Effects of anti-CD20 therapy on circulating and intrathecal follicular helper T cell subsets in multiple sclerosis.

Follicular helper T (Tfh) cells and their interplay with B cells likely contribute to the pathogenesis of relapsing-remitting multiple sclerosis (RRMS). Tfh cells are enriched in cerebrospinal fluid (CSF) in RRMS, but effects of anti-CD20 therapy are unknown. We investigated Tfh cells in controls, untreated and anti-CD20-treated patients with RRMS using flow cytometry. CSF Tfh cells were increased in untreated patients. Compared to paired blood samples, CD25- Tfh cells were enriched in CSF in RRMS, but not in controls. Contrast-enhancing brain MRI lesions and IgG index correlated with CSF CD25- Tfh cell frequency in untreated patients with RRMS. Anti-CD20 therapy reduced the numbers of circulating PD1+ Tfh cells and CD25- Tfh cells, and the frequency of CSF CD25- Tfh cells. The study suggests that CD25- Tfh cells are recruited to the CSF in RRMS, associated with focal inflammation, and are reduced by anti-CD20 therapy.

Cluster headache polygenetic risk and known functional variants of CYP3A4 are not associated with treatment response.

BACKGROUND AND PURPOSE: The response to cluster headache treatments has a high interindividual variation. To date, treatment response has only been assessed by a candidate gene approach and no investigations into metabolic pathways have been performed. Our aim was to investigate the association between the polygenetic risk of cluster headache and treatment response to first-line cluster headache treatments as well as known functional variants of CYP3A4 and the response to verapamil. Further, it was aimed to replicate previous single nucleotide polymorphisms found to be associated with treatment response in cluster headache and/or migraine. METHODS: In, 508 cluster headache patients diagnosed according to the International Classification of Headache Disorders were genotyped and participated in a semi-structured interview to evaluate treatment response. Polygenetic risk scores were calculated by the effect retrieved from a meta-analysis of the latest two genome-wide association studies on cluster headache. RESULTS: Inferior treatment response to oxygen, triptans and verapamil is associated with chronicity of cluster headache were confirmed but no evidence was found that a response could be predicted by a high genetic risk of cluster headache. Likewise, verapamil response was not associated with functional variants of CYP3A4. No support of the genetic variants previously reported to be associated with treatment response to triptans or verapamil was found. CONCLUSION: The clinically relevant variation in treatment response for cluster headache was not influenced by genetic factors in the present study.

Evidence for oestrogen sensitivity in perinatal depression: pharmacological sex hormone manipulation study.

BACKGROUND: Enhanced sensitivity to oestrogen signalling may drive increased risk for depressive symptoms when exposed to peripartum sex-steroid hormone fluctuations. AIM: Testing if 116 pre-identified sex steroid-responsive transcripts that predicted perinatal depression (PND) translates to a pharmacological model of hormone-induced mood changes. METHOD: We generated longitudinal, genome-wide gene-expression and DNA-methylation data from 60 women exposed to a gonadotrophin-releasing hormone agonist (GnRHa) or placebo. We used linear mixed-effect models to assess differences between baseline and follow-up for gene expression and DNA methylation in the biphasic ovarian response to GnRHa. RESULTS: Of the 116 PND-predictive transcripts, a significant (19%) overlap was observed with those differentially expressed post-GnRHa at both early and later follow-up, indicating sustained effects. Similarly, 49% of tested genes were differentially methylated post-GnRHa at the late follow-up. Within the GnRHa group, a large proportion of PND genes were significantly associated (gene expression; DNA methylation) with changes in depressive symptoms (28%; 66%), oestradiol levels (49%; 66%) and neocortex serotonin transporter binding (8%; 45%) between baseline and follow-up. CONCLUSIONS: Our data bridge clinical PND biomarkers with a pharmacological model of sex hormone-induced mood changes and directly relate oestrogen-induced biological changes with depressive symptoms and associated serotonin-signalling changes. Our data highlight that individual variations in molecular sensitivity to oestrogen associate with susceptibility to hormone-induced mood changes and hold promise for candidate biomarkers. DECLARATION OF INTEREST: V.G.F. received honorarium for being a speaker for H. Lundbeck A/S. E.B.B. receives research funding from Böhringer Ingelheim to investigate FKBP5 as a potential drug target for depression.

