Spinal Cord Of Experimental Autoimmune Encephalomyelitis Mice Research Articles

Eugenol ameliorates uveitis in mice with experimental autoimmune encephalomyelitis through the suppression of key inflammatory genes

ABSTRACT Visual impairment associated with uveitis is among the potential complications in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Bioinformatics analyses have shown that some hub genes are closely associated with the molecular mechanisms underlying uveitis in EAE. This study evaluated whether 4-allyl-2-methoxyphenol (eugenol) can mitigate the pathogenesis of uveitis in EAE through the interruption of key uveitogenic gene expression. Myelin oligodendrocyte glycoprotein35–55 (MOG) peptide-immunized C57BL/6 mice were injected intraperitoneally with eugenol. The eyeballs and spinal cords of EAE mice with or without eugenol treatment were collected simultaneously and immunohistochemical and molecular biological analyses were conducted. Eugenol treatment significantly ameliorated hindlimb paralysis. Ionized calcium-binding adapter molecule 1 (Iba-1) immunohistochemistry showed that the inflammatory response was significantly reduced in the uvea of eugenol-treated EAE mice compared with vehicle-treated controls. Eugenol also significantly reduced the expression of key uveitogenic genes including C1qb and Tyrobp. The suppressive effect of eugenol on inflammation was also observed in the spinal cord, as determined by the suppression of Iba-1-positive microglial cells. Together, these results suggest that the ameliorative effect of eugenol against EAE uveitis is associated with the suppression of key proinflammatory genes, which may represent targets for the treatment of uveitis.

A High MCT-Based Ketogenic Diet Suppresses Th1 and Th17 Responses to Ameliorate Experimental Autoimmune Encephalomyelitis in Mice by Inhibiting GSDMD and JAK2-STAT3/4 Pathways.

Inflammation and pyroptosis play important roles in the pathogenesis of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). In this study, we evaluated the therapeutic potential of ketogenic diet (KD) in EAE. The administration of KD reduces demyelination and microglial activation in the spinal cord of EAE mice. Meanwhile, KD decreases the levels of Th1 and Th17 associated cytokines/transcription factors production (T-bet, IFN-γ, RORγt, and IL-17) and increases those of Th2 and Treg cytokines/transcription factors (GATA3, IL-4, Foxp3, and IL-10) in the spinal cord and spleen. Corresponding, KD reduces the expression of chemokines in EAE, which those chemokines associate with T-cell infiltration into central nervous system (CNS). In addition, KD inhibits the GSDMD activation in microglia, oligodendrocyte, CD31+ cells, CCR2+ cells, and T cells in the spinal cord. Moreover, KD significantly decreases the ratios of p-JAK2/JAK2, p-STAT3/STAT3, and p-STAT4/STAT4, as well as GSDMD in EAE mice. this study demonstrates that KD reduces the activation and differentiation of T cells in the spinal cord and spleen and prevents T cell infiltration into CNS of EAE via modulating the GSDMD and STAT3/4 pathways, suggesting that KD is a potentially effective strategy in the treatment of MS.

Nogo-A antibody delivery through the olfactory mucosa mitigates experimental autoimmune encephalomyelitis in the mouse CNS

Systemic administration of Nogo-A-neutralizing antibody ameliorates experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. However, the blood-brain barrier (BBB) is a major obstacle limiting the passage of systemically applied antibody to the CNS. To bypass the BBB, in the present study we tested the intranasal route of administration by targeting the olfactory mucosa with the Nogo-A-blocking antibody 11C7 mAb in myelin oligodendrocyte glycoprotein-induced EAE. Antibodies were specifically administered onto the olfactory mucosa using a microcatheter. Antibody distribution was examined in the CNS by ELISA and light-sheet microscopy. The effects of 11C7 mAb on Nogo-A signaling were assessed by Western blotting. EAE-induced deficits were monitored daily. Demyelination was observed on spinal cord histological sections. Gene expression changes were followed by trancriptomic analyses. A sensitive capture ELISA revealed a rapid and widespread distribution of 11C7 mAb in the CNS, including the olfactory bulb, the cerebellum and the lumbar spinal cord, but not in the CSF. Light-sheet microscopy allowed to observe antibody accumulation in the parenchyma, thus demonstrating nose-to-brain transfer of IgG. At the functional level, the widespread penetration of 11C7 mAb in the CNS, including the thoracolumbar spinal cord, resulted in the improvement of motor symptoms and in the preservation of myelin in the spinal cord of EAE mice. This was accompanied by Nogo-A signaling downregulation, as reflected by the decreased level of phosphorylated cofilin observed by Western blotting in the cerebellum. In the brain of EAE score-matched animals, 11C7 modified the expression of genes that can influence neurotransmission and cognitive functions, independently of the demyelination phenotype in the spinal cord. In conclusion, our data show the feasibility of olfactory mucosa-directed administration for the delivery of therapeutic antibodies targeting CNS antigens in EAE mice.

Cerebrospinal fluid, brain, and spinal cord levels of L-aspartate signal excitatory neurotransmission abnormalities in multiple sclerosis patients and experimental autoimmune encephalomyelitis mouse model.

