Interferon-stimulated gene (ISG) is a class gene which can be stimulated by IFN [21]. Approximately 10% of the human genome is regulated by IFN in the human. Studies have uncovered that ISG inhibits virus invasion through a variety of pathways. IFITM3 blocks the membrane fusion of viruses through the endocytic pathway by the palmitoylated amphipathic α-helix[22]. NCOA7 can bind to vacuolar H+-ATPase to promote the degradation of the endocytosed virus[23] and VP30 regulates viral RNA synthesis by interacting with RBBP6, a ubiquitin ligase that plays a role in cell cycle progression and transcription[24]. In short, ISG can inhibit virus entry, virus proliferation in cells, and virus release. Studies have found that in addition to resisting viruses, ISG is also abnormally expressed in many diseases. The concentration of bile acid precursor 25-hydroxycholesterol (25-HC) in cerebrospinal fluid of multiple sclerosis (MS) is increased, which may due to the up-regulation of ISG-CH25H in macrophages[25]. ISG15 was significantly increased in the cerebral ischemia model without obvious signs of inflammation and the cortical shock-induced brain injury model[26]. Aberrant expression of ISG has been found in the synovium of rheumatoid arthritis (RA), and recent studies, including RNA-seq analysis, have further identified the expression of ISG in other tissues of RA lesions, such as joints[27] and these are sensitive to JAK inhibitors[28]. These results suggest that the efficacy of these compounds may be related to the inhibition of ISG. In SLE, microarray analysis data show that hundreds of ISG are up-regulated, suggesting that ISG may play an important role in the course of SLE[29].
In this study, we investigated the role of RSAD2 in B cells from SLE. Firstly, 18 genes were found to be highly expressed in B cells of SLE by RNA-seq which included RSAD2. Further screening the genes, we found a key module in the pathogenesis of SLE. Enrichment analysis of these genes revealed that they play vital roles in SLE mainly through IFN-related pathways. We found that RSAD2 was highly expressed in all B cell subsets of SLE, but the difference was more significant in PC. These results suggested that the presence of RSAD2 may increase the proliferation of B cells and be more closely related to the function of PC and the results were consistent with the bioinformatics results in single cell sequencing from public data. We also found that the expression level of mRNA of BLIMP1 in B cells from PBMCs was positively correlated with of RSAD2. BLIMP1 promotes the differentiation of B cells into PC[30], which further confirmed that RSAD2 may be related to the differentiation of B cells.
RSAD2 can be used as a preferred biomarker for disease activity in Aicardi-Goutières syndrome (AGS)[31], and as a specific biomarker for onset of antibody-mediated rejection (ABMR) after renal transplantation[32]. It is also a potential biomarker and therapeutic target for post-traumatic acute respiratory distress syndrome[33]. The disease severity of SLE has an important relationship with disease progression which even affects the prognosis of patients, but there is no sensitive and simple index to distinguish the progression of SLE now. Therefore, we analyzed the expression of RSAD2 in patients with active and inactive SLE. Unfortunately, it was no significant difference in the expression of RSAD2 between active and inactive SLE patients, and there was no clear correlation between RSAD2 and SLEDAI scores. What's interesting was that RSAD2 was generally more differentially expressed in active SLE, so we can further expand our sample to explore the role of RSAD2 as an indicator of active SLE. In addition to its diagnostic role, RSAD2 can also be used as a prognostic marker for IFN-β therapy in MS[34]. RSAD2 also can predict the prognosis of melanoma in the prediction model of CD8+ T lymphocyte involvement[35]. Therefore, whether RSAD2 can be used as a marker for SLE specific drug treatment warrants further study.
After the treatment of malignant tumors or hepatitis with IFN-I, individuals will produce autoantibodies and autoimmunity, which can induce SLE and SSc[36]. Anilumab, as a receptor antagonist of IFN-I, has a certain effect in the treatment of patients with moderate to severe SLE, which confirms the role of IFN in the pathogenesis of SLE from another perspective. IFN-I can induce the maturation and activation of dendritic cells (DC), increase the expression of MHC class I and II molecules of DC[37], and increase the presentation of self-antigens to B cells. It can also activate T cells and induce autoreactive PC to secrete autoantibodies. In this study, the frequency of PC and the expression of RSAD2 were increased after the sorted CD19+ B cells were stimulated with IFN-β in vitro, which provided a new idea that IFN-I could promote B cell differentiation by regulating ISG. Previous studies mainly focused on the effect of IFN-α on SLE and B cells, but in this study, the function of IFN-β was more intense on RSAD2, which uncovered a new role of IFN-β. In contrast to peripheral blood mRNA expression, BLIMP1 was not differentially expressed, but BCL6 was significantly decreased. BCL6 and BLIMP1 are antagonistic transcription factors in the differentiation and maturation of B cells[38], and the protein translated by BCL6 can inhibit the effect of BLIMP1[39]. Although there was no significant change in mRNA of BLIMP1, BCL6 was decreased and the final result was similar to that of increased BLIMP1 expression. However, the reasons for this difference in expression are still unclear. The expression of transcription factors IRF4, PAX5 and MTA3 did not change significantly in IFN-β stimulated sorted B cells.
It was found that the frequency of PC was lower than that of the control group after silencing RSAD2 in vitro, which further indicated that IFN-β could regulate the function of B cells through RSAD2. IRF4 belongs to interferon regulatory factors (IRF) [40]. In the upstream of BLIMP1, IRF4 can promote the transformation of B cells to PC. PAX5 regulates the expression of genes related to tissue development[41]. In the process of B cell development, PAX5 regulates the formation of BCR in immature B cells[42]. PAX5 can also activate cytidylate deaminase (AID) gene, which is essential for somatic hypermutation and antibody class switching[43], but PAX5 inhibits BLIMP1 expression and B cell differentiation towards PC[44, 45]. MTA3 is a member of the transfer-related family, and its encoded protein can activate histone deacetylase to change the acetylation state of BCL6 and cooperate with BCL6 to inhibit PC differentiation[46]. After RSAD2 silencing, the mRNA levels of IRF4, PAX5 and MTA3 decreased, suggesting that RSAD2 may be upstream. When RSAD2 is silenced, the coordinating effect of their RNA changes is no obvious change of BLIMP1 and BCL6. Nevertheless, the specific regulation methods need to be further explored.
IFN-I signaling is the result of the interaction of multiple molecules, and epigenetic modification is also a regulatory mechanism of IFN-I signaling. IFNs can activate the STAT pathway, and when IFN-α acts, STAT2 can recruit the histone acetyltransferase GCN5 to promote the acetylation of histone H3, which is a marker of general transcriptional activation[46]. Two studies have shown the genomic DNA hypomethylation signature of T cells and neutrophils from SLE[47, 48]. Similar to these results, RSAD2 hypomethylation sites were identified in B cells from SLE and corresponding hypomethylation was also observed after IFN-β treatment.