The Role And Mechanism Of HERVs-Derived DsRNA In The Pathogenesis Of Systemic Lupus Erythematosus

Systemic lupus erythematosus（SLE）is a complex autoimmune disease involving damage to multiple organs and systems.The pathogenesis of SLE is complicated,which includes disruption of immune tolerance to self-antigens,deposition of autoantibodies and infiltration of inflammatory cells in target organs such as kidney and brain,maladjustment of cellular and humoral immune response elements,and abnormal activation of T lymphocytes caused by abnormal signal transduction.CD4⁺T lymphocytes regulate the overactivity of B lymphocytes to produce autoantibodies,thus forming antigen-antibody complexes and activating complement in SLE patients.Meanwhile,effector T lymphocytes,such as Th1,Th2 and Th17 cells,release a large number of inflammatory cytokines（IFN-γ,IL-4 and IL-17）,which ultimately lead to organ damage in patients.However,the molecular mechanism that causes abnormal differentiation of CD4⁺T cells and inflammatory cytokines in SLE has not been clarified.Human endogenous retroviruses（HERVs）are genetic remnants of retroviruses that were integrated into the Human genome millions of years ago.Most of HERVs are unable to complete replication and protein coding and assemble into infectious particles due to the accumulation of mutations and omissions during evolution.Only a few of HERVs retain intact open reading frames,which are transcriptionally active and even translated into viral proteins.Studies on the effects of HERVs on the immune system mainly focus on viral proteins translated by HERVs.Multiple HERVs-translated proteins have been detected in SLE patients,which activate the autoimmune response through molecular simulation of autoantigen and participate in the inflammatory response in SLE patients.However,the expression and regulation of HERVs in the immune system of SLE patients remain unclear.In recent years,it has been found that the expression of HERVs LTR promoted the accumulation of cytosolic dsRNA,which can activate the RIG-I-like receptors（RLRs）pathway and drive enhanced type I IFN responses.It has been found that CD4⁺T cells express RLRs.However,the transcription of HERVs LTR in CD4⁺T cells of SLE patients and its pathogenesis involved in autoimmune response still need to be further studied.In this study,RNA-seq and whole transcriptome sequencing combined with RT-qPCR were used to confirm that the expression of HERVs in peripheral blood CD4⁺T cells of SLE patients was significantly increased,which promoted the accumulation of cytosolic dsRNA.Meanwhile,we found that the RLRs pathway in peripheral blood CD4⁺T cells of SLE patients were significantly up-regulated.RLRs signaling pathway was activated by dsRNA（Poly（I:C）or HERV-dsRNA ERV3-16A3-I-Int）,which promoted Th1 cell differentiation and inhibited Treg cell differentiation.Blocking RLRs signaling pathway by silencing RIG-I and MDA5 can inhibit Th1 cell differentiation and promote Treg cell differentiation.This study exposed the molecular mechanism of abnormal differentiation of CD4⁺T cells caused by elevated HERVs expression in SLE patients,providing important theoretical basis for the prevention and treatment of SLE.Objective:1.To detect the expression of HERVs and RLRs pathway in CD4⁺T cells of lupus and different CD4⁺T cell subtypes,and identify HERVs that can form dsRNA.2.To investigate the effect of dsRNA-RLRs on the differentiation of CD4⁺T cells.3.To explore the effects of environmental factors（UVB）on the regulation of HERV-RLRs.Methods:1.RNA-seq and whole transcriptome sequencing were used to perform transcriptional analysis of CD4⁺T cells in peripheral blood of SLE patients and healthy volunteers,and the differentially expressed HERVs were subjected to analysis in the Repeat Masker database.2.Magnetic beads were used to sort CD4⁺T cells in the peripheral blood of SLE patients and healthy volunteers,in spleen of MRL/Lpr and MRL/Mp J mice.Human peripheral blood naive CD4⁺T cells were screened by magnetic beads,and induced differentiation into Th0,Th1,Th2,Th17,Tfh and Treg cells.Total RNA of cells were extracted by Trizol.The expression of HERVs and RLRs in CD4⁺T cells of lupus and different subtypes of CD4⁺T cells were detected by RT-qPCR.3.Ds RNA formation ability of HERVs was screened by J2-dsRNA pull-down and RNase A enzyme digestion assay.The expression of HERV-dsRNA in naive CD4⁺T cells,Th0 cells,Th1 cells and peripheral blood CD4⁺T cells of SLE patients and healthy volunteers was detected by FISH.The level of dsRNA in CD4⁺T cells from SLE patients and healthy volunteers was detected by J2-dsRNA staining.4.The expression of RIG-I protein in peripheral blood CD4⁺T cells of SLE patients and healthy volunteers was detected by WB,and in naive CD4⁺T cells,Th0 cells,and Th1 cells was detected by immunofluorescence.5.The expression of RLRs pathway genes in naive CD4⁺T cells treated with dsRNA（Poly（I:C）or ERV3-16A3-I-Int）was detected by RT-qPCR.The expression of marker genes in different subclasses of CD4⁺T cells in naive CD4⁺T cells treated with Poly（I:C）was detected by RNA-seq and RT-qPCR.Poly（I:C）,ERV3-16A3-I-Int RNA,ERV3-16A3-I-Int antisense oligonucleotide（Smart Silence）,siRNA of RIG-I and MDA5 were respectively electrotransfected into Th1 cells and Treg cells during differentiation of Th1 cells and Treg cells,and the cell differentiation