Transcriptome alterations in peripheral blood B cells of patients with multiple sclerosis receiving immune reconstitution therapy.

BACKGROUND: Multiple sclerosis (MS) is a chronic, inflammatory and neurodegenerative disease that leads to irreversible damage to the brain and spinal cord. The goal of so-called "immune reconstitution therapies" (IRTs) is to achieve long-term disease remission by eliminating a pathogenic immune repertoire through intense short-term immune cell depletion. B cells are major targets for effective immunotherapy in MS. OBJECTIVES: The aim of this study was to analyze the gene expression pattern of B cells before and during IRT (i.e., before B-cell depletion and after B-cell repopulation) to better understand the therapeutic effects and to identify biomarker candidates of the clinical response to therapy. METHODS: B cells were obtained from blood samples of patients with relapsing-remitting MS (n = 50), patients with primary progressive MS (n = 13) as well as healthy controls (n = 28). The patients with relapsing MS received either monthly infusions of natalizumab (n = 29) or a pulsed IRT with alemtuzumab (n = 15) or cladribine (n = 6). B-cell subpopulation frequencies were determined by flow cytometry, and transcriptome profiling was performed using Clariom D arrays. Differentially expressed genes (DEGs) between the patient groups and controls were examined with regard to their functions and interactions. We also tested for differences in gene expression between patients with and without relapse following alemtuzumab administration. RESULTS: Patients treated with alemtuzumab or cladribine showed on average a > 20% lower proportion of memory B cells as compared to before IRT. This was paralleled by profound transcriptome shifts, with > 6000 significant DEGs after adjustment for multiple comparisons. The top DEGs were found to regulate apoptosis, cell adhesion and RNA processing, and the most highly connected nodes in the network of encoded proteins were ESR2, PHB and RC3H1. Higher mRNA levels of BCL2, IL13RA1 and SLC38A11 were seen in patients with relapse despite IRT, though these differences did not pass the false discovery rate correction. CONCLUSIONS: We show that B cells circulating in the blood of patients with MS undergoing IRT present a distinct gene expression signature, and we delineated the associated biological processes and gene interactions. Moreover, we identified genes whose expression may be an indicator of relapse risk, but further studies are needed to verify their potential value as biomarkers.

Genetic risk variants for multiple sclerosis are linked to differences in alternative pre-mRNA splicing.

Background: Multiple sclerosis (MS) is a chronic immune-mediated disease of the central nervous system to which a genetic predisposition contributes. Over 200 genetic regions have been associated with increased disease risk, but the disease-causing variants and their functional impact at the molecular level are mostly poorly defined. We hypothesized that single-nucleotide polymorphisms (SNPs) have an impact on pre-mRNA splicing in MS. Methods: Our study focused on 10 bioinformatically prioritized SNP-gene pairs, in which the SNP has a high potential to alter alternative splicing events (ASEs). We tested for differential gene expression and differential alternative splicing in B cells from MS patients and healthy controls. We further examined the impact of the SNP genotypes on ASEs and on splice isoform expression levels. Novel genotype-dependent effects on splicing were verified with splicing reporter minigene assays. Results: We were able to confirm previously described findings regarding the relation of MS-associated SNPs with the ASEs of the pre-mRNAs from GSDMB and SP140. We also observed an increased IL7R exon 6 skipping when comparing relapsing and progressive MS patients to healthy subjects. Moreover, we found evidence that the MS risk alleles of the SNPs rs3851808 (EFCAB13), rs1131123 (HLA-C), rs10783847 (TSFM), and rs2014886 (TSFM) may contribute to a differential splicing pattern. Of particular interest is the genotype-dependent exon skipping of TSFM due to the SNP rs2014886. The minor allele T creates a donor splice site, resulting in the expression of the exon 3 and 4 of a short TSFM transcript isoform, whereas in the presence of the MS risk allele C, this donor site is absent, and thus the short transcript isoform is not expressed. Conclusion: In summary, we found that genetic variants from MS risk loci affect pre-mRNA splicing. Our findings substantiate the role of ASEs with respect to the genetics of MS. Further studies on how disease-causing genetic variants may modify the interactions between splicing regulatory sequence elements and RNA-binding proteins can help to deepen our understanding of the genetic susceptibility to MS.

