Stages Of Experimental Autoimmune Encephalomyelitis Research Articles

Interactions between Neutrophils, Th17 Cells, and Chemokines during the Initiation of Experimental Model of Multiple Sclerosis

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS) in which activated T cell and neutrophil interactions lead to neuroinflammation. In this study the expression of CCR6, CXCR2, and CXCR6 in Th17 cells and neutrophils migrating to the brain during EAE was measured, alongside an evaluation of the production of IL-17, IL-23, CCL-20, and CXCL16 in the brain. Next, inflammatory cell subpopulations accumulating in the brain after intracerebral injections of IL-17 or CXCL1, as well as during modulation of EAE with anti-IL-23R or anti-CXCR2 antibodies, were analyzed. Th17 cells upregulate CXCR2 during the preclinical phase of EAE and a significant migration of these cells to the brain was observed. Neutrophils upregulated CCR6, CXCR2, and CXCR6 during EAE, accumulating in the brain both prior to and during acute EAE attacks. Production of IL-17, IL-23, CCL20, and CXCL16 in the CNS was increased during both preclinical and acute EAE. Intracerebral delivery of CXCL1 stimulated the early accumulation of neutrophils in normal and preclinical EAE brains but reduced the migration of Th17 cells to the brain during the preclinical stage of EAE. Modulation of EAE by anti-IL-23R antibodies ameliorated EAE by decreasing the intracerebral accumulation of Th17 cells.

PERK Activation Preserves the Viability and Function of Remyelinating Oligodendrocytes in Immune-Mediated Demyelinating Diseases

Remyelination occurs in multiple sclerosis (MS) lesions but is generally considered to be insufficient. One of the major challenges in MS research is to understand the causes of remyelination failure and to identify therapeutic targets that promote remyelination. Activation of pancreatic endoplasmic reticulum kinase (PERK) signaling in response to endoplasmic reticulum stress modulates cell viability and function under stressful conditions. There is evidence that PERK is activated in remyelinating oligodendrocytes in demyelinated lesions in both MS and its animal model, experimental autoimmune encephalomyelitis (EAE). In this study, we sought to determine the role of PERK signaling in remyelinating oligodendrocytes in MS and EAE using transgenic mice that allow temporally controlled activation of PERK signaling specifically in oligodendrocytes. We demonstrated that persistent PERK activation was not deleterious to myelinating oligodendrocytes in young, developing mice or to remyelinating oligodendrocytes in cuprizone-induced demyelinated lesions. We found that enhancing PERK activation, specifically in (re)myelinating oligodendrocytes, protected the cells and myelin against the detrimental effects of interferon-γ, a key proinflammatory cytokine in MS and EAE. More important, we showed that enhancing PERK activation in remyelinating oligodendrocytes at the recovery stage of EAE promoted cell survival and remyelination in EAE demyelinated lesions. Thus, our data provide direct evidence that PERK activation cell-autonomously enhances the survival and preserves function of remyelinating oligodendrocytes in immune-mediated demyelinating diseases.

Effects of PGE2 EP3/EP4 receptors on bladder dysfunction in mice with experimental autoimmune encephalomyelitis

To investigate the expression of four subtypes of PGE2 E-prostanoid (EP) receptors (EP1-EP4) and the effects of EP3/EP4 on bladder dysfunction in a new neurogenic bladder model induced by experimental autoimmune encephalomyelitis (EAE), the mouse model of EAE was induced using a previously established method, and bladder function in mice with different defined levels of neurological impairment was then examined, including micturition frequencies and voiding weight. Bladders were then harvested for analysis of EP receptor expression by Western blot. Activities of agonists/antagonists of EP3 and EP4 receptors as well as PGE2 were also evaluated at different stages of EAE. The results showed that EAE mice developed profound bladder dysfunction characterized by significantly increased micturition and significantly decreased urine output per micturition. EAE-induced upregulation of EP3 and EP4 receptors in the bladder was accompanied by bladder dysfunction. However, EAE had no significant effect on EP1 and EP2 receptors. Moreover, PGE2 and agonists/antagonists of EP3 and EP4 receptors significantly affected bladder dysfunction in EAE mice. Thus, we believe that EAE mice are useful for investigations of the neurogenic bladder. In addition, EP3 and EP4 receptors play a role in EAE-induced bladder dysfunction, providing us with a new target for the treatment of neurogenic bladders.

