Experimental Autoimmune Encephalomyelitis Model Research Articles

ApTOLL: A new therapeutic aptamer for cytoprotection and (re)myelination after multiple sclerosis.

ApTOLL is an aptamer selected to antagonize toll-like receptor 4 (TLR4), a relevant actor for innate immunity involved in inflammatory responses in multiple sclerosis (MS) and other diseases. The currently available therapeutic arsenal to treat MS is composed of immunomodulators but, to date, there are no (re)myelinating drugs available in clinics. In our present study, we studied the effect of ApTOLL on different animal models of MS. The experimental autoimmune encephalomyelitis (EAE) model was used to evaluate the effect of ApTOLL on reducing the inflammatory component. A more direct effect on oligodendroglia was studied with the cuprizone model and purified primary cultures of murine and human oligodendrocyte precursor cells (OPCs) isolated through magnetic-activated cell sorting (MACS) from samples of brain cortex. Also, we tested these effects in an ex vivo model of organotypic cultures demyelinated with lysolecithin (LPC). ApTOLL treatment positively impacted the clinical symptomatology of mice in the EAE and cuprizone models, which was associated with better preservation plus restoration of myelin and oligodendrocytes in the demyelinated lesions of animals. Restoration was corroborated on purified cultures of rodent and human OPCs. Our findings reveal a new therapeutic approach for the treatment of inflammatory and demyelinating diseases such as MS. The molecular nature of the aptamer exerts not only an anti-inflammatory effect but also neuroprotective and remyelinating effects. The excellent safety profile demonstrated by ApTOLL in animals and humans opens the door to future clinical trials in MS patients.

Fibulin-2 is an extracellular matrix inhibitor of oligodendrocytes relevant to multiple sclerosis.

Impairment of oligodendrocytes and myelin contributes to neurological disorders including multiple sclerosis (MS), stroke and Alzheimer's disease. Regeneration of myelin (remyelination) decreases the vulnerability of demyelinated axons, but this repair process commonly fails with disease progression. A contributor to inefficient remyelination is the altered extracellular matrix (ECM) in lesions that remains to be better defined. We have identified fibulin-2 (FBLN2) as a highly upregulated ECM component in lesions of MS and stroke, and in proteome databases of Alzheimer's disease and traumatic brain injury. Focusing on MS, the inhibitory role of FBLN2 was suggested in the experimental autoimmune encephalomyelitis (EAE) model in which genetic FBLN2 deficiency improved behavioral recovery by promoting the maturation of oligodendrocytes and enhancing remyelination. Mechanistically, when oligodendrocyte progenitors were cultured in differentiation media, FBLN2 impeded their maturation into oligodendrocytes by engaging the Notch pathway, leading to cell death. Adeno-associated virus-deletion of FBLN2 in astrocytes improved oligodendrocyte numbers and functional recovery in EAE and generated new myelin profiles after lysolecithin-induced demyelination. Collectively, our findings implicate FBLN2 as a hitherto unrecognized injury-elevated ECM, and a therapeutic target, that impairs oligodendrocyte maturation and myelin repair.

Enhanced Therapeutic Effects of Human Mesenchymal stem Cells Transduced with Secreted Klotho in a Murine Experimental Autoimmune Encephalomyelitis Model.

Treatment of multiple sclerosis (MS) remains a major challenge. The aim of this study was to evaluate the therapeutic potential of mesenchymal stem cells (MSCs) engineered with secreted Klotho (SKL) in an experimental autoimmune encephalomyelitis (EAE) mouse model of MS. EAE was induced in mice. MSCs or MSCs engineered with SKL (SKL-MSCs) were administered to EAE mice at the onset of disease. Hematoxylin-eosin and luxol fast blue staining were performed to evaluate histopathological changes. Expression of pro-inflammatory (TNF-α, IFN-γ, and IL-17) and anti-inflammatory (IL-10) cytokines was determined in the spinal cord using real-time PCR. Spinal cords were then processed for immunohistochemistry of the aforementioned cytokines. The frequencies of Th1, Th17, and regulatory T (Treg) cells were evaluated by flow cytometry of the spleen. The results showed that SKL-MSCs decreased clinical scores and reduced demyelination and inflammatory infiltration in the spinal cord more significantly than MSCs. Compared to MSCs, SKL-MSCs also exhibited a more profound capability of decreasing expression of TNF-α, IFN-γ, and IL-17 and increasing expression of IL-10 in the spinal cord with an enhanced homing to the inflamed tissue. Moreover, SKL-MSCs decreased the frequencies of Th1 and Th17 cells and increased the frequency of Treg cells in the spleen more potently than MSCs. Taken together, these findings demonstrate that SKL overexpression enhances the therapeutic potential of MSCs, as evidenced by significantly improved disease severity, decreased inflammation and tissue damage in the spinal cord, and a promoted shift in the Th17/Treg balance towards the anti-inflammatory Treg side in the EAE mice.

