Over the past decades, epidemiological researches have shown that viruses infection are closely associated with the pathogenesis and development of MS, including EBV, CMV, HIV, measles virus and influenza virus[6–10]. In this study, we obtained the transcriptomics datasets of these viruses and MS patients from the GEO database, and the correlation between EBV infection and MS risk was the most significant according to systematic analysis. The result was in line with previous studies, Rosella et al found that three herpesviruses showed statistical significance through the analysis between MS susceptibility genes and 20 interactomes, with EBV showing higher levels of significance compared to Human Herpesvirus 8 and, more evidently, to CMV. Therefore, we dived deep into the roles of EBV in MS pathogenesis.
MS is caused by the interaction between genetic susceptibility and environmental triggers. International Multiple Sclerosis Genetics Consortium (IMSGC) has identified 284 MS susceptibility genes through GWAS of more than 100,000 MS patients and healthy controls, which can account for 25% of the perceived heritability of MS[2–4]. Thus, hereditary susceptibility did not solely dominate the alteration of gene expression during MS development, environmental factors also played an important role, suggesting both susceptibility genes and DEGs should be regarded as MS-related genes. We then obtained a total of 873 MS-related genes by integrating 284 susceptibility genes and 601 DEGs from B cells. After KEGG pathway enrichment analysis, we found that MS-related genes in B cells were mainly mapped into immune signaling pathways, including T helper type 17 (Th17) cell differentiation, Epstein-Barr virus infection, janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway and NF-kappa B (NF-κB) signaling pathway. Among them, Th17 cells have been confirmed as pathogenic cells in the pathogenesis of MS and experimental autoimmune encephalomyelitis (EAE), the main animal models of MS. The JAK/STAT and NF-κB signaling pathways played an important role in Th17 cells differentiation and the development of EAE[41, 42]. Inhibition of the JAK/STAT pathway can lead to amelioration of clinical symptoms in EAE models. In addition, EBV infection is a necessary environmental factor in the pathogenesis of MS. EBV can establish latency in the host memory B cells after primary infection, and be induced to activation when the immune system is impaired. However, the roles of EBV infection in the pathogenesis of MS have remained unclear.
Subsequently, we characterized the crucial roles of EBV infection in the pathogenesis of MS. Smith et al performed transcriptome analyses on resting B cells and two types of B blasts at 7 days after EBV infection or (CD40 Ligand) CD40L/interleukin (IL)-4 stimulation. There were more dysregulated genes in B blasts with EBV infection when compared to the original resting B cells, reflecting EBV infection played a facilitative role in B cell activation. Thus, we obtained a total of 3556 DEGs from primary B cells after EBV infection in vitro. Through intersecting these 3556 DEGs and 873 MS-related genes in B cells by the same trend, we identified 150 EBV-regulated, MS-related genes. Subsequently, a total of 18 closely associated MS risk pathways regulated by EBV were obtained through KEGG pathway enrichment analysis and hypergeometric test, including Viral protein interaction with cytokine and cytokine receptor, NF-κB signaling pathway and PD-L1 expression and PD-1 checkpoint pathway in cancer. After pathway-gene network analysis, HLA-DRB1 was confirmed as a vital factor in the development of MS that EBV was involved in. Recent studies revealed that B cells could express MHC class II and serve as antigen-presenting cells for activation of CD4+ T cells, which was critically involved in MS pathogenesis. Meanwhile, another two molecules in the MS risk pathway-gene network regulated by EBV, MALT1 and BCL10, were important parts of CARMA1-BCL10-MALT1 (CBM) complex, which was a central mediator of T cell receptor and B cell receptor-induced NF-κB activation, and in this way drove lymphocyte activation. These results indicated that EBV-infected B cells might lead to the activation of T cells via NF-κB pathway, which may point to the crucial molecular processes in the pathogenesis of MS.
In order to explore the regulatory effects and associated signaling pathways of EBV miRNAs in MS pathogenesis, we identified 42 MS-related genes regulated by 36 target EBV miRNAs. By constructing pathway-pathway and pathway-gene networks, a total of 5 hub genes (MALT1, BCL10, IFNGR2, STAT3 and CDK6) were obtained. Subsequently, a PPI network of these 42 target proteins were established by STRING database, and we found that FOXP1 had a close relationship with MALT1 and BCL10, which should also be a focus point in our study. Finally, we identified 15 target EBV miRNAs and their regulated, 6 MS-related genes. It is worth noting that ebv-miR-BHRF1-2-5p and ebv-miR-BHRF1-3 belonged to the same class cluster and had a synergistic effect. In this study, we found that ebv-miR-BHRF1-2-5p can target both MALT1 and BCL10, the components of CBM complex. MALT1 was a key molecule that promoted activation of NF-κB pathway, regulated regulatory T cells (Tregs) function and maintained immune tolerance. MALT1 knockout mice showed absence of Tregs, increased Th1 and Th2 cells, which consequently led to lymphocyte infiltration and multi-organ inflammation. Surprisingly, we firstly detected ebv-miR-BHRF1-2-5p and ebv-miR-BHRF1-3 were significantly increased in the circulation of RRMS patients. By luciferase reporter assay, MALT1 has been confirmed as a target gene of ebv-miR-BHRF1-2-5p in our research. The target gene of ebv-miR-BHRF1-3, phosphatase and tensin homolog (PTEN), has also been confirmed. PTEN was a key regulator of the phosphatidylinositol 3-kinase (PIK3)/AKT (protein kinase B) survival pathway. Furthermore, FOXP1 was closely associated with MALT1 and BCL10 in the PPI network and identified as a target gene of ebv-miR-BART11[38, 39]. FOXP1 was a critical regulator in maintaining Tregs homeostasis and suppressive function. Mice with FOXP1-deficient Tregs developed spontaneous inflammatory disease with age, which can lead to more severe EAE. In addition, we found another two EBV miRNAs-regulated, MS-related genes, interferon gamma receptor 2 (IFNGR2) and STAT3 were both the upstream genes of programmed cell death ligand 1 (PD-L1) and PD-1 in the PD-L1 expression and PD-1 checkpoint pathway in cancer. PD-L1 and PD-1 knockout mice showed more severe inflammatory in EAE models. Thus, EBV miRNAs could directly target MS-related genes, which consequently altered the expression and function of B cells, changed the autoimmune response of T cells and promoted the initiation and development of MS through NF-κB (MALT1 and BCL10) and PD-L1/PD-1 (IFNGR2 and STAT3) pathways (Fig. 6).
In this study, we have identified that 15 target EBV miRNAs and their regulated, 6 MS-related genes. Among them, Song et al reported that FOXP1 could be directly regulated by ebv-miR-BART11. Meanwhile, ebv-miR-BHRF1-2-5p directly targeting MALT1 was confirmed by our experiments. However, there is a paucity of studies about their roles in clinical application. In addition, future work is required to investigate other target EBV miRNAs and their regulated MS-related genes.