BACKGROUND:Dimethyl fumarate (DMF) alters the phenotype of circulating immune cells and causes lymphopenia in a subpopulation of treated multiple sclerosis (MS) patients.OBJECTIVE:To phenotypically characterize circulating leukocytes in DMF-treated MS patients.METHODS:Cross-sectional observational comparisons of peripheral blood from DMF-treated MS patients (n = 17 lymphopenic and n = 24 non-lymphopenic), untreated MS patients (n = 17) and healthy controls (n = 23); immunophenotyped using flow cytometry. Longitudinal samples were analyzed for 13 DMF-treated patients.RESULTS:Lymphopenic DMF-treated patients had significantly fewer circulating CD8(+) and CD4(+) T cells, CD56(dim) natural killer (NK) cells, CD19(+) B cells and plasmacytoid dendritic cells when compared to controls. CXCR3(+) and CCR6(+) expression was disproportionately reduced among CD4(+) T cells, while the proportion of T-regulatory (T-reg) cells was unchanged. DMF did not affect circulating CD56(hi) NKcells, monocytes or myeloid dendritic cells. Whether lymphopenic or not, DMF-treated patients had a lower proportion of circulating central and effector memory T cells and concomitant expansion of naïve T cells compared to the controls.CONCLUSIONS:DMF shifts the immunophenotypes of circulating T cells, causing a reduction of memory cells and a relative expansion of naïve cells, regardless of the absolute lymphocyte count. This may represent one mechanism of action of the drug. Lymphopenic patients had a disproportionate loss of CD8(+) T-cells, which may affect their immunocompetence.

Previous work demonstrated that EAE induced by recombinant human MOG was B cell-dependent. Data presented here reveal a T cell response to MOG61–85 in human rMOG-immunized B cell−/− mice not observed in WT mice. Further study revealed this peptide to be a cryptic epitope in WT mice. Co-immunization of B cell−/− mice with MOG35–55 and MOG61–85 peptides led to less severe disease compared to mice immunized with MOG35–55 alone. Disease amelioration was associated with decreased production of Interferon-γ by lymph node cells. Thus, MOG61–85 represents a protective epitope to human rMOG induced EAE in B cell−/− mice.

BACKGROUND: Neural stem cells (NSCs) have great potential in treating neurodegeneration. The self-renewal of endogenous NSCs can be increased during acute injury. We have shown that during EAE (the animal model of multiple sclerosis) chronic inflammation decreases the repair potential of subventricular zone (SVZ) NSCs. However, the specific pathways that are altered are largely unknown. OBJECTIVES: To identify mechanisms for neural stem cell failure in EAE. METHODS: We investigated the expression of STAT1 by qPCR and confocal microscope in EAE. Retrovirus STAT1-GFP to study self-renewal of NSCs. Stat1-/NSCs were analyzed in vivo and in vitro. Microarray analysis of IFN-g treated and Stat1-/NCSs with genomic pathway analysis, was used to identify new candidates and confirmed by western blot. Chromatin Immunoprecipitation (CHIP) assay and transfection of Sox9 luciferase constructs were used to refine regions of Sox9 involved in Stat1 binding. RESULTS: We report increase of STAT1 in the Sox2+ adult NSCs from SVZ of mice in the chronic and relapsing remitting models of EAE. Overexpressing STAT1 in NSCs by a Stat1 virus decreases NSCs self-renewal capacity; in contrast Stat1 -/mice exhibit an increase in proliferation and population doublings in neurospheres in vitro and an increase in the proliferation of Sox2 NSCs in vivo. Furthermore, Stat1-/NSCs showed increased self-renewal, neurogenesis, oligodendrogenesis and survival to deleterious signals. Activation of STAT1 during EAE is likely mediated by IFN-g as evidenced by increased IFN-g and not IL-17 producing cells in the CSF of EAE animals. In vitro, IFN-g was superior to IL-17 in reducing self-renewal of NSCs, inducing STAT1 phosphorylation and increased Stat1, p21Cip1, p16ink4A gene expression. Using genomic network analysis of IFN-g treated NSCs, we found that Stat1 gene network was induced by IFN-g in NSCs, and Stat1-/NSCs were resistant to the IFN-g deleterious effect. One of the increased genes in Stat1/NSCs was the core NSC transcription factor Sox9, critical for NSCs renewal and differentiation. IFN-g decreases Sox9 expression in a Stat1 dependent manner. Furthermore Sox9 upregulation associates with increased self-renewal and down-regulation with decreased self-renewal of NSCs. CHIP assay, revealed that Stat1 binds three different regions of the Sox9 promoter. Using multiple Sox9 luciferase constructs spanning the entire promoter region to refine the Sox9 region bound by Stat1, we demonstrated that Stat1 is a transcriptional repressor of Sox9 with maximum efficacy in a conserved 0.5 Kb region of the 5’ flanking region of the Sox9 promoter. CONCLUSIONS: We identify for the first time an immunological mechanism that negatively impacts NSCs, and a novel role of Stat1 as a checkpoint for self-renewal of NSCs limiting homeostatic proliferation. Thus, inactivation of Stat1 and promotion of Sox9 may offer a target to enhance neuro-regeneration.

