Objective Oryeongsan (Goreisan), a formula composed of five herbal medicines, has long been used to treat impairments of the regulation of body fluid homeostasis. Goreisan has been revealed to have anti-inflammatory actions and inhibit a water channel, the aquaporin (AQP). We herein report the therapeutic effect of Goreisan on experimental autoimmune encephalomyelitis (EAE in, an animal model of inflammatory demyelinating diseases. Materials and Methods EAE mice immunized with MOG35-55 peptide were divided into Goreisan- and sham-treated groups. The clinical EAE score and histopathological finding of the central nervous system (CNS) were analyzed. For the proliferation assay, prepared spleen cells from immunized mice were cultured and analyzed for the [3H]-thymidine uptake and cytokine concentrations of the culture supernatant. The relative quantification of AQP4 mRNA in the CNS of EAE mice was analyzed quantitatively. Results The EAE score of the Goreisan-treated mice was significantly lower than that of the sham-treated mice. The CD4-positive cell number in the CNS of Goreisan-treated mice was lower than that of sham-treated mice. In the recall response to MOG35-55 peptide, the cell proliferation did not differ markedly between the spleen cells from Goreisan- and sham-treated mice. Furthermore, Goreisan decreased the mRNA level of AQP4 in the spinal cord during EAE. Conclusion Goreisan prevented the disease activity of EAE by inhibiting the migration of pathogenic cells into the CNS by suppressing the AQP4 expression in the CNS. Goreisan may have a therapeutic effect on inflammatory demyelinating diseases.

MiR-150 regulates maturation and differentiation of T cells but how it functions in multiple sclerosis (MS) is unclear. In miR-150 knockout (KO) mice, we examined the effect of miR-150 deletion on disease severity of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. After deleting miR-150, EAE disease severity was reduced according to clinical score. Histological staining and MBP immunofluorescence staining revealed that miR-150 deletion limited the extent of inflammatory demyelination and axonal damage in the spinal cord. Flow cytometry showed that CD3+, CD4+, and CD8+ T cells were increased in WT-EAE mice, but miR-150 deletion significantly reversed EAE-mediated up-regulation of CD3+, CD4+, and CD8+ T cells and down-regulation of CD19+ B cells. In addition, miR-150 deletion reduced the mRNA expression of IL-1β, IL-6, IL-17, and TNF-α in spleen and spinal cord after EAE induction. Thus, miR-150 deletion reduces EAE severity and demyelination, probably through inhibiting the activated immune response and the inflammation in the central nervous system.

Anatabine, a naturally occurring alkaloid, is becoming a commonly used human food supplement, taken for its claimed anti-inflammatory properties although this has not yet been reported in human clinical trials. We have previously shown that anatabine does display certain anti-inflammatory properties and readily crosses the blood-brain barrier suggesting it could represent an important compound for mitigating neuro-inflammatory conditions. The present study was designed to determine whether anatabine had beneficial effects on the development of experimental autoimmune encephalomyelitis (EAE) in mice and to precisely determine its underlying mechanism of action in this mouse model of multiple sclerosis (MS). We found that orally administered anatabine markedly suppressed neurological deficits associated with EAE. Analyses of cytokine production in the periphery of the animals revealed that anatabine significantly reduced Th1 and Th17 cytokines known to contribute to the development of EAE. Anatabine appears to significantly suppress STAT3 and p65 NFκB phosphorylation in the spleen and the brain of EAE mice. These two transcription factors regulate a large array of inflammatory genes including cytokines suggesting a mechanism by which anatabine antagonizes pro-inflammatory cytokine production. Additionally, we found that anatabine alleviated the infiltration of macrophages/microglia and astrogliosis and significantly prevented demyelination in the spinal cord of EAE mice. Altogether our data suggest that anatabine may be effective in the treatment of MS and should be piloted in clinical trials.

