-249e-40c1-aa63-f5d58311eec0","owner":[],"postedDate":"February 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":28719149,"name":"Health sciences/Diseases/Immunological disorders/Autoimmune diseases/Systemic lupus erythematosus"},{"id":28719150,"name":"Health sciences/Pathogenesis/Immunopathogenesis/Adaptive immunity/Cellular immunity/Lymphocyte differentiation"}],"tags":[],"updatedAt":"2025-02-10T07:35:25+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-13 18:52:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3939567","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3939567","identity":"rs-3939567","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}