The neuroinflammatory process characterizing multiple sclerosis (MS) is associated with changes in excitatory synaptic transmission and altered central concentrations of the primary excitatory amino acid, L-glutamate (L-Glu). Recent findings report that cerebrospinal fluid (CSF) levels of L-Glu positively correlate with pro-inflammatory cytokines in MS patients. However, to date, there is no evidence about the relationship between the other primary excitatory amino acid, L-aspartate (L-Asp), its derivative D-enantiomer, D-aspartate, and the levels of pro-inflammatory and anti-inflammatory cytokines in the CSF of MS. In the present study, we measured by HPLC the levels of these amino acids in the cortex, hippocampus, cerebellum, and spinal cord of mice affected by experimental autoimmune encephalomyelitis (EAE). Interestingly, in support of glutamatergic neurotransmission abnormalities in neuroinflammatory conditions, we showed reduced L-Asp levels in the cortex and spinal cord of EAE mice and increased D-aspartate/total aspartate ratio within the cerebellum and spinal cord of these animals. Additionally, we found significantly decreased CSF levels of L-Asp in both relapsing-remitting (n = 157) MS (RR-MS) and secondary progressive/primary progressive (n = 22) (SP/PP-MS) patients, compared to control subjects with other neurological diseases (n = 40). Importantly, in RR-MS patients, L-Asp levels were correlated with the CSF concentrations of the inflammatory biomarkers G-CSF, IL-1ra, MIP-1β, and Eotaxin, indicating that the central content of this excitatory amino acid, as previously reported for L-Glu, reflects a neuroinflammatory environment in MS. In keeping with this, we revealed that CSF L-Asp levels were positively correlated with those of L-Glu, highlighting the convergent variation of these two excitatory amino acids under inflammatory synaptopathy occurring in MS.

Suppression of Experimental Autoimmune Encephalomyelitis in Mice by β-Hydroxy β-Methylbutyrate, a Body-Building Supplement in Humans.

Although several immunomodulatory drugs are available for multiple sclerosis (MS), most present significant side effects with long-term use. Therefore, delineation of nontoxic drugs for MS is an important area of research. β-Hydroxy β-methylbutyrate (HMB) is accessible in local GNC stores as a muscle-building supplement in humans. This study underlines the importance of HMB in suppressing clinical symptoms of experimental autoimmune encephalomyelitis (EAE) in mice, an animal model of MS. Dose-dependent study shows that oral HMB at a dose of 1 mg/kg body weight/d or higher significantly suppresses clinical symptoms of EAE in mice. Accordingly, orally administered HMB attenuated perivascular cuffing, preserved the integrity of the blood-brain barrier and blood-spinal cord barrier, inhibited inflammation, maintained the expression of myelin genes, and blocked demyelination in the spinal cord of EAE mice. From the immunomodulatory side, HMB protected regulatory T cells and suppressed Th1 and Th17 biasness. Using peroxisome proliferator-activated receptor (PPAR)α-/- and PPARβ-/- mice, we observed that HMB required PPARβ, but not PPARα, to exhibit immunomodulation and suppress EAE. Interestingly, HMB reduced the production of NO via PPARβ to protect regulatory T cells. These results describe a novel anti-autoimmune property of HMB that may be beneficial in the treatment of MS and other autoimmune disorders.

Therapeutic effect of the total saponin from Panax Japonicus on experimental autoimmune encephalomyelitis by attenuating inflammation and regulating gut microbiota in mice

Ethnopharmacological relevanceRhizomes of Panax japonicus (RPJ), a traditional herbal medicine, was used for treating arthritis and physical weakness in China from the Ming dynasty. Triterpene saponins are the main bioactive components of RPJ. In this work, for the first time, we evaluate the therapeutic effect of the total saponin from RPJ (TSPJ) on experimental autoimmune encephalomyelitis (EAE) mice induced by myelin oligodendrocyte glycoprotein (MOG) 35-55, a commonly used animal model of Multiple sclerosis (MS). Aim of the studyTo evaluate the therapeutic effect of TSPJ on EAE and explored its possible underlying mechanisms. Materials and methodsEAE was induced by MOG 35-55. Mice were administrated with TSPJ (36.5 mg/kg, 73 mg/kg) and prednisone acetate (positive control) orally once daily up to 28 days postimmunization, and their neurological deficit was scored. Hematoxylin and Eosin (HE), Luxol Fast Blue (LFB), and transmission electron microscopy (TEM) were carried out to evaluate the EAE-induced pathological changes in the brain and spinal cord. IL-17a and Foxp3 levels in central nervous system (CNS)were evaluated by immunohistochemical staining. The changes in IL-1β, IL-6, and TNF-α levels in serum and CNS were measured with ELISA. Quantitative reverse transcription PCR (qRT-PCR) was used to access mRNA expression in CNS of the above indices. The percentages of Th1, Th2, Th17and Treg cells in spleen were determined by Flow Cytometry (FCM). Furthermore, 16S rDNA sequencing was used to detect the intestinal flora of mice in each group. In vitro studies, lipopolysaccharides (LPS)-induced BV2 microglia cells were used and the expression of TLR4, MyD88, p65, and p-p65 in cells was detected by Western blot. ResultsTSPJ treatment significantly alleviated neurological impairment caused by EAE. Histological examination confirmed the protective effects of TSPJ on myelin sheath and the reduction of inflammatory cell infiltration in the brain and spinal cord of EAE mice. TSPJ notably downregulated the ratio of IL-17a/Foxp3 at protein and mRNA levels in CNS, as well as Th17/Treg and Th1/Th2 cell ratios in the spleen of EAE mice. The levels of TNF-α, IL-6, and IL-1β in CNS and peripheral serum also decreased post-TSPJ treatment. In vitro, TSPJ suppressed LPS-induced production of inflammatory factors in BV2 cells via TLR4-MyD88-NF-κB signaling pathway. More importantly, TSPJ interventions altered the composition of gut microbiota and restored the ratio of Firmicutes to Bacteroidetes in EAE mice. Furthermore, Spearman's correlation analysis revealed that a relationship existed between statistically significantly altered genera and CNS inflammatory indices. ConclusionOur results demonstrated TSPJ had therapeutic effects on EAE. Its anti-neuroinflammation property in EAE was related to modulating gut microbiota and inhibiting TLR4-MyD88-NF-κB signaling pathway. Our study indicated that TSPJ may be a potential candidate for the treatment of MS.