Discovery of a brain penetrant small molecule antagonist targeting LPA1 receptors to reduce neuroinflammation and promote remyelination in multiple sclerosis

Multiple sclerosis (MS) is a chronic neurological disease characterized by inflammatory demyelination that disrupts neuronal transmission resulting in neurodegeneration progressive disability. While current treatments focus on immunosuppression to limit inflammation and further myelin loss, no approved therapies effectively promote remyelination to mitigate the progressive disability associated with chronic demyelination. Lysophosphatidic acid (LPA) is a pro-inflammatory lipid that is upregulated in MS patient plasma and cerebrospinal fluid (CSF). LPA activates the LPA1 receptor, resulting in elevated CNS cytokine and chemokine levels, infiltration of immune cells, and microglial/astrocyte activation. This results in a neuroinflammatory response leading to demyelination and suppressed remyelination. A medicinal chemistry effort identified PIPE-791, an oral, brain-penetrant, LPA1 antagonist. PIPE-791 was characterized in vitro and in vivo and was found to be a potent, selective LPA1 antagonist with slow receptor off-rate kinetics. In vitro, PIPE-791 induced OPC differentiation and promoted remyelination following a demyelinating insult. PIPE-791 further mitigated the macrophage-mediated inhibition of OPC differentiation and inhibited microglial and fibroblast activation. In vivo, the compound readily crossed the blood–brain barrier and blocked LPA1 in the CNS after oral dosing. Direct dosing of PIPE-791 in vivo increased oligodendrocyte number, and in the mouse experimental autoimmune encephalomyelitis (EAE) model of MS, we observed that PIPE-791 promoted myelination, reduced neuroinflammation, and restored visual evoked potential latencies (VEP). These findings support targeting LPA1 for remyelination and encourage development of PIPE-791 for treating MS patients with advantages not seen with current immunosuppressive disease modifying therapies.

Therapeutic potential of EAE mice with sodium oligomannate and effects of intestinal flora and microglia polarization

Objective: To investigate the therapeutic effect of sodium oligomannate on experimental autoimmune encephalomyelitis (EAE) mice and its effect on intestinal flora and microglia polarization. Methods: Fifty female C57BL/6 mice were randomly divided by the random number table method into the control group, EAE model group and low-dose, medium-dose and high-dose group of sodium oligomannate with 10 mice each. The EAE model group and each dose group of sodium oligomannate were induced by subcutaneous multi-point injection of MOG35-55 peptide for the EAE model. Mice in the low-dose, medium-dose and high-dose group of sodium oligomannate were gavaged sodium oligomannate 40, 80, and 160 mg/kg twice a day, respectively, starting from the day after modeling. The intervention continued until the mice were euthanized. Observe the incidence of disease, infiltration of inflammatory cells in spinal cord tissue, and demyelination in each group of mice.. The mice feces were collected and tested for intestinal flora by 16S rRNA sequencing. Immunofluorescence staining was used to observe the expression of Iba-1 protein, an activation indicator of microglia, in spinal cord tissue. The protein levels of M1 type markers iNOS, CD16, and M2 type markers Arg1 and CD206 were tsested in the spinal cord by Western blotting and immunofluorescence staining. Results: None of the mice in the control group developed any disease, while the mice in other groups showed varying degrees of disease, including tail sag, unstable walking, and hind limb weakness. Compared with the EAE model group, the incubation period was prolonged, the peak was delayed and the peak neurological dysfunction score was reduced (3.6±0.6 vs 3.0±0.6, 2.8±0.5, 1.8±0.6, P<0.05) in all sodium oligomannate groups, with milder symptoms at higher doses. The differences in pairwise comparisons between the groups were all statistically significant (all P<0.05). In the control group, no inflammatory cell infiltration or demyelinating changes were observed in spinal cord tissue. In the EAE model group, inflammatory cell infiltration and demyelination changes were evident in the spinal cord tissues at the onset peak. Compared with the EAE model group, inflammatory cell infiltration and demyelination were ameliorated in all sodium oligomannate groups. Compared with the control group, the relative abundance of Bacteroidota decreased and that of Firmicutes increased in the EAE model group. Compared with the EAE model group, the relative abundance of Bacteroidota increased and that of Firmicutes decreased, the ratio of Bacteroidetes to Firmicutes increased (0.20±0.05 vs 0.37±0.02,0.61±0.03,0.91±0.08,P<0.01) in the respective dose groups. The difference in pairwise comparison between groups was statistically significant (P<0.01), with greater changes at higher doses. Compared with the control group, the levels of Iba-1、CD16 and iNOS increased, while the levels of Arg-1 and CD206 decreased in the EAE model group. Compared with the EAE model group, the levels of Iba-1、CD16 and iNOS decreased, while the levels of Arg-1 and CD206 increased in all sodium oligomannate groups(P<0.01), with greater changes at higher doses. The difference between groups was statistically significant (P<0.01). Conclusions: Sodium oligomannate has a therapeutic effect on EAE and is dose-dependent. Its mechanism of action may be related toimproving intestinal microecology and the modulation of microglial polarization.

