Author Of 6 Presentations
P0136 - Profiling of small non-coding RNAs across cellular and biofluid compartments in patients with Multiple Sclerosis (ID 1195)
Abstract
Background
Small non-coding RNAs (sncRNAs) are important regulators of gene expression at the transcriptional and post-transcriptional levels in various biological contexts, such as regulation of immune system functions, homeostasis, and autoimmunity development. While the role of microRNAs (miRNAs) in multiple sclerosis (MS) has attained major interest, studies investigating other sncRNA classes, especially in the target central nervous system compartment, are still scarce.
Objectives
We aimed to perform a comprehensive, comparative analysis of all classes of sncRNAs in matching peripheral blood mononuclear cells (PBMCs), plasma, cerebrospinal fluid (CSF) cells, and cell-free CSF from MS patients and controls.
Methods
23 relapsing-remitting (RRMS, n=12 in relapse, n=11 in remission), 6 secondary progressive (SPMS) MS patients and 16 non-inflammatory and inflammatory neurological disease controls (NINDC, n=11; INDC, n=5) were included in the analysis. We utilized Small-seq (Faridani et al., Nat Biotechnol. 2016) to quantify sncRNA transcripts.
Results
We observed distinct and variable profiles of sncRNA classes across all analyzed cellular and biofluid compartments. While miRNAs were the most abundant class of sncRNAs in PBMCs and plasma, transfer RNAs represented the most abundant class in CSF cells and cell-free CSF. Furthermore, we observed an opposing quantitative pattern of small nuclear, nucleolar, transfer RNAs, and miRNAs changes between the blood and CNS compartments. In CSF cells, 133/133 and 115/117 differentially expressed sncRNAs were increased in RRMS relapse compared to remission and RRMS compared to NINDC, respectively. In contrast, 65/67 differentially expressed PBMC sncRNAs were decreased in RRMS compared to NINDC. These findings underscore the importance of including both the peripheral and intrathecal compartments in studies investigating the role of sncRNAs in MS.
Conclusions
Our findings demonstrate widespread alterations of several classes of sncRNAs, particularly during the relapse phase in CSF cells. Genome-wide small non-coding RNA profiling provides therefore an informative moleculer panel for addressing MS pathogenesis, where further research may lead to the identification of novel biomarkers and possible treatment targets.
P0525 - Investigating a role of B cells and their depletion in relapsing-remitting Multiple Sclerosis using DNA methylation patterns (ID 892)
Abstract
Background
Multiple Sclerosis (MS) is a chronic inflammatory disease characterized by autoimmune attack and destruction of myelin and neuroaxonal degeneration in the central nervous system (CNS). Previous reports have indicated that widespread differences in DNA methylation between MS cases and healthy controls in B cells. Importantly, B cells have come under renewed interest due to the effectiveness of treatments that deplete B cells, such as rituximab.
Objectives
To characterize epigenetic changes and their functional consequences in CD19+ B cells from MS patients, and to investigate changes in CD4+ T cells and CD14+ monocytes following B cell depletion with rituximab.
Methods
We measured DNA methylation in CD19+ B cells sorted from peripheral blood from relapsing-remitting (RRMS) (n= 26) and healthy controls (HC) (n = 15), which we compared and integrated with previously analyzed cohort (RRMS n = 12, HC n = 10). We also quantified DNA methylation in CD4+ T cells (n = 17) and CD14+ monocytes (n = 17) sorted from peripheral blood at baseline and 6 months of rituximab treatment. DNA methylation was measured using Infinium HumanMethylationEPIC arrays. Rituximab results were compared with results from a dimethyl fumarate treated cohort.
Results
Meta-analysis of the two B cell cohorts revealed 3 003 differentially methylated CpGs between RRMS and HC (with adjusted p-value < 0.05). Pathway analyses of the cohorts implicated dysregulation of genes involved in cell-to-cell communication, cell migration and activation. Rituximab treatment did not yield genome-wide significant changes in DNA methylation in CD4+ and CD14+ cells likely due to the indirect action of the drugs. Nevertheless pathway analysis of candidate differentially methylated CpGs associated with changes in activation immune activation, subset differentiation and motility being affected by B cell depletion.