The costimulatory CD40L-CD40 dyad plays a major role in multiple sclerosis (MS). CD40 is highly expressed on MHCII+ B cells, dendritic cells and macrophages in human MS lesions. Here we investigated the role of the CD40 downstream signaling intermediates TNF receptor-associated factor 2 (TRAF2) and TRAF6 in MHCII+ cells in experimental autoimmune encephalomyelitis (EAE). Both MHCII-CD40-Traf2-/- and MHCII-CD40-Traf6-/- mice showed a reduction in clinical signs of EAE and prevented demyelination. However, only MHCII-CD40-Traf6-/- mice displayed a decrease in myeloid and lymphoid cell infiltration into the CNS that was accompanied by reduced levels of TNF-α, IL-6 and IFN-γ. As CD40-TRAF6 interactions predominantly occur in macrophages, we subjected CD40flfl LysMcre mice to EAE. This myeloid-specific deletion of CD40 resulted in a significant reduction in EAE severity, reduced CNS inflammation and demyelination. In conclusion, the CD40-TRAF6 signaling pathway in MHCII+ cells plays a key role in neuroinflammation and demyelination during EAE. Concomitant with the fact that CD40-TRAF6 interactions are predominant in macrophages, depletion of myeloid CD40 also reduces neuroinflammation. CD40-TRAF6 interactions thus represent a promising therapeutic target for MS. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Exercise training (ET) has beneficial effects on multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). However, the intensity-dependent effects of ET on the systemic immune system in EAE remain undefined.

Multiple sclerosis (MS) is the most common disabling neurological disease of young adults. The pathophysiological mechanism of MS remains largely unknown and no cure is available. Current clinical treatments for MS modulate the immune system, with the rationale that autoimmunity is at the core of MS pathophysiology.

Pannexin1 (Panx1) is a plasma membrane channel permeable to relatively large molecules, such as ATP. In the central nervous system (CNS) Panx1 is found in neurons and glia and in the immune system in macrophages and T-cells. We tested the hypothesis that Panx1-mediated ATP release contributes to expression of Experimental Autoimmune Encephalomyelitis (EAE), an animal model for multiple sclerosis, using wild-type (WT) and Panx1 knockout (KO) mice. Panx1 KO mice displayed a delayed onset of clinical signs of EAE and decreased mortality compared to WT mice, but developed as severe symptoms as the surviving WT mice. Spinal cord inflammatory lesions were also reduced in Panx1 KO EAE mice during acute disease. Additionally, pharmacologic inhibition of Panx1 channels with mefloquine (MFQ) reduced severity of acute and chronic EAE when administered before or after onset of clinical signs. ATP release and YoPro uptake were significantly increased in WT mice with EAE as compared to WT non-EAE and reduced in tissues of EAE Panx1 KO mice. Interestingly, we found that the P2X7 receptor was upregulated in the chronic phase of EAE in both WT and Panx1 KO spinal cords. Such increase in receptor expression is likely to counterbalance the decrease in ATP release recorded from Panx1 KO mice and thus contribute to the development of EAE symptoms in these mice. The present study shows that a Panx1 dependent mechanism (ATP release and/or inflammasome activation) contributes to disease progression, and that inhibition of Panx1 using pharmacology or gene disruption delays and attenuates clinical signs of EAE.

Brain atrophy measured by MRI is an important correlate with clinical disability and disease duration in multiple sclerosis (MS). Unfortunately, neuropathologic mechanisms which lead to this grey matter atrophy remain unknown. The objective of this study was to determine whether brain atrophy occurs in the mouse model, experimental autoimmune encephalomyelitis (EAE). Postmortem high-resolution T2-weighted magnetic resonance microscopy (MRM) images from 32 mouse brains (21 EAE and 11 control) were collected. A minimum deformation atlas was constructed and a deformable atlas approach was used to quantify volumetric changes in neuroanatomical structures. A significant decrease in the mean cerebellar cortex volume in mice with late EAE (48-56 days after disease induction) as compared to normal strain, gender, and age-matched controls was observed. There was a direct correlation between cerebellar cortical atrophy and disease duration. At an early time point in disease, 15 days after disease induction, cerebellar white matter lesions were detected by both histology and MRM. These data demonstrate that myelin-specific autoimmune responses can lead to grey matter atrophy in an otherwise normal CNS. The model described herein can now be used to investigate neuropathologic mechanisms that lead to the development of gray matter atrophy in this setting.

Multiple sclerosis (MS) is an autoimmune, neurodegenerative disease but the molecular mechanisms underlying neurodegenerative aspects of the disease are poorly understood. microRNAs (miRNAs) are powerful regulators of gene expression that regulate numerous mRNAs simultaneously and can thus regulate programs of gene expression. Here, we describe miRNA expression in neurons captured from mice subjected to experimental autoimmune encephalomyelitis (EAE), a model of central nervous system (CNS) inflammation. Lumbar motor neurons and retinal neurons were laser captured from EAE mice and miRNA expression was assessed by next-generation sequencing and validated by qPCR. We describe 14 miRNAs that are differentially regulated in both neuronal subtypes and determine putative mRNA targets though in silico analysis. Several upregulated neuronal miRNAs are predicted to target pathways that could mediate repair and regeneration during EAE. This work identifies miRNAs that are affected by inflammation and suggests novel candidates that may be targeted to improve neuroprotection in the context of pathological inflammation.

