Suppression of experimental autoimmune encephalomyelitis by dermatan sulfate

The effect of dermatan sulfate (DS) on the treatment of Lewis rats with experimental autoimmune encephalomyelitis (EAE) was examined. DS, a sulfated glycosaminoglycan, has been reported to exhibit anticoagulant and fibrinolytic activities. DS treatment (50 mg/kg/day) facilitates recovery from the clinical manifestations of EAE. In this study, the fibrinolytic activity was higher in DS-treated rats than in saline-treated rats. Although the degree of perivascular mononuclear cell infiltration in the spinal cord was not suppressed in DS-treated rats compared to that in saline-treated rats, perivascular fibrin deposition was markedly suppressed in DS-treated rats. These findings suggest that DS would act as an effective therapeutic agent for EAE by preventing fibrin deposition.     

Antibodies specific for VB8 receptor peptide suppress experimental autoimmune encephalomyelitis.

Recent studies from our laboratory have shown, for the first time, that a synthetic peptide from that TCR VB chain used preferentially by encephalitogenic T cells induced the formation of protective, MHC class I-restricted T cells and prevented the development of EAE in Lewis rats. In this report we 1) demonstrate that immunization with the TCR-VB8-39-59 peptide generated peptide-specific antibodies that protect against experimental autoimmune encephalomyelitis induced by either of the two distinct encephalitogenic epitopes of basic protein, and 2) characterize the production and biologic functions of rat and rabbit antibody responses to the TCR peptide. The antibodies in both species increased in titer over time, were highly specific for the immunogen by direct reaction and inhibition assays, stained only VB8+ T cells, and suppressed clinical signs and to lesser extent the number of histologic lesions of experimental autoimmune encephalomyelitis mediated by VB8+ T cells. Coupled with our previous work, these results indicate that both humoral and cellular responses to the TCR-VB8-39-59 peptide can contribute independent immunoregulatory effects on encephalitogenic T lymphocytes that use common V region genes in response to epitopes of myelin basic protein.     

Peripheral T cells are the therapeutic targets of glucocorticoids in experimental autoimmune encephalomyelitis

High-dose glucocorticoid (GC) therapy is widely used to treat multiple sclerosis (MS), but the underlying mechanisms remain debatable. In this study, we investigated the impact of GC administration on experimental autoimmune encephalomyelitis using different GC receptor (GR)-deficient mutants. Heterozygous GR knockout mice were less sensitive to dexamethasone therapy, indicating that the expression level of the receptor determines therapeutic efficacy. Mice reconstituted with homozygous GR knockout fetal liver cells showed an earlier onset of the disease and were largely refractory to GC treatment, indicating that the GR in hematopoietic cells is essential for the beneficial effects of endogenous GCs and dexamethasone. Using cell-type specific GR-deficient mice, we could demonstrate that GCs mainly act on T cells, while modulation of macrophage function was largely dispensable in this context. The therapeutic effects were achieved through induction of apoptosis and down-regulation of cell adhesion molecules in peripheral T(H)17 and bystander T cells, while similar effects were not observed within the spinal cord. In addition, dexamethasone inhibited T cell migration into the CNS, confirming that peripheral but not CNS-residing T lymphocytes are the essential targets of GCs. Collectively, our findings reveal a highly selective mechanism of GC action in experimental autoimmune encephalomyelitis and presumably multiple sclerosis.     

CNS neurotrophins are biologically active and expressed by multiple cell types

Neutrotrophins are increasingly appreciated as potential modulators of neuronal function in the adult central nervous system (CNS). To describe the neurotrophin environment within the adult CNS, mRNA and protein expression patterns of neurotrophins-3 and –4 and of brain-derived neurotrophin were investigated in adult rat spinal cord and brain. Co-localization studies with CNS cell type-specific markers demonstrates that multiple cell types, including both neurons and glia, express these neurotrophins in the normal adult CNS. Although widely implicated in important CNS functions such as synaptic plasticity, biological activity of endogenous CNS neurotrophins has not been directly demonstrated. With a sensitive neurite outgrowth bioassay we demonstrate that CNS neurotrophins elicit neurite outgrowth and are biologically active. Moreover, antibody-blocking studies suggest that these three neurotrophins may comprise the bulk of adult CNS neurotrophic activity.     