Lipid metabolism is dysregulated in endocrine glands upon autoimmune demyelination

Disturbance in neuroendocrine signaling has been consistently documented in multiple sclerosis (MS), a chronic autoimmune disorder of the central nervous system (CNS) representing the main cause of non-traumatic brain injury among young adults. In fact, MS patients display altered hormonal levels and psychiatric symptoms along with the pathologic hallmarks of the disease, which include demyelination, neuroinflammation and axonal injury. In addition, we have recently shown that extensive transcriptional changes take place in the hypothalamus of mice upon the MS model experimental autoimmune encephalomyelitis (EAE). We also detected structural and functional aberrancies in endocrine glands of EAE animals. Specifically, we described the hyperplasia of adrenal glands and the atrophy of ovaries at disease peak. To further expand the characterization of these phenotypes, here we profiled the transcriptomes of both glands by means of RNA-seq technology. Notably, we identified fatty acid and cholesterol biosynthetic pathways as the most dysregulated molecular processes in adrenals and ovaries, respectively. Furthermore, we demonstrated that key genes encoding neuropeptides and hormone receptors undergo distinct expression dynamics in the hypothalamus along disease progression. Altogether, our results corroborate the dysfunction of the neuroendocrine system as a major pathological event of autoimmune demyelination and highlight the crosstalk between the CNS and the periphery in mediating such disease phenotypes.

CPT-11 mitigates autoimmune diseases by suppressing effector T cells without affecting long-term anti-tumor immunity

The incidence of autoimmune diseases has significantly increased over the past 20 years. Excessive host immunoreactions and disordered immunoregulation are at the core of the pathogenesis of autoimmune diseases. The traditional anti-tumor chemotherapy drug CPT-11 is associated with leukopenia. Considering that CPT-11 induces leukopenia, we believe that it is a promising drug for the control of autoimmune diseases. Here, we show that CPT-11 suppresses T cell proliferation and pro-inflammatory cytokine production in healthy C57BL/6 mice and in complete Freund’s adjuvant-challenged mice. We found that CPT-11 effectively inhibited T cell proliferation and Th1 and Th17 cell differentiation by inhibiting glycolysis in T cells. We also assessed CPT-11 efficacy in treating autoimmune diseases in models of experimental autoimmune encephalomyelitis and psoriasis. Finally, we proved that treatment of autoimmune diseases with CPT-11 did not suppress long-term immune surveillance for cancer. Taken together, these results show that CPT-11 is a promising immunosuppressive drug for autoimmune disease treatment.

MicroRNAs targeting TGF-β signaling exacerbate central nervous system autoimmunity by disrupting regulatory T cell development and function.

