Experimental Autoimmune Encephalomyelitis Recovery Research Articles

Hepatocyte growth factor mediates mesenchymal stem cell–induced recovery in multiple sclerosis models

Mesenchymal stem cells have emerged as a potential therapy for a range of neural insults. In animal models of multiple sclerosis, an autoimmune disease that targets oligodendrocytes and myelin, treatment with human MSCs results in functional improvement that reflects both modulation of the immune response and myelin repair. Here we demonstrate that conditioned medium (CM) from human MSCs reduces functional deficits in mouse MOG35–55-induced EAE and promotes the development of oligodendrocytes and neurons. Functional assays identify a critical role for Hepatocyte Growth Factor (HGF) and its primary receptor cMet in MSCs stimulated recovery in EAE, neural cell development and remyelination. Active MSC-CM contains HGF and exogenously supplied HGF promotes recovery in EAE while cMet and anti-HGF antibodies block the functional recovery mediated by HGF and MSC-CM. Systemic treatment with HGF dramatically accelerated remyelination in lysolecithin-induced rat dorsal spinal cord lesions and in slice cultures. Together these data strongly implicate HGF in mediating MSC-stimulated functional recovery in animal models of multiple sclerosis.

Cannabinoid receptor-2-selective agonists improve recovery in experimental autoimmune encephalomyelitis (116.7)

Abstract Previous studies showed that cannabinoids have anti-inflammatory effects in experimental autoimmune encephalomyelitis (EAE), but the mechanism is elusive. In the present study, we investigated the effects of Gp1a (a new CB2 selective agonist) in EAE. EAE developed in a similar way in both vehicle and Gp1a treatment group, but mice treated with Gp1a recovered faster than the vehicle group. Cellular analysis was performed in spleen, brain and spinal cord. In spleen, there was no difference in numbers of CD3+ T cells, Th1 and Th17 cells, or macrophages. In contrast, CNS mononuclear cell analysis showed fewer CD3+ T cells, Th1 and Th17 cells, macrophages and activated microglia in spinal cord but not in brainin the Gp1a treatment group. Accordingly, for the Gp1a-treated group, histology studies also showed fewer CD3+ T cells, CD11b+ macrophages/microglia and less Iba-1 expression in spinal cord but not in brain. In vivo cell proliferation as measured through BrdU incorporation in CD3+CD45hi, CD11b+CD45hi and CD11b+CD45int mononuclear cells was not affected by Gp1a treatment. We hypothesize that Gp1a inhibits cell migration to the CNS. We also analyzed gene expression in brain and spinal cord following EAE development and found lower levels of VCAM-1, CXCL-10 and MMP9 in spinal cord. We also found that Gp1a inhibits MMP9 and TNFα production in BV.2 cells (a microglia cell line). We conclude that Gp1a improves EAE recovery through inhibiting immune cell migration into CNS.

A Novel IL-10–Independent Regulatory Role for B Cells in Suppressing Autoimmunity by Maintenance of Regulatory T Cells via GITR Ligand

B cells are important for the regulation of autoimmune responses. In experimental autoimmune encephalomyelitis (EAE), B cells are required for spontaneous recovery in acute models. Production of IL-10 by regulatory B cells has been shown to modulate the severity EAE and other autoimmune diseases. Previously, we suggested that B cells regulated the number of CD4(+)Foxp3(+) T regulatory cells (Treg) in the CNS during EAE. Because Treg suppress autoimmune responses, we asked whether B cells control autoimmunity by maintenance of Treg numbers. B cell deficiency achieved either genetically (μMT) or by depletion with anti-CD20 resulted in a significant reduction in the number of peripheral but not thymic Treg. Adoptive transfer of WT B cells into μMT mice restored both Treg numbers and recovery from EAE. When we investigated the mechanism whereby B cells induce the proliferation of Treg and EAE recovery, we found that glucocorticoid-induced TNF ligand, but not IL-10, expression by B cells was required. Of clinical significance is the finding that anti-CD20 depletion of B cells accelerated spontaneous EAE and colitis. Our results demonstrate that B cells play a major role in immune tolerance required for the prevention of autoimmunity by maintenance of Treg via their expression of glucocorticoid-induced TNFR ligand.