Loss of astrocytic leptin signaling worsens experimental autoimmune encephalomyelitis

Leptin is commonly thought to play a detrimental role in exacerbating experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis. Paradoxically, we show here that astrocytic leptin signaling has beneficial effects in reducing disease severity. In the astrocyte specific leptin receptor knockout (ALKO) mouse in which leptin signaling is absent in astrocytes, there were higher EAE scores (more locomotor deficits) than in the wildtype counterparts. The difference mainly occurred at a late stage of EAE when wildtype mice showed signs of recovery whereas ALKO mice continued to deteriorate. The more severe symptoms in ALKO mice coincided with more infiltrating cells in the spinal cord and perivascular brain parenchyma, more demyelination, more infiltrating CD4 cells, and a lower percent of neutrophils in the spinal cord 28 days after EAE induction. Cultured astrocytes from wildtype mice showed increased adenosine release in response to interleukin-6 and the hippocampus of wildtype mice had increased adenosine production 28 days after EAE induction, but the ALKO mutation abolished the increase in both conditions. This indicates a role of astrocytic leptin in normal gliotransmitter release and astrocyte functions. The worsening of EAE in the ALKO mice in the late stage suggests that astrocytic leptin signaling helps to clear infiltrating leukocytes and reduce autoimmune destruction of the CNS.

Saturable Leptin Transport Across the BBB Persists in EAE Mice

We have shown that mice with experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, have upregulated leptin receptor expression in reactive astrocytes of the hippocampus, a region involved in sickness behavior. Leptin can exacerbate EAE when its serum concentration is high. Although leptin receptors in astrocytes modulate leptin transport across cultured endothelial cell monolayers, it is not known how leptin transport in EAE mice is regulated. Here, we determined brain and cervical spinal cord uptake of leptin in early and recovery stages of EAE, after either intravenous delivery or in situ brain perfusion of (125)I-leptin and the vascular marker (131)I-albumin. While increased vascular space and general blood-brain barrier (BBB) permeability after EAE were expected, the specific saturable transport system for leptin crossing the BBB also persisted. Moreover, there was upregulation of leptin transport in hippocampus and cervical spinal cord in the early stage of EAE, shown by higher leptin uptake in these regions and by competitive inhibition with coadministered excess unlabeled leptin. We conclude that EAE induced a time- and region-specific increase of leptin transport. The results provide a link between circulating leptin and enhanced leptin signaling that may play a crucial role in disease progression.

Persistent Inflammation in the CNS during Chronic EAE Despite Local Absence of IL-17 Production

Experimental autoimmune encephalomyelitis (EAE) is an artificially induced demyelination of the central nervous system (CNS) that resembles multiple sclerosis in its clinical, histopathological, and immunological features. Activated Th1 and Th17 cells are thought to be the main immunological players during EAE development. This study was designed to evaluate peripheral and local contribution of IL-17 to acute and chronic EAE stages. C57BL/6 mice were immunized with MOG plus complete Freund's adjuvant followed by pertussis toxin. Mice presented an initial acute phase characterized by accentuated weight loss and high clinical score, followed by a partial recovery when the animals reached normal body weight and smaller clinical scores. Spleen cells stimulated with MOG produced significantly higher levels of IFN-γ during the acute period whereas similar IL-17 levels were produced during both disease stages. CNS-infiltrating cells stimulated with MOG produced similar amounts of IFN-γ but, IL-17 was produced only at the acute phase of EAE. The percentage of Foxp3+ Treg cells, at the spleen and CNS, was elevated during both phases. The degree of inflammation was similar at both disease stages. Partial clinical recovery observed during chronic EAE was associated with no IL-17 production and presence of Foxp3+ Treg cells in the CNS.