Neuroprotective effect of Vesatolimod in an experimental autoimmune encephalomyelitis mice model

BackgroundMultiple sclerosis is a chronic demyelinating autoimmune disease accompanied by inflammation and loss of axons and neurons. Toll-like receptors play crucial roles in the innate immune system and inflammation. However, few studies have explored the specific effects of toll-like receptor 7 signaling pathway in multiple sclerosis. To explore underlying effects to develop a new therapeutic target, we use Vesatolimod, a safe and well-tolerated agonist of toll-like receptor 7, to assess the possible effects in Experimental autoimmune encephalomyelitis (EAE) animal model. MethodsEAE animal model was induced by injection of MOG35–55 and monitored daily for clinical symptoms, and the treatment group was given Vesatolimod at the onset of illness. The therapeutic effects of Vesatolimod on EAE inflammation, demyelination, CD107b cells and T cells infiltration, and microglia activation was evaluated. Autophagy within the spinal cords of EAE mice was also preliminarily assessed. ResultsTreatment with Vesatolimod significantly alleviated clinical symptoms of EAE from day 18 post-immunization and decreased the expression levels of inflammatory cytokines, particularly Eotaxin and IL-12 (P40), in peripheral blood. It also inhibited demyelination in spinal cords. Moreover, VES treatment reduced activation of microglia, infiltration of CD3 + T cells and CD107b + cells, as well as inhibited the autophagy-related proteins expression in the spinal cords of EAE mice. ConclusionOur results indicate that Vesatolimod exhibits protective effects on EAE mice and is promising for treatment of MS.

Emerging role of neuregulin-1beta1 in pathogenesis and progression of multiple sclerosis