Implication of genetic variants in primary microRNA processing sites in the risk of multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system with a well-established genetic contribution to susceptibility. Over 200 genetic regions have been linked to the inherited risk of developing MS, but the disease-causing variants and their functional effects at the molecular level are still largely unresolved. We hypothesised that MS-associated single-nucleotide polymorphisms (SNPs) affect the recognition and enzymatic cleavage of primary microRNAs (pri-miRNAs). METHODS: Our study focused on 11 pri-miRNAs (9 primate-specific) that are encoded in genetic risk loci for MS. The levels of mature miRNAs and potential isoforms (isomiRs) produced from those pri-miRNAs were measured in B cells obtained from the peripheral blood of 63 MS patients and 28 healthy controls. We tested for associations between SNP genotypes and miRNA expression in cis using quantitative trait locus (cis-miR-eQTL) analyses. Genetic effects on miRNA stem-loop processing efficiency were verified using luciferase reporter assays. Potential direct miRNA target genes were identified by transcriptome profiling and computational binding site assessment. FINDINGS: Mature miRNAs and isomiRs from hsa-mir-26a-2, hsa-mir-199a-1, hsa-mir-4304, hsa-mir-4423, hsa-mir-4464 and hsa-mir-4492 could be detected in all B-cell samples. When MS patient subgroups were compared with healthy controls, a significant differential expression was observed for miRNAs from the 5' and 3' strands of hsa-mir-26a-2 and hsa-mir-199a-1. The cis-miR-eQTL analyses and reporter assays pointed to a slightly more efficient Drosha-mediated processing of hsa-mir-199a-1 when the MS risk allele T of SNP rs1005039 is present. On the other hand, the MS risk allele A of SNP rs817478, which substitutes the first C in a CNNC sequence motif, was found to cause a markedly lower efficiency in the processing of hsa-mir-4423. Overexpression of hsa-mir-199a-1 inhibited the expression of 60 protein-coding genes, including IRAK2, MIF, TNFRSF12A and TRAF1. The only target gene identified for hsa-mir-4423 was TMEM47. INTERPRETATION: We found that MS-associated SNPs in sequence determinants of pri-miRNA processing can affect the expression of mature miRNAs. Our findings complement the existing literature on the dysregulation of miRNAs in MS. Further studies on the maturation and function of miRNAs in different cell types and tissues may help to gain a more detailed functional understanding of the genetic basis of MS. FUNDING: This study was funded by the Rostock University Medical Center (FORUN program, grant: 889002), Sanofi Genzyme (grant: GZ-2016-11560) and Merck Serono GmbH (Darmstadt, Germany, an affiliate of Merck KGaA, CrossRef Funder ID: 10.13039/100009945, grant: 4501860307). NB was supported by the Stiftung der Deutschen Wirtschaft (sdw) and the FAZIT foundation. EP was supported by the Landesgraduiertenförderung Mecklenburg-Vorpommern.

Vaccination in Multiple Sclerosis: Friend or Foe?

Multiple sclerosis (MS) is a debilitating disease of the central nervous systems (CNS). Disease-modifying treatments (including immunosuppressive treatments) have shown positive effects on the disease course, but are associated with systemic consequences on the immune system and may increase the risk of infections and alter vaccine efficiency. Therefore, vaccination of MS patients is of major interest. Over the last years, vaccine hesitancy has steadily grown especially in Western countries, partly due to fear of sequelae arising from vaccination, especially neurological disorders. The interaction of vaccination and MS has been discussed for decades. In this review, we highlight the immunology of vaccination, provide a review of literature and discuss the clinical consideration of MS, vaccination and immunosuppression. In conclusion, there is consensus that MS cannot be caused by vaccines, neither by inactivated nor by live vaccines. However, particular attention should be paid to two aspects: First, in immunocompromised patients, live vaccines may lead to a stronger immune reaction with signs of the disease against which the patients have been vaccinated, albeit in weakened form. Second, protection provided by vaccination should be controlled in patients who have been vaccinated while receiving immunomodulatory or immunosuppressive treatment. In conclusion, there is evidence that systemic infections can worsen MS, thus vaccination will lower the risk of relapses by reducing the risk of infections. Therefore, vaccination should be in general recommended to MS patients.

Aberrant expression of alternative splicing variants in multiple sclerosis - A systematic review.

OBJECTIVE: Alternative splicing is an important form of RNA processing that affects nearly all human genes. The differential expression of specific transcript and protein isoforms holds the potential of novel biomarkers for complex diseases. In this systematic review, we compiled the existing literature on aberrant alternative splicing events in multiple sclerosis (MS). METHODS: A systematic literature search in the PubMed database was carried out and supplemented by screening the reference lists of the identified articles. We selected only MS-related original research studies which compared the levels of different isoforms of human protein-coding genes. A narrative synthesis of the research findings was conducted. Additionally, we performed a case-control analysis using high-density transcriptome microarray data to reevaluate the genes that were examined in the reviewed studies. RESULTS: A total of 160 records were screened. Of those, 36 studies from the last two decades were included. Most commonly, peripheral blood samples were analyzed (32 studies), and PCR-based techniques were usually employed (27 studies) for measuring the expression of selected genes. Two studies used an exploratory genome-wide approach. Overall, 27 alternatively spliced genes were investigated. Nine of these genes appeared in at least two studies (CD40, CFLAR, FOXP3, IFNAR2, IL7R, MOG, PTPRC, SP140 and TNFRSF1A). The microarray data analysis confirmed differential alternative pre-mRNA splicing for 19 genes. CONCLUSIONS: An altered RNA processing of genes mediating immune signaling pathways has been repeatedly implicated in MS. The analysis of individual exon-level expression patterns is stimulated by the advancement of transcriptome profiling technologies. In particular, the examination of genes encoded in MS-associated genetic regions may provide important insights into the pathogenesis of the disease and help to identify new biomarkers.

