Relapsing-remitting Experimental Autoimmune Encephalomyelitis Model Research Articles

Tyrphostin A9 protects axons in experimental autoimmune encephalomyelitis through activation of ERKs

AimsSmall molecule compound tyrphostin A9 (A9), an inhibitor of platelet-derived growth factor (PDGF) receptor, was previously reported by our group to stimulate extracellular signal-regulated kinase 1 (ERK1) and 2 (ERK2) in neuronal cells in a PDGF receptor-irrelevant manner. The study aimed to investigate whether A9 could protect axons in experimental autoimmune encephalomyelitis through activation of ERKs. Main methodsA9 treatment on the protection on neurite outgrowth in SH-SY5Y neuroblastoma cells and primary substantia nigra neuron cultures from the neurotoxin MPP+ were analyzed. Then, clinical symptoms as well as ERK1/2 activation, axonal protection induction, and the abundance increases of the regeneration biomarker GAP-43 in the CNS in the relapsing-remitting experimental autoimmune encephalomyelitis (EAE) model were verified. Key findingsA9 treatment could stimulate neurite outgrowth in SH-SY5Y neuroblastoma cells and protect primary substantia nigra neuron cultures from the neurotoxin MPP+. In the relapsing-remitting EAE model, oral administration of A9 successfully ameliorated clinical symptoms, activated ERK1/2, induced axonal protection, and increased the abundance of the regeneration biomarker GAP-43 in the CNS. Interestingly, gene deficiency of ERK1 or ERK2 disrupted the beneficial effects of A9 in MOG-35-55-induced EAE. SignificanceThese results demonstrated that small molecule compounds that stimulate persistent ERK activation in vitro and in vivo may be useful in protective or restorative treatment for neurodegenerative diseases.

Oral D-mannose treatment suppresses experimental autoimmune encephalomyelitis via induction of regulatory T cells

D-mannose (D-m) is a glucose epimer found in natural products, especially fruits. In mouse models of diabetes and airway inflammation, D-m supplementation via drinking water attenuated pathology by modifying cellular energy metabolism, leading to the activation of latent transforming growth factor beta (TGF-β), which in turn induced T regulatory cells (Tregs). Given that Tregs are important in controlling neuroinflammation in experimental autoimmune encephalomyelitis (EAE) and likely in multiple sclerosis (MS), we hypothesized that D-m could also suppress EAE. We found that D-m delayed disease onset and reduced disease severity in two models of EAE. Importantly, D-m treatment prevented relapses in a relapsing-remitting model of EAE, which mimics the most common clinical manifestation of MS. EAE suppression was accompanied by increased frequency of CD4+FoxP3+ Tregs in the central nervous system, suggesting that EAE suppression resulted from Treg cell induction by D-m. These findings suggest that D-m has the potential to be a safe and low-cost complementary therapy for MS.

Pharmacological Inhibition of STAT3 by Stattic Ameliorates Clinical Symptoms and Reduces Autoinflammation in Myeloid, Lymphoid, and Neuronal Tissue Compartments in Relapsing-Remitting Model of Experimental Autoimmune Encephalomyelitis in SJL/J Mice.

Multiple sclerosis (MS) is an immune-mediated inflammatory disease that leads to demyelination and neuronal loss in the central nervous system. Immune cells of lymphoid and myeloid origin play a significant role in the initiation and amplification of neuronal inflammation in MS. STAT3 signaling plays a pivotal role in both myeloid and lymphoid immune cells, such as neutrophils and CD4+ T cells, through regulation of their inflammatory potential. Dysregulation in STAT3 signaling in myeloid and lymphoid cell compartments has been reported in MS. In this report, we attempted to investigate the effect of a small molecular inhibitor of STAT3, i.e., Stattic, in a relapsing–remitting (RR) model of experimental autoimmune encephalomyelitis (EAE). The effect of Stattic was investigated for clinical features, oxidative stress parameters, and Th17-related signaling in both the periphery and brain of SJL/J mice. Our data report that p-STAT3 expression is elevated in granulocytes, CD4+ T cells, and brain tissue in myelin proteolipid protein (PLP)-immunized SJL/J mice, which is associated with the presence of clinical symptoms and upregulation of inflammatory markers in these cells/tissues. Treatment with Stattic leads to the amelioration of disease symptoms and attenuation of inflammatory markers in neutrophils (iNOS/nitrotyrosine/IL-1β), CD4+ T cells (IL-17A/IL-23R), and brain tissue (IL-17A/iNOS/IL-1β/MPO activity/lipid peroxides) in mice with EAE. These data suggest that the blockade of STAT3 signaling in cells of lymphoid and myeloid origin may cause the attenuation of systemic and neuronal inflammation, which could be responsible for the amelioration of disease symptoms in an RR model of EAE. Therefore, pharmacological inhibition of STAT3 in RRMS could be a potential therapeutic strategy.

