Chronic Progressive Experimental Autoimmune Encephalomyelitis Research Articles

Therapeutic role of interferon-γ in experimental autoimmune encephalomyelitis is mediated through a tolerogenic subset of splenic CD11b+ myeloid cells

Cumulative evidence has established that Interferon (IFN)-γ has both pathogenic and protective roles in Multiple Sclerosis and the animal model, Experimental Autoimmune Encephalomyelitis (EAE). However, the underlying mechanisms to the beneficial effects of IFN-γ are not well understood. In this study, we found that IFN-γ exerts therapeutic effects on chronic, relapsing-remitting, and chronic progressive EAE models. The frequency of regulatory T (Treg) cells in spinal cords from chronic EAE mice treated with IFN-γ was significantly increased with no effect on Th1 and Th17 cells. Consistently, depletion of FOXP3-expressing cells blocked the protective effects of IFN-γ, indicating that the therapeutic effect of IFN-γ depends on the presence of Treg cells. However, IFN-γ did not trigger direct in vitro differentiation of Treg cells. In vivo administration of blocking antibodies against either interleukin (IL)-10, transforming growth factor (TGF)-β or program death (PD)-1, revealed that the protective effects of IFN-γ in EAE were also dependent on TGF-β and PD-1, but not on IL-10, suggesting that IFN-γ might have an indirect role on Treg cells acting through antigen-presenting cells. Indeed, IFN-γ treatment increased the frequency of a subset of splenic CD11b+ myeloid cells expressing TGF-β-Latency Associated Peptide (LAP) and program death ligand 1 (PD-L1) in a signal transducer and activator of transcription (STAT)-1-dependent manner. Furthermore, splenic CD11b+ cells from EAE mice preconditioned in vitro with IFN-γ and myelin oligodendrocyte glycoprotein (MOG) peptide exhibited a tolerogenic phenotype with the capability to induce conversion of naïve CD4+ T cells mediated by secretion of TGF-β. Remarkably, adoptive transfer of splenic CD11b+ cells from IFN-γ-treated EAE mice into untreated recipient mice ameliorated clinical symptoms of EAE and limited central nervous system infiltration of mononuclear cells and effector helper T cells. These results reveal a novel cellular and molecular mechanism whereby IFN-γ promotes beneficial effects in EAE by endowing splenic CD11b+ myeloid cells with tolerogenic and therapeutic activities.

Oligodendrocyte progenitor cells differentiation induction with MAPK/ERK inhibitor fails to support repair processes in the chronically demyelinated CNS.

Remyelination failure is considered a major obstacle in treating chronic-progressive multiple sclerosis (MS). Studies have shown blockage in the differentiation of resident oligodendrocyte progenitor cells (OPC) into myelin-forming cells, suggesting that pushing OPC into a differentiation program might be sufficient to overcome remyelination failure. Others stressed the need for a permissive environment to allow proper activation, migration, and differentiation of OPC. PD0325901, a MAPK/ERK inhibitor, was previously shown to induce OPC differentiation, non-specific immunosuppression, and a significant therapeutic effect in acute demyelinating MS models. We examined PD0325901 effects in the chronically inflamed central nervous system. Treatment with PD0325901 induced OPC differentiation into mature oligodendrocytes with high morphological complexity. However, treatment of Biozzi mice with chronic-progressive experimental autoimmune encephalomyelitis with PD0325901 showed no clinical improvement in comparison to the control group, no reduction in demyelination, nor induction of OPC migration into foci of demyelination. PD0325901 induced a direct general immunosuppressive effect on various cell populations, leading to a diminished phagocytic capability of microglia and less activation of lymph-node cells. It also significantly impaired the immune-modulatory functions of OPC. Our findings suggest OPC regenerative function depends on a permissive environment, which may include pro-regenerative inflammatory elements. Furthermore, they indicate that maintaining a delicate balance between the pro-myelinating and immune functions of OPC is of importance. Thus, the highly complex mission of creating a pro-regenerative environment depends upon an appropriate immune response controlled in time, place, and intensity. We suggest the need to employ a multi-systematic therapeutic approach, which cannot be achieved through a single molecule-based therapy.