β-Lapachone is a naturally occurring quinine, originally isolated from the bark of the lapacho tree (Tabebuia avellanedae) which is currently being evaluated in clinical trials for the treatment of cancer. In addition, recent investigations suggest its potential application for treatment of inflammatory diseases. Multiple sclerosis (MS) is an autoimmune disorder characterized by CNS inflammation and demyelination. Reactive T cells including IL-17 and IFN-γ-secreting T cells are believed to initiate MS and the associated animal model system experimental autoimmune encephalomyelitis (EAE). IL-12 family cytokines secreted by peripheral dendritic cells (DCs) and CNS microglia are capable of modulating T-cell phenotypes. The present studies demonstrated that β-lapachone selectively inhibited the expression of IL-12 family cytokines including IL-12 and IL-23 by DCs and microglia, and reduced IL-17 production by CD4(+) T-cells indirectly through suppressing IL-23 expression by microglia. Importantly, our studies also demonstrated that β-lapachone ameliorated the development on EAE. β-Lapachone suppression of EAE was associated with decreased expression of mRNAs encoding IL-12 family cytokines, IL-23R and IL-17RA, and molecules important in Toll-like receptor signaling. Collectively, these studies suggest mechanisms by which β-lapachone suppresses EAE and suggest that β-lapachone may be effective in the treatment of inflammatory diseases such as MS.

Astrocytes are the main cells responsible for maintenance of brain homeostasis. Undisturbed action and signaling with other cells are crucial for proper functioning of the central nervous system (CNS). Dysfunctional astrocytes may determine the degree of neuronal injury and are associated with several brain pathologies, among which are multiple sclerosis (MS) and the animal model of this disease which is known as experimental autoimmune encephalomyelitis (EAE). One of the many functions of astrocytes is their response to CNS damage when they undergo reactive gliosis. Our data reveal that activation of astrocytes occurs in forebrains of immunized rats at a very early stage of EAE, well before the symptomatic phase of the disease. We have noted enhanced expression of GFAP and S100β starting from day 4 post-immunization. Temporal coincidence between the expression of astrocyte activation markers and the expression of connexin 43 and purinergic P2X7 receptor (P2X7R) was also observed. Administration of Brilliant blue G, an antagonist of P2X7R, significantly decreases astrogliosis as confirmed by immunohistochemical analysis and observation of decreased levels of GFAP and S100β. The condition of the treated animals was improved and the neurological symptoms of the disease were alleviated. With the knowledge that cerebral astroglia represent the main source of ATP and glutamate which are potentially neurotoxic substances released through P2X7R and connexin hemichannels, we suggest that astroglia may be involved in pathogenesis of MS/EAE at a very early stage through the purinergic/glutamatergic mechanisms.

Depletion of the enzyme cofactor, tetrahydrobiopterin (BH4), in T-cells was shown to prevent their proliferation upon receptor stimulation in models of allergic inflammation in mice, suggesting that BH4 drives autoimmunity. Hence, the clinically available BH4 drug (sapropterin) might increase the risk of autoimmune diseases. The present study assessed the implications for multiple sclerosis (MS) as an exemplary CNS autoimmune disease. Plasma levels of biopterin were persistently low in MS patients and tended to be lower with high Expanded Disability Status Scale (EDSS). Instead, the bypass product, neopterin, was increased. The deregulation suggested that BH4 replenishment might further drive the immune response or beneficially restore the BH4 balances. To answer this question, mice were treated with sapropterin in immunization-evoked autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. Sapropterin-treated mice had higher EAE disease scores associated with higher numbers of T-cells infiltrating the spinal cord, but normal T-cell subpopulations in spleen and blood. Mechanistically, sapropterin treatment was associated with increased plasma levels of long-chain ceramides and low levels of the poly-unsaturated fatty acid, linolenic acid (FA18:3). These lipid changes are known to contribute to disruptions of the blood-brain barrier in EAE mice. Indeed, RNA data analyses revealed upregulations of genes involved in ceramide synthesis in brain endothelial cells of EAE mice (LASS6/CERS6, LASS3/CERS3, UGCG, ELOVL6, and ELOVL4). The results support the view that BH4 fortifies autoimmune CNS disease, mechanistically involving lipid deregulations that are known to contribute to the EAE pathology.