Chronic mild hypoxia accelerates recovery from preexisting EAE by enhancing vascular integrity and apoptosis of infiltrated monocytes

While several studies have shown that hypoxic preconditioning suppresses development of the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS), no one has yet examined the important clinically relevant question of whether mild hypoxia can impact the progression of preexisting disease. Using a relapsing-remitting model of EAE, here we demonstrate that when applied to preexisting disease, chronic mild hypoxia (CMH, 10% O2) markedly accelerates clinical recovery, leading to long-term stable reductions in clinical score. At the histological level, CMH led to significant reductions in vascular disruption, leukocyte accumulation, and demyelination. Spinal cord blood vessels of CMH-treated mice showed reduced expression of the endothelial activation molecule VCAM-1 but increased expression of the endothelial tight junction proteins ZO-1 and occludin, key mechanisms underlying vascular integrity. Interestingly, while equal numbers of inflammatory leukocytes were present in the spinal cord at peak disease (day 14 postimmunization; i.e., 3 d after CMH started), apoptotic removal of infiltrated leukocytes during the remission phase was markedly accelerated in CMH-treated mice, as determined by increased numbers of monocytes positive for TUNEL and cleaved caspase-3. The enhanced monocyte apoptosis in CMH-treated mice was paralleled by increased numbers of HIF-1α+ monocytes, suggesting that CMH enhances monocyte removal by amplifying the hypoxic stress manifest within monocytes in acute inflammatory lesions. These data demonstrate that mild hypoxia promotes recovery from preexisting inflammatory demyelinating disease and suggest that this protection is primarily the result of enhanced vascular integrity and accelerated apoptosis of infiltrated monocytes.

MR Elastography-Based Assessment of Matrix Remodeling at Lesion Sites Associated With Clinical Severity in a Model of Multiple Sclerosis.

Magnetic resonance imaging (MRI) with gadolinium based contrast agents (GBCA) is routinely used in the clinic to visualize lesions in multiple sclerosis (MS). Although GBCA reveal endothelial permeability, they fail to expose other aspects of lesion formation such as the magnitude of inflammation or tissue changes occurring at sites of blood-brain barrier (BBB) disruption. Moreover, evidence pointing to potential side effects of GBCA has been increasing. Thus, there is an urgent need to develop GBCA-independent imaging tools to monitor pathology in MS. Using MR-elastography (MRE), we previously demonstrated in both MS and the animal model experimental autoimmune encephalomyelitis (EAE) that inflammation was associated with a reduction of brain stiffness. Now, using the relapsing-remitting EAE model, we show that the cerebellum—a region with predominant inflammation in this model—is especially prone to loss of stiffness. We also demonstrate that, contrary to GBCA-MRI, reduction of brain stiffness correlates with clinical disability and is associated with enhanced expression of the extracellular matrix protein fibronectin (FN). Further, we show that FN is largely expressed by activated astrocytes at acute lesions, and reflects the magnitude of tissue remodeling at sites of BBB breakdown. Therefore, MRE could emerge as a safe tool suitable to monitor disease activity in MS.