Smoking is associated with increased disease activity during natalizumab treatment in multiple sclerosis.

BACKGROUND: Smoking has been associated with increased multiple sclerosis (MS) risk, disease worsening, and progression in MS patients. Furthermore, interactions between smoking and human leukocyte antigen (HLA) genes have been shown for MS risk. Recently, we found that smoking was associated with an increased relapse rate in interferon-beta-treated relapsing-remitting multiple sclerosis (RRMS) patients. OBJECTIVES: We examined the association between smoking and relapses in natalizumab-treated RRMS patients. Second, we investigated if an interaction between smoking and HLA-DRB1*15:01 or HLA-A*02:01 affected the number of relapses during treatment. METHODS: In this observational cohort study, 355 natalizumab-treated RRMS patients were assessed. Prespecified criteria excluded 62 patients. Clinical data from the starting of treatment to the two-year follow-up visit were collected. Smoking status was obtained by a questionnaire survey. TaqMan allelic discrimination was used for genotyping of tag single-nucleotide polymorphisms (SNPs) for HLA-DRB1*15:01 and HLA-A*02:01. Negative binomial regression analysis was used to analyze the association between relapse rate and smoking intensity and HLA. RESULTS: One pack of cigarettes (20 cigarettes) per day during natalizumab treatment increased the relapse rate during treatment with 38% (incidence rate ratio (IRR) = 1.38, 95% confidence interval (CI): 1.08-1.77, p = 0.01). No association or interaction was found between smoking and HLA-DRB1*15:01 or HLA-A*02:01, respectively. CONCLUSION: Smoking intensity was significantly associated with the number of relapses during natalizumab treatment.

Alcohol consumption in adolescence is associated with a lower risk of multiple sclerosis in a Danish cohort.

BACKGROUND AND OBJECTIVE: Due to the possible existence of a vulnerable period of multiple sclerosis (MS) susceptibility in adolescence and because Danish teenagers have a high alcohol consumption, we investigated the association between alcohol consumption at ages 15-19 and the risk of developing MS. METHODS: A total of 1717 patients with MS and 4685 healthy blood donors filled in a comprehensive environmental and lifestyle questionnaire. Data were analysed by logistic regression models and adjusted for selected confounders. RESULTS: We found an inverse association between alcohol consumption in adolescence and risk of developing MS in both women (p < 0.001) and men (p = 0.012). Women with low alcohol consumption had an odds ratio (OR) of 0.56 (95% confidence interval (CI): 0.47-0.66) compared with non-drinking women. The ORs were similar for women with moderate (OR = 0.49, 95% CI: 0.38-0.62) and high consumption (OR = 0.57, 95% CI: 0.38-0.84). Men with low alcohol consumption had an OR of 0.69 (95% CI: 0.53-0.89) compared with non-drinking men but no decreased risk was found for men with moderate and high consumption. CONCLUSION: Alcohol consumption in adolescence was associated with lower risk of developing MS among both sexes.

The transcriptome of peripheral blood mononuclear cells in patients with clinical subtypes of late age-related macular degeneration.

BACKGROUND: Peripheral blood mononuclear cells (PBMCs) are implicated in the pathogenesis of age-related macular degeneration (AMD). We here mapped the global gene transcriptome of PBMCs from patients with different clinical subtypes of late AMD. RESULTS: We sampled fresh venous blood from patients with geographic atrophy (GA) secondary to AMD without choroidal neovascularizations (n = 19), patients with neovascular AMD without GA (n = 38), patients with polypoidal choroidal vasculopathy (PCV) (n = 19), and aged control individuals with healthy retinae (n = 20). We isolated PBMCs, extracted RNA, and used microarray to investigate gene expression. Volcano plots identified statistically significant differentially expressed genes (P < 0.05) at a high magnitude (≥30% higher/lower) for GA (62 genes), neovascular AMD (41 genes), and PCV (41 genes). These clinical subtypes differed substantially across gene expression and the following pathways identified in enrichment analyses. In a subgroup analysis, we investigated presence vs. absence of subretinal fibrosis and found 826 differentially expressed genes (≥30% higher/lower, P < 0.05) with relation to mRNA splicing, endothelial migration, and interleukin-1 signaling. CONCLUSIONS: We here map the global gene transcriptome of PBMCs related to clinical subtypes of late AMD and find evidence of subtype-specific immunological involvement. Our findings provide a transcriptomic insight into the systemic immunity associated with AMD.