Extracellular adenosine signaling induces CX3CL1 expression in the brain to promote experimental autoimmune encephalomyelitis

BackgroundMultiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE) are debilitating neuroinflammatory diseases mediated by lymphocyte entry into the central nervous system (CNS). While it is not known what triggers lymphocyte entry into the CNS during neuroinflammation, blockade of lymphocyte migration has been shown to be effective in controlling neuroinflammatory diseases. Since we have previously shown that extracellular adenosine is a key mediator of lymphocyte migration into the CNS during EAE progression, we wanted to determine which factors are regulated by adenosine to modulate EAE development.MethodsWe performed a genetic analysis of wild type and CD73−/− (that are unable to produce extracellular adenosine and are protected from EAE development) to identify factors that are both important for EAE development and controlled by extracellular adenosine signaling.ResultsWe show that extracellular adenosine triggered lymphocyte migration into the CNS by inducing the expression of the specialized chemokine/adhesion molecule CX3CL1 at the choroid plexus. In wild type mice, CX3CL1 is upregulated in the brain on Day 10 post EAE induction, which corresponds with initial CNS lymphocyte infiltration and the acute stage of EAE. Conversely, mice that cannot synthesize extracellular adenosine (CD73−/− mice) do not upregulate CX3CL1 in the brain following EAE induction and are protected from EAE development and its associated lymphocyte infiltration. Additionally, blockade of the A2A adenosine receptor following EAE induction prevents disease development and the induction of brain CX3CL1 expression. The CX3CL1 induced during EAE is found on the choroid plexus, which is the barrier between the blood and cerebral spinal fluid in the brain and is a prime entry point into the CNS for immune cells. Furthermore, CX3CL1 expression can be induced in the brains of mice and in choroid plexus cell line following A2A adenosine receptor agonist administration. Most importantly, we show that CX3CL1 blockade protects against EAE development and inhibits lymphocyte entry into the CNS.ConclusionsWe conclude that extracellular adenosine is an endogenous modulator of neuroinflammation during EAE that induces CX3CL1 at the choroid plexus to trigger lymphocyte entry into the brain.

Active induction of experimental autoimmune encephalomyelitis by MOG35-55 peptide immunization is associated with differential responses in separate compartments of the choroid plexus

BackgroundThere is increasing awareness that, aside from producing cerebrospinal fluid, the choroid plexus (CP) might be a key regulator of immune activity in the central nervous system (CNS) during neuroinflammation. Specifically, the CP has recently been posited to control entry of sentinel T cells into the uninflamed CNS during the early stages of neuroinflammatory diseases, like multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). As the CP is compartmentalized into a stromal core containing fenestrated capillaries devoid of typical blood–brain barrier properties, surrounded by a tight junction-expressing choroidal epithelium, each of these compartments might mount unique responses that instigate the neuroinflammatory process.MethodsTo discern responses of the respective CP stromal capillary and choroidal epithelial tissues during evolving neuroinflammation, we investigated morphology and in situ expression of 93 immune-related genes during early stages of EAE induced by immunization with myelin oligodendrocyte glycoprotein peptide (MOG35-55). Specifically, 3-D immunofluorescent imaging was employed to gauge morphological changes, and laser capture microdissection was coupled to an Immune Panel TaqMan Low Density Array to detail alterations in gene expression patterns at these separate CP sites on days 9 and 15 post-immunization (p.i.). To resolve CP effects due to autoimmunity against MOG peptide, from those due to complete Freund’s adjuvant (CFA) and pertussis toxin (PTX) included in the immunization, analysis was performed on MOG-CFA/PTX-treated, CFA/PTX-treated, and naïve cohorts.ResultsThe CP became swollen and displayed significant molecular changes in response to MOG-CFA/PTX immunization. Both stromal capillary and choroidal epithelial tissues mounted vigorous, yet different, changes in expression of numerous genes over the time course analyzed - including those encoding adhesion molecules, cytokines, chemokines, statins, interleukins, T cell activation markers, costimulatory molecules, cyclooxygenase, pro-inflammatory transcription factors and pro-apoptotic markers. Moreover, CFA/PTX-treatment, alone, resulted in extensive, though less robust, alterations in both CP compartments.ConclusionsMOG-CFA/PTX immunization significantly affects CP morphology and stimulates distinct expression patterns of immune-related genes in CP stromal capillary and epithelial tissues during evolving EAE. CFA/PTX treatment, alone, causes widespread gene alterations that could prime the CP to unlock the CNS to T cell infiltration during neuroinflammatory disease.