Transforming growth factor beta (TGF-β) signaling is essential for a balanced immune response by mediating the development and function of regulatory T cells (Tregs) and suppressing autoreactive T cells. Disruption of this balance can result in autoimmune diseases, including multiple sclerosis (MS). MicroRNAs (miRNAs) targeting TGF-β signaling have been shown to be upregulated in naïve CD4 T cells in MS patients, resulting in a limited in vitro generation of human Tregs. Utilizing the murine model experimental autoimmune encephalomyelitis, we show that perinatal administration of miRNAs, which target the TGF-β signaling pathway, enhanced susceptibility to central nervous system (CNS) autoimmunity. Neonatal mice administered with these miRNAs further exhibited reduced Treg frequencies with a loss in T cell receptor repertoire diversity following the induction of experimental autoimmune encephalomyelitis in adulthood. Exacerbated CNS autoimmunity as a result of miRNA overexpression in CD4 T cells was accompanied by enhanced Th1 and Th17 cell frequencies. These findings demonstrate that increased levels of TGF-β-associated miRNAs impede the development of a diverse Treg population, leading to enhanced effector cell activity, and contributing to an increased susceptibility to CNS autoimmunity. Thus, TGF-β-targeting miRNAs could be a risk factor for MS, and recovering optimal TGF-β signaling may restore immune homeostasis in MS patients.

Adult Neoneurogenesis and Oligodendrogenesis in Multiple Sclerosis: A Systematic Review of Human and Animal Studies.

Introduction: The subventricular zone promotes remyelination through activation differentiation of oligodendroglial precursor cells (OPCs) and neural stem cells (NSCs) into mature oligodendrocytes and thus in the adult brain. In multiple sclerosis (MS) this regenerative capability is halted resulting in neurodegeneration. We aimed to systematically search and synthesize evidence on mechanisms and phenomena associated with subventricular zone (SVZ) dysfunction in MS. Materials and Methods: Our systematic review was reported according to the PRISMA-ScR statement. MEDLINE, SCOPUS, ProQuest, and Google Scholar were searched using the terms "subventricular zone" and "multiple sclerosis," including English-written invivo and postmortem studies. Results: Twenty studies were included. Thirteen studies on models of experimental autoimmune encephalomyelitis (EAE) reported among others strong stathmin immunoreactivity in the SVZ of EAE models, the role of MOG immunization in neurogenesis impairment, the effect of parenchymal OPCs and NSCs in myelin repair, and the importance of ependymal cells (E1/E2) and ciliated B1 cells in SVZ stem cell signaling. CXCR4 signaling and transcriptional profiles of SVZ microglia, Gli1 pathway, and galactin-3 were also explored. Studies in humans demonstrated microstructural SVZ damage in progressive MS and the persistence of black holes near the SVZ, whereas postmortem confirmed the generation of polysialic acid-neural cell adhesion molecule and NG2-positive progenitors through SVZ activation, SVZ stathmin immunoreactivity, Shh pathway, and Gal-3 upregulation. Discussion: Oligodendrogenesis defects translate to reduced remyelination, a hallmark of MS that determines its end-phenotype and disease course. Conclusion: The role of inflammation and subsequent SVZ microenvironment disruption is evident in MS pathology.

Short report of potential Myelinogenesis effects of taper up-off of opium tincture in rodent model of multiple sclerosis

Multiple Sclerosis (MS) is one of the most common demyelinating autoimmune diseases that affects the central nervous system and is characterized by major immune-mediated myelin and axonal damage or axonal loss explicable to the absence of myelin sheaths. Here we present the early findings of the gene expression study of myelinogenesis-related genes of MS rat models which were treated with a novel protocol of taper up-off of opium tincture. The study included normal Lewis rats, MS rat models by induction of experimental autoimmune encephalomyelitis (EAE) without treatment, and MS rat models with a novel protocol of taper up-off treatment of opium tincture called Dezhakam-step-time (DST) in different dosages. RNA was extracted and cDNA was synthesized from the spinal cord tissue. Gene expression analysis was conducted for eight genes as markers of myelinogenesis (OLIG1, OLIG2, MBP, MYRF, PLP1, PMP22, EGF, and UGT8) using the Real-time PCR. All eight genes were down-regulated in EAE models vs. healthy controls and all eight genes were up-regulated after the taper up-off treatment of opium tincture. The most over-expression of myelinogenesis-related genes was revealed at higher dosages of opium tincture. These are the early results of a gene expression study in a multiple sclerosis model treated with opium tincture. It seems that the opium tincture method may induce the activation of myelinogenesis in EAE models which could lead to a potential treatment for improvement of neural dysfunctions in MS patients.