Evobrutinib therapeutic response is associated with an increase in the number and maturation of peripheral and central classical dendritic cells (P7-3.005)

<h3>Objective:</h3> To assess the impact of evobrutinib on classical dendritic cells (cDCs) in the MOG_35–55-induced chronic-progressive experimental autoimmune encephalomyelitis (EAE) Multiple sclerosis (MS) model (with limited B cell contribution). <h3>Background:</h3> Evobrutinib is an oral, highly selective covalent Bruton’s tyrosine kinase (BTK) inhibitor with promising results in a Phase II trial for relapsing MS. Lately, the role of BTK in myeloid cell activity, including cDCs, has been gaining importance in MS pathogenesis. <h3>Design/Methods:</h3> Female EAE mice received individualized daily oral treatment of vehicle (EAE-Veh) or evobrutinib (EAE-Evo) for 4 days from the day of disease onset. EAE was scored clinically each day and flow cytometry analyses of spleens and spinal cords were performed. EAE-Evo mice with a clinical course indistinguishable from that of EAE-Veh were classified as non-responders (Evo-NR), whereas those with less severe disease were considered responders (Evo-R). <h3>Results:</h3> The therapeutic response in Evo-R was associated with an increase in MHC-II⁺ cDC (mature cDCs) in the spleen. Evobrutinib shifted the correlation between CD69⁺ T cells/cDCs from positive to inverse. However, Evo-NR mice showed myeloid content similar to EAE-Veh mice and Evo-R mice exhibited a lower myeloid cell infiltrate in the spinal cord, albeit enriched in mature cDCs. Evobrutinib induced specific modifications in the immunological synapse components in cDCs. Whereas both Evo-R and Evo-NR showed a decrease in MHC-II in splenic cDCs, only Evo-R exhibited a decrease in CD80. In contrast, spinal cord cDCs showed decreased CD80 with no modification of MHC-II brightness. <h3>Conclusions:</h3> In the EAE model, evobrutinib therapeutic response shows a clear relationship with the enrichment of mature cDCs at both the peripheral and central levels, with a decrease in activated T cell presence. The interpretation of the immunological alteration of cDCs by evobrutinib will be further investigated using functional <i>ex vivo</i> assays. <b>Disclosure:</b> The institution of Dr. Clemente has received research support from EMD Serono Research &amp; Development Institute, Inc.. The institution of Dr. Clemente has received research support from Bristol Myers Squibb. The institution of Dr. Clemente has received research support from Instituto de Salud Carlos III. Dr. Clemente has received personal compensation in the range of $500-$4,999 for serving as a Reviewer of Grants with Spanish National Investigation Agency. Dr. Serrano Regal has nothing to disclose. Dr. Calahorra has nothing to disclose. Miss Alonso García has nothing to disclose. Ursula Boschert has nothing to disclose. Philipp Haselmayer has received personal compensation for serving as an employee of Merck Healthcare KGaA. Dr. Ortega has nothing to disclose. Miss Díaz has nothing to disclose. Mrs. Camacho Toledano has nothing to disclose. Mrs. Arocha has nothing to disclose.

MOG-Specific T Cells Lead to Spontaneous EAE with Multilocular B Cell Infiltration in the GF-IL23 Model

Although IL-23 and downstream signal transduction play essential roles in neuroinflammation, the local impact of IL-23 in multiple sclerosis is still not fully understood. Our previous study revealed that the central nervous system (CNS)-restricted expression of IL-23 in a mouse model with astrocyte-specific expression of IL-23, called GF-IL23 mice, leads to spontaneous formation of infiltrates in the brain, especially in the cerebellum. To further investigate the impact of CNS-specific IL-23-expression on neuroinflammation, we studied the GF-IL23 model in mice expressing a myelin oligodendrocyte glycoprotein (MOG)-specific T cell receptor (GF23-2D2 mice). The GF23-2D2 mice developed a chronic progressive experimental autoimmune encephalomyelitis with myelitis and ataxia without requiring additional immunization. CNS-production of IL-23 alone induced pronounced neuroinflammation in the transgenic MOG-specific T cell receptor model. The GF23-2D2 mice spontaneously developed multilocular infiltrates with a high number of B cells, demyelination and a proinflammatory cytokine milieu indicating that the interaction of encephalitogenic T cells and B cells via co-stimulatory factors seemed to be crucial.