Upregulation of β-1,4-Galactosyltransferase I in Rat Spinal Cord with Experimental Autoimmune Encephalomyelitis

Inflammatory infiltration has been recently emphasized in the demyelinating diseases of the central nervous system including multiple sclerosis. β-1,4-Galactosyltransferase I (β-1,4-GalT-I) is a major galactosyltransferase responsible for selectin-ligand biosynthesis, mediating rolling of the inflammatory lymphocytes. In the present study, Western blot showed that expression of β-1,4-GalT-I was low in normal or complete Freund's adjuvant (CFA) control rats' spinal cords, and it began to increase since early stage and peaked at E4 stage of experimental autoimmune encephalomyelitis (EAE) and restored approximately at normal level in the recovery stage. Immunohistochemisty revealed that upregulation of β-1,4-GalT-I was predominantly distributed in the white matter of spinal cord , while there was also some increased staining of β-1,4-GalT-I in the grey matter. Meanwhile, the expression of E-selectin, the substrate of β-1,4-GalT-I, was significantly increased, with a peak at E4 stage of EAE, and gradually decreased thereafter. Lectin blot showed that the protein bands with molecular weights of 65-25kDa reacted a remarkable increase at the peak stage of EAE when compared with the normal and CFA control. Ricinus Communis Agglutinin-I (RCA-I) histochemistry revealed that RCA-Ι-positive signals were most intense in white matter of lumbosacral spinal cord at the peak stage of EAE (E4). Immunohistochemistry showed that β-1,4-GalT-I and CD62E, a marker for E-selectin stainings located in a considerable number of ED1 (+) macrophages in perivascular or in the white matter in EAE lesions, and a good co-localization of ED1 (+) cells with CD62E was observed. All these results suggest that β-1,4-GalT-I might serve as an inflammatory mediator regulating adhesion and migration of inflammatory cells in EAE, possibly through influencing the modification of galactosylated carbohydrate chains to modulate selectin-ligand biosynthesis and interaction with E-selectin.

Limiting multiple sclerosis related axonopathy by blocking Nogo receptor and CRMP-2 phosphorylation

Multiple sclerosis involves demyelination and axonal degeneration of the central nervous system. The molecular mechanisms of axonal degeneration are relatively unexplored in both multiple sclerosis and its mouse model, experimental autoimmune encephalomyelitis. We previously reported that targeting the axonal growth inhibitor, Nogo-A, may protect against neurodegeneration in experimental autoimmune encephalomyelitis; however, the mechanism by which this occurs is unclear. We now show that the collapsin response mediator protein 2 (CRMP-2), an important tubulin-associated protein that regulates axonal growth, is phosphorylated and hence inhibited during the progression of experimental autoimmune encephalomyelitis in degenerating axons. The phosphorylated form of CRMP-2 (pThr555CRMP-2) is localized to spinal cord neurons and axons in chronic-active multiple sclerosis lesions. Specifically, pThr555CRMP-2 is implicated to be Nogo-66 receptor 1 (NgR1)-dependent, since myelin oligodendrocyte glycoprotein (MOG)(35-55)-induced NgR1 knock-out (ngr1(-)(/)(-)) mice display a reduced experimental autoimmune encephalomyelitis disease progression, without a deregulation of ngr1(-)(/)(-) MOG(35-55)-reactive lymphocytes and monocytes. The limitation of axonal degeneration/loss in experimental autoimmune encephalomyelitis-induced ngr1(-)(/)(-) mice is associated with lower levels of pThr555CRMP-2 in the spinal cord and optic nerve during experimental autoimmune encephalomyelitis. Furthermore, transduction of retinal ganglion cells with an adeno-associated viral vector encoding a site-specific mutant T555ACRMP-2 construct, limits optic nerve axonal degeneration occurring at peak stage of experimental autoimmune encephalomyelitis. Therapeutic administration of the anti-Nogo(623-640) antibody during the course of experimental autoimmune encephalomyelitis, associated with an improved clinical outcome, is demonstrated to abrogate the protein levels of pThr555CRMP-2 in the spinal cord and improve pathological outcome. We conclude that phosphorylation of CRMP-2 may be downstream of NgR1 activation and play a role in axonal degeneration in experimental autoimmune encephalomyelitis and multiple sclerosis. Blockade of Nogo-A/NgR1 interaction may serve as a viable therapeutic target in multiple sclerosis.