Multiple sclerosis (MS) is a chronic immune-mediated disorder of the central nervous system (CNS) that causes focal demyelinating lesions, followed by axonal and neuronal degeneration. Several genetic and environmental factors are found to be associated with MS incidence. While MS etiology seems to be multifactorial and needs further elucidation, it is understood that the response of an immune system to specific myelin antigens triggers the onset of MS (Dendrou et al., 2015). However, how the autoimmune response initiates against myelin, and the cellular and molecular mechanisms underpinning the development and progression of MS are not fully understood. Thus, deconstructing MS pathogenesis is of paramount importance for identifying novel diagnostic and therapeutic targets for this complex disease. Different components of the adaptive and innate immune responses are implicated in the pathogenesis of MS (Dendrou et al., 2015). An adaptive immune response is traditionally known to play a major role in the initiation of demyelination in MS and in its animal model, experimental autoimmune encephalomyelitis (EAE). Autoreactive T cells of both helper T cell 1 (Th1) and Th17 subsets, along with CD8+ cytotoxic T cells are among the main effector cells in this process (van Langelaar et al., 2020). Growing evidence suggests that regulatory T cells (Tregs) also play a crucial modulatory role in the autoimmune response in the EAE mice. Tregs are involved in the resolution of neuroinflammation and recovery in EAE through their anti-inflammatory properties. Recent studies on the motility differences in Tregs and effector Th17 cells have proposed three putative mechanisms for an immunomodulatory role of Tregs in MS that include reducing the number of antigen-presenting cells, limiting the access of Th17 to these cells, and inhibiting calcium signaling in Th17 cells (Othy et al., 2020). Autoreactive T cells are also known to cause neuropathologic effects through their interactions with the cells of the innate immune responses (Dong and Yong, 2019). Therefore, in recent years, there has been increasing emphasis on the role of peripheral and CNS innate immunity in the pathogenesis of MS and EAE. Infiltrating monocyte-derived macrophages (MDMs) and resident microglia are two important members of the innate immune response in the CNS of EAE animals. Microglia and MDMs demonstrate diverse phenotypes in EAE and are polarized along a spectrum of pro-inflammatory/neurodegenerative to anti-inflammatory, neuroprotective/pro-regenerative phenotype, based on the stage of the disease (Wang et al., 2019). Since microglia and MDMs are not readily distinguishable in the CNS tissue morphologically or immunocytologically, understanding their specific functional roles in regulating MS pathogenesis and recovery has been historically challenging. However, with recent technical advances, new evidence shows distinct gene expression as well as temporal and spatial modulatory roles for microglia and MDMs in EAE mice or in response to the same inflammatory stimuli. Studies by Yamasaki and colleagues demonstrate that MDMs are predominantly implicated in the initiation of demyelination at the onset of EAE, while microglia are mainly involved in myelin clearance (Yamasaki et al., 2014). Intriguingly, subsequent fate mapping and single-cell RNA sequencing of microglia by Plemel and colleagues unraveled distinct subpopulations of microglia with diverse activation states, and suggested a complicated cross-talk between microglia and MDMs in demyelinating lesions that is critical to prevent MDMs from invading the CNS tissue (Plemel et al., 2020). These studies, among others, have collectively pointed to the critical role of the innate immune response in modulating both the injury and repair processes in MS. However, the endogenous mechanisms that modulate the neuro-degenerative or neuro-regenerative nature of the immune responses during the pathologic events of MS are not fully understood. In efforts to uncover the endogenous mechanisms that regulate the phenotype of innate and adaptive immune responses in MS, our group has recently uncovered an important immunomodulatory role for Neuregulin-1β1 (Nrg-1β1) in MS pathogenesis (Kataria et al., 2021; Figure 1). This article provides a perspective on these new findings. The Neuregulins belong to a family of signaling proteins with diverse regulatory roles in development and physiology of neurons, axons, myelin, and glial cells in the CNS. Nrg-1β1 has been emerged as a mediator of CNS injury and repair in recent years (Kataria et al., 2019). Nrg-1β1 is mainly expressed by neurons and cells of the oligodendrocyte lineage, while its receptors, ErbB-2, ErbB-3, ErbB-4, are expressed widely by various cell types in the CNS (Kataria et al., 2019). Our studies in the EAE mice showed that Nrg-1β1 protein is down-regulated in the spinal cord, and peripherally in the blood and spleen of these mice (Kataria et al., 2021). Nrg-1β1 down-regulation preceded the disease onset as its levels were significantly reduced in the pre-symptomatic phase of EAE that persisted after the peak of the disease. Previously, we also found significantly dysregulated levels of Nrg-1β1 protein in demyelinating lesions of spinal cord injury and lysolecithin-induced focal demyelination model (Alizadeh et al., 2018; Kataria et al., 2018), suggesting its broader impact on CNS demyelination. Nrg-1β1 dysregulation had functional ramifications on the onset and progression of EAE as restoring its levels with peptide treatment using recombinant human Nrg-1β1 was sufficient to attenuate disease severity and progression in EAE mice. Nrg-1 treatment delivered at pre-symptomatic, onset, peak, and post-peak of the EAE was effective in improving disease outcomes. Pre-symptomatic administration of Nrg-1 delayed the onset of symptoms in EAE mice, and when appeared, the symptoms were less severe. These findings suggest dysregulation of Nrg-1 is an early pathology in EAE and its promise as a treatment with a wide therapeutic window of opportunity for MS.Figure 1: The current proposed role and mechanisms of Nrg-1β1 in pathophysiology of MS and EAE.(1) We have demonstrated that expression level of Nrg-1β1 protein is dysregulated in the spinal cord lesions, peripheral blood, and spleen of EAE mice, as well as active plaques of individuals with MS and plasma of CIS individuals. (2) Restoration of Nrg-1β1 levels using recombinant human Nrg-1β1 peptide therapy has been beneficial for recovery in animal models of MS through various mechanisms: (3) Modulation of the immune responses through the regulation of both adaptive and innate components of neuroinflammation. (4) In adaptive immune response, while Nrg-1β1 treatment does not affect the total number of T helper cells in the blood or to the spinal cord of EAE, it significantly decreases the population of CD4+/IFNγ+ Th1 cells that leads to reduced levels of inflammatory cytokines such as IFNγ, IL-2 and IL-16. Nrg-1β1 treatment increases anti-inflammatory Tregs within the EAE lesions in the spinal cord. (5) In innate immune response, restoring Nrg-1β1 levels reduces monocytes in the blood and their infiltration into the CNS and consequently a decrease in the number of MDMs, while the recruitment of microglia remains unaffected. This was correlated with a reduction in the production of CSPGs and MMPs that are known mediators involved in the blood-CNS permeability in EAE and MS. Availability of Nrg-1β1 also supports a phenotype shift from pro-inflammatory M1 to neuroprotective M2 in both microglia and MDM populations. (6) Using LPC model, we have demonstrated that Nrg-1β1 treatment promotes differentiation of OPCs to mature myelinating oligodendrocytes that results in accelerated and improved remyelination of demyelinating lesions of the spinal cord. CIS: Clinically isolated syndrome; CNS: central nervous system; CSPGs: chondroitin sulfate proteoglycans; EAE: experimental autoimmune encephalomyelitis; IFNγ: interferon γ; IL: interleukin; LPC: lysolecithin induced focal demyelination; MDM: monocyte-derived macrophage; MMP9: matrix metalloproteinase-9; MS: multiple sclerosis; Nrg-1β1: neuregulin-1beta1; OPC: oligodendrocyte progenitor cell; Th1: helper T cell type 1; Tregs: regulatory T cells. This figure is created with BioRender.com.Relevance of preclinical findings in animal models to human MS pathology is of paramount importance to validate the translational impact of these discoveries for diagnosis and therapeutic development. Assessment of MS brain confirmed down-regulation of Nrg-1 in active demyelinating plaques as compared to normal-appearing white matter in the same patients. Importantly, Nrg1-β1 protein levels were declined in the plasma of individuals with clinically isolated syndrome (CIS). CIS individuals present with the first episode of neurological impairment characterized by inflammation or demyelination on magnetic resonance imaging consistent with an MS relapse. These individuals have a high probability of transition to relapsing-remitting MS in a few years. Analysis of the plasma levels of Nrg-1 in CIS and healthy control individuals showed a significant reduction in Nrg-1β1 plasma levels of CIS individuals who eventually developed relapsing-remitting MS. These findings suggest that dysregulation of Nrg-1β1 in the early stages of MS pathogenesis may present a potential disease biomarker, as well as a promising therapeutic target for MS. This opens a new direction for future research with a larger number of human samples of all MS subtypes to verify the association between Nrg-1β1 levels and disease onset and progression both peripherally and within the CNS. From the mechanistic point of view, these studies unraveled that increasing the availability of Nrg-1β1 can modulate both the innate and adaptive immune responses in the spinal cord of EAE mice (Kataria et al., 2021; Figure 1). Interestingly, Nrg-1β1 treatment did not affect the overall number of CD3+/CD4+ T cells in the blood or the spinal cord of EAE mice, while it significantly changed the phenotype of T cell population in the EAE lesion. Availability of Nrg-1β1 reduced the number of effector CD4+/interferon γ (IFNγ) expressing Th1 cells while increasing anti-inflammatory Tregs. This modulatory effect was associated with a concomitant reduction in the spinal cord levels of Th1 related cytokines including IFNγ, interleukin 2 (IL-2), and IL-16. Intriguingly, this work demonstrated that Th1 regulatory action of Nrg-1β1 treatment happens within the CNS lesions and not in the periphery, although dysregulation of Nrg-1β1 occurs both peripherally and within the CNS during disease pathogenesis. To decipher further information concerning the cellular source of Nrg-1β1 effects and peripheral verses CNS mediated mechanisms of Nrg-1β1 in pathogenesis of EAE, passive transfer studies in EAE mice and CNS specific deletion of Nrg-1β1 is warranted. Therapeutic restoration of Nrg-1β1 exert a more pronounced effect on monocyte infiltration in EAE mice resulting in a robust decrease in MDMs in the spinal cord lesions with no effects on the recruitment of resident microglia to EAE lesions (Kataria et al., 2021; Figure 1). Interestingly, this phenomenon was associated with reduced levels of chondroitin sulfate proteoglycans (CSPGs) and matrix metalloproteinase 9 (MMP9) in EAE lesions. Of note, MMP9 and CSPGs play critical roles in the integrity of the blood-CNS-barrier and leukocyte trafficking in MS and EAE. In the perivascular cuff, CSPGs are known to aid accumulation of leukocytes and their entry to the CNS parenchyma of EAE and MS (Stephenson et al., 2018). MMP9 also has an established role in pathogenesis of MS by increasing permeability of blood-CNS-barrier and myelin damage. Hence, the ability of Nrg-1β1 in attenuating CSPG and MMP9 production is an important therapeutic effect. Overall, these findings suggest that Nrg-1β1 potentially influences the recruitment of MDM in EAE lesions by regulating the permeability of the blood-CNS-barrier. Notably, MMP9 and CSPGs are primarily produced by pro-inflammatory leukocytes, microglia and astrocytes in EAE. Thus, downregulation of MMP9 and CSPGs by Nrg-1β1 treatment in EAE may reflect the immunomodulatory role of Nrg-1β1 in supporting the phenotype shift of both MDMs and microglia from pro-inflammatory M1 to neuroprotective M2 (Kataria et al., 2021). This phenotype shift in microglia and MDMs by Nrg-1β1 treatment was also reported previously by our group in rat spinal cord injury lesions (Alizadeh et al., 2018). Key pro-inflammatory cytokines associated with the M1 phenotype of microglia and MDMs in MS and EAE include IL-1β, IL-6, and tumor necrosis factor α, and these cells are also known to produce higher levels of reactive oxygen species (Wang et al., 2019). The phenotype shift in microglia and MDMs under Nrg-1 treatment was also accompanied by a decrease in the spinal cord levels of IL-1β, IL-6, tumor necrosis factor α and reactive oxygen species in EAE (Kataria et al., 2021). Pro-regenerative M2 macrophages and microglia play a supportive role in remyelination through the secretion of mediators that promote oligodendrocyte progenitor cell differentiation and maturation (Wang et al., 2019). In our previous work using lysolecithin-induced focal demyelination model, we showed Nrg-1β1 can promote the generation of mature oligodendrocytes that subsequently accelerated and enhanced remyelination in demyelinating lesions of the spinal cord (Kataria et al., 2018; Figure 1). These results along with the observations from the recent study propose that Nrg-1β1 may promote oligodendrocyte maturation through modulation of microglia and MDMs along the spectrum of pro-inflammatory to anti-inflammatory, pro-regenerative phenotype. Altogether, these findings have provided new insights into the putative mechanisms by which Nrg-1β1 dysregulation regulates responses in EAE. The current data propose that Nrg-1β1 can modulate both the innate and adaptive immune responses at the CNS levels, and its effects peripherally are mainly through the regulation of monocytes and their infiltration into the CNS. This, however, raises the possibility that Nrg-1β1 regulation of T cell responses in the CNS lesions of EAE might be indirectly through modulation of microglia and MDMs phenotype and function, which provides new directions for further investigations. In conclusion, understanding the underlying endogenous mechanisms that drive MS pathogenesis and progression is pivotal to deconstruct disease complexity, and unravel specific targets for MS diagnosis and treatment. Our new findings highlighted in Kataria et al. (2021) have proposed Nrg-1β1 dysregulation as a disease mechanism implicated in the early phase of MS pathogenesis and a potential predictive marker for disease progression. Importantly, the functional relevance of Nrg-1β1 dysregulation was confirmed by its therapeutic potential to ameliorate disease severity and progression in EAE mice. Mechanistically, the availability of Nrg-1β1 appears to play a role in immune homeostasis in the CNS as its dysregulation is associated with “monocyte extravasation” and promoting T helper 1 effector cell response in EAE. Further investigation using animal models with Nrg-1 genetic deletion is warranted to verify whether absence of Nrg-1 could trigger immune dysregulation peripherally and/or at the CNS. Moreover, to establish Nrg-1β1 as a potential disease marker for MS with both diagnostic and prognostic values in clinical settings, further investigation of its plasma levels in a larger size of MS patient samples is required. SMZ was supported by a University of Manitoba Graduate Fellowship, Graduate Enhancement of Tri-council Supplement (GETS) and Hillary Kaufman Lerner Memorial Funds. SKA was supported by the grants from Multiple Sclerosis Society of Canada (EGDI-2936, EGDI-3742) and the Canadian Institute of Health Research (133721 and 156218). Availability of data and materials:All data generated or analyzed during this study are included in this published article and its supplementary information files. Open peer reviewer:Victor M. Rivera, Baylor College of Medicine, USA.