Conclusions
Our data establish that B cells from MS patients acquire a distinct epigenetic profile connected to changes in pathways of importance for B cell functions. Furthermore, we demonstrate changes in other cell types following B cell depletion as a therapeutic modality.
P0526 - Methylome and transcriptome analysis implicates NBPF locus in PPMS etiopathology (ID 880)
Abstract
Background
Multiple Sclerosis is characterized by autoimmune destruction of myelin and neurons in the CNS leading to a variety of neurological symptoms. Primary progressive multiple sclerosis (PPMS) is characterized by accumulation of clinical disability from the onset, without relapses or remissions. The mechanisms underpinning MS progression are still largely unknown and specific clinical translations are lacking.
Objectives
To identify DNA methylation and gene expression changes that associate with progressive MS states using genetic, epigenetic and network analysis approaches.
Methods
Our methylome analysis in blood showed a striking increase in methylation at the NBPF locus specifically in PPMS (n=279, p=5x10-6), which was validated in independent samples. We then discovered that genetic variants determine methylation levels at this locus (p-val. range 10-21-10-13) and that the strongest variant potentially associates with the risk of developing PPMS (nPPMS=482, ncontrols=11718, p<0.03, OR=1.2). The same variant associated with reduced expression of FMO5, PRKAB2 and CHD1L in blood (n=156, p-val. range 10-7-10-2).
Results
Notably, a large body of evidence strongly implicate the identified locus in nervous processes involved in brain size and neuropsychiatric disorders. Thus, we hypothesize that it harbors the gene(s) that predispose for progressive disability in PPMS. To functionally confirm the identified differentially methylated region (DMR) can potentially regulate gene expression in a DNA methylation-dependent manner, we have used an in-vitro epigenetic reporter system and our data showed that the DMR region has properties of a gene-regulatory region. Moreover, we investigated the putative relevance of the genes included in the NBPF locus in PPMS brain pathology by constructing an unbiased correlation network analysis using RNA-sequencing data from brain tissue samples of MS patients (nPPMS=5 and nSPMS=7) and controls (n=10). Strikingly, identified gene modules were found centered on genes from the NBPF locus in PPMS. Indeed, exploration of the biggest module, revealed CHD1L as a major central node within this network. Gene ontology analysis of each module underscored implication in nervous processes. Thus, this unbiased in-silico approach further supports the potential implication of genes of NBPF locus in nervous processes in PPMS patients.
Conclusions
Our DNA methylation studies along with the unbiased network analysis approach using the transcriptome data independently suggest that the locus on chromosome 1 predisposes for PPMS.
P0956 - DNA methylation of cerebrospinal fluid cells in Multiple Sclerosis (ID 1193)
Abstract
Background
Multiple Sclerosis (MS) is a chronic inflammatory disease of a yet unknown cause characterized by autoimmune destruction of myelin and neurons in the central nervous system. Immune cells from the cerebrospinal fluid (CSF) can provide valuable insight into the pathogenic processes occurring in the typically inaccessible target organ; however, they have not been sufficiently utilized due to low numbers. DNA methylation is an epigenetic modification that without altering the genetic code can stably change the expression of genes and thus may play an important role in MS development. Moreover, due to stability, high specificity and the ability to be measured in limited amounts of material genome-wide, methylation changes may assist in studying MS pathogenesis.
Objectives
We aimed to establish and optimize a genome-wide methylation approach to investigate methylation changes that render immune cells pathogenic in MS with the prospect of better understanding disease pathogenesis.
Methods
Cells from the CSF of relapsing-remitting MS (RRMS n=5) and age- and sex-matched non-inflammatory controls (NINDC n=5) were extracted. Whole-genome bisulfite sequencing (WGBS) libraries were generated using the post-bisulfite adaptor tagging (PBAT) protocol, which originally was designed for single-cell and adapted to the limited cell amount as previously described (1). Results were replicated in a larger cohort (RRMS n=18 and NINDC n=7), while the reliability of the methodology was confirmed with technical replication of the original cohort.