Gray matter atrophy observed by brain MRI is an important correlate to clinical disability and disease duration in multiple sclerosis. The objective of this study was to link brain atrophy visualized by neuroimaging to its underlying neuropathology using the MS model, experimental autoimmune encephalomyelitis (EAE). Volumetric changes in brains of EAE mice, as well as matched healthy normal controls, were quantified by collecting post-mortem high-resolution T2-weighted magnetic resonance microscopy and actively stained magnetic resonance histology images. Anatomical delineations demonstrated a significant decrease in the volume of the whole cerebellum, cerebellar cortex, and molecular layer of the cerebellar cortex in EAE as compared to normal controls. The pro-apoptotic marker caspase-3 was detected in Purkinje cells and a significant decrease in Purkinje cell number was found in EAE. Cross modality and temporal correlations revealed a significant association between Purkinje cell loss on neuropathology and atrophy of the molecular layer of the cerebellar cortex by neuroimaging. These results demonstrate the power of using combined population atlasing and neuropathology approaches to discern novel insights underlying gray matter atrophy in animal models of neurodegenerative disease.

Using metagenomic analysis, we identified a novel picornavirus in young preweaned lambs with neurologic signs associated with severe nonsuppurative encephalitis and sensory ganglionitis in 2016 and 2017 in the United Kingdom. In situ hybridization demonstrated intralesional neuronotropism of this virus, which was also detected in archived samples of similarly affected lambs (1998-2014).

Cyclophilins have diverse functions that may affect the course of central nervous system (CNS) inflammatory disorders. Anti-inflammatory and neuroprotective mechanisms may be targeted by inhibition of cyclophilin A-dependent and cyclophilin D-dependent functions, respectively. We tested the effect of cyclophilin inhibition on CNS inflammation by administering N-methyl-4-isoleucine-cyclosporin (NIM811) to mice undergoing experimental allergic encephalomyelitis (EAE). Treatment with NIM811 resulted in significant reduction of EAE clinical severity. Analysis of mitochondrial calcium retention capacity and the course of EAE in cyclophilin D knockout mice indicated that the effect of NIM811 on EAE was not entirely cyclophilin D-dependent. NIM811-treated EAE animals showed reduction in interleukin-2 expression and reduction in CNS inflammatory infiltrates. These results indicate that anti-inflammatory rather than neuroprotective mechanisms associated with cyclophilins are likely involved in the mechanism of NIM811 in EAE.

Studies have correlated excessive S100B, a small inflammatory molecule, with demyelination and associated inflammatory processes occurring in multiple sclerosis. The relevance of S100B in multiple sclerosis pathology brought an emerging curiosity highlighting its use as a potential therapeutic target to reduce damage during the multiple sclerosis course, namely during inflammatory relapses. We examined the relevance of S100B and further investigated the potential of S100B-neutralizing small-molecule pentamidine in chronic experimental autoimmune encephalomyelitis. S100B depletion had beneficial pathological outcomes and based on promising results of a variety of S100B blockade strategies in an ex vivo demyelinating model, we choose pentamidine to assay its role in the in vivo experimental autoimmune encephalomyelitis. We report that pentamidine prevents more aggressive clinical symptoms and improves recovery of chronic experimental autoimmune encephalomyelitis. Blockade of S100B by pentamidine protects against oligodendrogenesis impairment and neuroinflammation by reducing astrocyte reactivity and microglia pro-inflammatory phenotype. Pentamidine also increased regulatory T cell density in the spinal cord suggesting an additional immunomodulatory action. These results showed the relevance of S100B as a main driver of neuroinflammation in experimental autoimmune encephalomyelitis and identified an uncharacterized mode of action of pentamidine, strengthening the possibility to use this drug as an anti-inflammatory and remyelinating therapy for progressive multiple sclerosis.