Ketogenic diet attenuates neuroinflammation and induces conversion of M1 microglia to M2 in an EAE model of multiple sclerosis by regulating the NF-κB/NLRP3 pathway and inhibiting HDAC3 and P2X7R activation.

Multiple sclerosis (MS) is an autoimmune disorder characterized by demyelination and neurodegeneration in the central nervous system (CNS); severe symptoms lead MS patients to use complementary treatments. Ketogenic diet (KD) shows wide neuroprotective effects, but the precise mechanisms underlying the therapeutic activity of KD in MS are unclear. The present study established a continuous 24 days experimental autoimmune encephalomyelitis (EAE) mouse model with or without KD. The changes in motor function, pathological hallmarks of EAE, the status of microglia, neuroinflammatory response and intracellular signaling pathways in mice were detected by the rotarod test, histological analysis, real-time PCR (RT-PCR) and western blotting. Our results showed that KD could prevent motor deficiency, reduce clinical scores, inhibit demyelination, improve pathological lesions and suppress microglial activation in the spinal cord of EAE mice. Meanwhile, KD shifted microglial polarization toward the protective M2 phenotype and modified the inflammatory milieu by downregulating the production of pro-inflammatory cytokines, including TNF-α, IL-1β and IL-6, as well as upregulating the release of anti-inflammatory cytokines such as TGF-β. Furthermore, KD decreased the expression levels of CCL2, CCR2, CCL3, CCR1, CCR5, CXCL10 and CXCR3 in the spinal cord and spleen with reduced monocyte/macrophage infiltration in the CNS. In addition, KD inhibits NLRP3 activation in the microglia, as revealed by the significantly decreased co-expression of NLRP3+ and Iba-1+ in the KD + EAE group. Further studies demonstrated that KD suppresses inflammatory response and M1 microglial polarization by inhibiting the TLR4/MyD88/NF-κB/NLRP3 pathway, the JAK1/STAT1 pathway, HDAC3 and P2X7R activation, as well as up-regulation of JAK3/STAT6.