Relationship between soluble CD25 and gene expression in healthy individuals and patients with multiple sclerosis.

Genome wide association studies and fine mapping has established a firm link between the IL2RA gene, encoding the interleukin-2 receptor α-chain CD25, and susceptibility to multiple sclerosis (MS). We hypothesized that gene expression in peripheral blood mononuclear cells (PBMCs) from healthy controls (HCs) and MS patients are associated with IL2RA SNP rs2104286 and that gene expression levels correlate with soluble CD25 (sCD25) concentrations - that are affected by rs2104286. We used the Affymetrix Human Gene ST 1.0 microarray to analyze gene expression levels in PBMCs from 18 HCs and 51MS patients. Plasma concentrations of sCD25 were measured by ELISA in all individuals. In HCs 266 genes correlated with sCD25 with Spearman's rho≥0.707; 70 of these genes had a false discovery rate (FDR) value of q<0.05. These genes were highest expressed in cells belonging to the innate immune system. Gene-networks were focused around NFKB1, TNF, BCL6 and STAT1. Eighteen genes correlated with sCD25 with rho≥0.707 in relapsing remitting MS versus 33 in secondary progressive and 34 in primary progressive MS. None had a FDR<0.05. Thirty-eight and 23 genes were differentially expressed between rs2104286 genotype-groups in MS patients and HCs respectively, however they were not significant after FDR correction. Our study indicates that rs2104286 influences gene expression in PBMCs in HCs as shown by the high correlations with the rs2104286-affected sCD25 protein. Correlations were strongest in HCs suggesting that immunological alterations may obscure the role of the IL2RA SNP rs2104286 in established MS.

The interaction between smoking and HLA genes in multiple sclerosis: replication and refinement.

Interactions between environment and genetics may contribute to multiple sclerosis (MS) development. We investigated whether the previously observed interaction between smoking and HLA genotype in the Swedish population could be replicated, refined and extended to include other populations. We used six independent case-control studies from five different countries (Sweden, Denmark, Norway, Serbia, United States). A pooled analysis was performed for replication of previous observations (7190 cases, 8876 controls). Refined detailed analyses were carried out by combining the genetically similar populations from the Nordic studies (6265 cases, 8401 controls). In both the pooled analyses and in the combined Nordic material, interactions were observed between HLA-DRB*15 and absence of HLA-A*02 and between smoking and each of the genetic risk factors. Two way interactions were observed between each combination of the three variables, invariant over categories of the third. Further, there was also a three way interaction between the risk factors. The difference in MS risk between the extremes was considerable; smokers carrying HLA-DRB1*15 and lacking HLA-A*02 had a 13-fold increased risk compared with never smokers without these genetic risk factors (OR 12.7, 95% CI 10.8-14.9). The risk of MS associated with HLA genotypes is strongly influenced by smoking status and vice versa. Since the function of HLA molecules is to present peptide antigens to T cells, the demonstrated interactions strongly suggest that smoking alters MS risk through actions on adaptive immunity.

JC polyomavirus infection is strongly controlled by human leucocyte antigen class II variants.