Intestinal MAdCAM-1 imaging as biomarker for prognostic in murine models of multiple sclerosis

IntroductionMultiple sclerosis (MS) is an autoimmune disease of the central nervous system. Recent evidence suggests that lymphocyte trafficking in the intestines could play a key role in its etiology. Nevertheless, it is not clear how intestinal tissue is involved in the disease onset nor its evolution. In the present study, we aimed to evaluate intestinal inflammation dynamic throughout the disease course and its potential impact on disease progression. MethodsWe used tissue immunophenotyping (immunohistofluorescence and flow cytometry) and a recently described molecular magnetic resonance imaging (MRI) method targeting mucosal addressin cell adhesion molecule-1 (MAdCAM-1) to assess intestinal inflammation in vivo in two distinct animal models of MS (Experimental Autoimmune Encephalomyelitis − EAE) at several time points of disease progression. ResultsWe report a positive correlation between disease severity and MAdCAM-1 MRI signal in two EAE models. Moreover, high MAdCAM-1 MRI signal during the asymptomatic phase is associated with a delayed disease onset in progressive EAE and to a lower risk of conversion to a secondary-progressive form in relapsing-remitting EAE. During disease evolution, in line with a bi-directional immune communication between the gut and the central nervous system, we observed a decrease in T-CD4+ and B lymphocytes in the ileum concomitantly with their increase in the spinal cord. ConclusionAltogether, these data unveil a crosstalk between intestinal and central inflammation in EAE and support the use of molecular MRI of intestinal MAdCAM-1 as a new biomarker for prognostic in MS patients.

Antibody-Mediated Depletion of Autoreactive T Lymphocytes through PD-1 Improves Disease Outcomes and Visualizes T Cell Activation in Experimental Autoimmune Encephalomyelitis.

Long-term therapeutic outcomes of multiple sclerosis (MS) remain hindered by the chronic nature of immune cell stimulation toward self-antigens. Development of novel methods to target and deplete autoreactive T lymphocytes remains an attractive target for therapeutics for MS. We developed a programmed cell death 1 (PD-1)-targeted radiolabeled mAb and assessed its ability to deplete activated PD-1+ T lymphocytes invitro and its ability to reduce disease burden of the myelin oligodendrocyte glycoprotein 35-55 experimental autoimmune encephalomyelitis (EAE) model in C57BL/6 mice. We also investigated the upregulation of PD-1 on infiltrating lymphocytes in an animal model of MS. Finally, we demonstrate the (to our knowledge) first reported positron-emission tomography/computed tomography imaging of activated PD-1+ cells in the EAE animal model of MS. We found that the 177Lu radioisotope-labeled anti-PD-1 mAb demonstrated significant invitro cytotoxicity toward activated CD4+PD-1+ T lymphocytes and led to significant reduction in overall disease progression in the EAE animal model. Our results show high expression of PD-1 on infiltrating lymphocytes in the spinal cords of EAE diseased animals. Positron-emission tomography/computed tomography imaging of the anti-PD-1 mAb demonstrated significant uptake in the cervical draining lymph nodes highlighting accumulation of activated lymphocytes. Targeted depletion of T lymphocytes using T cell activation markers such as PD-1 may present a novel method to reduce autoimmune attack and inflammation in autoimmune diseases such as MS. Development of multimodal nuclear theranostic agents may present the opportunity to monitor T cell activation via imaging radioisotopes and simultaneously treat MS using therapeutic radioisotopes.

Endoplasmic reticulum stress response in immune cells contributes to experimental autoimmune encephalomyelitis pathogenesis in rats

