Rikke Ratzer

Systemic inflammation in progressive multiple sclerosis involves follicular T-helper, Th17- and activated B-cells and correlates with progression.

Pathology studies of progressive multiple sclerosis (MS) indicate a major role of inflammation including Th17-cells and meningeal inflammation with ectopic lymphoid follicles, B-cells and plasma cells, the latter indicating a possible role of the newly identified subset of follicular T-helper (TFH) cells. Although previous studies reported increased systemic inflammation in progressive MS it remains unclear whether systemic inflammation contributes to disease progression and intrathecal inflammation. This study aimed to investigate systemic inflammation in progressive MS and its relationship with disease progression, using flow cytometry and gene expression analysis of CD4+ and CD8+T-cells, B-cells, monocytes and dendritic cells. Furthermore, gene expression of cerebrospinal fluid cells was studied. Flow cytometry studies revealed increased frequencies of ICOS+TFH-cells in peripheral blood from relapsing-remitting (RRMS) and secondary progressive (SPMS) MS patients. All MS subtypes had decreased frequencies of Th1 TFH-cells, while primary progressive (PPMS) MS patients had increased frequency of Th17 TFH-cells. The Th17-subset, interleukin-23-receptor+CD4+T-cells, was significantly increased in PPMS and SPMS. In the analysis of B-cells, we found a significant increase of plasmablasts and DC-SIGN+ and CD83+B-cells in SPMS. ICOS+TFH-cells and DC-SIGN+B-cells correlated with disease progression in SPMS patients. Gene expression analysis of peripheral blood cell subsets substantiated the flow cytometry findings by demonstrating increased expression of IL21, IL21R and ICOS in CD4+T-cells in progressive MS. Cerebrospinal fluid cells from RRMS and progressive MS (pooled SPMS and PPMS patients) had increased expression of TFH-cell and plasmablast markers. In conclusion, this study is the first to demonstrate the potential involvement of activated TFH-cells in MS. The increased frequencies of Th17-cells, activated TFH- and B-cells parallel findings from pathology studies which, along with the correlation between activated TFH- and B-cells and disease progression, suggest a pathogenic role of systemic inflammation in progressive MS. These observations may have implications for the treatment of progressive MS.

High-dose erythropoietin in patients with progressive multiple sclerosis: A randomized, placebo-controlled, phase 2 trial

Background: Erythropoietin (EPO) is a part of an endogenous neuroprotective system in the brain and may address pathophysiological mechanisms in progressive multiple sclerosis (MS). Objective: To evaluate a treatment effect of EPO on progressive MS. Methods: This was a single-center, randomized, double-blind, placebo-controlled phase 2 trial, in which 52 patients with secondary or primary progressive MS were allocated to treatment with recombinant EPO (48,000 IU) or placebo, administered intravenously 17 times during 24 weeks. Patients had an Expanded Disability Status Score (EDSS) from 4 to 6.5 and clinical progression without relapses in the 2 preceding years. The primary outcome was the change in a composite measure of maximum gait distance, hand dexterity, and cognition from baseline to 24 weeks. Results: A total of 50 patients completed the study. Venesection was performed often but no thromboembolic events occurred. We found no difference in the primary outcome between the EPO and the placebo group using the intention-to-treat principle (p = 0.22). None of the secondary outcomes, neither clinical nor magnetic resonance imaging (MRI) measures showed any significant differences. Conclusion: This study provides class II evidence that treatment with high-dose EPO is not an effective treatment in patients with moderately advanced progressive MS.

Endogenous Interferon-β-Inducible Gene Expression and Interferon-β-Treatment Are Associated with Reduced T Cell Responses to Myelin Basic Protein in Multiple Sclerosis