CSF SERPINA3 Levels Are Elevated in Patients With Progressive MS.

ObjectiveTo identify biomarkers associated with progressive phases of MS and with neuroprotective potential.MethodsCombined analysis of the transcriptional and proteomic profiles obtained in CNS tissue during chronic progressive phases of experimental autoimmune encephalomyelitis (EAE) with the transcriptional profile obtained during the differentiation of murine neural stem cells into neurons. Candidate biomarkers were measured by ELISA in the CSF of 65 patients with MS (29 with relapsing-remitting MS [RRMS], 20 with secondary progressive MS, and 16 with primary progressive MS [PPMS]) and 30 noninflammatory neurologic controls (NINCs).ResultsIntegrative analysis of gene and protein expression data identified 2 biomarkers, the serine protease inhibitor Serpina3n and the calcium-binding protein S100A4, which were upregulated in chronic progressive EAE and whose expression was induced during neuronal differentiation. Immunofluorescence studies revealed a primarily neuronal expression of S100A4 and Serpina3n during EAE. CSF levels of SERPINA3, the human ortholog of murine Serpina3n, and S100A4 were increased in patients with MS compared with NINCs (SERPINA3: 1,320 vs 838.6 ng/mL, p = 0.0001; S100A4: 1.6 vs 0.8 ng/mL, p = 0.02). Within the MS group, CSF SERPINA3 levels were significantly elevated in patients with progressive forms, mainly patients with PPMS compared with patients with RRMS (1,617 vs 1,129 ng/mL, p = 0.02) and NINCs (1,617 vs 838.6 ng/mL, p = 0.0001). Of interest, CSF SERPINA3 levels significantly correlated with CSF neurofilament light chain levels only in the PPMS group (r = 0.62, p = 0.01).ConclusionThese results point to a role of SERPINA3 as a biomarker associated with the progressive forms of MS, particularly PPMS.

Tolerogenic nanoparticles suppress central nervous system inflammation

Therapeutic approaches for the induction of immune tolerance remain an unmet clinical need for the treatment of autoimmune diseases, including multiple sclerosis (MS). Based on its role in the control of the immune response, the ligand-activated transcription factor aryl hydrocarbon receptor (AhR) is a candidate target for novel immunotherapies. Here, we report the development of AhR-activating nanoliposomes (NLPs) to induce antigen-specific tolerance. NLPs loaded with the AhR agonist ITE and a T cell epitope from myelin oligodendrocyte glycoprotein (MOG)35-55 induced tolerogenic dendritic cells and suppressed the development of experimental autoimmune encephalomyelitis (EAE), a preclinical model of MS, in preventive and therapeutic setups. EAE suppression was associated with the expansion of MOG35-55-specific FoxP3+ regulatory T cells (Treg cells) and type 1 regulatory T cells (Tr1 cells), concomitant with a reduction in central nervous system-infiltrating effector T cells (Teff cells). Notably, NLPs induced bystander suppression in the EAE model established in C57BL/6 × SJL F1 mice. Moreover, NLPs ameliorated chronic progressive EAE in nonobese diabetic mice, a model which resembles some aspects of secondary progressive MS. In summary, these studies describe a platform for the therapeutic induction of antigen-specific tolerance in autoimmune diseases.

Tryptamine treatment results in the amelioration of EAE by inducing an anti-inflammatory shift in the Th17/Treg balance mediated by regulation of miRNA expression profile

Abstract In this study we focus on understanding the novel immunotherapeutic potential of tryptamine, a bioactive indole compound derived from microorganisms and various botanicals, in the context of ameliorating symptoms of Experimental Autoimmune Encephalomyelitis (EAE). EAE is used in the laboratory setting to replicate the etiology as well as pathology of Multiple Sclerosis (MS) in murine organisms for research purposes. Globally MS, an incurable autoimmune disorder, is experiencing increases in incidence and treatment costs which places emphasis on the discovery of novel immunosuppressents with limited toxicity. In this study we show that tryptamine poses potent immunosuppressive activity and ameliorates the clinical parameters of EAE by shifting the balance of CD4+ T cells from a pro-inflammatory T helper 17 cell (Th17) towards an anti-inflammatory T regulatory cell (Treg) dominated landscape in chronic progressive EAE. Based on preliminary miRNA array data, we have found that the observed shift in the Th17/Treg balance following tryptamine treatment is due to an anti-inflammatory miRNA expression profile within the lymphocytes of treated mice. MiR-3473g and miR-140-3p, putative targets of the enzyme SIRT1, were found to be upregulated within the lymphocytes of tryptamine treated mice. SIRT1 in the context of inflammatory disorders works as a marker of pro-inflammatory responses in lymphocytes due to SIRT1’s activation of the master transcription factor of Th17 cells known as RORγt. This epigenetic regulation of gene expression provides a clearly linkable mechanism explaining the shift in the Th17/Treg balance as well as the amelioration of symptoms observed following tryptamine treatment in EAE mice.