We examined the role of endoplasmic reticulum (ER) stress and the ensuing unfolded protein response (UPR) in the development of the central nervous system (CNS)-directed immune response in the rat model of experimental autoimmune encephalomyelitis (EAE). The induction of EAE with syngeneic spinal cord homogenate in complete Freund's adjuvant (CFA) caused a time-dependent increase in the expression of ER stress/UPR markers glucose-regulated protein 78 (GRP78), X-box-binding protein 1 (XBP1), C/EBP homologous protein (CHOP), and phosphorylated eukaryotic initiation factor 2α (eIF2α) in the draining lymph nodes of both EAE-susceptible Dark Agouti (DA) and EAE-resistant Albino Oxford (AO) rats. However, the increase in ER stress markers was more pronounced in AO rats. CFA alone also induced ER stress, but the effect was weaker and less sustained compared to full immunization. The ultrastructural analysis of DA lymph node tissue by electron microscopy revealed ER dilatation in lymphocytes, macrophages, and plasma cells, while immunoblot analysis of CD3-sorted lymph node cells demonstrated the increase in ER stress/UPR markers in both CD3+ (T cell) and CD3− (non-T) cell compartments. A positive correlation was observed between the levels of ER stress/UPR markers in the CNS-infiltrated mononuclear cells and the clinical activity of the disease. Finally, the reduction of EAE clinical signs by ER stress inhibitor ursodeoxycholic acid was associated with the decrease in the expression of mRNA encoding pro-inflammatory cytokines TNF and IL-1β, and encephalitogenic T cell cytokines IFN-γ and IL-17. Collectively, our data indicate that ER stress response in immune cells might be an important pathogenetic factor and a valid therapeutic target in the inflammatory damage of the CNS.

CD4+CD25+ regulatory T cells ex vivo generated from autologous naïve CD4+ T cells suppress EAE progression

CD4+CD25+ regulatory T cells (Tregs) play an important role in maintaining immune homeostasis in multiple sclerosis (MS). Hence, we aimed to explore the therapeutic efficacy and safety of adoptive cell therapy (ACT) utilizing induced antigen-specific Tregs in an animal model of MS, that is, in an experimental autoimmune encephalomyelitis (EAE) model. B cells from EAE model that were activated with soluble CD40L were used as antigen-presenting cells (APCs) to induce the differentiation of antigen-specific Tregs from naïve CD4 precursors, and then, a stepwise isolation of CD4+CD25highCD127low Tregs was performed using a flow sorter. All EAE mice were divided into Treg-treated group (2 × 104 cells in 0.2 mL per mouse, n = 14) and sham-treated group (0.2 mL normal saline (NS), n = 20), which were observed daily for clinical assessment, and for abnormal appearance for 6 weeks. Afterward, histological analysis, immunofluorescence and real-time PCR were performed. Compared to sham-treated mice, Treg-treated mice exhibited a significant decrease in disease severity scores and reduced inflammatory infiltration and demyelination in the spinal cord. Additionally, Tregs-treated mice demonstrated higher CCN3 protein and mRNA levels than sham-treated mice. The results of this preclinical study further support the therapeutic potential of this ACT approach in the treatment of MS.

Neuroprotection by acrolein sequestration through exogenously applied scavengers and endogenous enzymatic enabling strategies in mouse EAE model

We have previously shown that the pro-oxidative aldehyde acrolein is a critical factor in MS pathology. In this study, we found that the acrolein scavenger hydralazine (HZ), when applied from the day of induction, can suppress acrolein and alleviate motor and sensory deficits in a mouse experimental autoimmune encephalomyelitis (EAE) model. Furthermore, we also demonstrated that HZ can alleviate motor deficits when applied after the emergence of MS symptoms, making potential anti-acrolein treatment a more clinically relevant strategy. In addition, HZ can reduce both acrolein and MPO, suggesting a connection between acrolein and inflammation. We also found that in addition to HZ, phenelzine (PZ), a structurally distinct acrolein scavenger, can mitigate motor deficits in EAE when applied from the day of induction. This suggests that the likely chief factor of neuroprotection offered by these two structurally distinct acrolein scavengers in EAE is their common feature of acrolein neutralization. Finally, up-and-down regulation of the function of aldehyde dehydrogenase 2 (ALDH2) in EAE mice using either a pharmacological or genetic strategy led to correspondent motor and sensory changes. This data indicates a potential key role of ALDH2 in influencing acrolein levels, oxidative stress, inflammation, and behavior in EAE. These findings further consolidate the critical role of aldehydes in the pathology of EAE and its mechanisms of regulation. This is expected to reinforce and expand the possible therapeutic targets of anti-aldehyde treatment to achieve neuroprotection through both endogenous and exogenous manners.

Resting-state functional magnetic resonance imaging reveals functional connectivity alteration in the experimental autoimmune encephalomyelitis model of multiple sclerosis.