Neuroprotective effects of bornyl acetate on experimental autoimmune encephalomyelitis via anti-inflammatory effects and maintaining blood-brain-barrier integrity

Background: Bornyl acetate (BA), a chemical component of essential oil in the Pinus family, has yet to be actively studies in terms of its therapeutic effect on numerous diseases, including autoimmune diseases.Purpose: This study aimed to investigate the pharmacological effects and molecular mechanisms of BA on myelin oligodendrocyte glycoprotein (MOG35-55)-induced experimental autoimmune encephalomyelitis (EAE) mice in an animal model of multiple sclerosis (MS), a representative autoimmune disease in central nervous system.Methods: BA (100, 200, or 400 mg/kg) was orally treated to EAE mice once daily for 30 days after immunization for the behavioral test and for the 16th-18th days for the histopathological and molecular analyses, from the onset stage (8th day) of EAE symptoms.Results: BA mitigated behavioral dysfunction (motor disability) and demyelination in the spinal cord that were associated with the down-regulation of representative pro-inflammatory cytokines (interleukin (IL)-1 beta, IL-6, and tumor necrosis factor-alpha), enzymes (cyclooxygenase-2 and inducible nitric oxide synthase), and chemokines (monocyte chemotactic protein-1, macrophage inflammatory protein-1 alpha, and regulated on activation), and decreased infiltration of microglia (CD11b+/CD45+(low)) and macrophages (CD11b+/CD45+(high)). The anti-inflammatory effect of BA was related to the inhibition of mitogen-activated protein kinases and nuclear factor-kappa B pathways. BA also reduced the recruitment/infiltration rates of CD4+ T, Th1, and Th17 cells into the spinal cords of EAE mice, which was related to reduced blood-spinal cord barrier (BSCB) disruption.Conclusion: These findings strongly suggest that BA may alleviate EAE due to its anti-inflammatory and BSCB protective activities. This indicates that BA is a potential therapeutic agent for treating autoimmune demyelinating diseases including MS.

Anti-inflammatory mechanisms and pharmacological actions of phycocyanobilin in a mouse model of experimental autoimmune encephalomyelitis: A therapeutic promise for multiple sclerosis

Cytokines, demyelination and neuroaxonal degeneration in the central nervous system are pivotal elements implicated in the pathogenesis of multiple sclerosis (MS) and its nonclinical model of experimental autoimmune encephalomyelitis (EAE). Phycocyanobilin (PCB), a chromophore of the biliprotein C-Phycocyanin (C-PC) from Spirulina platensis, has antioxidant, immunoregulatory and anti-inflammatory effects in this disease, and it could complement the effect of other Disease Modifying Treatments (DMT), such as Interferon-β (IFN-β). Here, our main goal was to evaluate the potential PCB benefits and its mechanisms of action to counteract the chronic EAE in mice. MOG35-55-induced EAE was implemented in C57BL/6 female mice. Clinical signs, pro-inflammatory cytokines levels by ELISA, qPCR in the brain and immunohistochemistry using precursor/mature oligodendrocytes cells antibodies in the spinal cord, were assessed. PCB enhanced the neurological condition, and waned the brain concentrations of IL-17A and IL-6, pro-inflammatory cytokines, in a dose-dependent manner. A down- or up-regulating activity of PCB at 1 mg/kg was identified in the brain on three (LINGO1, NOTCH1, and TNF-α), and five genes (MAL, CXCL12, MOG, OLIG1, and NKX2-2), respectively. Interestingly, a reduction of demyelination, active microglia/macrophages density, and axonal damage was detected along with an increase in oligodendrocyte precursor cells and mature oligodendrocytes, when assessed the spinal cords of EAE mice that took up PCB. The studies in vitro in rodent encephalitogenic T cells and in vivo in the EAE mouse model with the PCB/IFN-β combination, showed an enhanced positive effect of this combined therapy. Overall, these results demonstrate the anti-inflammatory activity and the protective properties of PCB on the myelin and support its use with IFN-β as an improved DMT combination for MS.

Ferroptosis as a mechanism of oligodendrocyte loss and demyelination in experimental autoimmune encephalomyelitis

Ferroptosis, distinct from necrosis, autophagy and apoptosis, is a unique form of regulated cell death，and is a potential pathogenic mechanism of neuronal loss and defunction in many neurodegenerative disorders. Recent studies have shown a presence of iron deposition in the central nervous system (CNS) of patients with multiple sclerosis (MS). However, whether ferroptosis is involved in the pathogenesis of MS remains unclear. In the present study, we tested certain classical biomarkers of ferroptosis in the spinal cord of mice with experimental autoimmune encephalomyelitis (EAE), an animal model of MS, to substantiate the relationship between ferroptosis and oligodendrocyte (OL) loss and demyelination. Our results revealed decreased levels of key molecules in glutathione antioxidant mechanisms, including system xC (xCT) and glutathione peroxidase 4 (GPX4) in spinal cord of EAE mice, with evident lipid peroxidation in OLs. Moreover, transferrin receptor and ferritinophagy further catalyzed the generation of lipid reactive oxygen species through the fenton reaction, which induced OL death and demyelination at disease peak of EAE. This phenomenon was largely reversed by administering Fer-1, an inhibitor of ferritin phagocytosis, further validating the key role of ferritin phagocytosis in EAE. Taken together, these findings demonstrate that OL loss and demyelination may be induced in EAE through, at least in part, a mechanism of ferroptosis.