MK2-Deficiency Fails To Protect Mice from Experimental Autoimmune Encephalomyelitis (P05.114)

Objective: Investigation of the role of the mitogen-activated proteinkinase activated proteinkinase 2 in experimental autoimmune encephalomyelitis. Background The p38 mitogen-activated protein kinase pathway mediates cellular responses to injurious stress and immune signaling. The downstream targets of p38 MAPK include the mitogen-activated proteinkinase activated proteinkinase (MK) 2. Recently, the role of MK2 has been studied in models of inflammatory or degenerative diseases. The findings suggest that a lack of MK2 reduces inflammation and protects against destruction of the brain and other tissues. Thus, we investigated the role of MK2 in experimental autoimmune encephalomyelitis (EAE). Design/Methods: EAE was induced in MK2 knockout mice (MK2-/-) and wildtype controls by immunization with MOG35-55 in CFA. Animals were observed over a period of 24 days. The inflammation and demyelination was estimated by histology and immunohistochemistry in the acute phase of the disease. Moreover, cells isolated from the CNS were counted and TNFα serum level was assigned by ELISA in different stages of EAE. Results: Contrary to expectance, MK2-/- had more severe disease signs correlating with an increased number of cells in the CNS. There was no difference in the onset of EAE and both groups developed paresis in the early phase of the disease. From day 18 post immunization wildtype controls showed significantly improved locomotor recovery compared to MK2-/-. The histological examination of CNS sections in the acute phase showed that there was no difference in demyelination but MK2-/- had more CD4 and CD8 positive cells in the CNS. Moreover, we found a reduced level of the inflammation mediator TNFα. Conclusions: In contrast to the expectations, the MK2-/- aggravated the disease course and was associated with more inflammation. The reason for these findings may partially be explained by differences in TNFα biosynthesis and therefore a changed apoptosis characteristic in autoreactive cells in the CNS of MK2-/- mice. Disclosure: Dr. Tietz has nothing to disclose. Dr. Viard has nothing to disclose. Dr. Thomas has nothing to disclose. Dr. Gaestel has nothing to disclose. Dr. Berghoff has received personal compensation for activities with Bayer Vital GmbH, Merck Serono, TEVA Neuroscience and Biogen Idec as a speaker. Dr. Berghoff has received research support from Bayer Vital GmhB, TEVA Neuroscience, Merck Serono and Biogen Idec.

Immunohistochemical study of arginase-1 in the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis

Arginase-1, a marker for M2 phenotype alternatively activated macrophages, inhibits inflammation and is associated with phagocytosis of cell debris and apoptotic cells. We analyzed the expression of arginase-1, a competitive enzyme of inducible nitric oxide synthase (iNOS), in the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis (EAE). Western blot analysis showed that both arginase-1 and iNOS significantly increased in the spinal cords of rats at the peak stage of EAE compared with the expression level in control animals (p<0.05) and declined thereafter. Immunofluorescent staining demonstrated that increased expression of arginase-1 in EAE spinal cords was confirmed in macrophages as well as in some neurons and astrocytes that were constitutively positive for arginase-1 in normal spinal cords. A semiquantitative analysis by immunofluorescence showed that in EAE lesions, an increased level of arginase-1 immunoreactivity was matched with ED1-positive macrophages, which were also positive for activin A, a marker for the M2 phenotype. Taking all of these findings into consideration, we postulate that the increased level of arginase-1, which is partly from M2 macrophages, contributes to the modulation of neuroinflammation in EAE lesions, possibly through the reduction of nitric oxide in the lesion via competition with iNOS for the use of l-arginine.