Intercellular adhesion molecule-1 expression is required on multiple cell types for the development of experimental autoimmune encephalomyelitis

Many members of the Ig superfamily of adhesion molecules, such as ICAM-1 and VCAM-1, have been implicated in the pathogenesis of multiple sclerosis. Although it is well-established that VCAM-1/VLA-4 interactions can play important roles in mediating CNS inflammatory events in multiple sclerosis patients and during the development of experimental allergic encephalomyelitis (EAE), the contributions of ICAM-1 are poorly understood. This is due in large part to conflicting results from Ab inhibition studies and the observation of exacerbated EAE in ICAM-1 mutant mice that express a restricted set of ICAM-1 isoforms. To determine ICAM-1-mediated mechanisms in EAE, we analyzed ICAM-1 null mutant mice (ICAM-1(null)), which express no ICAM-1 isoforms. ICAM-1(null) mice had significantly attenuated EAE characterized by markedly reduced spinal cord T cell infiltration and IFN-gamma production by these cells. Adoptive transfer of Ag-restimulated T cells from wild-type to ICAM-1(null) mice or transfer of ICAM-1(null) Ag-restimulated T cells to control mice failed to induce EAE. ICAM-1(null) T cells also showed reduced proliferative capacity and substantially reduced levels of IFN-gamma, TNF-alpha, IL-4, IL-10, and IL-12 compared with that of control T cells following myelin oligodendrocyte glycoprotein 35-55 restimulation in vitro. Our results indicate that ICAM-1 expression is critical on T cells and other cell types for the development of demyelinating disease and suggest that expression of VCAM-1 and other adhesion molecules cannot fully compensate for the loss of ICAM-1 during EAE development.     

Regulatory T cell vaccination without autoantigen protects against experimental autoimmune encephalomyelitis

Regulatory T (T(reg)) cells show promise for treating autoimmune diseases, but their induction to elevated potency has been problematic when the most optimally derived cells are from diseased animals. To circumvent reliance on autoantigen-reactive T(reg) cells, stimulation to myelin-independent Ags may offer a viable alternative while maintaining potency to treat experimental autoimmune encephalomyelitis (EAE). The experimental Salmonella vaccine expressing colonization factor Ag I possesses anti-inflammatory properties and, when applied therapeutically, reduces further development of EAE in SJL mice. To ascertain T(reg) cell dependency, a kinetic analysis was performed showing increased levels of FoxP3(+)CD25(+)CD4(+) T cells. Inactivation of these T(reg) cells resulted in loss of protection. Adoptive transfer of the vaccine-induced T(reg) cells protected mice against EAE with greater potency than naive or Salmonella vector-induced T(reg) cells, and cytokine analysis revealed enhanced production of TGF-beta, not IL-10. The development of these T(reg) cells in conjunction with immune deviation by Th2 cells optimally induced protective T(reg) cells when compared those induced in the absence of Th2 cells. These data show that T(reg) cells can be induced to high potency to non-disease-inducing Ags using a bacterial vaccine.     

ERK1-deficient mice show normal T cell effector function and are highly susceptible to experimental autoimmune encephalomyelitis