Multiple sclerosis (MS) is an autoimmune degenerative disease targeting white matter in the central nervous system. The most common animal model that mimics MS is experimental autoimmune encephalomyelitis (EAE) and it plays a crucial role in pharmacological research, from the identification of a therapeutic target to the in vivo validation of efficacy. Magnetic resonance imaging (MRI) is largely used to detect MS lesions, and resting-state functional MRI (rsfMRI) to investigate alterations in the brain functional connectivity (FC). MRI was mainly used in EAE studies to detect lesions in the spinal cord and brain. The current longitudinal MRI study aims to validate rsfMRI as a biomarker of the disease progression in the myelin oligodendrocyte glycoprotein 35-55 induced EAE animal model of MS. MR images were acquired 14, 25, and 50 days postimmunization. Seed-based analysis was used to investigate the whole-brain FC with some predefined areas, such as the thalamic regions, cerebellum, motor and somatosensory cortex. When compared with the control group, the EAE group exhibited a slightly altered FC and a decreasing trend in the total number of activated voxels along the disease progression. The most interesting result regards the whole-brain FC with the cerebellum. A hyperconnectivity behavior was found at an early phase and a significant reduced connectivity at a late phase. Moreover, we found a negative correlation between the total number of activated voxels during the late phase and the cumulative disease index. The results obtained provide a clinically relevant experimental platform that may be pivotal for the elucidation of the key mechanisms of accumulation of irreversible disability, as well as the development of innovative therapies for MS. Moreover, the negative correlation between the disease severity and the size of the activated area suggests a possible research pathway to follow for the resolution of the clinico-radiological paradox.

B cell depletion with anti-CD20 promotes neuroprotection in a BAFF-dependent manner in mice and humans.

Anti-CD20 therapy to deplete B cells is highly efficacious in preventing new white matter lesions in patients with relapsing-remitting multiple sclerosis (RRMS), but its protective capacity against gray matter injury and axonal damage is unclear. In a passive experimental autoimmune encephalomyelitis (EAE) model whereby TH17 cells promote brain leptomeningeal immune cell aggregates, we found that anti-CD20 treatment effectively spared myelin content and prevented myeloid cell activation, oxidative damage, and mitochondrial stress in the subpial gray matter. Anti-CD20 treatment increased B cell survival factor (BAFF) in the serum, cerebrospinal fluid, and leptomeninges of mice with EAE. Although anti-CD20 prevented gray matter demyelination, axonal loss, and neuronal atrophy, co-treatment with anti-BAFF abrogated these benefits. Consistent with the murine studies, we observed that elevated BAFF concentrations after anti-CD20 treatment in patients with RRMS were associated with better clinical outcomes. Moreover, BAFF promoted survival of human neurons in vitro. Together, our data demonstrate that BAFF exerts beneficial functions in MS and EAE in the context of anti-CD20 treatment.

Role of autoreactive Tc17 cells in the pathogenesis of experimental autoimmune encephalomyelitis

AbstractBackgroundThe pathogenesis of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE—an animal model of MS) is primarily mediated by T cells. However, recent studies have only focused on interleukin (IL)‐17‐secreting CD4+ T‐helper cells, also known as Th17 cells. This study aimed to compare Th17 cells and IL‐17‐secreting CD8+ T‐cytotoxic cells (Tc17) in the context of MS/EAE.MethodsFemale C57BL/6 mice were immunized with myelin oligodendrocyte glycoprotein peptides 35–55 (MOG35–55), pertussis toxin, and complete Freund's adjuvant to establish the EAE animal model. T cells were isolated from the spleen (12–14 days postimmunization). CD4+ and CD8+ T cells were purified using isolation kit and then differentiated into Th17 and Tc17, respectively, using MOG35–55 and IL‐23. The secretion levels of interferon‐γ (IFN‐γ) and IL‐17 were measured via enzyme‐linked immunosorbent assay using cultured CD4+ and CD8+ T cell supernatants. The pathogenicity of Tc17 and Th17 cells was assessed through adoptive transfer (tEAE), with the clinical course assessed using an EAE score (0–5). Hematoxylin and eosin as well as Luxol fast blue staining were used to examine the spinal cord. Purified CD8+ CD3+ and CD4+ CD3+ cells differentiated into Tc17 and Th17 cells, respectively, were stimulated with MOG35–55 peptide for proliferation assays.ResultsThe results showed that Tc17 cells (15,951 ± 1985 vs. 55,709 ± 4196 cpm; p &lt; 0.050) exhibited a weaker response to highest dose (20 μg/mL) MOG35–55 than Th17 cells. However, this response was not dependent on Th17 cells. After the 48 h stimulation, at the highest dose (20 μg/mL) of MOG35–55. Tc17 cells secreted lower levels of IFN‐γ (280.00 ± 15.00 vs. 556.67 ± 15.28 pg/mL, p &lt; 0.050) and IL‐17 (102.67 ± 5.86 pg/mL vs. 288.33 ± 12.58 pg/mL; p &lt; 0.050) than Th17 cells. Similar patterns were observed for IFN‐γ secretion at 96 and 144 h. Furthermore, Tc17 cell‐induced tEAE mice exhibited similar EAE scores to Th17 cell‐induced tEAE mice and also showed similar inflammation and demyelination.ConclusionThe degree of pathogenicity of Tc17 cells in EAE is lower than that of Th17 cells. Future investigation on different immune cells and EAE models is warranted to determine the mechanisms underlying MS.