JC polyomavirus (JCV) carriers with a compromised immune system, such as in HIV, or subjects on immune-modulating therapies, such as anti VLA-4 therapy may develop progressive multifocal leukoencephalopathy (PML) which is a lytic infection of oligodendrocytes in the brain. Serum antibodies to JCV mark infection occur only in 50-60% of infected individuals, and high JCV-antibody titers seem to increase the risk of developing PML. We here investigated the role of human leukocyte antigen (HLA), instrumental in immune defense in JCV antibody response. Anti-JCV antibody status, as a surrogate for JCV infection, were compared to HLA class I and II alleles in 1621 Scandinavian persons with MS and 1064 population-based Swedish controls and associations were replicated in 718 German persons with MS. HLA-alleles were determined by SNP imputation, sequence specific (SSP) kits and a reverse PCR sequence-specific oligonucleotide (PCR-SSO) method. An initial GWAS screen displayed a strong HLA class II region signal. The HLA-DRB1*15 haplotype was strongly negatively associated to JCV sero-status in Scandinavian MS cases (OR = 0.42, p = 7×10(-15)) and controls (OR = 0.53, p = 2×10(-5)). In contrast, the DQB1*06:03 haplotype was positively associated with JCV sero-status, in Scandinavian MS cases (OR = 1.63, p = 0.006), and controls (OR = 2.69, p = 1×10(-5)). The German dataset confirmed these findings (OR = 0.54, p = 1×10(-4) and OR = 1.58, p = 0.03 respectively for these haplotypes). HLA class II restricted immune responses, and hence CD4+ T cell immunity is pivotal for JCV infection control. Alleles within the HLA-DR1*15 haplotype are associated with a protective effect on JCV infection. Alleles within the DQB1*06:03 haplotype show an opposite association. These associations between JC virus antibody response and human leucocyte antigens supports the notion that CD4+ T cells are crucial in the immune defence to JCV and lays the ground for risk stratification for PML and development of therapy and prevention.

Genetic variants are major determinants of CSF antibody levels in multiple sclerosis.

Immunological hallmarks of multiple sclerosis include the production of antibodies in the central nervous system, expressed as presence of oligoclonal bands and/or an increased immunoglobulin G index-the level of immunoglobulin G in the cerebrospinal fluid compared to serum. However, the underlying differences between oligoclonal band-positive and -negative patients with multiple sclerosis and reasons for variability in immunoglobulin G index are not known. To identify genetic factors influencing the variation in the antibody levels in the cerebrospinal fluid in multiple sclerosis, we have performed a genome-wide association screen in patients collected from nine countries for two traits, presence or absence of oligoclonal bands (n = 3026) and immunoglobulin G index levels (n = 938), followed by a replication in 3891 additional patients. We replicate previously suggested association signals for oligoclonal band status in the major histocompatibility complex region for the rs9271640*A-rs6457617*G haplotype, correlated with HLA-DRB1*1501, and rs34083746*G, correlated with HLA-DQA1*0301 (P comparing two haplotypes = 8.88 × 10(-16)). Furthermore, we identify a novel association signal of rs9807334, near the ELAC1/SMAD4 genes, for oligoclonal band status (P = 8.45 × 10(-7)). The previously reported association of the immunoglobulin heavy chain locus with immunoglobulin G index reaches strong evidence for association in this data set (P = 3.79 × 10(-37)). We identify two novel associations in the major histocompatibility complex region with immunoglobulin G index: the rs9271640*A-rs6457617*G haplotype (P = 1.59 × 10(-22)), shared with oligoclonal band status, and an additional independent effect of rs6457617*G (P = 3.68 × 10(-6)). Variants identified in this study account for up to 2-fold differences in the odds of being oligoclonal band positive and 7.75% of the variation in immunoglobulin G index. Both traits are associated with clinical features of disease such as female gender, age at onset and severity. This is the largest study population so far investigated for the genetic influence on antibody levels in the cerebrospinal fluid in multiple sclerosis, including 6950 patients. We confirm that genetic factors underlie these antibody levels and identify both the major histocompatibility complex and immunoglobulin heavy chain region as major determinants.

Genetic and environmental determinants of 25-hydroxyvitamin D levels in multiple sclerosis.