Autoreactive CD4+ T-cells are considered to play a major role in the pathogenesis of multiple sclerosis. In experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, exogenous and endogenous type I interferons restrict disease severity. Recombinant interferon-β is used for treatment of multiple sclerosis, and some untreated multiple sclerosis patients have increased expression levels of type I interferon-inducible genes in immune cells. The role of endogenous type I interferons in multiple sclerosis is controversial: some studies found an association of high expression levels of interferon-β-inducible genes with an increased expression of interleukin-10 and a milder disease course in untreated multiple sclerosis patients, whereas other studies reported an association with a poor response to treatment with interferon-β. In the present study, we found that untreated multiple sclerosis patients with an increased expression of interferon-β-inducible genes in peripheral blood mononuclear cells and interferon-β-treated multiple sclerosis patients had decreased CD4+ T-cell reactivity to the autoantigen myelin basic protein ex vivo. Interferon-β-treated multiple sclerosis patients had increased IL10 and IL27 gene expression levels in monocytes in vivo. In vitro, neutralization of interleukin-10 and monocyte depletion increased CD4+ T-cell reactivity to myelin basic protein while interleukin-10, in the presence or absence of monocytes, inhibited CD4+ T-cell reactivity to myelin basic protein. Our findings suggest that spontaneous expression of interferon-β-inducible genes in peripheral blood mononuclear cells from untreated multiple sclerosis patients and treatment with interferon-β are associated with reduced myelin basic protein-induced T-cell responses. Reduced myelin basic protein-induced CD4+ T-cell autoreactivity in interferon-β-treated multiple sclerosis patients may be mediated by monocyte-derived interleukin-10.

Monthly oral methylprednisolone pulse treatment in progressive multiple sclerosis

Background: There is a large unmet need for treatments for patients with progressive multiple sclerosis (MS). Phase 2 studies with cerebrospinal fluid (CSF) biomarker outcomes may be well suited for the initial evaluation of efficacious treatments. Objective: To evaluate the effect of monthly oral methylprednisolone pulse treatment on intrathecal inflammation in progressive MS. Methods: In this open-label phase 2A study, 15 primary progressive and 15 secondary progressive MS patients received oral methylprednisolone pulse treatment for 60 weeks. Primary outcome was changes in CSF concentrations of osteopontin. Secondary outcomes were other CSF biomarkers of inflammation, axonal damage and demyelination; clinical scores; magnetic resonance imaging measures of disease activity, magnetization transfer ratio (MTR) and diffusion tensor imaging (DTI); motor evoked potentials; and bone density scans. Results: We found no change in the CSF concentration of osteopontin, but we observed significant improvement in clinical scores, MTR, DTI and some secondary CSF outcome measures. Adverse events were well-known side effects to methylprednisolone. Conclusion: Monthly methylprednisolone pulse treatment was safe, but had no effect on the primary outcome. However, improvements in secondary clinical and MRI outcome measures suggest that this treatment regimen may have a beneficial effect in progressive MS.

Smoking reduces circulating CD26hiCD161hi MAIT cells in healthy individuals and patients with multiple sclerosis

Upon chronic cigarette smoke exposure, inhaled antigens and irritants cause altered lung immune homeostasis. Circulating immune cells are affected, and smoking is associated with an increased risk of developing various disorders, including multiple sclerosis (MS). This study was conducted to determine the impact of smoking on circulating immune cell subsets. Furthermore, we determined whether any smoking‐associated changes were related to MS. With the use of flow cytometry, CFSE assays, and ELISpot assays, we analyzed circulating immune cell phenotypes and quantified antigen‐induced proliferation and cytokine secretion in smokers and nonsmokers in a cohort of 100 healthy individuals (HI). In addition, we analyzed immune cell subsets associated with smoking in 2 independent cohorts of patients with MS. In HI smokers compared with nonsmokers, we found increased blood cell counts of granulocytes, monocytes, and lymphocytes. These cells were not more proinflammatory, autoreactive, or EBV reactive compared with cells from nonsmokers. Phenotypic differences were seen in plasmacytoid dendritic cells (pDCs) and CD8+ T cells as higher percentages of ICOS ligand (ICOSL)+ pDCs and lower percentages of CD26hiCD161hi CD8+ T cells and CCR6+ CD8+ T cells in smokers compared with nonsmokers. In supplemental analyses, we showed that CD26hiCD161hi CD8+ T cells were mainly mucosal‐associated invariant T cells (MAITs). Comparable frequencies of ICOSL+ pDCs, CCR6+ CD8+ T cells, and CD26hiCD161hi CD8+ T cells were found between HI and MS patients who were nonsmokers. Our findings suggest general proinflammatory effects from smoking combined with skewing of specific cell populations in HI and MS patients. The function of these cell populations needs further investigation.