T cell activation engages multiple intracellular signaling cascades, including the ERK1/2 (p44/p42) pathway. It has been suggested that ERKs integrate TCR signal strength, and are important for thymocyte development and positive selection. However, the requirement of ERKs for the effector functions of peripheral mature T cells and, specifically, for T cell-mediated autoimmunity has not been established. Moreover, the specific requirements for ERK1 vs ERK2 in T cells have not been resolved. Therefore, we investigated the role of ERK1 in T cell immunity to foreign and self Ags and in the induction of experimental autoimmune encephalomyelitis. The results show that in ERK1-deficient (ERK1-/-) mice, the priming, proliferation, and cytokine secretion of T cells to the self Ag myelin oligodendrocyte glycoprotein peptide 35-55 and to the prototypic foreign Ag OVA are not impaired as compared with wild-type mice. Furthermore, ERK1-/- mice are highly susceptible to experimental autoimmune encephalomyelitis induced with myelin oligodendrocyte glycoprotein peptide 35-55. Finally, thymocyte development and mitogen-induced proliferation were not impaired in ERK1-/- mice on the inbred 129 Sv and C57BL/6 backgrounds. Collectively, the data show that ERK1 is not critical for the function of peripheral T cells in the response to self and foreign Ags and in T cell-mediated autoimmunity, and suggest that its loss can be compensated by ERK2.     

Identification of differentially expressed proteins in experimental autoimmune encephalomyelitis (EAE) by proteomic analysis of the spinal cord

The present study used isobaric tags for relative and absolute quantitation (iTRAQ) to identify novel targets in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. The expression of 41 proteins was significantly altered in the inflamed spinal cord. Twenty of these are implicated in EAE for the first time and many have previously been shown to play a role in antigen processing, inflammation, neuroprotection, or neurodegeneration.     

Impaired autoimmune T helper 17 cell responses following DNA vaccination against rat experimental autoimmune encephalomyelitis

Background

We have previously shown that vaccination with DNA encoding the encephalitogenic peptide myelin oligodendrocyte glycoprotein (MOG)_91–108 (pMOG) suppresses MOG_91–108-induced rat Experimental Autoimmune Encephalomyelitis (EAE), a model for human Multiple Sclerosis (MS). The suppressive effect of pMOG is dependent on inclusion of CpG DNA in the plasmid backbone and is associated with early induction of Interferon (IFN)-β.

Principal Findings

In this study we examined the mechanisms underlying pMOG-induced protection. We found that in the DNA vaccinated cohort proinflammatory Interleukin (IL)-17 and IL-21 responses were dramatically reduced compared to in the control group, but that the expression of Foxp3 and Tumor Growth Factor (TGF)-β1, which are associated with regulatory T cells, was not enhanced. Moreover, genes associated with Type I IFNs were upregulated. To delineate the role of IFN-β in the protective mechanism we employed short interfering RNA (siRNA) to IFN-β in the DNA vaccine. SiRNA to IFN-β completely abrogated the protective effects of the vaccine, demonstrating that a local early elaboration of IFN-β is important for EAE protection. IL-17 responses comparable to those in control rats developed in rats injected with the IFN-β-silencing DNA vaccine.

Conclusions

We herein demonstrate that DNA vaccination protects from proinflammatory Th17 cell responses during induction of EAE. The mechanism involves IFN-β as IL-17 responses are rescued by silencing of IFN-β during DNA vaccination.     

CD8+ T cells specific for cryptic apoptosis-associated epitopes exacerbate experimental autoimmune encephalomyelitis

The autoimmune immunopathology occurring in multiple sclerosis (MS) is sustained by myelin-specific and -nonspecific CD8⁺ T cells. We have previously shown that, in MS, activated T cells undergoing apoptosis induce a CD8⁺ T cell response directed against antigens that are unveiled during the apoptotic process, namely caspase-cleaved structural proteins such as non-muscle myosin and vimentin. Here, we have explored in vivo the development and the function of the immune responses to cryptic apoptosis-associated epitopes (AEs) in a well-established mouse model of MS, experimental autoimmune encephalomyelitis (EAE), through a combination of immunization approaches, multiparametric flow cytometry, and functional assays. First, we confirmed that this model recapitulated the main findings observed in MS patients, namely that apoptotic T cells and effector/memory AE-specific CD8⁺ T cells accumulate in the central nervous system of mice with EAE, positively correlating with disease severity. Interestingly, we found that AE-specific CD8⁺ T cells were present also in the lymphoid organs of unprimed mice, proliferated under peptide stimulation in vitro, but failed to respond to peptide immunization in vivo, suggesting a physiological control of this response. However, when mice were immunized with AEs along with EAE induction, AE-specific CD8⁺ T cells with an effector/memory phenotype accumulated in the central nervous system, and the disease severity was exacerbated. In conclusion, we demonstrate that AE-specific autoimmunity may contribute to immunopathology in neuroinflammation.Subject terms: Cell death and immune response, Immunological disorders     