BACKGROUND: Evidence is accumulating supporting a beneficial effect of vitamin D in multiple sclerosis (MS). Genome-wide association studies (GWAS) have shown significant associations between 25-hydroxyvitamin D (25(OH)D) and single nucleotide polymorphisms (SNPs) in key genes in the vitamin D metabolism. OBJECTIVE: To examine the association between 25(OH)D and six GWAS SNPs and environmental factors in 1497 MS patients. METHODS: Blood samples and lifestyle questionnaires were collected between 2009 and 2012. Genotyping of GC-, CYP2R1- and NADSYN1-SNPs was performed by TaqMan allelic discrimination (Life Technologies). RESULTS: We found significant associations between 25(OH)D and SNPs in GC (rs7041, p = 0.01 and rs2282679, p = 0.03) and CYP2R1 (rs10741657, p =1.8 × 10(-4)). Season of blood sampling (p = 2.8 × 10(-31)), sex (p = 1.9 × 10(-5)), BMI (p = 2.3 × 10(-5)), vitamin supplements (p = 7.0 × 10(-22)), and fish intake (p = 0.02) also had significant effects on 25(OH)D. CONCLUSION: In this cross-sectional study, we found significant effects of environmental factors and SNPs in GC and CYP2R1 on 25(OH)D in MS patients. Since 25(OH)D might have protective effects in MS, and vitamin D supply is a modifiable factor, it may be important to include this in the MS treatment regimen.

Genome-wide association study in a high-risk isolate for multiple sclerosis reveals associated variants in STAT3 gene.

Genetic risk for multiple sclerosis (MS) is thought to involve both common and rare risk alleles. Recent GWAS and subsequent meta-analysis have established the critical role of the HLA locus and identified new common variants associated to MS. These variants have small odds ratios (ORs) and explain only a fraction of the genetic risk. To expose potentially rare, high-impact alleles, we conducted a GWAS of 68 distantly related cases and 136 controls from a high-risk internal isolate of Finland with increased prevalence and familial occurrence of MS. The top 27 loci with p < 10(-4) were tested in 711 cases and 1029 controls from Finland, and the top two findings were validated in 3859 cases and 9110 controls from more heterogeneous populations. SNP (rs744166) within the STAT3 gene was associated to MS (p = 2.75 x 10(-10), OR 0.87, confidence interval 0.83-0.91). The protective haplotype for MS in STAT3 is a risk allele for Crohn disease, implying that STAT3 represents a shared risk locus for at least two autoimmune diseases. This study also demonstrates the potential of special isolated populations in search for variants contributing to complex traits.

Differential microRNA expression in blood in multiple sclerosis.

BACKGROUND: microRNAs (miRNAs) regulate the expression of the genome at the post-transcriptional level. They play a role in autoimmunity and inflammation, and show potential for use as therapeutic targets in many diseases. With the recent detection of miRNAs in body fluids, the possibility for using miRNAs as diagnostic biomarkers has emerged. OBJECTIVE: We assessed whether miRNAs contribute to the altered immune activation state in relapsing-remitting multiple sclerosis (RRMS) patients and investigated the possible use of miRNAs as diagnostic biomarkers in multiple sclerosis (MS). METHODS: We performed global miRNA expression profiling analysis in peripheral blood mononuclear cells (PBMCs) and selected miRNAs were measured in plasma. We detected expression of miRNAs by real-time qPCR and compared results with cytokines related to inflammation and disease activity. Selected miRNAs were analyzed in PBMC subpopulations, after isolating them by magnetic bead separation. RESULTS: We found that among validated miRNAs, let-7d correlated with the pro-inflammatory cytokine interleukin-1B. The miR-145 was 3-fold up-regulated in MS patients; its possible use as a diagnostic biomarker in PBMCs, plasma and serum was confirmed by ROC-curve analysis (Area under the curve (AUC) 0.785, p = 0.0004; 0.785, p = 0.004; 0.981, P < 0.0001, respectively). CONCLUSIONS: RRMS patients in remission had altered expression of miRNAs. We validated miR-145 as a potential diagnostic biomarker for the diagnosis of MS in blood, plasma and serum.

Neurofilament light chain levels in serum among a large mixed memory clinic cohort: Confounders and diagnostic usefulness.