Interleukin-25 Expressed by Brain Capillary Endothelial Cells Maintains Blood-Brain Barrier Function in a Protein Kinase Cϵ-dependent Manner

Interleukin (IL)-25, a member of the IL-17 family of cytokines, is expressed in the brains of normal mice. However, the cellular source of IL-25 and its function in the brain remain to be elucidated. Here, we show that IL-25 plays an important role in preventing infiltration of the inflammatory cells into the central nervous system. Brain capillary endothelial cells (BCECs) express IL-25. However, it is down-regulated by inflammatory cytokines, including tumor necrosis factor (TNF)-alpha, IL-17, interferon-gamma, IL-1beta, and IL-6 in vitro, and is also reduced in active multiple sclerosis (MS) lesions and in the inflamed spinal cord of experimental autoimmune encephalomyelitis, an animal model of MS. Furthermore, IL-25 restores the reduced expression of tight junction proteins, occludin, junction adhesion molecule, and claudin-5, induced by TNF-alpha in BCECs and consequently repairs TNF-alpha-induced blood-brain barrier (BBB) permeability. IL-25 induces protein kinase Cepsilon (PKCepsilon) phosphorylation, and up-regulation of claudin-5 is suppressed by PKCepsilon inhibitor peptide in the IL-25-stimulated BCECs. These results suggest that IL-25 is produced by BCECs and protects against inflammatory cytokine-induced excessive BBB collapse through a PKCepsilon-dependent pathway. These novel functions of IL-25 in maintaining BBB integrity may help us understand the pathophysiology of inflammatory brain diseases such as MS.

Thymic Regulation of Autoimmune Disease by Accelerated Differentiation of Foxp3+ Regulatory T Cells through IL-7 Signaling Pathway

The exact role of adult thymus in autoimmune disease state is poorly understood. We show here that thymus regulated experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, as evidenced by loss of spontaneous recovery in thymectomized EAE mice. There was progressive enrichment for CD4 single-positive Foxp3(+) regulatory T cells in thymocytes during the course of EAE and they suppressed the disease when adoptively transferred. Thymus was shown to undergo an active process characterized by accelerated differentiation and proliferation of regulatory T (Treg) cells through a mechanism involving increased expression of IL-7 in stromal cells and dynamic expression of IL-7 receptor in thymic Treg cells. This process preceded EAE recovery and selectively affected Treg over non-Treg cells in the thymus, leading to increased output of thymic Treg cells and self-regulation of EAE. The study reveals a novel role of thymus in self-regulation of autoimmune condition.

Transgenic inhibition of astroglial NF-κB improves functional recovery in EAE by suppressing chronic neuroinflammation. (50.7)

Abstract In the CNS the transcription factor NF-κB is a key regulator of inflammation and secondary injury processes. Following trauma or disease, the expression of NF-κB-dependent genes is activated, leading to both protective and detrimental effects. Here we show that transgenic inactivation of astroglial NF-κB (GFAP-IκBα-dn mice) resulted in reduced disease severity and improved functional recovery following experimental autoimmune encephalomyelitis (EAE). At the chronic stage of the disease, transgenic mice exhibited an overall higher presence of leukocytes in spinal cord and brain, and a markedly higher percentage of CD8+CD122+ T regulatory cells compared to WT, which correlated with the timing of clinical recovery. We also observed that expression of proinflammatory genes in both spinal cord and cerebellum was delayed and reduced, while the loss of neuronal-specific molecules essential for synaptic transmission was limited compared to WT mice. Furthermore, death of retinal ganglion cells in affected retinas was almost abolished, suggesting the activation of neuroprotective mechanisms. Our data indicate that inhibiting NF-κB in astrocytes results in neuroprotective effects following EAE, directly implicating astrocytes in the pathophysiology of this disease.