Differential regulation of CD4+ T helper cell responses by curcumin in experimental autoimmune encephalomyelitis

Nutraceuticals and phytochemicals are important regulators of human health and diseases. Curcumin is a polyphenolic phytochemical isolated from the rhizome of the plant Curcuma longa (turmeric) that has been traditionally used for the treatment of inflammation and wound healing for centuries. Systematic analyses have shown that curcumin exerts its beneficial effects through antioxidant, antiproliferative and anti-inflammatory properties. We and others have shown earlier that curcumin ameliorates experimental autoimmune encephalomyelitis (EAE) model for multiple sclerosis. In this study, we show that C57BL/6 mice induced to develop EAE express elevated levels of interferon (IFN) γ and interleukin (IL)-17 in the central nervous system (CNS) and lymphoid organs that decreased significantly following in vivo treatment with curcumin. The EAE mice also showed elevated expression of IL-12 and IL-23 that decreased after treatment with curcumin. Ex vivo and in vitro treatment with curcumin resulted in a dose-dependent decrease in the secretion of IFNγ, IL-17, IL-12 and IL-23 in culture. The inhibition of EAE by curcumin was also associated with an up-regulation of IL-10, peroxisome proliferator activated receptor γ and CD4⁺CD25⁺Foxp3⁺ Treg cells in the CNS and lymphoid organs. These findings highlight that curcumin differentially regulates CD4⁺ T helper cell responses in EAE.     

Alterations in the spinal cord T cell repertoire during relapsing experimental autoimmune encephalomyelitis

The CNS T cell repertoire was analyzed by RT-PCR, spectratyping, and nucleotide sequencing of the amplified products at different times following adoptive transfer of a CD4+, Th1, VB2+ encephalitogenic SJL/J proteolipid protein peptide 139-151-specific T cell clone. The third complementarity-determining region of TCR B chains in the spinal cord was used as an indicator of T cell heterogeneity. Spectratypic analysis revealed that a single peak corresponding to the third complementarity-determining region of the initiating T cell clone predominated during the acute phase. During recovery and relapse the complexity of the spectratype increased. DNA sequence analysis revealed that the donor clone predominated at the acute phase. By the first relapse the donor clone, although represented most frequently, was a minority of the total. Spectratypic analysis of the same samples for several other VB families revealed their presence during acute disease or relapses but, with the exception of VB17, their absence during the recovery stage.     

Lithium prevents and ameliorates experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) models, in animals, many characteristics of multiple sclerosis, for which there is no adequate therapy. We investigated whether lithium, an inhibitor of glycogen synthase kinase-3 (GSK3), can ameliorate EAE in mice. Pretreatment with lithium markedly suppressed the clinical symptoms of EAE induced in mice by myelin oligodendrocyte glycoprotein peptide (MOG35-55) immunization and greatly reduced demyelination, microglia activation, and leukocyte infiltration in the spinal cord. Lithium administered postimmunization, after disease onset, reduced disease severity and facilitated partial recovery. Conversely, in knock-in mice expressing constitutively active GSK3, EAE developed more rapidly and was more severe. In vivo lithium therapy suppressed MOG35-55-reactive effector T cell differentiation, greatly reducing in vitro MOG35-55- stimulated proliferation of mononuclear cells from draining lymph nodes and spleens, and MOG35-55-induced IFN-gamma, IL-6, and IL-17 production by splenocytes isolated from MOG35-55-immunized mice. In relapsing/remitting EAE induced with proteolipid protein peptide139-151, lithium administered after the first clinical episode maintained long-term (90 days after immunization) protection, and after lithium withdrawal the disease rapidly relapsed. These results demonstrate that lithium suppresses EAE and identify GSK3 as a new target for inhibition that may be useful for therapeutic intervention of multiple sclerosis and other autoimmune and inflammatory diseases afflicting the CNS.     