Multiple sclerosis (MS) is a presumed autoimmune disease directed against central nervous system (CNS) myelin, in which diet and obesity are implicated as risk factors. Immune responses can be influenced by molecules produced by fat cells, called adipokines. Adiponectin is an adipokine with anti-inflammatory effects. We tested the hypothesis that adiponectin has a protective role in the EAE model for MS, that can be induced by immunization with myelin antigens or transfer of myelin-specific T lymphocytes. Adiponectin deficient (ADPKO) mice developed worse EAE with greater CNS inflammation, demyelination, and axon injury. Lymphocytes from myelin-immunized ADPKO mice proliferated more, produced higher amounts of IFN-γ, IL-17, TNF-α, IL-6, and transferred more severe EAE than wild type (WT) lymphocytes. At EAE peak, the spleen and CNS of ADPKO had fewer regulatory T (Treg) cells than WT mice and during EAE recovery, Foxp3, IL-10 and TGF-β expression levels in the CNS were reduced in ADPKO compared with WT mice. Treatment with globular adiponectin in vivo ameliorated EAE, and was associated with an increase in Treg cells. These data indicate that adiponectin is an important regulator of T-cell functions during EAE, suggesting a new avenue of investigation for MS treatment.

The role of B cells and antibody in experimental autoimmune encephalomyelitis (EAE) appears to differ based on the identity and state (protein vs. encephalitogenic peptide) of the inducing antigen and the strain of mouse utilized. The involvement of B cells in the induction of EAE by peptides of proteolipid protein (PLP) in BALB/c mice was investigated. Wild-type and B cell-deficient (B cell-/-) mice on the BALB/c background were immunized with overlapping PLP peptides, and the disease course was followed. Although incidence and onset of PLP(180-199)-induced EAE was similar in WT and B cell-/- mice, the clinical course was more severe in B cell-/- mice. During acute disease, proliferation and interferon-gamma production by lymphoid cells from both strains were similar and were elicited predominantly in response to the immunizing antigen. However, during chronic disease lymphoid cells isolated from B cell-/- mice proliferated to a greater extent and produced more interferon-gamma in response to the overlapping peptide PLP185-206 and to the smaller internal peptide PLP185-199 than did WT mice. These data suggest that B cells regulate PLP-induced EAE in BALB/c mice through control of epitope spreading.

Inducible nitric oxide synthase (NOS2) expression in the central nervous system correlates with EAE disease activity. Inhibition of NOS2 ameliorates adoptively transferred EAE, yet exacerbates actively induced EAE. Herein, the encephalitogenicity of T cells induced by immunization in the presence or absence of NOS2 was examined. Upon passive transfer, T cells from myelin oligodendrocyte glycoprotein-immunized NOS2-deficient C57BL/6 mice induced more severe EAE than T cells from wild-type mice. The heightened encephalitogenicity of NOS2−/− T cells correlated with enhanced expression of VLA-4 (CD49d) and increased production of interferon gamma and tumor necrosis factor. NO plays an important regulatory role in autoimmune T cell induction.