The Aryl Hydrocarbon Receptor as a regulator of CNS autoimmunity

Abstract Therapeutic approaches aimed at controlling the balance between effector T cells and regulatory T cells in an antigen-specific manner are of high clinical significance in the field of autoimmunity. The aryl hydrocarbon receptor (AhR) has been identified as a key regulator of the adaptive immune system and thus represents a strong targetable candidate for the induction of immune tolerance. Here, we report the generation of a novel liposomal platform to co-administer a tolerogenic AhR ligand, ITE, and an autoimmune disease-specific T cell antigen. Liposomes containing ITE and a T-cell epitope from myelin oligodendrocyte glycoprotein (MOG)35–55 are taken up by dendritic cells and induce a tolerogenic transcriptional program which promotes the differentiation and expansion of long-lasting antigen-specific regulatory T cells. Treatment with these liposomes almost completely prevented the development of experimental autoimmune encephalomyelitis (EAE), an experimental model of relapsing-remitting multiple sclerosis (MS), and reversed symptoms in mice with ongoing EAE. This disease suppression was associated with the expansion of antigen-specific FoxP3+ regulatory T cells (Treg cells) and type 1 regulatory T cells (Tr1 cells) as well as the suppression of CNS-infiltrating effector T cells. Furthermore, treatment with liposomes containing ITE and MOG reversed symptoms of disease in chronic progressive EAE in nonobese diabetic mice, an experimental model resembling several aspects of secondary progressive MS. Taken together, these results suggest that our liposomes represent a novel therapeutic platform to induce antigen-specific tolerance for the treatment of autoimmune diseases.

Featured Article: Serum [Met5]-enkephalin levels are reduced in multiple sclerosis and restored by low-dose naltrexone.

Low-dose naltrexone is a widely used off-label therapeutic prescribed for a variety of immune-related disorders. The mechanism underlying low-dose naltrexone's efficacy for fatigue, Crohn's disease, fibromyalgia, and multiple sclerosis is, in part, intermittent blockade of opioid receptors followed by upregulation of endogenous opioids. Short, intermittent blockade by naltrexone specifically blocks the opioid growth factor receptor resulting in biofeedback events that increase production of the endogenous opioid growth factor (OGF) (chemically termed [Met5]-enkephalin) facilitating interactions between opioid growth factor and opioid growth factor receptor that ultimately, result in inhibited cell proliferation. Preclinical studies have reported that enkephalin levels are deficient in animal models of experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. Our hypothesis is that serum enkephalin levels are diminished in humans with multiple sclerosis and experimental autoimmune encephalomyelitis mice, and that change in serum opioid growth factor levels may serve as a reasonable candidate biomarker for the onset of experimental autoimmune encephalomyelitis and response to therapy. To address this, we designed a two-part study to measure endogenous opioids in multiple sclerosis patients, and to investigate the temporal pattern of decline in serum enkephalin concentrations in mice with chronic progressive experimental autoimmune encephalomyelitis and treated with low-dose naltrexone. For comparison, we investigated whether low-dose naltrexone exposure in normal mice also resulted in altered enkephalin levels. In both animal models, we monitored tactile and heat sensitivity, as well as differential white blood cell counts as indicators of inflammation. Serum [Met5]-enkephalin levels were lower in humans with multiple sclerosis relative to non-multiple sclerosis patients, and low-dose naltrexone restored their levels. In experimental autoimmune encephalomyelitis mice, [Met5]-enkephalin levels were depressed prior to the appearance of clinical disease, and were restored with low-dose naltrexone treatment. Low-dose naltrexone therapy had no effect on serum [Met5]-enkephalin or β-endorphin in normal mice. Thus, [Met5]-enkephalin (i.e. opioid growth factor) may be a reasonable candidate biomarker for multiple sclerosis, and may signal new pathways for treatment of autoimmune disorders. Impact statement This report presents human and animal data identifying a novel biomarker for the onset and progression of multiple sclerosis (MS). Humans diagnosed with MS have reduced serum levels of OGF (i.e. [Met5]-enkephalin) relative to non-MS neurologic patients, and low-dose naltrexone (LDN) therapy restored their enkephalin levels. Serum OGF levels were reduced in mice immunized with MOG35-55 prior to any clinical behavioral sign of experimental autoimmune encephalomyelitis, and LDN therapy restored their serum OGF levels. β-endorphin concentrations were not altered by LDN in humans or mice. Thus, blood levels of OGF may serve as a new, selective biomarker for the progression of MS, as well as response to therapy.

