Petra Bergman

Circulating miR-150 in CSF is a novel candidate biomarker for multiple sclerosis

Objective: To explore circulating microRNAs (miRNAs) in cell-free CSF as novel biomarkers for multiple sclerosis (MS). Methods: Profiling of miRNAs in CSF of pooled patients with clinically isolated syndrome (CIS), patients with relapsing-remitting MS, and inflammatory and noninflammatory neurologic disease controls was performed using TaqMan miRNA arrays. Two independent patient cohorts (n = 142 and n = 430) were used for validation with real-time PCR. Results: We reliably detected 88 CSF miRNAs in the exploratory cohort. Subsequent validation in 2 cohorts demonstrated significantly higher levels of miR-150 in patients with MS. Higher miR-150 levels were also observed in patients with CIS who converted to MS compared to nonconverters, and in patients initiating natalizumab treatment. Levels of miR-150 correlated with immunologic parameters including CSF cell count, immunoglobulin G index, and presence of oligoclonal bands, and with candidate protein biomarkers C-X-C motif chemokine 13, matrix metallopeptidase 9, and osteopontin. Correlation with neurofilament light chain (NFL) was observed only when NFL was adjusted for age using a method that requires further validation. Additionally, miR-150 discriminated MS from controls and CIS converters from nonconverters equally well as the most informative protein biomarkers. Following treatment with natalizumab, but not fingolimod, CSF levels of miR-150 decreased, while plasma levels increased with natalizumab and decreased with fingolimod, suggesting immune cells as a source of miR-150. Conclusions: Our findings demonstrate miR-150 as a putative novel biomarker of inflammatory active disease with the potential to be used for early diagnosis of MS. Classification of evidence: This study provides Class II evidence that CSF miR-150 distinguishes patients with MS from patients with other neurologic conditions.

Next-Generation Sequencing Identifies MicroRNAs that Associate with Pathogenic Autoimmune Neuroinflammation in Rats

MicroRNAs (miRNAs) are known to regulate most biological processes and have been found dysregulated in a variety of diseases, including multiple sclerosis (MS). In this study, we characterized miRNAs that associate with susceptibility to develop experimental autoimmune encephalomyelitis (EAE) in rats, a well-established animal model of MS. Using Illumina next-generation sequencing, we detected 544 miRNAs in the lymph nodes of EAE-susceptible Dark Agouti and EAE-resistant Piebald Virol Glaxo rats during immune activation. Forty-three miRNAs were found differentially expressed between the two strains, with 81% (35 out of 43) showing higher expression in the susceptible strain. Only 33% of tested miRNAs displayed differential expression in naive lymph nodes, suggesting that a majority of regulated miRNAs are EAE dependent. Further investigation of a selected six miRNAs indicates differences in cellular source and kinetics of expression. Several of the miRNAs, including miR-146a, miR-21, miR-181a, miR-223, and let-7, have previously been implicated in immune system regulation. Moreover, 77% (33 out of 43) of the miRNAs were associated with MS and other autoimmune diseases. Target genes likely regulated by the miRNAs were identified using computational predictions combined with whole-genome expression data. Differentially expressed miRNAs and their targets involve functions important for MS and EAE, such as immune cell migration through targeting genes like Cxcr3 and cellular maintenance and signaling by regulation of Prkcd and Stat1. In addition, we demonstrated that these three genes are direct targets of miR-181a. Our study highlights the impact of multiple miRNAs, displaying diverse kinetics and cellular sources, on development of pathogenic autoimmune inflammation.

Parent-of-Origin Effects Implicate Epigenetic Regulation of Experimental Autoimmune Encephalomyelitis and Identify Imprinted Dlk1 as a Novel Risk Gene

Parent-of-origin effects comprise a range of genetic and epigenetic mechanisms of inheritance. Recently, detection of such effects implicated epigenetic mechanisms in the etiology of multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system. We here sought to dissect the magnitude and the type of parent-of-origin effects in the pathogenesis of experimental neuroinflammation under controlled environmental conditions. We investigated inheritance of an MS-like disease in rat, experimental autoimmune encephalomyelitis (EAE), using a backcross strategy designed to identify the parental origin of disease-predisposing alleles. A striking 37–54% of all detected disease-predisposing loci depended on parental transmission. Additionally, the Y chromosome from the susceptible strain contributed to disease susceptibility. Accounting for parent-of-origin enabled more powerful and precise identification of novel risk factors and increased the disease variance explained by the identified factors by 2-4-fold. The majority of loci displayed an imprinting–like pattern whereby a gene expressed only from the maternal or paternal copy exerts an effect. In particular, a locus on chromosome 6 comprises a well-known cluster of imprinted genes including the paternally expressed Dlk1, an atypical Notch ligand. Disease-predisposing alleles at the locus conferred lower Dlk1 expression in rats and, together with data from transgenic overexpressing Dlk1 mice, demonstrate that reduced Dlk1 drives more severe disease and modulates adaptive immune reactions in EAE. Our findings suggest a significant epigenetic contribution to the etiology of EAE. Incorporating these effects enables more powerful and precise identification of novel risk factors with diagnostic and prognostic implications for complex disease.