Immunohistochemical studies on disabled-2 protein in the spinal cords of rats with experimental autoimmune encephalomyelitis

Disabled-2 (Dab-2), an adaptor protein of transforming growth factor beta (TGF-β) signaling, was studied in the spinal cords of rats with experimental autoimmune encephalomyelitis (EAE) to evaluate the possible involvement of Dab-2 in the pathogenesis of EAE using Western blot and immunohistochemical analyses. Western blot analysis showed that two isoforms (p96 kDa and p67 kDa) of Dab-2 were detected in the spinal cords of rats used as controls. Both isoforms of Dab-2 were significantly elevated in the EAE spinal cord at the peak stage of EAE ( P < 0.05) and declined at the recovery stage. However, only the p96 kDa isoform was markedly phosphorylated in the EAE spinal cord. Immunohistochemistry showed that Dab-2 and p-Dab-2 were detected in some vascular endothelial cells, glial cells, and some neurons in the rat spinal cords of normal and immunized CFA-alone controls. In EAE lesions, Dab-2 and p-Dab-2 were immunodetected in some inflammatory cells (mainly in ED1-positive macrophages and R73-positive T cells), while the enhanced immunoreactivity of Dab-2 in spinal cord cells suggested constitutive expression. Additionally, TGF-β1 immunoreactivity showed a similar expression pattern of Dab-2 in EAE lesions. These findings suggest that Dab-2 is transiently upregulated and phosphorylated (particularly the p96 kDa isoform) in EAE, a CNS autoimmune disease, and may be involved in TGF- β signaling.

PI3Kδ drives the pathogenesis of experimental autoimmune encephalomyelitis by inhibiting effector T cell apoptosis and promoting Th17 differentiation

The Class IA phosphoinositide 3-kinase delta (PI3Kδ) has been implicated in multiple signaling pathways involved in leukocyte activation and hence is an attractive target in many human autoimmune diseases, including multiple sclerosis (MS). Here, using mice expressing a catalytically inactive form of the PI3Kδ subunit p110δ, we show that signaling through PI3Kδ is required for full and sustained pathology of experimental autoimmune encephalomyelitis (EAE), a Th17-driven model of MS. In p110δ-inactivated mice, T cell activation and function during EAE was markedly reduced and fewer T cells were observed in the central nervous system (CNS). The decrease in T cell activation is unlikely to be due to defects in dendritic cell (DC) function, as p110δ-inactivated DCs migrate and present antigen normally. However, significant increases in the proportion of T cells undergoing apoptosis at early stages of EAE were evident in the absence of PI3Kδ activity. Furthermore, a profound defect in Th17 cellular responses during EAE was apparent in the absence of PI3Kδ activity while Th1 responses were less affected. A highly selective PI3Kδ inhibitor, IC87114, also had greater inhibitory effects on Th17 cell generation in vitro than it did on Th1 cell generation. Thus, PI3Kδ plays an important role in Th17 responses in EAE, suggesting that small molecule inhibitors of PI3Kδ may be useful therapeutics for treatment of MS and other autoimmune diseases.

Angiogenesis is present in experimental autoimmune encephalomyelitis and pro-angiogenic factors are increased in multiple sclerosis lesions