Time-course expression of CNS inflammatory, neurodegenerative tissue repair markers and metallothioneins during experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an animal model for multiple sclerosis (MS). EAE and MS are characterized by CNS inflammation, demyelination and neurodegeneration. The inflammatory response occurring within the CNS leads to glial activation, dysfunction and death, as well as axonal damage and neurological deficit. Although the pathogenic mechanisms involved in EAE/MS are not well understood, accumulating data suggest that oxidative stress plays a major role in lesion development, and contributes to axonal dysfunction and degeneration. Metallothionein-I and -II are anti-inflammatory, neuroprotective, antioxidant proteins expressed during EAE and MS, in which they might play a protective role. The present study aimed to describe the expression profile of a group of inflammatory, neurodegenerative and tissue repair markers as well as metallothioneins during proteolipid protein-induced EAE, and to establish the time-relationships these molecules had during EAE. Interestingly, we found two marker expression profiles. In the first, marker expression increased as clinical signs worsened and reverted to baseline expression during recovery; in the second, marker expression increased at a later point during relapse, peaked at highest clinical score, and remained elevated throughout recovery. Of note, metallothionein expression was found to be related to the second profile, which would suggest that metallothionein proteins are implicated in the clinical recovery of EAE and perhaps these antioxidant proteins may provide therapeutic benefits in MS.

Dendritic and Synaptic Pathology in Experimental Autoimmune Encephalomyelitis

Evidence has shown that excitotoxicity may contribute to the loss of central nervous system axons and oligodendrocytes in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). Because dendrites and synapses are vulnerable to excitotoxicity, we examined these structures in acute and chronic models of EAE. Immunostaining for microtubule-associated protein-2 showed that extensive dendritic beading occurred in the white matter of the lumbosacral spinal cord (LSSC) during acute EAE episodes and EAE relapses. Retrograde labeling confirmed that most motoneuron dendrites were beaded in the white matter of the LSSC in acute EAE. In contrast, only mild swelling was observed in the gray matter of the LSSC. Dendritic beading showed marked recovery during EAE remission and after EAE recovery. In addition, synaptophysin, synapsin I, and PSD-95 immunoreactivities were significantly reduced in both the gray and white matter of the LSSC during acute EAE episodes and EAE relapses, but showed partial recovery during EAE remission and after EAE recovery. Pathologically, both dendritic beading and the reduction in synaptic protein immunoreactivity were well correlated with inflammatory cell infiltration in the LSSC at different EAE stages. We propose that dendritic and synaptic damage in the spinal cord may contribute to the neurological deficits in EAE.

Expression of the Long Form of Human FLIP by Retroviral Gene Transfer of Hemopoietic Stem Cells Exacerbates Experimental Autoimmune Encephalomyelitis

Subsidence of inflammation and clinical recovery in experimental autoimmune encephalomyelitis (EAE) is postulated to involve apoptosis of inflammatory cells. To test this concept, we examined the effects of overexpressing the long form of human FLICE-inhibitory protein, a potent inhibitor of death receptor-mediated apoptosis, in myelin oligodendrocyte glycoprotein-induced EAE in DBA/1 mice. We found that overexpression of the long form of human FLICE-inhibitory protein by retroviral gene transfer of hemopoietic stem cells led to a clinically more severe EAE in these mice compared with control mice receiving the retroviral vector alone. The exacerbated disease was evident by an enhanced and prolonged inflammatory reaction in the CNS of these animals compared with control mice. The acute phase of EAE was characterized by a massive infiltration of macrophages and granulocytes and a simultaneous increase in TNF-alpha production in the CNS. In the chronic phase of the disease, there was a prolonged inflammatory response in the form of persistent CD4(+) T and B cells in the CNS and a peripheral Th1 cytokine bias caused by elevated levels of IFN-gamma and reduced levels of IL-4 in the spleen. Our findings demonstrate that death receptor-mediated apoptosis can be important in the pathogenesis of EAE and further emphasize the need for effective apoptotic elimination of inflammatory cells to achieve disease remission.