Effects of B cell depletion by rituximab, a monoclonal antibody to CD20, were studied in patients with relapsing MS that had not responded optimally to standard immunomodulatory therapies. Flow cytometry demonstrated reduced cerebrospinal fluid (CSF) B cells and T cells in most patients at 6 months post-treatment. ELISAs demonstrated modest reductions in serum antibodies to myelin oligodendrocyte glycoprotein and myelin basic protein in some subjects. Beta-interferon neutralizing antibodies were reduced in three subjects, but developed anew after treatment in three others, suggesting caution in considering rituximab as a means to eliminate NABs. In summary, rituximab depleted B cells from CSF at 24 weeks after initial treatment, and this B cell depletion was associated with a reduction in CSF T cells as well.

Myelin loss and axonal damage are both observed in white matter injuries. Each may have significant impact on the long-term disability of patients. Currently, there does not exist a noninvasive biological marker that enables differentiation between myelin and axonal injury. We describe herein the use of magnetic resonance diffusion tensor imaging (DTI) to quantify the effect of dysmyelination on water directional diffusivities in brains of shiverer mice in vivo. The principal diffusion eigenvalues of eight axonal fiber tracts that can be identified with certainty on DTI maps were measured. The water diffusivity perpendicular to axonal fiber tracts, lambda(perpendicular), was significantly higher in shiverer mice compared with age-matched controls, reflecting the lack of myelin and the increased freedom of cross-fiber diffusion in white matter. The water diffusivity parallel to axonal fiber tracts, lambda(parallel), was not different, which is consistent with the presence of intact axons. It is clear that dysmyelination alone does not impact lambda(parallel). The presence of intact axons in the setting of incomplete myelination was confirmed by electron microscopy. Although further validation is still needed, our finding suggests that changes in lambda(perpendicular) and lambda(parallel) may potentially be used to differentiate myelin loss versus axonal injury.

The role of B cells and antibody in the pathogenesis of experimental autoimmune encephalomyelitis (EAE) remains controversial. We previously demonstrated that B cells are required for EAE to be induced by the 120-amino acid extracellular domain of myelin oligodendrocyte glycoprotein (MOG). In the present study, the role of B cells in MOG-induced EAE was further characterized. Passive transfer of activated B cells or serum from MOG-primed wild-type (WT) mice was found to reconstitute the ability for clinical and histological EAE to be induced in MOG-immunized B cell-deficient mice. MOG-induced EAE did not occur with transfer of B cells that had been nonspecifically activated by lipopolysaccharide or isolated from naive or myelin basic protein (MBP)-primed WT mice. Likewise, MOG-primed serum, but not naive serum or serum from MBP-, Hen egg Iysozyme-, or MOG(35-55)-primed mice, led to EAE in B cell(-/-) animals. While both MOG-primed B cells and serum reconstituted the ability for disease induction, MOG-primed serum was much more efficient, leading to clinical and histological EAE similar to that seen in the WT. Injection of MOG serum into healthy B cell(-/-) mice 30 days after MOG immunization led to rapid appearance of clinical signs and CNS inflammation, indicating that an antigen-specific factor is necessary for initiation of CNS inflammation, and not just demyelination. These data strongly suggest that MOG-specific antibody is critical to the initiation of MOG-induced murine EAE.

It was previously shown that BALB/c mice were susceptible to experimental autoimmune encephalomyelitis induced by immunization with proteolipid protein (PLP). To determine the encephalitogenic epitopes of PLP in BALB/c mice, mice were immunized with successively smaller pools of 20-mer peptides spanning the PLP molecule from amino acid 30 to amino acid 206. Immunization with PLP180–199 resulted in clinical EAE in 9/15 mice (mean max clinical score of 3.3), and immunization with PLP185–206 induced clinical EAE in 7/21 BALB/c mice (mean maximum score of 3.7). No relapses in disease were observed. No EAE was observed in BALB/c mice immunized with PLP185–199 (n=15), PLP178–191 (n=13) or other regions of PLP (n=15). Passive transfer of PLP180–199-primed lymph node cells into naı̈ve BALB/c mice resulted in EAE (2/2 mice, max score of 4.0). One-micron toluidine blue stained sections from the spinal cord of EAE-affected BALB/c mice revealed features typical of EAE in other strains, including mononuclear cell infiltration, myelin loss, and axonal loss.