BackgroundAngiogenesis is a common finding in chronic inflammatory diseases; however, its role in multiple sclerosis (MS) is unclear. Central nervous system lesions from both MS and experimental autoimmune encephalomyelitis (EAE), the animal model of MS, contain T cells, macrophages and activated glia, which can produce pro-angiogenic factors. Previous EAE studies have demonstrated an increase in blood vessels, but differences between the different phases of disease have not been reported. Therefore we examined angiogenic promoting factors in MS and EAE lesions to determine if there were changes in blood vessel density at different stages of EAE.MethodsIn this series of experiments we used a combination of vascular casting, VEGF ELISA and immunohistochemistry to examine angiogenesis in experimental autoimmune encephalomyelitis (EAE). Using immunohistochemistry we also examined chronic active MS lesions for angiogenic factors.ResultsVascular casting and histological examination of the spinal cord and brain of rats with EAE demonstrated that the density of patent blood vessels increased in the lumbar spinal cord during the relapse phase of the disease (p < 0.05). We found an increased expression of VEGF by inflammatory cells and a decrease in the recently described angiogenesis inhibitor meteorin. Examination of chronic active human MS tissues demonstrated glial expression of VEGF and glial and blood vessel expression of the pro-angiogenic receptor VEGFR2. There was a decreased expression of VEGFR1 in the lesions compared to normal white matter.ConclusionsThese findings reveal that angiogenesis is intimately involved in the progression of EAE and may have a role in MS.

Immunohistochemical Study of Netrin-1 in the Spinal Cord with Rat Experimental Autoimmune Encephalomyelitis

To investigate whether netrin-1 is involved in autoimmune injury of the central nervous system, the expression of netrin-1 protein was analyzed in the spinal cord of Lewis rats with experimental autoimmune encephalomyelitis (EAE). Western blot analysis revealed significantly increased content of netrin-1 in the spinal cords of rats at the peak stage of EAE, as compared with the levels in normal control animals (p < 0.01). Immunohistochemistry detected the netrin-1 protein in neurons, oligodendrocytes, astrocytes and vascular endothelial cells in the spinal cords of normal controls. In EAE-affected spinal cords, netrin-1 immunoreactivity was detected in infiltrating inflammatory cells at the peak stage as well as in neurons, oligodendrocytes and astrocytes. These results suggest that netrin-1 is transiently increased in rat EAE lesions, where it contributes to the modulation of rat acute EAE.

Targeting ganglioside epitope 3G11 on the surface of CD4+ T cells suppresses EAE by altering the Treg/Th17 cell balance

Loss of expression of the 3G11 epitope, present on disialoceramide that is predominantly found on CD4(+) T cells, has been associated with a regulatory T cell (Treg) phenotype and tolerance induction in experimental autoimmune encephalomyelitis (EAE). Here we report that treatment with anti-3G11 mAb shifts the immune response from pro-inflammatory to tolerogenic and suppresses both chronic-progressive and relapsing-remitting EAE. This therapeutic effect can be achieved at different stages of EAE. Treatment with anti-3G11 mAb increased the proportion of Foxp3(+)CD25(+)CD4(+) Tregs and IL-10 production while inhibiting production of pro-inflammatory cytokines and responsiveness to IL-2 and decreasing the proportion of T(h)17 cells. The effect of anti-3G11 mAb was diminished in IL-10(-/-) mice, indicating that this cytokine mediates some of its effects. As 3G11 belongs to the ganglioside family, which is expressed on the surface of both murine and human CD4(+) T cells, targeting this class of molecules may provide a novel approach for treating autoimmune diseases.

Research on the mechanism of Zuogui Pill (左归丸) and Yougui Pill (右归丸) in promoting axonal regeneration in model rats of autoimmune encephalomyelitis