Enhanced expression of constitutive and inducible forms of nitric oxide synthase in autoimmune encephalomyelitis

To elucidate the role of nitric oxide synthase (NOS) in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), we analyzed the expression of constitutive neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS) in the spinal cords of rats with EAE. We further examined the structural interaction between apoptotic cells and spinal cord cells including neurons and astrocytes, which are potent cell types of nitric oxide (NO) production in the brain. Western blot analysis showed that three forms of NOS significantly increased in the spinal cords of rats at the peak stage of EAE, while small amounts of these enzymes were identified in the spinal cords of rats without EAE. Immunohistochemical study showed that the expression of either nNOS or eNOS increased in the brain cells including neurons and astrocytes during the peak and recovery stages of EAE, while the expression of iNOS was found mainly in the inflammatory macrophages in the perivascular EAE lesions. Double labeling showed that apoptotic cells had intimate contacts with either neurons or astrocytes, which are major cell types to express nNOS and eNOS constitutively. Our results suggest that the three NOS may play an important role in the recovery of EAE.

Neonatal injection of Lewis rats with recombinant Vβ8.2 induces T cell but not B cell tolerance and increased severity of experimental autoimmune encephalomyelitis

In Lewis rats with experimental autoimmune encephalomyelitis (EAE) mediated by Vβ8.2 effector cells, anti-idiotypic T cells and antibodies could be boosted by injection of Vβ8.2 peptides, inducing both T cells and antibodies that reduced the severity and shortened the course of disease. However, EAE in Lewis rats is self-limiting, and we sought to determine if the anti-idiotypic response contributed to the natural recovery process. In a previous study, we found that adult tolerance induced to one of the regulatory idiotopes, Vβ8.2-44-54, caused worsening of EAE, implicating response to this epitope in recovery from EAE. However, neonatally-induced tolerance to Vβ8.2-44-54 did not alter the course of EAE, suggesting either compensation by additional Vβ8.2 determinants, or mechanistic differences in tolerization protocols. In this report, we reevaluate the role of Vβ8.2 determinants in recovery from EAE, using two recombinant Vβ8.2 constructs to induce neonatal tolerance to the comprehensive set of Vβ8.2 epitopes prior to adult induction of EAE. We found that neonatal exposure to either of the recombinant Vβ8.2 molecules induced “split” tolerance - specific T cell tolerance but enhanced antibody responses - and a more severe course of EAE. In contrast, neonatal exposure to a Vβ8.2 + T cell hybridoma or a control protein did not induce T cell tolerance to Vβ8.2 determinants and did not alter the EAE disease course. These results are consistent with those obtained by inducing adult tolerance, and suggest that our previous result (normal recovery from EAE in rats neonatally tolerized to Vβ8.2-44-54) was probably due to a compensatory response to other Vβ8.2 determinants. In both studies, the data clearly implicate T cell recognition of Vβ8.2 determinants in the natural EAE recovery process. © 1996 Wiley-Liss, Inc.