Sphingosine 1-phosphate receptor modulation suppresses pathogenic astrocyte activation and chronic progressive CNS inflammation

Multiple sclerosis (MS) is an autoimmune inflammatory demyelinating disease of the CNS that causes disability in young adults as a result of the irreversible accumulation of neurological deficits. Although there are potent disease-modifying agents for its initial relapsing-remitting phase, these therapies show limited efficacy in secondary progressive MS (SPMS). Thus, there is an unmet clinical need for the identification of disease mechanisms and potential therapeutic approaches for SPMS. Here, we show that the sphingosine 1-phosphate receptor (S1PR) modulator fingolimod (FTY720) ameliorated chronic progressive experimental autoimmune encephalomyelitis in nonobese diabetic mice, an experimental model that resembles several aspects of SPMS, including neurodegeneration and disease progression driven by the innate immune response in the CNS. Indeed, S1PR modulation by FTY720 in murine and human astrocytes suppressed neurodegeneration-promoting mechanisms mediated by astrocytes, microglia, and CNS-infiltrating proinflammatory monocytes. Genome-wide studies showed that FTY720 suppresses transcriptional programs associated with the promotion of disease progression by astrocytes. The study of the molecular mechanisms controlling these transcriptional modules may open new avenues for the development of therapeutic strategies for progressive MS.

Distinct Immunomodulatory and Migratory Mechanisms Underpin the Therapeutic Potential of Human Mesenchymal Stem Cells in Autoimmune Demyelination

Mesenchymal stem cells (MSCs) are efficacious in a variety of intractable diseases. While bone marrow (BM)-derived MSCs (BM-MSCs) have been widely investigated, MSCs from other tissue sources have also been shown to be effective in several autoimmune and inflammatory disorders. In the present study, we simultaneously assessed the therapeutic efficacy of human BM-MSCs, as well as MSCs isolated from adipose tissue (Ad-MSCs) and umbilical cord Wharton's jelly (UC-MSCs), in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). Prior to in vivo experiments, we characterized the phenotype and function of all three MSC types. We show that BM-MSCs were more efficient at suppressing the in vitro proliferation of mitogen or antigen-stimulated T-cell responses compared to Ad-MSCs and UC-MSCs. Notably BM-MSCs induced the differential expression of cytokines from normal and stimulated T-cells. Paradoxically, intravenous transplantation of BM-MSCs into C57Bl/6 mice with chronic progressive EAE had a negligible effect on the disease course, even when multiple MSC injections were administered over a number of time points. In contrast, Ad-MSCs had the most significant impact on clinical and pathological disease outcomes in chronic progressive and relapsing-remitting EAE models. In vivo tracking studies revealed that Ad-MSCs were able to migrate to the central nervous system (CNS), a property that most likely correlated with their broader expression of homing molecules, while BM-MSCs were not detected in this anatomic region. Collectively, this comparative investigation demonstrates that transplanted Ad-MSCs play a significant role in tissue repair processes by virtue of their ability to suppress inflammation coupled with their enhanced ability to home to the injured CNS. Given the access and relatively ease for harvesting adipose tissue, these data further implicate Ad-MSCs as a cell therapeutic that may be used to treat MS patients.