INTRODUCTION: Early and accurate diagnosis of neurocognitive disorders including neurodegenerative dementia remains challenging. This study explores the impact of biological factors on serum neurofilament light chain (NfL) levels and clinical usefulness for the detection of neurocognitive disorders in a mixed memory clinic. METHODS: Serum samples and clinical data were obtained from 1188 patients who underwent diagnostic investigations for memory complaints between January 2018 and September 2019. Serum NfL was measured using single molecule array technology. RESULTS: NfL exhibited a moderate association with age, estimated glomerular filtration rate (eGFR), and Fazekas score. NfL was able to differentiate between patients with neurocognitive disorders and those without with a sensitivity and specificity of 80%. NfL could, however, not distinguish between different dementia etiologies. DISCUSSION: Serum NfL could aid early diagnostic triage by identifying patients requiring further diagnostic procedures and therefore aid in a more focused use of health-care resources.

Aerobic exercise does not affect serum neurofilament light in patients with mild Alzheimer's disease.

Introduction: Aerobic exercise has been shown to modify Alzheimer pathology in animal models, and in patients with multiple sclerosis to reduce neurofilament light (NfL), a biomarker of neurodegeneration. Objective: To investigate whether a 16-week aerobic exercise program was able to reduce serum NfL in patients with mild Alzheimer's disease (AD). Methods: This is a secondary analysis of data from the multi-center Preserving Cognition, Quality of Life, Physical Health, and Functional Ability in Alzheimer's disease: The Effect of Physical Exercise (ADEX) study. Participants were randomized to 16 weeks of moderate intensity aerobic exercise or usual care. Clinical assessment and measurement of serum NfL was done at baseline and after the intervention. Results: A total of 136 participants were included in the analysis. Groups were comparable at baseline except for APOEε4 carriership which was higher in the usual care group (75.3 versus 60.2%; p = 0.04). There was no effect of the intervention on serum NfL [intervention: baseline NfL (pg/mL) 25.76, change from baseline 0.87; usual care: baseline 27.09, change from baseline -1.16, p = 0.09]. Conclusion: The findings do not support an effect of the exercise intervention on a single measure of neurodegeneration in AD. Further studies are needed using other types and durations of exercise and other measures of neurodegeneration. Clinical trial registration: clinicaltrials.gov, identifier NCT01681602.

Cellular sources of dysregulated cytokines in relapsing-remitting multiple sclerosis.

BACKGROUND: Numerous cytokines are implicated in the immunopathogenesis of multiple sclerosis (MS), but studies are often limited to whole blood (WB) or peripheral blood mononuclear cells (PBMCs), thereby omitting important information about the cellular origin of the cytokines. Knowledge about the relation between blood and cerebrospinal fluid (CSF) cell expression of cytokines and the cellular source of CSF cytokines is even more scarce. METHODS: We studied gene expression of a broad panel of cytokines in WB from relapsing-remitting multiple sclerosis (RRMS) patients in remission and healthy controls (HCs). Subsequently we determined the gene expression of the dysregulated cytokines in isolated PBMC subsets (CD4+, CD8+T-cells, NK-cells, B-cells, monocytes and dendritic cells) from RRMS patients and HCs and in CSF-cells from RRMS patients in clinical relapse and non-inflammatory neurological controls (NIND). RESULTS: RRMS patients had increased expression of IFN-gamma (IFNG), interleukin (IL) 1-beta (IL1B), IL7, IL10, IL12A, IL15, IL23, IL27, lymphotoxin-alpha (LTA) and lymphotoxin-beta (LTB) in WB. In PBMC subsets the main sources of pro-inflammatory cytokines were T- and B-cells, whereas monocytes were the most prominent source of immunoregulatory cytokines. In CSF-cells, RRMS patients had increased expression of IFNG and CD19 and decreased expression of IL10 and CD14 compared to NINDs. CD19 expression correlated with expression of IFNG, IL7, IL12A, IL15 and LTA whereas CD14 expression correlated with IL10 expression. CONCLUSIONS: Using a systematic approach, we show that expression of pro-inflammatory cytokines in peripheral blood primarily originates from T- and B-cells, with an important exception of IFNG which is most strongly expressed by NK-cells. In CSF-cell studies, B-cells appear to be enriched in RRMS and associated with expression of pro-inflammatory cytokines; contrarily, monocytes are relatively scarce in CSF from RRMS patients and are associated with IL10 expression. Thus, our findings suggest a pathogenetic role of B-cells and an immunoregulatory role of monocytes in RRMS.