Neonatal injection of Lewis rats with recombinant Vβ8.2 induces T cell but not B cell tolerance and increased severity of experimental autoimmune encephalomyelitis

In Lewis rats with experimental autoimmune encephalomyelitis (EAE) mediated by V beta 8.2 effector cells, anti-idiotypic T cells and antibodies could be boosted by injection of V beta 8.2 peptides, inducing both T cells and antibodies that reduced the severity and shortened the course of disease. However, EAE in Lewis rats is self-limiting, and we sought to determine if the anti-idiotypic response contributed to the natural recovery process. In a previous study, we found that adult tolerance induced to one of the regulatory idiotopes, V beta 8.2-44-54, caused worsening of EAE, implicating response to this epitope in recovery from EAE. However, neonatally-induced tolerance to V beta 8.2-44-54 did not alter the course of EAE, suggesting either compensation by additional V beta 8.2 determinants, or mechanistic differences in tolerization protocols. In this report, we reevaluate the role of V beta 8.2 determinants in recovery from EAE, using two recombinant V beta 8.2 constructs to induce neonatal tolerance to the comprehensive set of V beta 8.2 epitopes prior to adult induction of EAE. We found that neonatal exposure to either of the recombinant V beta 8.2 molecules induced "split" tolerance-specific T cell tolerance but enhanced antibody responses- and a more severe course of EAE. In contrast, neonatal exposure to a V beta 8.2 + T cell hybridoma or a control protein did not induce T cell tolerance to V beta 8.2 determinants and did not alter the EAE disease course. These results are consistent with those obtained by inducing adult tolerance, and suggest that our previous result (normal recovery from EAE in rats neonatally tolerized to V beta 8.2-44-54) was probably due to a compensatory response to other V beta 8.2 determinants. In both studies, the data clearly implicate T cell recognition of V beta 8.2 determinants in the natural EAE recovery process.

T Cell Receptor Peptide Therapy Triggers Autoregulation of Experimental Encephalomyelitis

Encephalitogenic T cells specific for myelin basic protein share common V beta 8 peptide sequences in their T cell receptor (TCR) that can induce autoregulatory T cells and antibodies that prevent clinical signs of experimental autoimmune encephalomyelitis (EAE). It is not known, however, if TCR peptides can treat established disease. To test its therapeutic value, TCR-V beta 8-39-59 peptide was injected into rats with clinical signs of EAE. This treatment reduced disease severity and speeded recovery, apparently by boosting anti-V beta 8 T cells and antibodies raised naturally in response to encephalitogenic V beta 8+ T cells. These results demonstrate that synthetic TCR peptides can be used therapeutically, and implicate the TCR-V beta 8-39-59 sequence as a natural idiotope involved in EAE recovery. Similarly, human TCR peptides may be effective in enhancing natural regulation of autoreactive T cells that share common V genes.

Monoamines and related substances in brainstem and spinal cord of Lewis rats during the attack and recovery of experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis influenced catecholaminergic and indoleaminergic neurotransmitter systems in the central nervous system of Lewis rats. During paralysis, serotonin and noradrenaline were significantly reduced compared to animals injected with complete Freund's adjuvant in the posterior dorsomedial brainstem and in lower spinal cord segments. These diminutions remained after recovery from neurological signs in T 11-S 1. The serotonin metabolite 5-hydroxyindoleacetic acid was greatly augmented during the attack in all segments but was depleted during recovery in the lumbar spinal cord, which may indicate a normalized turnover at a reduced serotonin level. These results suggest functional impairment of monoaminergic neurons of the brainstem and spinal cord followed by permanent damage to some monoaminergic fibers in the spinal cord.

Measurement of free radical scavengers in the spinal cord of rats with experimental autoimmune encephalomyelitis

Antioxidants (ascorbic acid, glutathione, cysteine, α-tocopherol) and uric acid were measured using two high-pressure liquid chromatographic methods in 3 regions (cervical, thoracic, lumbar) of the spinal cord and in blood of Lewis rats during the attack and recovery of experimental autoimmune encephalomyelitis (EAE). Uric acid, which is thought to be a marker of free radical release, was greatly increased and glutathione correspondingly decreased in lumbar and thoracic regions. Cysteine and ascorbic acid were practically unchanged, whereas α-tocopherol was significantly increased during attack and recovery. Results, which could have therapeutic implications, generally support the hypothesis that free radicals are released during EAE.