Opioid growth factor (OGF) prevents relapses in mice with relapsing‐remitting EAE, a model for multiple sclerosis

Relapse‐remitting multiple sclerosis (MS) is a life‐long, inflammatory disease of the CNS that affects about 350,000 people in the U.S. [Met5]‐enkephalin, termed opioid growth factor (OGF) administered daily to C57BL/6 mice with established chronic progressive experimental autoimmune encephalomyelitis (EAE) reversed the progression of behavioral disease within 6 days of treatment. To determine whether administration of OGF modulates the course of relapsing‐remitting EAE (RR‐EAE) when OGF is administered at the time of induction, female SJL/J mice received proteolipid protein 139–151 emulsified in complete Freund's adjuvant, and were injected daily (i.p.) with OGF (10 mg/kg) or sterile saline. OGF treated RR‐EAE mice had a 40% reduction in peak disease score and significantly fewer relapses over the course of 55 days relative to controls. The duration of relapse for OGF‐treated mice was approximately 1 day in comparison to 13 days for controls. Evaluation of overall disease scores revealed that only 15% of RR‐EAE mice receiving OGF ever expressed disease scores ≥ 4 in comparison to 40% of controls. Histological examination of spinal cords revealed that OGF‐treated mice had markedly less demyelination and fewer activated astrocytes than controls. The results indicate that OGF treatment reduces the progression of RR‐EAE, and may be a novel biological therapy for the treatment of MS. Supported by the Shockey Family Foundation.

Fasudil Ameliorates Disease Progression in Experimental Autoimmune Encephalomyelitis, Acting Possibly Through Antiinflammatory Effect

The purpose of this investigation was to further explore the mechanism(s) underlying the amelioration in EAE caused by Fasudil, particularly focusing on anti-inflammatory effect. We induced a chronic-progressive experimental autoimmune encephalomyelitis (EAE) in B6 mice immunized with myelin oligodendrocyte glycoprotein(35-55) and performed Fasudil intervention in early and late stages of the disease. The administration of Fasudil (40mg/kg, i.p) had a therapeutic effect in delaying the onset and ameliorating the severity of EAE, accompanied by the improvement in myelination and the decrease in inflammatory cells in spinal cords. Fasudil inhibited TLR-4, p-NF-kB/p65, and inflammatory cytokines (IL-1β, IL-6, and TNF-α) and enhanced IL-10 production in spinal cords. The ratio of arginase/iNOS was enhanced mainly in the spinal cords of EAE mice treated with Fasudil, reflecting a shift toward the M2 (antiinflammation) macrophage/microglia phenotype. The administration of Fasudil also induced the upregulation of CB2 receptor in spinal cords, but did not significantly trigger CB1 receptor. Levels of neurotrophic factors NGF, BDNF, and GDNF in the CNS were not altered by Fasudil. Fasudil ameliorates disease progression in EAE, acting possibly through antiinflammatory pathway.

Targeting ganglioside epitope 3G11 on the surface of CD4+ T cells suppresses EAE by altering the Treg/Th17 cell balance

Loss of expression of the 3G11 epitope, present on disialoceramide that is predominantly found on CD4(+) T cells, has been associated with a regulatory T cell (Treg) phenotype and tolerance induction in experimental autoimmune encephalomyelitis (EAE). Here we report that treatment with anti-3G11 mAb shifts the immune response from pro-inflammatory to tolerogenic and suppresses both chronic-progressive and relapsing-remitting EAE. This therapeutic effect can be achieved at different stages of EAE. Treatment with anti-3G11 mAb increased the proportion of Foxp3(+)CD25(+)CD4(+) Tregs and IL-10 production while inhibiting production of pro-inflammatory cytokines and responsiveness to IL-2 and decreasing the proportion of T(h)17 cells. The effect of anti-3G11 mAb was diminished in IL-10(-/-) mice, indicating that this cytokine mediates some of its effects. As 3G11 belongs to the ganglioside family, which is expressed on the surface of both murine and human CD4(+) T cells, targeting this class of molecules may provide a novel approach for treating autoimmune diseases.