ratio was detected by flow cytometry.6.Naive CD4⁺T cells in the spleen of C57BL/6J（WT）and DDX58^-/-mice were obtained by magnetic beads sorting.The cells were induced to differentiate into Th1,Th2,Th17,Treg and Tfh cells in vitro.Differentiation proportion of cell were detected by flow cytometry.7.Rag2 mice were respectively injected with naive CD4⁺T cells from the spleen of WT and DDX58^-/-mice respectively via tail vein,and the mice were indued into EAE modeling.When the disease score of mice was the highest,the proportions of Th1 and Th17 cells in spleen and lymph nodes of mice were detected by flow cytometry.The spinal cord and brain tissues of mice were stained with H&E and LFB.8.RNA-seq and RT-qPCR were used to analyze the expression of HERVs,RLRs signaling pathway and I-IFN in HaCaT cells with UVB irradiation.RNase A digestion assay was used to screen HERVs that could form dsRNA,and J2 immunofluorescence was used to detect the expression level of dsRNA in HaCaT cells after UVB irradiation.HaCaT cells were transfected with siRNA（RIG-I and MDA5）,then irradiated UVB.The proliferation ability of HaCaT cells was detected by Ed U,and the apoptosis of HaCaT cells was detected by Annexin V/PI staining.9.The two-sided unpaired t-test is adopted to compare the two independent samples which conform to normal distribution and homogeneity test of variance.The means of multiple groups of samples are compared using one-way analysis of variance（ANOVA）and post-correlation test.non-parametric test is used to analyze the data which does not conform to normal distribution or homogeneity test of variance is not uniform.P<0.05 was considered statistically significant（*P<0.05,**P<0.01,***P<0.001）.Results:1.The results of RNA-seq and whole transcriptome sequencing transcriptional analysis showed that HERVs and RLRs signaling pathway were highly expressed in peripheral blood CD4⁺T cells of SLE patients.RT-qPCR results confirmed that the expression levels of MER21C,ERV3-16A3-I-Int,THE1C,MLT1B,MLT2A2 and RIG-I and MDA5were significantly increased in peripheral blood CD4⁺T cells of SLE patients compared with healthy control.The expression of ERV3-16A3-I-Int was positively correlated with the expression of RIG-I and MDA5 in peripheral blood CD4⁺T cells of SLE patients and healthy volunteers.RIG-I and MDA5 were significantly increased in CD4⁺T cells of lupus-susceptible MRL/Lpr mice compared with MRL/Mp J mice.2.The results showed that the expression of ERV3-16A3-I-Int,MLT1B RIG-I and MDA5 in Th1 cells was significantly higher than that in other CD4⁺T cell subtypes.The results of FISH showed that transcription of ERV3-16A3-I-Int was activated during naive CD4⁺T cells into Th0 cells,and transcription of ERV3-16A3-I-Int was further increased during Th0cell differentiation into Th1 cells.3.The levels of dsRNA in CD4⁺T cells of SLE patients were significantly increased.Meanwhile,ERV3-16A3-I-Int and MLT1B had the characteristics of dsRNA formation.4.Compared with the control group,the expression of RIG-I,MDA5,IRF7,IFNγ,T-BET,and RORγT was significantly increased.The expression of Fox P3 was significantly decreased in naive CD4⁺T cells with dsRNA treatment.Ds RNA promoted the differentiation of Th1 cells and inhibited the differentiation of Treg cells.Inhibition of ERV3-16A3-I-Int,RIG-I or MDA5 expression could inhibit Th1 cell differentiation,and inhibition of RIG-I expression could promote Treg cell differentiation.5.Compared with control,proportion of Th1 cell differentiation was significantly lower in cells from DDX58^-/-mice.There was no significant difference in the proportion of Th2 and Th17 cells differentiation in cells from WT and DDX58^-/-mice.The differentiation ratio of Treg cells was significantly increased in cells from DDX58^-/-mice.The proportion of Tfh cell differentiation was significantly decreased in cells from DDX58^-/-mice.6.WT-Rag2 mice began to show symptoms on the 10th day after immunization,and DDX58^-/-Rag2 mice began to show symptoms on the11th day.On day 16,compared with WT-Rag2 mice,DDX58^-/-Rag2 mice had significantly lower disease score and lower lost weight.The percentages of Th1 and Th17 cells in the spleen of DDX58^-/-Rag2 mice were significantly lower compared with WT-Rag2 mice.H&E staining and LFB staining showed less inflammatory cell infiltration and less severe demyelination in the spinal cord of DDX58^-/-Rag2 mice.7.The expression of HERVs and the level of dsRNA was significantly increased in HaCaT cells exposed with UVB.RLRs signaling pathway（the expression of RIG-I,MDA5 and IRF7,phosphorylation of IRF3 and IFN-β）in HaCaT cells was activated after UVB irradiation.The proliferating HaCaT cells were inhibited and apoptosis of HaCaT cells was increased.Inhibition of RIG-I or MDA5 expression significantly alleviated UVB-induced proliferation inhibition and pro-apoptotic effects of HaCaT cells.Conclusion:1.The expression of HERVs in peripheral blood CD4⁺T cells of SLE patients was increased.HERVs in the form of dsRNA was associated with the expression of RLRs pathway genes in peripheral blood CD4+T cells of SLE patients.2.HERVs-dsRNA promotes Th1 cell differentiation and inhibit Treg cell differentiation by activating RLRs pathway genes.3.Environmental factors（UVB）aggravate skin damage by upregulating HERV-RLRs pathway.Reducing HERV-RLRs pathway could be a potential therapeutic targets of SLE...