Functional genomics analysis of vitamin D effects on CD4+ T cells in vivo in experimental autoimmune encephalomyelitis

Significance Vitamin D has been suggested to be associated with beneficial immunomodulation in autoimmune diseases. We demonstrate that the protective effect of vitamin D in an animal model of multiple sclerosis (MS) is linked to multiple signaling and metabolic pathways critical for T-cell activation and differentiation into pathogenic T helper (Th) 1 and Th17 subsets in vivo. This effect is mediated by epigenetic mechanisms as reflected by genome-wide reduction of DNA methylation and upregulation of microRNAs, with concomitant downregulation of their protein-coding target genes. Our data support the role of vitamin D in modulating risk for human disease, because orthologues of nearly 50% of MS candidate risk genes changed their expression in vivo in CD4+ T cells upon vitamin D supplementation. Vitamin D exerts multiple immunomodulatory functions and has been implicated in the etiology and treatment of several autoimmune diseases, including multiple sclerosis (MS). We have previously reported that in juvenile/adolescent rats, vitamin D supplementation protects from experimental autoimmune encephalomyelitis (EAE), a model of MS. Here we demonstrate that this protective effect associates with decreased proliferation of CD4+ T cells and lower frequency of pathogenic T helper (Th) 17 cells. Using transcriptome, methylome, and pathway analyses in CD4+ T cells, we show that vitamin D affects multiple signaling and metabolic pathways critical for T-cell activation and differentiation into Th1 and Th17 subsets in vivo. Namely, Jak/Stat, Erk/Mapk, and Pi3K/Akt/mTor signaling pathway genes were down-regulated upon vitamin D supplementation. The protective effect associated with epigenetic mechanisms, such as (i) changed levels of enzymes involved in establishment and maintenance of epigenetic marks, i.e., DNA methylation and histone modifications; (ii) genome-wide reduction of DNA methylation, and (iii) up-regulation of noncoding RNAs, including microRNAs, with concomitant down-regulation of their protein-coding target RNAs involved in T-cell activation and differentiation. We further demonstrate that treatment of myelin-specific T cells with vitamin D reduces frequency of Th1 and Th17 cells, down-regulates genes in key signaling pathways and epigenetic machinery, and impairs their ability to transfer EAE. Finally, orthologs of nearly 50% of candidate MS risk genes and 40% of signature genes of myelin-reactive T cells in MS changed their expression in vivo in EAE upon supplementation, supporting the hypothesis that vitamin D may modulate risk for developing MS.

The Calcitonin Receptor Gene Is a Candidate for Regulation of Susceptibility to Herpes simplex Type 1 Neuronal Infection Leading to Encephalitis in Rat

Herpes simplex encephalitis (HSE) is a fatal infection of the central nervous system (CNS) predominantly caused by Herpes simplex virus type 1. Factors regulating the susceptibility to HSE are still largely unknown. To identify host gene(s) regulating HSE susceptibility we performed a genome-wide linkage scan in an intercross between the susceptible DA and the resistant PVG rat. We found one major quantitative trait locus (QTL), Hse1, on rat chromosome 4 (confidence interval 24.3–31 Mb; LOD score 29.5) governing disease susceptibility. Fine mapping of Hse1 using recombinants, haplotype mapping and sequencing, as well as expression analysis of all genes in the interval identified the calcitonin receptor gene (Calcr) as the main candidate, which also is supported by functional studies. Thus, using unbiased genetic approach variability in Calcr was identified as potentially critical for infection and viral spread to the CNS and subsequent HSE development.

A Silent Exonic SNP in Kdm3a Affects Nucleic Acids Structure but Does Not Regulate Experimental Autoimmune Encephalomyelitis

Defining genetic variants that predispose for diseases is an important initiative that can improve biological understanding and focus therapeutic development. Genetic mapping in humans and animal models has defined genomic regions controlling a variety of phenotypes known as quantitative trait loci (QTL). Causative disease determinants, including single nucleotide polymorphisms (SNPs), lie within these regions and can often be identified through effects on gene expression. We previously identified a QTL on rat chromosome 4 regulating macrophage phenotypes and immune-mediated diseases including experimental autoimmune encephalomyelitis (EAE). Gene analysis and a literature search identified lysine-specific demethylase 3A (Kdm3a) as a potential regulator of these phenotypes. Genomic sequencing determined only two synonymous SNPs in Kdm3a. The silent synonymous SNP in exon 15 of Kdm3a caused problems with quantitative PCR detection in the susceptible strain through reduced amplification efficiency due to altered secondary cDNA structure. Shape Probability Shift analysis predicted that the SNP often affects RNA folding; thus, it may impact protein translation. Despite these differences in rats, genetic knockout of Kdm3a in mice resulted in no dramatic effect on immune system development and activation or EAE susceptibility and severity. These results provide support for tools that analyze causative SNPs that impact nucleic acid structures.

miR-31 regulates energy metabolism and is suppressed in T cells from patients with Sjögren's syndrome