Prevention of axonal injury using calpain inhibitor in chronic progressive experimental autoimmune encephalomyelitis

Axonal injury is the major correlate of permanent disability in neurodegenerative diseases such as multiple sclerosis (MS), especially in secondary-progressive MS which follows relapsing–remitting disease course. Proteolytic enzyme, calpain, is a potential candidate for causing axonal injury. Most current treatment options only target the inflammatory component of MS. Previous work using calpain inhibitor CYLA in our laboratory showed significant reduction in clinical sign, demyelination and tissue calpain content in acute experimental autoimmune encephalomyelitis (EAE). Here we evaluated markers of axonal injury (amyloid precursor protein, Na v1.6 channels), neuronal calpain content and the effect of CYLA on axonal protection using histological methods in chronic EAE [myelin oligodendrocyte glycoprotein (MOG)-induced disease model of MS]. Intraperitoneal application of CYLA (2 mg/mouse/day) significantly reduced the clinical signs, tissue calpain content, demyelination and inflammatory infiltration of EAE. Similarly, markers for axonal injury were barely detectable in the treated mice. Thus, this novel drug, which markedly suppresses the disease course, axonal injury and its progression, is a candidate for the treatment of a neurodegenerative disease such as multiple sclerosis.

Autoimmunity Against Myelin Oligodendrocyte Glycoprotein Is Dispensable for the Initiation Although Essential for the Progression of Chronic Encephalomyelitis in Common Marmosets

To elucidate the pathogenetic significance of myelin/oligodendrocyte glycoprotein (MOG)-specific autoreactivity in a genetically and immunologically heterogeneous nonhuman primate model of multiple sclerosis, we analyzed experimental autoimmune encephalomyelitis (EAE) in the outbred common marmoset (Callithrix jacchus). One sibling each of 5 bone marrow chimeric marmoset twins was immunized with myelin derived from wild-type (WT) C57BL/6 mice (WT myelin); the other sibling was immunized with myelin from MOG-deficient C57BL/6 mice (MOG -/- myelin). One twin pair developed acute EAE simultaneously; the 4 remaining twin siblings immunized with WT myelin developed chronic progressive EAE, whereas siblings of these 4 monkeys remained free of clinical disease signs. Many EAE-related abnormalities were identified in the CNS of both groups by magnetic resonance imaging and histologic analysis, but mean percentages of spinal cord demyelination were lower in monkeys immunized with MOG -/- myelin (8.2%) than in WT myelin-immunized animals (40.5%). There was a strong correlation between the development of overt clinical EAE and seropositivity for anti-MOG antibodies, but blood and lymph node T-cell proliferative responses showed no relationship to disease. These results indicate that the initiation of CNS inflammation and demyelination can take place in the absence of detectable autoimmunity against MOG, but the clinical progression and histopathologic severity depends on the presence of antibodies against MOG in this multiple sclerosis model.

A Suppressive Oligodeoxynucleotide Enhances the Efficacy of Myelin Cocktail/IL-4-Tolerizing DNA Vaccination and Treats Autoimmune Disease

Targeting pathogenic T cells with Ag-specific tolerizing DNA vaccines encoding autoantigens is a powerful and feasible therapeutic strategy for Th1-mediated autoimmune diseases. However, plasmid DNA contains abundant unmethylated CpG motifs, which induce a strong Th1 immune response. We describe here a novel approach to counteract this undesired side effect of plasmid DNA used for vaccination in Th1-mediated autoimmune diseases. In chronic relapsing experimental autoimmune encephalomyelitis (EAE), combining a myelin cocktail plus IL-4-tolerizing DNA vaccine with a suppressive GpG oligodeoxynucleotide (GpG-ODN) induced a shift of the autoreactive T cell response toward a protective Th2 cytokine pattern. Myelin microarrays demonstrate that tolerizing DNA vaccination plus GpG-ODN further decreased anti-myelin autoantibody epitope spreading and shifted the autoreactive B cell response to a protective IgG1 isotype. Moreover, the addition of GpG-ODN to tolerizing DNA vaccination therapy effectively reduced overall mean disease severity in both the chronic relapsing EAE and chronic progressive EAE mouse models. In conclusion, suppressive GpG-ODN effectively counteracted the undesired CpG-induced inflammatory effect of a tolerizing DNA vaccine in a Th1-mediated autoimmune disease by skewing both the autoaggressive T cell and B cell responses toward a protective Th2 phenotype. These results demonstrate that suppressive GpG-ODN is a simple and highly effective novel therapeutic adjuvant that will boost the efficacy of Ag-specific tolerizing DNA vaccines used for treating Th1-mediated autoimmune diseases.