IL2RA Methylation and Gene Expression in Relation to the Multiple Sclerosis-Associated Gene Variant rs2104286 and Soluble IL-2Rα in CD8+ T Cells.

CD8+ T cells are involved in the pathogenesis of multiple sclerosis (MS). The interleukin-2 receptor α (IL-2Rα) is important for CD8+ T cell function, and single nucleotide polymorphisms (SNPs) in the IL2RA gene encoding IL-2Rα increase the risk of MS. Therefore, in isolated CD8+ T cells we investigated IL2RA gene methylation and gene expression in relation to the MS-associated IL2RA SNP rs2104286 and soluble IL-2Rα (sIL-2Rα). We have identified allele specific methylation of the CpG-site located in intron 1 that is perturbed by the rs2104286 SNP in CD8+ T cells from genotype-selected healthy subjects (HS). However, methylation of selected CpG-sites in the promotor or 5'UTR region of the IL2RA gene was neither associated with the rs2104286 SNP nor significantly correlated with IL2RA gene expression in HS. In CD8+ T cells from HS, we explored expression of immune relevant genes but observed only few associations with the rs2104286 SNP. However, we found that sIL-2Rα correlated negatively with expression of 55 immune relevant genes, including the IL-7 receptor gene, with Spearman's rho between -0.49 and -0.32. Additionally, in HS by use of flow cytometry we observed that the IL-7 receptor on naïve CD8+ T cells correlated negatively with sIL-2Rα and was downregulated in carriers of the rs2104286 MS-associated risk genotype. Collectively, our study of resting CD8+ T cells indicates that the rs2104286 SNP has a minor effect and sIL-2Rα may negatively regulate the CD8+ T cell response.

Biomarkers of systemic inflammation, soluble IL-2Rα and the multiple sclerosis-associated IL2RA SNP rs2104286 in healthy subjects and multiple sclerosis patients.

Soluble interleukin-2 (IL-2) receptor α (sIL-2Rα) antagonizes IL-2 signaling and is involved in the pathogenesis of several immune-mediated diseases including multiple sclerosis (MS). The level of sIL-2Rα is affected by the MS-associated single nucleotide polymorphism (SNP) rs2104286. By use of ELISA and electrochemiluminescence, we investigated if 26 biomarkers of systemic inflammation were associated with sIL-2Rα and rs2104286 in cohorts of healthy subjects and MS patients in serum and heparin plasma. We found that sIL-2Rα significantly correlated with the level of tumor necrosis factor-α (TNFα) (r = 0.391, p = 0.002) in healthy subjects and the association was validated in a separate cohort. Additional, in healthy subjects we confirmed a previous report indicating that C-reactive protein (CRP) correlates with sIL-2Rα (r = 0.278, p = 0.034). None of the biomarkers of systemic inflammation were significantly associated with sIL-2Rα in MS patients. Furthermore, the MS-associated SNP rs2104286 was not significantly associated with any of the biomarkers of systemic inflammation in neither healthy subjects nor MS patients. We conclude that sIL-2Rα is associated with TNFα and CRP in healthy subjects. However, further research is required to confirm the use of sIL-2Rα as biomarker of systemic inflammation as well as to assess the mechanism underlying the observed correlation between levels of sIL-2Rα and TNFα.

Multiplex assessment of cerebrospinal fluid biomarkers in multiple sclerosis.