Astrocytic SARM1 promotes neuroinflammation and axonal demyelination in experimental autoimmune encephalomyelitis through inhibiting GDNF signaling

Astrocytes are important components of the innate immune response in the central nervous system (CNS), involving in the inflammatory and neurotoxic responses that occur in CNS diseases, such as multiple sclerosis (MS). Recent studies have shown that SARM1 plays a critical role in axonal degeneration and inflammation. However, the detailed role of astrocytic SARM1 in MS remains unclear. Here, we established the MS model of mice - experimental autoimmune encephalomyelitis (EAE) and found that SARM1 was upregulated in astrocytes of the spinal cords of EAE mice. Moreover, conditional knockout of astrocytic SARM1 (SARM1GFAP-CKO mice, SARM1Aldh1L1-CKO mice) delayed EAE with later onset, alleviated the inflammatory infiltration, and inhibited the demyelination and neuronal death. Mechanically, RNA-seq revealed that the expression of glial-derived neurotrophic factor (GDNF) was upregulated in SARM1−/− astrocytes. Western blot and immunostaining further confirmed the upregulation of GDNF in spinal cord astrocytes of SARM1GFAP-CKO EAE mice. Interestingly, the downregulation of GDNF by streptozotocin (STZ, a drug used to downregulate GDNF) treatment worsened the deficits of SARM1GFAP-CKO EAE mice. These findings identify that astrocytic SARM1 promotes neuroinflammation and axonal demyelination in EAE by inhibiting the expression of GDNF, reveal the novel role of SARM1/GDNF signaling in EAE, and provide new therapeutic ideas for the treatment of MS.

Curcumin regulates anti-inflammatory responses by AXL/JAK2/STAT3 signaling pathway in experimental autoimmune encephalomyelitis

Microglia-mediated neuroinflammation plays an important role in multiple sclerosis (MS). This study explored whether curcumin has a protective effect on experimental autoimmune encephalomyelitis (EAE), and the specific mechanism was investigated. We found that curcumin attenuates the severity of EAE mice. It inhibits the activation of microglia in the spinal cord of EAE mice and LPS-stimulated BV-2 cells. The findings clarify that curcumin may inhibit the inflammatory response mediated by microglia by inactivating the AXL/JAK2/STAT3 signaling pathway, which laid a theoretical foundation for the clinical management of MS.

Cathelicidin-related antimicrobial peptide promotes neuroinflammation through astrocyte-microglia communication in experimental autoimmune encephalomyelitis.

Cathelicidin-related antimicrobial peptide (CRAMP) is an effector molecule of the innate immune system with direct antimicrobial and immunomodulatory activities; however, its role in neuroinflammatory responses and related diseases is not clearly understood. In particular, the expression of CRAMP and its functional role has not been previously studied in experimental autoimmune encephalomyelitis (EAE) or multiple sclerosis (MS). Here, we investigated the role of CRAMP in neuroinflammation, using an EAE mouse model of MS and postmortem patient tissues. We found that the CRAMP expression was increased in the spinal cords of EAE-induced mice. Immunofluorescence analysis revealed that CRAMP is mainly induced in reactive astrocytes in the inflamed spinal cord of EAE mice. A similar pattern of the LL-37 (human CRAMP) expression was observed in the brain and spinal cord tissues of patients with MS. An intrathecal injection of the CRAMP peptide in EAE mice accelerated the onset of symptoms and increased disease severity with augmented expression of inflammatory mediators, glial activation, infiltration of inflammatory cells, and demyelination. In addition, shRNA-mediated knockdown of Cramp in the spinal cord resulted in a milder disease course with less inflammation in EAE mice. We identified FPR2 on microglia as a CRAMP receptor and demonstrated that CRAMP potentiates IFN-γ-induced microglial activation via the STAT3 pathway. Taken together, our findings suggest that CRAMP is a novel mediator of astrocyte-microglia interactions in neuroinflammatory conditions such as EAE. Thus, CRAMP could be exploited as a biomarker or therapeutic target for the diagnosis or treatment of MS.

Mapping of neuroinflammation-induced hypoxia in the spinal cord using optoacoustic imaging

Recent studies suggest that metabolic changes and oxygen deficiency in the central nervous system play an important role in the pathophysiology of multiple sclerosis (MS). In our present study, we investigated the changes in oxygenation and analyzed the vascular perfusion of the spinal cord in a rodent model of MS. We performed multispectral optoacoustic tomography of the lumbar spinal cord before and after an oxygen enhancement challenge in mice with experimental autoimmune encephalomyelitis (EAE), a model for MS. In addition, mice were transcardially perfused with lectin to label the vasculature and their spinal columns were optically cleared, followed by light sheet fluorescence microscopy. To analyze the angioarchitecture of the intact spine, we used VesSAP, a novel deep learning-based framework. In EAE mice, the spinal cord had lower oxygen saturation and hemoglobin concentration compared to healthy mice, indicating compromised perfusion of the spinal cord. Oxygen administration reversed hypoxia in the spinal cord of EAE mice, although the ventral region remained hypoxic. Additionally, despite the increased vascular density, we report a reduction in length and complexity of the perfused vascular network in EAE. Taken together, these findings highlight a new aspect of neuroinflammatory pathology, revealing a significant degree of hypoxia in EAE in vivo that is accompanied by changes in spinal vascular perfusion. The study also introduces optoacoustic imaging as a tractable technique with the potential to further decipher the role of hypoxia in EAE and to monitor it in MS patients.