Systemic autoimmune diseases are characterized by the overexpression of type I IFN stimulated genes, and accumulating evidence indicate a role for type I IFNs in these diseases. However, the underlying mechanisms for this are still poorly understood. To explore the role of type I IFN regulated miRNAs in systemic autoimmune disease, we characterized cellular expression of miRNAs during both acute and chronic type I IFN responses. We identified a T cell‐specific reduction of miR‐31‐5p levels, both after intramuscular injection of IFNβ and in patients with Sjögrens syndrome (SjS). To interrogate the role of miR‐31‐51p in T cells we transfected human CD4+ T cells with a miR‐31‐5p inhibitor and performed metabolic measurements. This identified an increase in basal levels of glucose metabolism after inhibition of miR‐31‐5p. Furthermore, treatment with IFN‐α also increased the basal levels of human CD4+ T‐cell metabolism. In all, our results suggest that reduced levels of miR‐31‐5p in T cells of SjS patients support autoimmune T‐cell responses during chronic type I IFN exposure.

Methylome characterization of CD4+ T cells in multiple sclerosis — Establishing a role for miR-21 in autoimmune disease

expression of Ly6C and Ly6G, very important in infectious, autoimmune and tumor models. The present work will further characterize the potential role of miR-223 in the EAE model and MS. First we found an upregulation of miR-233 in the Peripheral Blood Mononuclear Cell (PBMC) of 20 MS samples vs. 20 controls (fold change over controls 1.64 ± 1.25 vs. 1.20 ± 0.95, P = 0.018). This result was confirmed in a different cohort of subjects, including 15 untreated MS subjects (population from Italy: 11 RRMS, 4 PPMS) and 12 healthy controls. In this cohort, miR-233 was upregulated in MS vs. control subjects (fold change over controls 0.81 ± 0.65 vs. 0.40 ± 0.26, P = 0.010). We also performed several active EAE experiments in miR-223 knockout (miR-223 KO) mice and littermate control mice. MiR-223 KO mice developed a significantly less severe disease (P b 0.0001 by two-way ANOVA) with a significantly higher percentage of PMN-MDSC (CD11b/Ly6G positive cells) and MO-MDSC (CD11b/Ly6C positive cells) in the spleens and spinal cords compared to control mice. We found also that MO-MDSC from miR-223 KO mice had greater immune-suppressive effects on CD4 T cell proliferation than controls in antigen T cell stimulatory conditions. It is established that MO-MDSCs inhibit CD4 and CD8 T cell proliferation mostly via ARG1 action. ARG1 was promptly upregulated in MO-MDSC from miR-223 KO cells corresponding to their high immunosuppressive function. These results demonstrate altered levels of miR 223 in the PBMC of MS patients and suggest that miR-223 plays a role in EAE. This may lead to the identification of new disease biomarkers of therapeutic targets.

A novel connection of autophagy related gene 7 to multiple sclerosis and experimental autoimmune encephalomyelitis

Aim: The role of inflammation in stroke lesion development is currently intensively investigated. Most experimental models make use of young animals with normal nutritional status despite the fact that ageing and obesity are important risk factors for patients to suffer from cerebrovascular disease. Both ageing and nutritional customs are known to alter inflammatory responses and to affect stroke outcome. However, no data are available on the effect of nutrition regimenon stroke outcome and brain inflammation in aged animals. The objective of this study was to determine the effect of nutrition on neurogenesis in the subventricular zone (SVZ) and inflammatory responses in stroke in aged rats. Material and methods: 12 month old Sprague–Dawley rats were divided into an ad libitum (AL; n = 20) and a caloric restricted (CR; n = 20) feeding regimen. The CR group was fed 65 % of the calculated average food intake for a period of 12 months until the surgery. Animals from the AL and CR group were randomly subjected to middle cerebral artery occlusion (MCAO) or sham operation. Newly generated cells were labelled by BrdU injection. Three days after surgery rats were perfused with 4% PFA. Neurogenesis (BrdU, DCX) and inflammation (CD45, CD22, CD4) were quantified using Abercrombies cell counting method. NeuN staining was used to determine the infarct volume. Results: The infarct volume in CR tended to be lower compared to AL rats. Quantification of CD45 expression demonstrated enhanced expression of CD45 on cells in the corpus callosum and the ischemic border in CRcompared to AL rats. Some CD45 cells in the border of the infarct stained double positive for CD22 or CD4 demonstrating the infiltration of Band T cells into the ischemic area. Assessment of proliferation, differentiation and neurogenesis following stroke revealed reduced expression of BrdU, DCX and BrdU/DCX cells in the SVZ of CR compared to AL fed rats. Conclusion: In aged rats long-term CR tended to reduce infarct volume despite impaired neurogenesis in the SVZ. Furthermore the inflammatory response in the brain of CR animals was enhanced compared to AL rats. These data suggest that mechanisms independent of neurogenesis and inflammation determine infarct development in aged rats. Furthermore the effect of nutrition on inflammation may differ in young and aged animals.