BACKGROUND: Several roles for biomarkers in multiple sclerosis (MS) exist, including aiding in the diagnosis of MS, predicting disease activity or progression, and defining individuals who may be responsive to specific treatments. Cerebrospinal fluid (CSF) concentrations of soluble B cell maturation antigen (sBCMA) and soluble CD27 (sCD27) have been shown to be sensitive biomarkers of inflammation in MS and are thought to reflect B and T cell activity, respectively. Furthermore, chitinase 3-like 1 (CHI3L1) and soluble CD14 (sCD14) have been suggested as measures of innate immune cell activity in MS. In this study we sought to validate measurements of these CSF biomarkers of inflammation using multiplex bead-based immunoassays. METHODS: By using commercially available multiplex bead-based assays, concentrations of sBCMA, sCD27, sCD14 and CHI3L1 were determined in CSF from 22 patients with either untreated clinically isolated syndromes (CIS) or relapsing-remitting MS (RRMS), 13 patients with RRMS treated with either natalizumab or alemtuzumab, and 35 symptomatic controls (SC). RESULTS: Increased CSF concentrations of sBCMA, sCD27 and CHI3L1 were observed in untreated MS patients compared to symptomatic controls (all p < 0.001). Concentrations of sBCMA (p = 0.02) and sCD27 (p = 0.0003) were higher in treated MS patients than in SC, and levels of sBCMA (p = 0.02) and sCD27 (p = 0.01) were even higher in untreated compared to treated patients. sCD14 levels did not differ between the groups. Levels of sBCMA and sCD27 correlated strongly with each other (Spearman's rho: 0.98, p < 0.0001) as well as with the IgG index (Spearman's rho: 0.91, p < 0.0001 and 0.90, p < 0.0001, respectively). ROC curve analysis showed a high discriminatory potential for sBCMA and sCD27 with areas under the curve of 0.88 and 0.93, respectively. CONCLUSION: We confirm reports of elevated concentrations of sBCMA, sCD27 and CHI3L1 in CSF from untreated MS patients compared to SC. sBCMA and sCD27 levels were elevated in both treated and untreated MS patients compared to SC, but highest in untreated patients. Finally, CSF concentrations of sBCMA, sCD27 and the IgG index correlated strongly, suggesting that the cellular source of sCD27 and sBCMA includes memory B cells, plasmablasts and plasma cells.

Pregnancy-Induced Changes in microRNA Expression in Multiple Sclerosis.

Pregnancy affects the disease course in multiple sclerosis (MS), particularly in the third trimester, where the relapse rate is reduced by as much as two thirds. This study aimed at identifying changes in microRNA (miRNA) and immune cell phenotypes in pregnant MS patients. Discovery and validation studies to detect differentially expressed miRNAs were performed with quantitative real-time PCR on peripheral blood mononuclear cells (PBMC). Flow cytometry analysis was performed on PBMC stained with antibodies directed against surface markers of antigen presenting cells (APCs), NK-cells, NKT cells, CD4+ and CD8+ T cells and subsets of these cell types, including PDL1 and PDL2 expressing subsets. RNA was extracted from whole blood, monocytes, and NK-cells to investigate expression and correlation between regulated miRNAs and mRNAs. In total, 15 miRNAs were validated to be differentially expressed between third trimester pregnant and postpartum MS patients (Benjamini-Hochberg false discovery rate from p = 0.03-0.00004). Of these, 12 miRNAs were downregulated in pregnancy and 6 of the 15 miRNAs were altered by more than ±2-fold (+2.99- to -6.38-fold). Pregnant MS patients had a highly significant increase in the percentage of monocytes and a decrease of NK-cells and myeloid dendritic cells compared to non-pregnant MS patients. We confirm previous reports of a relative increase in CD56-bright NK-cells and a decrease in CD56-dim NK-cells in third trimester of pregnancy and report an increase in non-committed follicular helper cells. PDL1 and PDL2 expression was increased in pregnant patients together with IL10. Also, in monocytes IL10, PDL1, and PDL2 were upregulated whereas miR-1, miR-20a, miR-28, miR-95, miR-146a, miR-335, and miR-625 were downregulated between pregnant and untreated MS patients. IL10, PDL1, and PDL2 were predicted targets of MS pregnancy-changed miRNAs, further supported by their negative correlations. Additionally, previously identified pregnancy-regulated mRNAs were identified as predicted targets of the miRNAs. PDL1 and PDL2 bind PD-1 expressed on T cells with an inhibitory effect on T-cell proliferation and increase in IL10 production. These results indicate that some of the effects behind the disease-ameliorating third trimester of pregnancy might be caused by changed expression of miRNAs and immunoregulatory molecules in monocytes.