Administration of Maresin-1 ameliorates the physiopathology of experimental autoimmune encephalomyelitis

BackgroundResolution of inflammation is an active and regulated process that leads to the clearance of cell debris and immune cells from the challenged tissue, facilitating the recovery of homeostasis. This physiological response is coordinated by endogenous bioactive lipids known as specialized pro-resolving mediators (SPMs). When resolution fails, inflammation becomes uncontrolled leading chronic inflammation and tissue damage, as occurs in multiple sclerosis (MS).MethodsSPMs and the key biosynthetic enzymes involved in SPM production were analysed by metabololipidomics and qPCR in active brain lesions, serum and peripheral blood mononuclear cells (PBMC) of MS patients as well as in the spinal cord of mice with experimental autoimmune encephalomyelitis (EAE). We also tested the therapeutic actions of the SPM coined Maresin-1 (MaR1) in EAE mice and studied its impact on inflammation by doing luminex and flow cytometry analysis.ResultsWe show that levels of MaR1 and other SPMs were below the limit of detection or not increased in the spinal cord of EAE mice, whereas the production of pro-inflammatory eicosanoids was induced during disease progression. Similarly, we reveal that SPMs were undetected in serum and active brain lesion samples of MS patients, which was linked to impaired expression of the enzymes involved in the biosynthetic pathways of SPMs. We demonstrate that exogenous administration of MaR1 in EAE mice suppressed the protein levels of various pro-inflammatory cytokines and reduced immune cells counts in the spinal cord and blood. MaR1 also decreased the numbers of Th1 cells but increased the accumulation of regulatory T cells and drove macrophage polarization towards an anti-inflammatory phenotype. Importantly, we provide clear evidence that administration of MaR1 in mice with clinical signs of EAE enhanced neurological outcomes and protected from demyelination.ConclusionsThis study reveals that there is an imbalance in the production of SPMs in MS patients and in EAE mice, and that increasing the bioavailability of SPMs, such as MaR1, minimizes inflammation and mediates therapeutic actions. Thus, these data suggest that immunoresolvent therapies, such as MaR1, could be a novel avenue for the treatment of MS.

Cordycepin prevents and ameliorates experimental autoimmune encephalomyelitis by inhibiting leukocyte infiltration and reducing neuroinflammation

Multiple sclerosis (MS) is a neuroinflammatory autoimmune disease characterized by multifocal perivascular infiltration of immune cells in the central nervous system (CNS). Cordycepin (3′-deoxyadenosine), an adenosine analogue initially extracted from the fungus Cordyceps militarisa, is one of the candidates that has multiple actions. We investigated that cordycepin attenuated the activation of LPS-induced mouse bone marrow-derived dendritic cells (BMDCs) and human monocyte-derived dendritic cells (MoDCs) through the inhibition of the AKT, ERK, NFκB, and ROS pathways and impaired the migration of BMDCs through the downregulation of adhesion molecules and chemokine receptors in vitro. In experimental autoimmune encephalomyelitis (EAE) model, preventive treatment with cordycepin decreased the expression of trafficking factors in the CNS, inhibited the secretion of inflammatory cytokines (IFN-γ, IL-6, TNF-α, and IL-17), and attenuated disease symptoms. A chemokine array indicated that cordycepin treatment reversed the high levels of CCL6, PARRES2, IL-16, CXCL10, and CCL12 in the brain and spinal cord of EAE mice, consistent with the RNA-seq data. Moreover, cordycepin suppressed the release of neuroinflammatory cytokines by activated microglial cells, macrophages, Th17 cells, Tc1 cells, and Th1 cells in vitro. Furthermore, cordycepin treatment exerted therapeutic effects on attenuating the disease severity in the early disease onset stage and late disease progression stage. Our study suggests that cordycepin treatment may not only prevent the occurrence of MS by inhibiting DC activation and migration but also potentially ameliorates the progression of MS by reducing neuroinflammation, which may provide insights into the development of new approaches for the treatment of MS.

Potential role of BAY11-7082, a NF-κB blocker inhibiting experimental autoimmune encephalomyelitis in C57BL/6J mice via declining NLRP3 inflammasomes.

Multiple sclerosis (MS) is an inflammatory autoimmune demyelinating disease of the central nervous system. NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasome is implicated in the pathogenesis of MS and its animal model, experimental autoimmune encephalomyelitis (EAE). However, the exact mechanism by which NLRP3 inflammasome is involved in the development of MS and EAE is not clear. NF-kappaB (NF-κB) is associated with the activity of NLRP3 inflammasomes, but the role of NF-κB is controversial. We sought to demonstrate that both NF-κB and NLRP3 contribute to development of MS and EAE, and NF-κB pathway is positively correlated with NLRP3 activation in EAE. The inhibitor of NF-κB and NLRP3, BAY11-7082, can prevent and treat EAE. BAY11-7082 (5 and 20mg/kg/i.p.) was intraperitoneally administered to EAE mice at the time of second injection of pertussis toxin (BAY11-7082 prevention group) or at the onset of symptoms (BAY11-7082 treatment group). mRNA expressions of NLRP3 were determined by qPCR. Protein expressions of NLRP3, NF-κB p65, and phosphorylated p65 were determined by western blotting. Serum levels of inflammatory cytokines were measured by cytometric bead array. Mice treated with BAY11-7082 (both prevention and treatment groups) showed lower clinical scores and attenuated pathological changes. NLRP3 inflammasome and activity of NF-κB in spinal cord of EAE mice was higher than that in control group. However, the level of NLRP3 inflammasome decreased in BAY11-7082 prevention and treatment groups. BAY11-7082 is a promising therapeutic agent for MS. NLRP3 activation in EAE maybe related with NF-κB pathway.

(R)-ketamine ameliorates the progression of experimental autoimmune encephalomyelitis in mice

Multiple sclerosis (MS) is an immune-mediated neurological disease that attacks the central nervous system, including spinal cord and brain. Experimental autoimmune encephalomyelitis (EAE) is the most commonly used model for MS. Depression is the most prevalent comorbidity in MS patients. We previously demonstrated that (R)-ketamine would be a novel antidepressant without side effects of ketamine. This study was undertaken to investigate whether (R)-ketamine could attenuate disease progression in EAE mouse model. (R)-ketamine (10 mg/kg/day for 15 days) significantly attenuated the reduction of body weight in EAE model mice compared to saline-treated mice. Furthermore, (R)-ketamine ameliorated the clinical EAE scores compared to saline-treated mice. Moreover, (R)-ketamine significantly attenuated the marked increases in the pathological scores, microglial activation, and blood-brain barrier integrity in the spinal cord compared to saline-treated mice. In conclusion, the current study suggests that (R)-ketamine could ameliorate EAE clinical scores and pathological changes in the spinal cord of EAE mice. Therefore, it is likely that (R)-ketamine would be a new potential prophylactic drug for MS.