Dysregulation of Inflammasome Priming and Activation by MicroRNAs in Human Immune-Mediated Diseases.

Inflammasomes are protein complexes that respond to a wide range of pathogens and cellular damage signals. Their activation prompts the caspase-1-mediated cleavage of the proinflammatory cytokines IL-1ß and IL-18. Inflammasome dysregulation has been demonstrated to play a role in a range of diseases involving the adaptive immune system like multiple sclerosis, rheumatic diseases, and type 1 diabetes. Priming and activation of inflammasomes can be modulated by microRNAs (miRNAs), small noncoding RNAs that regulate gene expression posttranscriptionally. miRNAs, such as miR-223-3p, have been demonstrated to directly target the inflammasome components NLRP3, caspase-1, and caspase-8. Other miRNAs like miR-155-5p modulate TLR-, IL-1R-, TNFR-, and IFNAR-mediated signaling pathways upstream of the inflammasomes. In this study, we discuss how a more detailed elucidation of miRNA-driven inflammasome regulation helps in understanding the molecular processes underlying immune-mediated human diseases, holds potential for the identification of biomarkers and may offer novel targets for the development of future therapeutics.

A genetic variant associated with multiple sclerosis inversely affects the expression of CD58 and microRNA-548ac from the same gene.

Genome-wide association studies have identified more than 200 genetic variants to be associated with an increased risk of developing multiple sclerosis (MS). Still, little is known about the causal molecular mechanisms that underlie the genetic contribution to disease susceptibility. In this study, we investigated the role of the single-nucleotide polymorphism (SNP) rs1414273, which is located within the microRNA-548ac stem-loop sequence in the first intron of the CD58 gene. We conducted an expression quantitative trait locus (eQTL) analysis based on public RNA-sequencing and microarray data of blood-derived cells of more than 1000 subjects. Additionally, CD58 transcripts and mature hsa-miR-548ac molecules were measured using real-time PCR in peripheral blood samples of 32 MS patients. Cell culture experiments were performed to evaluate the efficiency of Drosha-mediated stem-loop processing dependent on genotype and to determine the target genes of this underexplored microRNA. Across different global populations and data sets, carriers of the MS risk allele showed reduced CD58 mRNA levels but increased hsa-miR-548ac levels. We provide evidence that the SNP rs1414273 might alter Drosha cleavage activity, thereby provoking partial uncoupling of CD58 gene expression and microRNA-548ac production from the shared primary transcript in immune cells. Moreover, the microRNA was found to regulate genes, which participate in inflammatory processes and in controlling the balance of protein folding and degradation. We thus uncovered new regulatory implications of the MS-associated haplotype of the CD58 gene locus, and we remind that paradoxical findings can be encountered in the analysis of eQTLs upon data aggregation. Our study illustrates that a better understanding of RNA processing events might help to establish the functional nature of genetic variants, which predispose to inflammatory and neurological diseases.

Transcriptome profiling of peripheral blood immune cell populations in multiple sclerosis patients before and during treatment with a sphingosine-1-phosphate receptor modulator.

AIMS: Fingolimod is a sphingosine-1-phosphate (S1P) receptor modulator approved for the treatment of the relapsing form of multiple sclerosis (MS). It prevents the egress of lymphocyte subpopulations from lymphoid tissues into the circulation. Here, we explored the broad effects of fingolimod on gene expression in different immune cell subsets. METHODS: Utilizing 150 high-resolution microarrays from Affymetrix, we obtained the transcriptome profiles of 5 cell populations, which were separated from the peripheral blood of MS patients prior to and following oral administration of fingolimod. RESULTS: After 3 months of treatment, significant transcriptome shifts were seen in CD4+ and CD8+ cells, which is mainly attributable to the selective homing of naive T cells and central memory T cells. Although the number of B cells was greatly reduced in the blood of fingolimod-treated MS patients, the analysis of differential expression in CD19+ cells identified only a small set of 42 genes, which indicated a slightly higher frequency of transitional B cells. The transcriptome signatures of CD14+ monocytes and CD56+ natural killer cells were not affected. CONCLUSION: Our study corroborates changes in the composition of circulating immune cells in response to fingolimod and delineates the respective implications at the RNA level. Our data may be valuable for comparing the effects of novel S1P receptor modulating agents, which may be a therapeutic option for patients with secondary progressive MS as well.