Acylated and deacylated quillaja saponin-21 adjuvants have opposite roles when utilized for immunization of C57BL/6 mice model with MOG35-55 peptide

BackgroundThe majority of patients with multiple sclerosis (MS) suffer from central neuropathic pain (CNP). Using experimental autoimmune encephalomyelitis (EAE) model, only a few experiments were performed to assess pain behaviors in MS. To address this issue, complete Freund's adjuvant (CFA) was replaced with an acylated triterpene glycoside saponin adjuvant named quillaja saponin-21 (QS-21) to develop CNP in the EAE mouse model. The deacylated form of QS-21, named QT-0101, has been suggested to have an immunomodulatory effect. Thus, QT-0101 was used as a vaccine adjuvant to modulate the immune system against myelin oligodendrocyte glycoprotein (MOG35-55) antigen. MethodsIn this study, C57BL/6 mice, except for mice in the negative control (PBS) and MOG groups, were divided into three groups and immunized by MOG35-55 emulsified with CFA, QS-21, or QT-0101 adjuvants, respectively. Thermal hyperalgesia, as a CNP clinical manifestation, through the Hot Plate test and the clinical signs, was assessed for 60 days after immunization. On days 21 and 60, mice were sacrificed and the frequency of TCD4+, TCD8+, IL-17+, IL-4+, and CD25+/FoxP3+ cells population in the total splenocytes population was assessed by flow cytometry. Infiltration of Leukocytes into the brain and demyelination of white matter were also evaluated by histopathologic studies. ResultsOur results revealed that unlike the MOG+QT-0101 group, the MOG+QS-21 and MOG+CFA groups represented clinical symptoms that mimic the mild relapsing-remitting and monophasic models, respectively. Thermal hyperalgesia, as a CNP clinical manifestation, developed in the bilateral hind paws in the MOG+CFA and MOG+QS-21 mice groups during the onset of neurologic deficits, but it is maintained until completion of the study only in MOG+QS-21 mice group. The frequency of TCD4+, TCD8+ and IL-17+ cells population in the MOG+QS-21 and MOG+CFA mice groups, as well as IL-4+ and CD25+/Foxp3+ cells population in the MOG+QT-0101 mice group, significantly increased in comparison with the PBS mice group. Infiltration of inflammatory cells increased significantly in the MOG+QS-21 and MOG+CFA mice groups compared with the PBS mice group. Demyelination of white matter was identified significantly only in the MOG+CFA mice group compared with the PBS mice group. ConclusionThese results showed that QS-21 is a suitable adjuvant for the establishment of a mild relapsing-remitting EAE model for CNP development and open a new avenue to future pre-clinical and clinical research studies related to CNP treatment. Nevertheless, QT-0101 seems to have the potential to act as a vaccine adjuvant with immunomodulatory property against auto-antigens.

Multiple rodent models and behavioral measures reveal unexpected responses to FTY720 and DMF in experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a widely-used rodent model for multiple sclerosis (MS), but a single model can hardly capture all features of MS. We investigated whether behavioral parameters in addition to clinical motor function scores could be used to assess treatment efficacy during score-free intervals in the relapsing-remitting EAE model in SJL/J mice. We studied the effects of the clinical reference compounds FTY720 (fingolimod, 0.5mg/kg/day) and dimethyl fumarate (DMF, 20–30mg/kg/day) on clinical scores in several rodent EAE models in order to generate efficacy profiles. SJL/J mice with relapsing-remitting EAE were studied using behavioral tests, including rotarod, gait analysis, locomotor activity and grip strength. SJL/J mice were also examined according to Crawley’s sociability and preference for social novelty test. Prophylactic treatment with FTY720 prevented clinical scores in three of the four EAE rodent models: Dark Agouti (DA) and Lewis rats and C57BL/6J mice. Neither prophylactic nor late-therapeutic treatment with FTY720 reduced clinical scores or reversed deficits in the rotarod test in SJL/J mice, but we observed effects on motor functions and sociability in the absence of clinical scores. Prophylactic treatment with FTY720 improved the gait of SJL/J mice whereas late-therapeutic treatment improved manifestations of reduced social (re)cognition or preference for social novelty. DMF was tested in three EAE models and did not improve clinical scores at the dose used. These data indicate that improvements in behavioral deficits can occur in absence of clinical scores, which indicate subtle drug effects and may have translational value for human MS.

Inhibition of Gli1 mobilizes endogenous neural stem cells for remyelination.