To study the molecular mechanism of Zuogui Pill (ZGP) and Yougui Pill (YGP) on axonal regeneration in rats with experimental autoimmune encephalomyelitis (EAE). EAE rat model was established by bilateral rear pedes subcutaneous injection of antigen made by mixing myelin basic protein (MBP) and complete Freud's adjuvant (CFA) in the volume ratio of 1:1. The pathological changes of axonal injury and regeneration in the brain and the spinal cord were observed on the 14th (the acute stage) and the 28th day (the remission stage) after modeling, with hematoxylin-eosin (HE) staining, silver stain, and immunohistochemical staining. The rats treated with prednisone acetate were taken as controls. Observation under the light microscope with HE staining showed a sleeve-like change in rats' cerebrospinal parenchyma with inflammatory cell infiltration around the small vessels and neuronic denaturation, while silver staining showed excessive tumefaction and abscission of axon, and immunohistochemical analysis showed decreasing of nerve growth factor (NGF) expression at the acute stage of EAE, which was even more remarkable at the remission stage, showing significant difference as compared with the normal control (P<0.05). And the expressions of Nogo A, an axon growth inhibitor, and its receptor (Nogo-66 receptor, Ng R) were significantly higher than those in the normal control at the acute stage (P<0.01). However, after the intervention of ZGP and YGP, the pathological changes and axon damage in rats' brain and spinal cord were much more alleviated, and the NGF expression was significantly higher than that in the model group at the acute stage (P<0.05). The expression of NGF was even stronger during the remission stage, and a better effect was shown by YGP. As for Nogo A and Ng R expressions, they were significantly lower than those in the model group at the acute stage (P<0.05), but a better effect was shown by ZGP. ZGP and YGP can prevent axonal injury and promote the axonal regeneration in rats of EAE, and the possible mechanism is to increase the expression of NGF and reduce the expression of Nogo A and its receptor. However, some differences are observed between the two Chinese preparations in their acting times and points, which provides a certain basis for revealing the modern connotation of the Chinese medicine theory on tonifying Shen ()-yin and Shen-yang.

CD200R1 Agonist Attenuates Mechanisms of Chronic Disease in a Murine Model of Multiple Sclerosis

To assess the effects and mechanisms of a CD200R1 agonist administered during the progressive stage of a multiple sclerosis model, we administered CD200R1 agonist (CD200Fc) or control IgG2a during the chronic phase of disease (days 10-30) in mice with experimental autoimmune encephalomyelitis (EAE), induced using myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) peptide. We found that administration of CD200Fc during the chronic stages of EAE reduced disease severity, demyelination, and axonal damage, through the modulation of several key disease mechanisms. CD200Fc treatment suppressed macrophage and microglial accumulation within the CNS, in part through downregulation of adhesion molecules VLA-4 and LFA-1, which are necessary for macrophage migration. Additionally, expression of activation markers MHC-II and CD80 and production of proinflammatory cytokines IL-6, tumor necrosis factor-alpha, and nitric oxide by CD11b(+) cells were decreased in both the spleen and CNS in CD200Fc-treated animals. Antigen-presenting cell function in the spleen and CNS was suppressed in CD200Fc-treated mice, but there were no significant alterations on T cell activation or phenotype. CD200Fc increased apoptosis of CD11b(+) cells but not astrocytes. In contrast, addition of CD200Fc treatment protected oligodendrocytes from apoptosis in vitro and in vivo. Our results demonstrate that CD200R1 agonists modulate both myeloid- and non-myeloid-related mechanisms of chronic disease in the EAE model and may be effective in the treatment of progressive multiple sclerosis and other neurodegenerative diseases.

Regulation of Oligodendrocyte Progenitor Cell Maturation by PPARδ: Effects on Bone Morphogenetic Proteins

In EAE (experimental autoimmune encephalomyelitis), agonists of PPARs (peroxisome proliferator-activated receptors) provide clinical benefit and reduce damage. In contrast with PPARγ, agonists of PPARδ are more effective when given at later stages of EAE and increase myelin gene expression, suggesting effects on OL (oligodendrocyte) maturation. In the present study we examined effects of the PPARδ agonist GW0742 on OPCs (OL progenitor cells), and tested whether the effects involve modulation of BMPs (bone morphogenetic proteins). We show that effects of GW0742 are mediated through PPARδ since no amelioration of EAE clinical scores was observed in PPARδ-null mice. In OPCs derived from E13 mice (where E is embryonic day), GW0742, but not the PPARγ agonist pioglitazone, increased the number of myelin-producing OLs. This was due to activation of PPARδ since process formation was reduced in PPARδ-null compared with wild-type OPCs. In both OPCs and enriched astrocyte cultures, GW0742 increased noggin protein expression; however, noggin mRNA was only increased in astrocytes. In contrast, GW0742 reduced BMP2 and BMP4 mRNA levels in OPCs, with lesser effects in astrocytes. These findings demonstrate that PPARδ plays a role in OPC maturation, mediated, in part, by regulation of BMP and BMP antagonists.