Lack of adiponectin leads to increased lymphocyte activation and increased disease severity in a mouse model of multiple sclerosis.

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-ß 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.

NOS2 regulates cytokine production and VLA-4 expression in experimental autoimmune encephalomyelitis.

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.

Rituximab reduces B cells and T cells in cerebrospinal fluid of multiple sclerosis patients.

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.

Amelioration of EAE by a cryptic epitope of myelin oligodendrocyte glycoprotein.

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.

B cells limit epitope spreading and reduce severity of EAE induced with PLP peptide in BALB/c mice.

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.

Critical role of antigen-specific antibody in experimental autoimmune encephalomyelitis induced by recombinant myelin oligodendrocyte glycoprotein.

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 naïve or myelin basic protein (MBP)-primed WT mice. Likewise, MOG-primed serum, but not naive serum or serum from MBP-, Hen egg lysozyme-, 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.

Identification of the encephalitogenic epitopes of CNS proteolipid protein in BALB/c mice.

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 PLP(180-199) resulted in clinical EAE in 9/15 mice (mean max clinical score of 3.3), and immunization with PLP(185-206) 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 PLP(185-199) (n=15), PLP(178-191) (n=13) or other regions of PLP (n=15). Passive transfer of PLP(180-199)-primed lymph node cells into nai;ve 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.

Dysmyelination revealed through MRI as increased radial (but unchanged axial) diffusion of water.

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.