SummaryEnhancing repair of myelin is an important, but still elusive therapeutic goal in many neurological disorders1. In Multiple Sclerosis (MS), an inflammatory demyelinating disease, endogenous remyelination does occur but is frequently insufficient to restore function. Both parenchymal oligodendrocyte progenitor cells (OPCs) and endogenous adult neural stem cells (NSCs) resident within the subventricular zone (SVZ) are known sources of remyelinating cells2. Here, we characterize the contribution to remyelination of a subset of adult NSCs, identified by their expression of Gli1, a transcriptional effector of the Sonic Hedgehog (Shh) pathway. We show that these cells are recruited from the SVZ to populate demyelinated lesions in the forebrain but never enter healthy, white matter tracts. Unexpectedly, recruitment of this pool of NSCs, and their differentiation into oligodendrocytes, is significantly enhanced by genetic or pharmacological inhibition of Gli1. Importantly, complete inhibition of canonical hedgehog signaling was ineffective indicating that Gli1’s role in both augmenting hedgehog signaling and retarding myelination is specialized. Indeed, inhibition of Gli1 improves the functional outcome in a relapsing/remitting model of experimental autoimmune encephalomyelitis (RR-EAE) and is neuroprotective. Thus, endogenous NSCs can be mobilized for the repair of demyelinated lesions by inhibiting Gli1, identifying a new therapeutic avenue for the treatment of demyelinating disorders.

Immunosuppressive potential of human amnion epithelial cells in the treatment of experimental autoimmune encephalomyelitis.

BackgroundMultiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system (CNS). In recent years, it has been found that cells such as human amnion epithelial cells (hAECs) have the ability to modulate immune responses in vitro and in vivo and can differentiate into multiple cell lineages. Accordingly, we investigated the immunoregulatory effects of hAECs as a potential therapy in an MS-like disease, EAE (experimental autoimmune encephalomyelitis), in mice.MethodsUsing flow cytometry, the phenotypic profile of hAECs from different donors was assessed. The immunomodulatory properties of hAECs were examined in vitro using antigen-specific and one-way mixed lymphocyte proliferation assays. The therapeutic efficacy of hAECs was examined using a relapsing-remitting model of EAE in NOD/Lt mice. T cell responsiveness, cytokine secretion, T regulatory, and T helper cell phenotype were determined in the peripheral lymphoid organs and CNS of these animals.ResultsIn vitro, hAECs suppressed both specific and non-specific T cell proliferation, decreased pro-inflammatory cytokine production, and inhibited the activation of stimulated T cells. Furthermore, T cells retained their naïve phenotype when co-cultured with hAECs. In vivo studies revealed that hAECs not only suppressed the development of EAE but also prevented disease relapse in these mice. T cell responses and production of the pro-inflammatory cytokine interleukin (IL)-17A were reduced in hAEC-treated mice, and this was coupled with a significant increase in the number of peripheral T regulatory cells and naïve CD4+ T cells. Furthermore, increased proportions of Th2 cells in the peripheral lymphoid organs and within the CNS were observed.ConclusionThe therapeutic effect of hAECs is in part mediated by inducing an anti-inflammatory response within the CNS, demonstrating that hAECs hold promise for the treatment of autoimmune diseases like MS.

Beneficial Effects of Blueberries in Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an animal model of autoimmune disease that presents with pathological and clinical features similar to those of multiple sclerosis (MS) including inflammation and neurodegeneration. This study investigated whether blueberries, which possess immunomodulatory, anti-inflammatory, and neuroprotective properties, could provide protection in EAE. Dietary supplementation with 1% whole, freeze-dried blueberries reduced disease incidence by >50% in a chronic EAE model (p < 0.01). When blueberry-fed mice with EAE were compared with control-fed mice with EAE, blueberry-fed mice had significantly lower motor disability scores (p = 0.03) as well as significantly greater myelin preservation in the lumbar spinal cord (p = 0.04). In a relapsing-remitting EAE model, blueberry-supplemented mice showed improved cumulative and final motor scores compared to control diet-fed mice (p = 0.01 and 0.03, respectively). These data demonstrate that blueberry supplementation is beneficial in multiple EAE models, suggesting that blueberries, which are easily administered orally and well-tolerated, may provide benefit to MS patients.

