Jeppe Romme Christensen

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.

Natalizumab in progressive MS: Results of an open-label, phase 2A, proof-of-concept trial

Objective: Natalizumab inhibits the migration of systemic immune cells to the CNS and may be beneficial in progressive multiple sclerosis (MS). The objective of the study was to examine the effects of natalizumab in progressive MS. Methods: In an open-label phase 2A study, 24 patients with progressive MS were included to receive natalizumab treatment for 60 weeks. Response to natalizumab was assessed in CSF and MRI studies. The primary endpoint was change in CSF osteopontin, a biomarker of intrathecal inflammation, from baseline to week 60. Results: Seventeen patients completed the study. No new safety issues were encountered. CSF osteopontin decreased by 65 ng/mL (95% confidence interval 34–96 ng/mL; p = 0.0004) from baseline to week 60 in conjunction with decreases in other CSF biomarkers of inflammation, axonal damage, and demyelination. Magnetization transfer ratio increased in both cortical gray and normal-appearing white matter and correlated with decreases in CSF neurofilament light chain. Conclusions: Natalizumab treatment of progressive MS reduces intrathecal inflammation and tissue damage, supporting a beneficial effect of natalizumab treatment in progressive MS and suggesting that systemic inflammation contributes to the pathogenesis. Moreover, the study establishes the feasibility of using CSF biomarkers in proof-of-concept trials, allowing a low number of participants and short study duration. Classification of evidence: This study provides Class IV evidence that in patients with progressive MS, natalizumab reduces biomarkers of intrathecal inflammation.

CSF inflammation and axonal damage are increased and correlate in progressive multiple sclerosis.

Background: The mechanism underlying disease progression in progressive multiple sclerosis (MS) is uncertain. Pathological studies found widespread inflammation in progressive MS brains correlating with disease progression and axonal damage. Objectives: To study cerebrospinal fluid (CSF) biomarkers and clarify whether inflammation and axonal damage are associated in progressive MS. Methods: Using enzyme-linked immunosorbent assay (ELISA), we analysed CSF from 40 secondary progressive (SPMS), 21 primary progressive (PPMS), and 36 relapsing–remitting (RRMS) and 20 non-inflammatory neurological disease (NIND) patients. Twenty-two of the SPMS patients participated in an MBP8298 peptide clinical trial and had CSF follow-up after one year. Results: Compared to NIND patients, inflammatory biomarkers osteopontin and matrix metalloproteinase-9 (MMP9) were increased in all MS patients while CXCL13 was increased in RRMS and SPMS patients. Biomarkers of axonal damage (NFL) and demyelination (MBP) were increased in all MS patients. In progressive MS patients CSF levels of osteopontin and CXCL13 correlated with NFL while osteopontin and MMP9 correlated with MBP. MBP8298 treatment did not affect the levels of the biomarkers after one year of treatment. All biomarkers were continuously increased after one year of follow-up except MBP, which decreased. Conclusion: CSF biomarkers of inflammation, axonal damage and demyelination are continuously increased in progressive MS patients and correlate. These findings parallel pathology studies, emphasise a relationship between inflammation, axonal damage and demyelination and support the use of CSF biomarkers in progressive MS clinical trials.

Cellular sources of dysregulated cytokines in relapsing-remitting multiple sclerosis

BackgroundNumerous 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.MethodsWe 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).ResultsRRMS 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.ConclusionsUsing 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.

Effect of natalizumab on circulating CD4+ T-cells in multiple sclerosis.

In multiple sclerosis (MS), treatment with the monoclonal antibody natalizumab effectively reduces the formation of acute lesions in the central nervous system (CNS). Natalizumab binds the integrin very late antigen (VLA)-4, expressed on the surface of immune cells, and inhibits VLA-4 dependent transmigration of circulating immune-cells across the vascular endothelium into the CNS. Recent studies suggested that natalizumab treated MS patients have an increased T-cell pool in the blood compartment which may be selectively enriched in activated T-cells. Proposed causes are sequestration of activated T-cells due to reduced extravasation of activated and pro-inflammatory T-cells or due to induction of VLA-4 mediated co-stimulatory signals by natalizumab. In this study we examined how natalizumab treatment altered the distribution of effector and memory T-cell subsets in the blood compartment and if T-cells in general or myelin-reactive T-cells in particular showed signs of increased immune activation. Furthermore we examined the effects of natalizumab on CD4+ T-cell responses to myelin in vitro. Natalizumab-treated MS patients had significantly increased numbers of effector-memory T-cells in the blood. In T-cells from natalizumab-treated MS patients, the expression of TNF-α mRNA was increased whereas the expression of fourteen other effector cytokines or transcription factors was unchanged. Natalizumab-treated MS patients had significantly decreased expression of the co-stimulatory molecule CD134 on CD4+CD26HIGH T-cells, in blood, and natalizumab decreased the expression of CD134 on MBP-reactive CD26HIGHCD4+ T-cells in vitro. Otherwise CD4+ T-cells from natalizumab-treated and untreated MS patients showed similar responses to MBP. In conclusion natalizumab treatment selectively increased the effector memory T-cell pool but not the activation state of T-cells in the blood compartment. Myelin-reactive T-cells were not selectively increased in natalizumab treated 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.

A common representation of spatial features drives action and perception: grasping and judging object features within trials.

Spatial features of an object can be specified using two different response types: either by use of symbols or motorically by directly acting upon the object. Is this response dichotomy reflected in a dual representation of the visual world: one for perception and one for action? Previously, symbolic and motoric responses, specifying location, has been shown to rely on a common representation. What about more elaborate features such as length and orientation? Here we show that when motoric and symbolic responses are made within the same trial, the probability of making the same symbolic and motoric response is well above chance for both length and orientation. This suggests that motoric and symbolic responses to length and orientation are driven by a common representation. We also show that, for both response types, the spatial features of an object are processed independently. This finding of matching object-processing characteristics is also in agreement with the idea of a common representation driving both response types.

Lipocalin-2 is increased in progressive multiple sclerosis and inhibits remyelination

Objective: We aimed to examine the regulation of lipocalin-2 (LCN2) in multiple sclerosis (MS) and its potential functional relevance with regard to myelination and neurodegeneration. Methods: We determined LCN2 levels in 3 different studies: (1) in CSF and plasma from a case-control study comparing patients with MS (n = 147) with controls (n = 50) and patients with relapsing-remitting MS (n = 75) with patients with progressive MS (n = 72); (2) in CSF and brain tissue microdialysates from a case series of 7 patients with progressive MS; and (3) in CSF at baseline and 60 weeks after natalizumab treatment in a cohort study of 17 patients with progressive MS. Correlation to neurofilament light, a marker of neuroaxonal injury, was tested. The effect of LCN2 on myelination and neurodegeneration was studied in a rat in vitro neuroglial cell coculture model. Results: Intrathecal production of LCN2 was increased predominantly in patients with progressive MS (p < 0.005 vs relapsing-remitting MS) and displayed a positive correlation to neurofilament light (p = 0.005). Levels of LCN2 in brain microdialysates were severalfold higher than in the CSF, suggesting local production in progressive MS. Treatment with natalizumab in progressive MS reduced LCN2 levels an average of 13% (p < 0.0001). LCN2 was found to inhibit remyelination in a dose-dependent manner in vitro. Conclusions: LCN2 production is predominantly increased in progressive MS. Although this moderate increase does not support the use of LCN2 as a biomarker, the correlation to neurofilament light and the inhibitory effect on remyelination suggest that LCN2 might contribute to neurodegeneration through myelination-dependent pathways.