AMP-activated protein kinase as a mediator of mitochondrial dysfunction of multiple sclerosis in animal models: A systematic review.

Multiple sclerosis (MS) is a chronic central nervous system (CNS) disorder characterized by demyelination, neuronal damage, and oligodendrocyte depletion. Reliable biomarkers are essential for early diagnosis and disease management. Emerging research highlights the role of mitochondrial dysfunction and oxidative stress in CNS disorders, including MS, in which mitochondria are central to the degenerative process. Adenosine monophosphate-activated protein kinase (AMPK) regulates the mitochondrial energy balance and initiates responses in neurodegenerative conditions. This systematic review, following Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, aimed to comprehensively assess the literature on AMPK pathways, mitochondrial dysfunction, and in vivo studies using MS animal models. The search strategy involved the use of AMPK syntaxes, MS syntaxes, and animal model syntaxes. The PubMed, Scopus, Web of Science, and Google Scholar databases were systematically searched onAugust 26, 2023 without publication year restrictions. The review identified and analyzed relevant papers to provide a comprehensive overview of the current state of related research. Eight studies utilizing various interventions and methodological approaches were included. Risk of bias assessment revealed some areas of low risk but lacked explicit reporting in others. These studies collectively revealed a complex relationship between AMPK, mitochondrial dysfunction, and MS pathogenesis, with both cuprizone and experimental autoimmune encephalomyelitis models demonstrating associations between AMPK and mitochondrial disorders, including oxidative stress and impaired expression of mitochondrial genes. These studies illuminate the multifaceted role of AMPK in MS animal models, involving energy metabolism, inflammatory processes, oxidative stress, and gene regulation leading to mitochondrial dysfunction. However, unanswered questions about its mechanisms and clinical applications underscore the need for further research to fully harness its potential in addressing MS-related mitochondrial dysfunction.

Scoring Central Nervous System Inflammation, Demyelination, and Axon Injury in Experimental Autoimmune Encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a common immune-based model of multiple sclerosis (MS). This disease can be induced in rodents by active immunization with protein components of the myelin sheath and Complete Freund's adjuvant (CFA) or by the transfer of myelin-specific T effector cells from rodents primed with myelin protein/CFA into naïve rodents. The severity of EAE is typically scored on a 5-point clinical scale that measures the degree of ascending paralysis, but this scale is not optimal for assessing the extent of recovery from EAE. For example, clinical scores remain high in some EAE models (e.g., myelin oligodendrocyte glycoprotein [MOG] peptide-induced model of EAE) despite the resolution of inflammation. Thus, it is important to complement clinical scoring with histological scoring of EAE, which also provides a means to study the underlying mechanisms of cellular injury in the central nervous system (CNS). Here, a simple protocol is presented to prepare and stain spinal cord and brain sections from mice and to score inflammation, demyelination, and axonal injury in the spinal cord. The method for scoring leukocyte infiltration in the spinal cord can also be applied to score brain inflammation in EAE. A protocol for measuring soluble neurofilament light (sNF-L) in the serum of mice using a Small Molecule Assay (SIMOA) assay is also described, which providesfeedback on the extent of overall CNS injury in live mice.