We wished to study how infections might trigger relapses of autoimmune diseases such as multiple sclerosis (MS) and encephalomyelitis (EAE). We hypothesized that immune responses to heat shock proteins (hsp) induced by an infection could modulate responses to autoantigens. We induced extra-neuraxial inflammation in SJL mice housed either in specific-pathogen free (SPF) or conventional facilities. Mice in conventional housing are continuously exposed to large numbers of infectious agents. Spleen cell proliferative responses to human HSP60 and bacterial HSP65 were measured as were numbers of cells secreting IFN-gamma or IL-5. Proliferative responses to HSP60 were increased in conventionally housed mice compared to SPF mice and this was associated with skewing of secreted cytokines toward a Th2 pattern. Skewing toward a Th1 pattern was noted in SPF mice. Acute and relapsing EAE was induced in both groups of mice. Acute EAE was, in general, equivalent in all groups. However, SPF mice had more severe relapses than did conventionally housed animals and these differences were amplified by extra-neuraxial inflammation. Immunocytochemical analyses of brains from mice with relapsing EAE showed that increased numbers of brain gamma/delta cells were associated with disease remission. Our data suggest that frequent exposure to infectious agents leads to a relative Th2 skewing of immune responses to hsp and that this is associated with milder, less frequent relapses of EAE. They also support the concept that immune responses to hsp are of potential importance in exacerbating and perpetuating organ-restricted autoimmune diseases.

Heat shock protein (Hsp)70 is one of the most important stress-inducible proteins. Intracellular Hsp70 not only mediates chaperone-cytoprotective functions but can also block multiple steps in the apoptosis pathway. In addition, Hsp70 is actively released into the extracellular milieu, thereby promoting innate and adaptive immune responses. Thus, Hsp70 may be a critical molecule in multiple sclerosis (MS) pathogenesis and a potential target in this disease due to its immunological and cytoprotective functions. To investigate the role of Hsp70 in MS pathogenesis, we examined its immune and cytoprotective roles using both in vitro and in vivo experimental procedures. We found that Hsp70.1-deficient mice were more resistant to developing experimental autoimmune encephalomyelitis (EAE) compared with their wild-type (WT) littermates, suggesting that Hsp70.1 plays a critical role in promoting an effective myelin oligodendrocyte glycoprotein (MOG)-specific T cell response. Conversely, Hsp70.1-deficient mice that developed EAE showed an increased level of autoreactive T cells to achieve the same production of cytokines compared with the WT mice. Although a neuroprotective role of HSP70 has been suggested, Hsp70.1-deficient mice that developed EAE did not exhibit increased demyelination compared with the control mice. Accordingly, Hsp70 deficiency did not influence the vulnerability to apoptosis of oligodendrocyte precursor cells (OPCs) in culture. Thus, the immunological role of Hsp70 may be relevant in EAE, and specific therapies down-regulating Hsp70 expression may be a promising approach to reduce the early autoimmune response in MS patients.

Multiple sclerosis (MS) is a complex autoimmune condition with firmly established genetic and environmental components. Genome-wide association studies (GWAS) have revealed a large number of genetic polymorphisms in the vicinity of, and within, genes that associate to disease. However, the significance of these single-nucleotide polymorphisms in disease and possible mechanisms of action remain, with a few exceptions, to be established. While the animal model for MS, experimental autoimmune encephalomyelitis (EAE), has been instrumental in understanding immunity in general and mechanisms of MS disease in particular, much of the translational information gathered from the model in terms of treatment development (glatiramer acetate and natalizumab) has been extensively summarized. In this review, we would thus like to cover the work done in EAE from a GWAS perspective, highlighting the research that has addressed the role of different GWAS genes and their pathways in EAE pathogenesis. Understanding the contribution of these pathways to disease might allow for the stratification of disease subphenotypes in patients and in turn open the possibility for new and individualized treatment approaches in the future.

Experimental autoimmune encephalomyelitis (EAE) is a T-cell-mediated autoimmune central nervous system disease characterized by inflammation with oxidative stress. The aim of this study was to evaluate an anti-inflammatory effect of Ishige okamurae on EAE-induced paralysis in rats. An ethanolic extract of I. okamurae significantly delayed the first onset and reduced the duration and severity of hind-limb paralysis. The neuropathological and immunohistochemical findings in the spinal cord were in agreement with these clinical results. T-cell proliferation assay revealed that the ethyl-acetate fraction of I. okamurae suppressed the proliferation of myelin basic protein reactive T cells from EAE affected rats. Flow cytometric analysis showed TCRαβ+ T cells was significantly reduced in the spleen of EAE rats with I. okamurae treatment with concurrent decrease of inflammatory mediators including tumor necrosis factor-α and cyclooxygenase-2. Collectively, it is postulated that I. okamurae ameliorates EAE paralysis with suppression of T-cell proliferation as well as decrease of pro-inflammatory mediators as far as rat EAE is concerned.