Results
We first explored the global landscape of CpG methylation levels, as well as specific genomic features including promoters, enhancers and CTCF binding sites. We conducted statistical analysis based on three distinct methods, which identified differentially methylated CpG (DMCs). Methylation levels calculated in the sequencing data were well correlated with the independent technical replicates where the sequencing depths were comparatively lower which gave more evidence the less sequenced data can reflect the true values to some degree. Besides, in the replicate sample groups, a large proportion of DMCs changed in the same direction when applying a minimal 0.2 difference. Moreover, NFATC1, NFKBID, MAPK1, MAP2K2 and other genes participated in the regulation of the immune response were differentially modified. In addition, the differentially modified genes were significantly enriched in pathways including integrin signaling pathway which is important for cell movement and activation, CD40 signaling pathway which is related with the lymphocyte activation, as well as various cytokine signaling pathways such as IL-1, IL-6, IL-17.
Conclusions
Genome-wide methylation analysis, based on a low cell-number library preparation, will enable us to study MS pathogenesis using samples that are difficult to comprehensively study by many other methods.
P0979 - Methylome and transcriptome signature of bronchoalveolar cells from Multiple Sclerosis patients in relation to smoking (ID 1145)
Abstract
Background
Despite compelling evidence of the contribution of smoking in MS susceptibility and progression, little is known about smoking-associated changes in the primary exposed lung cells of patients.
Objectives
We aimed to examine molecular changes occurring in bronchoalveolar lavage (BAL) cells from MS patients in relation to smoking and in comparison to healthy controls (HC).
Methods
We profiled DNA methylation in BAL cells from female MS (n=17) and HC (n=22) individuals, using Illumina Infinium EPIC and performed RNA-sequencing in non-smokers.
Results
The most prominent changes were found in relation to smoking, with 1376 CpG sites (adjusted P < 0.05) differing between MS smokers and non-smokers. Approximately 30% of the affected genes overlapped with smoking-associated changes in HC, leading to a strong common smoking signature in both MS and HC after gene ontology analysis. Smoking in MS patients resulted in additional discrete changes related to neuronal processes. Methylome and transcriptome analyses in non-smokers MS patients compared to HC suggest that BAL cells from MS patients display very subtle (not reaching adjusted P < 0.05) but concordant changes in genes connected to reduced transcriptional/translational processes and enhanced cellular motility. This molecular signature was consistent with findings from animal studies of MS-like disease.
Conclusions
Our study provides insights into the molecular impact of smoking on lung inflammation and the immunopathogenesis of MS.
P0980 - MicroRNA-150 controls experimental autoimmune encephalomyelitis by regulating CD4 T cell differentiation and function (ID 1079)
Abstract
Background
MicroRNAs are small non-coding RNA molecules that have an important role in the fine tuning of all biological processes and are often found to be dysregulated in diseases, such as multiple sclerosis (MS). MS is an immune-mediated disease of the central nervous system characterized by demyelination, axonal loss and neurodegeneration. We have previously shown microRNA-150 (miR-150) levels to be elevated in cell-free cerebrospinal fluid (CSF) of MS patients compared to controls.
Objectives
The aim of this study is to further understand the physiopathological function of miR-150 using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS.
Methods
To establish its role in-vivo, we generated miR-150 knock-out (KO) and knock-in (KI) mice using CRISPR/Cas9. Immune profiling using flow cytometry as well as RNA sequencing were used to understand underlying mechanisms.
Results
After induction of EAE, miR-150 KO mice showed ameliorated disease compared to WT littermate controls while miR-150 KI mice presented with exacerbated disease. An ameliorated disease in miR-150 KO was accompanied by a decreased infiltration of CD4 T cells compared to WT and KI. At priming stage of EAE we found that miR-150 KO had an increase in regulatory CD4 T cells (TREGS). Furthermore, after reconstitution of T cell deficient animals, CD4 T cells from miR-150 KO mice could protect against EAE and also showed an increased FOXP3 expression. A role of miR-150 in regulating TREG cells was further substantiated by transcriptome profiling, where miR-150 KO CD4 T cells suggested an enhancement of TREG phenotype as well as a diminished translation in miR-150 KO CD4 T cells. Moreover the results implicated miR-150 with mechanisms such as translation, autophagy and metabolism as well T cell proliferation and differentiation.
Conclusions
miR-150 deficiency ameliorated EAE and favored a more anti-inflammatory environment while miR-150 expression promoted pathogenic CD4 T cells subsets, potentially associated with metabolic mechanisms.