Intrathecal delivery of IFN-gamma protects C57BL/6 mice from chronic-progressive experimental autoimmune encephalomyelitis by increasing apoptosis of central nervous system-infiltrating lymphocytes.

The exclusive detrimental role of proinflammatory cytokines in demyelinating diseases of the CNS, such as multiple sclerosis, is controversial. Here we show that the intrathecal delivery of an HSV-1-derived vector engineered with the mouse IFN-gamma gene leads to persistent (up to 4 wk) CNS production of IFN-gamma and inhibits the course of a chronic-progressive form of experimental autoimmune encephalomyelitis (EAE) induced in C57BL/6 mice by myelin oligodendrocyte glycoprotein (MOG)(35-55). Mice treated with the IFN-gamma-containing vector before EAE onset showed an earlier onset but a milder course of the disease compared with control mice treated with the empty vector. In addition, 83% of IFN-gamma-treated mice completely recovered within 25 days post immunization, whereas control mice did not recover up to 60 days post immunization. Mice treated with the IFN-gamma-containing vector within 1 wk after EAE onset partially recovered from the disease within 25 days after vector injection, whereas control mice worsened. Recovery from EAE in mice treated with IFN-gamma was associated with a significant increase of CNS-infiltrating lymphocytes undergoing apoptosis. During the recovery phase, the mRNA level of TNFR1 was also significantly increased in CNS-infiltrating cells from IFN-gamma-treated mice compared with controls. Our results further challenge the exclusive detrimental role of IFN-gamma in the CNS during EAE/multiple sclerosis, and indicate that CNS-confined inflammation may induce protective immunological countermechanisms leading to a faster clearance of encephalitogenic T cells by apoptosis, thus restoring the immune privilege of the CNS.

Peptide determinants of myelin proteolipid protein (PLP) in autoimmune demyelinating disease: a review.

This article reviews recent advances in understanding the role of myelin proteolipid protein (PLP) in autoimmune demyelination. It is drawn largely from work published within the last ten years and discusses the immunology of PLP in the historical context of what has been learned from extensive studies on the immune response to myelin basic protein (MBP). Despite the fact that PLP is the major protein constituent of mammalian myelin, its role in autoimmune demyelination has not been widely recognized. The lack of understanding about the immunology of PLP is a direct result of the biochemical characteristics of the protein. PLP is a highly hydrophobic membrane protein with limited aqueous solubility. The hydrophobicity of PLP has thwarted immunologic studies of the intact protein. Recent work has circumvented the technical obstacles of studying the intact protein by using soluble synthetic PLP peptides. This approach has rapidly resulted in a more definitive understanding of the immune response to PLP. Presently, the data indicate that: i) PLP is a major central nervous system (CNS) specific encephalitogen; ii) CD4+ T cell reactivity to discrete PLP peptide determinants can mediate the development of acute, chronic relapsing, and chronic progressive experimental autoimmune encephalomyelitis (EAE); and iii) T cell reactivity to multiple PLP determinants occurs in patients with multiple sclerosis (MS), the major human CNS demyelinating disease.

Spontaneous remission of autoimmune encephalomyelitis is inhibited by splenectomy, thymectomy or ageing.

Experimental autoimmune encephalomyelitis (EAE) can be induced in genetically susceptible animals by injecting them with basic protein of myelin (BP) in a suitable adjuvant. EAE in adult Lewis rats is expressed clinically by acute paralysis and histologically by mononuclear cell infiltration of the central nervous system. Most rats spontaneously recover from EAE and show little or no damage to myelin. We report here that chronic progressive EAE with marked myelin lesions can be induced by a single injection of BP in complete Freund's adjuvant in intact 13-month old rats, or in 4-month old rats provided they have been splenectomized. Juvenile 2 1/2-month old rats recover spontaneously despite splenectomy. Thymectomy of young adult rats leads to relapsing EAE. These results illustrate that the clinical course of EAE is markedly influenced by age and integrity of immune organs. Furthermore, they provide an experimental model with features similar to those of chronic relapsing disease of the nervous system of man.