Susceptibility to experimental autoimmune encephalomyelitis is associated with altered B-cell subsets distribution and decreased serum BAFF levels

B cells possess the ability to regulate either pathogenic or protective events in several autoimmune diseases such as multiple sclerosis (MS) and its experimental model, experimental autoimmune encephalomyelitis (EAE). Given the extensive use of B-cell-targeting treatments, it appears crucial to more precisely define the dual role of B cells in the progression of the disease. In the present study, we explored the impact of EAE induction on the distribution of potential regulatory B-cell subsets (CD5 + B1a, marginal zone and transitional 2 B cells) over critical time points in the relapsing-remitting EAE model, SJL/J (H2s). The same approach was carried out in B10.S mice that are resistant to EAE induction, (H2s). The comparative data obtained from these experiments showed that the homeostasis of the regulatory B-cell subsets is altered during the EAE preclinical and acute phases. These observations were associated with a distortion of the BAFF response. All these data suggest the existence of a close relationship between B-cell homeostasis, BAFF response and the susceptibility to develop EAE.

Inhibition of ERK MAPK Suppresses IL-23- and IL-1-Driven IL-17 Production and Attenuates Autoimmune Disease

IL-17-producing CD4(+) T (Th17) cells are pathogenic in many autoimmune diseases. The induction and expansion of Th17 cells is directed by cytokines, including IL-23 and IL-1beta, produced by innate immune cells through activation of pathogen recognition receptors. The NF-kappaB and IFN regulatory factor families of transcriptional factors mediate IL-12 production; however, distinct signaling pathways appear to be required for IL-23 production. In this study, we show that inhibition of ERK MAPK suppressed IL-23 and IL-1beta production by dendritic cells stimulated with TLR or dectin-1 agonists but did not affect IL-12p70 production. Furthermore, an ERK inhibitor suppressed the ability of Ag-pulsed TLR-activated dendritic cells to induce Ag-specific Th17 cells in vivo, but interestingly also inhibited the induction of Th1 cells. Treatment with an ERK inhibitor attenuated experimental autoimmune encephalomyelitis (EAE), when administered either at the induction phase of acute EAE or during remission in the relapsing-remitting EAE model. This was associated with significant suppression of autoantigen-specific Th17 and Th1 responses. The suppressive effect of the ERK inhibitor on attenuation of EAE was reversed by administration of IL-1beta and IL-23. Our findings suggest that ERK MAPK plays a critical and hitherto undescribed role in activating innate production of IL-23 and IL-1beta, which promote pathogenic T cell responses, and therefore represents an important target for therapeutic intervention against autoimmune diseases.

Quantification of Myelin and Axon Pathology During Relapsing Progressive Experimental Autoimmune Encephalomyelitis in the Biozzi ABH Mouse

Multiple sclerosis is an immune-mediated demyelinating disease, with axonal loss underlying long-term progressive disability. In this study, we have analyzed axonal and myelin pathology in a chronic relapsing-remitting experimental autoimmune encephalomyelitis model in Biozzi ABH mice induced by immunization with a syngeneic spinal cord homogenate. The animals were followed for3 months; inflammation, T-cell infiltration, demyelination, and axonal loss were examined at various time points throughout the disease course. We found that macrophage infiltration and microglia activation preceded detectable T-cell infiltration. Axonal loss was first evident at the acute phase of disease before demyelination was detected. Demyelination and axonal loss occurred after each relapse and correlated with increasing residual motor deficits in remission. The resulting lesions displayed evidence of demyelination, remyelination, axonal degeneration, and axon loss. After a series of 3 relapses, animals entered a chronic progressive phase with permanent paralysis and a relative absence of inflammation. Axonal loss continued in this phase, although demyelinated axons persisted. These findings indicate that experimental autoimmune encephalomyelitis in Biozzi ABH mice has important similarities to multiple sclerosis with a relapsing-remitting disease course followed by a secondary progressive phase; it is thus a suitable model in which to explore remyelination and neuroprotective therapies for multiple sclerosis.