Immunological effects of methylprednisolone pulse treatment in progressive multiple sclerosis.

OBJECTIVE To investigate the effect of monthly oral methylprednisolone pulse treatment in progressive MS. METHODS 30 progressive MS patients were treated with oral methylprednisolone every month. Peripheral blood mononuclear cells were analyzed by flow cytometry. RESULTS Out of 102 leukocyte phenotypes investigated, 25 changed at nominal significance from baseline to week 12 (p<0.05). After correction for multiple comparisons, we found 5 subpopulations that changed compared to baseline. No pattern were suggesting modulation of Th17 or TFH cells. CONCLUSION Methylprednisolone pulse treatment has some effects on circulating immune cells but does not modulate markers of Th17 and TFH cell activity in progressive MS.

Defining active progressive multiple sclerosis

Background: It is unknown whether disease activity according to consensus criteria (magnetic resonance imaging activity or clinical relapses) associate with cerebrospinal fluid (CSF) changes in progressive multiple sclerosis (MS). Objective: To compare CSF biomarkers in active and inactive progressive MS according to consensus criteria. Methods: Neurofilament light chain (NFL), myelin basic protein (MBP), IgG-index, chitinase-3-like-1 (CHI3L1), matrix metalloproteinase-9 (MMP-9), chemokine CXCL13, terminal complement complex, leukocyte counts and nitric oxide metabolites were measured in primary (n = 26) and secondary progressive MS (n = 26) and healthy controls (n = 24). Results: Progressive MS patients had higher CSF cell counts, IgG-index, CHI3L1, MMP-9, CXCL13, NFL and MBP concentrations. Active patients were younger and had higher NFL, CXCL13 and MMP-9 concentrations than inactive patients. Patients with active disease according to consensus criteria or detectable CXCL13 or MMP-9 in CSF were defined as having combined active progressive MS. These patients had increased CSF cell counts, IgG-index and MBP, NFL and CHI3L1 concentrations. Combined inactive patients only had increased IgG-index and MBP concentrations. Conclusion: Patients with combined active progressive MS show evidence of inflammation, demyelination and neuronal/axonal damage, whereas the remaining patients mainly show evidence of active demyelination. This challenges the idea that neurodegeneration independent of inflammation is crucial in disease progression.

Characterization of naïve, memory and effector T cells in progressive multiple sclerosis

We characterized naïve, central memory (CM), effector memory (EM) and terminally differentiated effector memory (TEMRA) CD4+ and CD8+ T cells and their expression of CD49d and CD26 in peripheral blood in patients with multiple sclerosis (MS) and healthy controls. CD26+ CD28+ CD4+ TEMRA T cells were increased in all subtypes of MS, and CD26+ CD28+ CD8+ TEMRA T cells were increased in relapsing-remitting and secondary progressive MS. Conversely, in progressive MS, CD49d+ CM T cells were decreased and natalizumab increased the circulating number of all six subsets but reduced the frequency of most subsets expressing CD49d and CD26.

Progressive multiple sclerosis, cognitive function, and quality of life

Patients with progressive multiple sclerosis (MS) often have cognitive impairment in addition to physical impairment. The burden of cognitive and physical impairment progresses over time, and may be major determinants of quality of life. The aim of this study was to assess to which degree quality of life correlates with physical and cognitive function in progressive MS.

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

HighlightsWe confirm that the IL2RA gene SNP rs2104286 affects sCD25 in HCs and MS patients.Levels of sCD25 correlate with gene expression in PBMCs from HCs.Fewer genes correlate with sCD25 in PBMCs from MS subtypes.The sCD25‐correlating genes in HCs are associated with innate immune cell subsets.The sCD25‐correlating genes in HCs are connected to NFKB1, TNF, BCL6 and STAT1. &NA; Genome wide association studies and fine mapping has established a firm link between the IL2RA gene, encoding the interleukin‐2 receptor &agr;‐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 51 MS patients. Plasma concentrations of sCD25 were measured by ELISA in all individuals. In HCs 266 genes correlated with sCD25 with Spearmans 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.