Taenia crassiceps infection abrogates experimental autoimmune encephalomyelitis

Helminth infections induce strong immunoregulation that can modulate subsequent pathogenic challenges. Taenia crassiceps causes a chronic infection that induces a Th2-biased response and modulates the host cellular immune response, including reduced lymphoproliferation in response to mitogens, impaired antigen presentation and the recruitment of suppressive alternatively activated macrophages (AAMФ). In this study, we aimed to evaluate the ability of T. crassiceps to reduce the severity of experimental autoimmune encephalomyelitis (EAE). Only 50% of T. crassiceps-infected mice displayed EAE symptoms, which were significantly less severe than uninfected mice. This effect was associated with both decreased MOG-specific splenocyte proliferation and IL-17 production and limited leukocyte infiltration into the spinal cord. Infection with T. crassiceps induced an anti-inflammatory cytokine microenvironment, including decreased TNF-α production and high MOG-specific production of IL-4 and IL-10. While the mRNA expression of TNF-α and iNOS was lower in the brain of T. crassiceps-infected mice with EAE, markers for AAMФ were highly expressed. Furthermore, in these mice, there was reduced entry of CD3⁺Foxp3⁻ cells into the brain. The T. crassiceps-induced immune regulation decreased EAE severity by dampening T cell activation, proliferation and migration to the CNS.     

Oral zinc aspartate treats experimental autoimmune encephalomyelitis

The essential trace element zinc plays a critical role in the regulation of immune homeostasis. Zinc deficiency or excess can cause severe impairment of the immune response, which points to the importance of the physiological and dietary control of zinc levels for a functioning immune system. We previously reported that injection of zinc aspartate suppresses experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), as well as effector T cell functions in vitro. Among the preferred characteristics of novel therapeutics for the treatment of autoimmune diseases such as MS are oral availability and a tolerable effective dose to minimize side effects. In this study, we investigated whether oral administration of zinc aspartate, an approved drug to treat zinc deficiency in humans, is effective in controlling EAE at clinically approved doses. We show that oral administration of 6 µg/day [0.3 mg/kg body weight (BW)] or 12 µg/day [0.6 mg/kg BW] of zinc aspartate reduces clinical and histopathological signs during the relapsing remitting phase of the disease in SJL mice. The clinical effect in mice was accompanied by suppression of IFN-γ, TNF-α, GM-CSF and IL-5 production in stimulated human T cells and mouse splenocytes in a dose-dependent manner. Furthermore, a large array of proinflammatory cytokines was modulated by zinc aspartate exposure in vitro. These data suggest that administration of oral zinc aspartate may have beneficial effects on autoimmune diseases like MS.     

Rituximab therapy reduces organ-specific T cell responses and ameliorates experimental autoimmune encephalomyelitis

Recent clinical trials have established B cell depletion by the anti-CD20 chimeric antibody Rituximab as a beneficial therapy for patients with relapsing-remitting multiple sclerosis (MS). The impact of Rituximab on T cell responses remains largely unexplored. In the experimental autoimmune encephalomyelitis (EAE) model of MS in mice that express human CD20, Rituximab administration rapidly depleted peripheral B cells and strongly reduced EAE severity. B cell depletion was also associated with diminished Delayed Type Hypersensitivity (DTH) and a reduction in T cell proliferation and IL-17 production during recall immune response experiments. While Rituximab is not considered a broad immunosuppressant, our results indicate a role for B cells as a therapeutic cellular target in regulating encephalitogenic T cell responses in specific tissues.