Myelin Oligodendrocyte Glycoprotein Peptide Research Articles

DUOC-01, a cord blood derived cell therapy product, ameliorates experimental autoimmune encephalomyelitis, a murine model for multiple sclerosis

Background & Aim We have developed an umbilical cord-blood derived, macrophage-like cell therapy, DUOC-01, to treat demyelinating conditions of the central nervous system. Previously, we showed that DUOC-01 accelerated remyelination, decreased gliosis, and reduced cellular infiltration in the brain of immune-incompetent mice exposed to the demyelinating agent, cuprizone. To further explore the mechanism of action and investigate whether DUOC-01 will be effective in other experimental models of demyelination, we tested DUOC-01 in the experimental autoimmune encephalomyelitis (EAE) model, an animal model used to study multiple sclerosis (MS). Methods, Results & Conclusion EAE was induced in C57BL/6 mice by immunizing with myelin oligodendrocyte glycoprotein peptide (MOG35-55) in complete Freund's adjuvant. We have previously established in a phase I trial of intrathecally administered DUOC-01 cells in patients with leukodystrophies, that DUOC-01 is formulated in hydrocortisone (HC) for infusion in the clinic. Therefore, to mimic the practice in the clinic, DUOC-01 cells were incubated in Ringer's Lactate with 3mg/mL HC at a dilution of 1 × 106 cells/ml for 2 hours at room temperature prior to injection. DUOC-01 cells were then administered at a dose of 3 × 105cells/mouse into the cerebrospinal fluid by a single intra-cisterna magna injection at the point when mice started showing early disease symptoms of EAE. Clinical score was recorded for next several days. Compared to mice injected with Ringer's (n=9), mice injected with DUOC-01 (n=9) had decreased severity of the disease, as indicated by lower clinical scores based on severity of ascending paralysis. The spinal cords of mice in the cohorts were studied histopathologically and those with mice injected with DUOC-01 had reduced inflammation and lower cellular infiltration compared to spinal cords of mice injected with Ringer's. The overall number of CD45+ immune cells were less in spinal cords of mice treated with DUOC-01 cells compared to the Ringer's injected mice. Most strikingly, among the various cell types, the number of neutrophils present in the spinal cords from the DUOC-01 treated group was less than the Ringer's treated samples. Presently we are exploring the mechanism(s) through which DUOC-01 cells promote remyelination and decrease immune cell infiltration. In brief, our data suggest that DUOC-01 cell therapy product could be beneficial in treating MS and other diverse neurological conditions with demyelination.

C-Met is expressed by highly autoreactive encephalitogenic CD8+ cells

BackgroundCD8+ T lymphocytes are critical mediators of neuroinflammatory diseases. Understanding the mechanisms that govern the function of this T cell population is crucial to better understanding central nervous system autoimmune disease pathology. We recently identified a novel population of highly cytotoxic c-Met-expressing CD8+ T lymphocytes and found that hepatocyte growth factor (HGF) limits effective murine cytotoxic T cell responses in cancer models. Here, we examined the role of c-Met-expressing CD8+ T cells by using a MOG35–55 T cell-mediated EAE model.MethodsMice were subcutaneously immunized with myelin oligodendrocyte glycoprotein peptide (MOG)35–55 in complete Freund’s adjuvant (CFA). Peripheral and CNS inflammation was evaluated at peak disease and chronic phase, and c-Met expression by CD8 was evaluated by flow cytometry and immunofluorescence. Molecular, cellular, and killing function analysis were performed by real-time PCR, ELISA, flow cytometry, and killing assay.ResultsIn the present study, we observed that a fraction of murine effector CD8+ T cells expressed c-Met receptor (c-Met+CD8+) in an experimental autoimmune encephalitis (EAE) model. Phenotypic and functional analysis of c-Met+CD8+ T cells revealed that they recognize the encephalitogenic epitope myelin oligodendrocyte glycoprotein37–50. We demonstrated that this T cell population produces higher levels of interferon-γ and granzyme B ex vivo and that HGF directly restrains the cytolytic function of c-Met+CD8+ T cells in cell-mediated cytotoxicity reactionsConclusionsAltogether, our findings suggest that the HGF/c-Met pathway could be exploited to modulate CD8+ T cell-mediated neuroinflammation.

Oligodendroglial connexin 47 regulates neuroinflammation upon autoimmune demyelination in a novel mouse model of multiple sclerosis

In multiple sclerosis plaques, oligodendroglial connexin (Cx) 47 constituting main gap junction channels with astroglial Cx43 is persistently lost. As mice with Cx47 single knockout exhibit no demyelination, the roles of Cx47 remain undefined. We aimed to clarify the effects of oligodendroglia-specific Cx47 inducible conditional knockout (icKO) on experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein peptide (MOG35-55) in PLP/CreERT;Cx47fl/fl mice at 14 d after tamoxifen injection. Cx47 icKO mice demonstrated exacerbation of acute and chronic relapsing EAE with more pronounced demyelination than Cx47 flox (fl)/fl littermates. CD3+ T cells more abundantly infiltrated the spinal cord in Cx47 icKO than in Cx47 fl/fl mice throughout the acute to chronic phases. CXCR3-CCR6+CD4+ and IL17+IFNγ-CD4+ helper T (Th) 17 cells isolated from spinal cord and brain tissues were significantly increased in Cx47 icKO mice compared with Cx47 fl/fl mice, while MOG35-55-specific proliferation and proinflammatory cytokine production of splenocytes were unaltered. Microarray analysis of isolated microglia revealed stronger microglial activation toward proinflammatory and injury-response phenotypes with increased expressions of chemokines that can attract Th17 cells, including Ccl2, Ccl3, Ccl4, Ccl7, and Ccl8, in Cx47 icKO mice compared with Cx47 fl/fl mice. In Cx47 icKO mice, NOS2+ and MHC class II+ microglia were more enriched immunohistochemically, and A1-specific astroglial gene expressions and astroglia immunostained for C3, a representative A1 astrocyte marker, were significantly increased at the acute phase, compared with Cx47 fl/fl mice. These findings suggest that oligodendroglia-specific Cx47 ablation induces severe inflammation upon autoimmune demyelination, underscoring a critical role for Cx47 in regulating neuroinflammation.

Actin-Binding Protein Cortactin Promotes Pathogenesis of Experimental Autoimmune Encephalomyelitis by Supporting Leukocyte Infiltration into the Central Nervous System.

Leukocyte entry into the central nervous system (CNS) is essential for immune surveillance but is also the basis for the development of pathologic inflammatory conditions within the CNS, such as multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). The actin-binding protein, cortactin, in endothelial cells is an important player in regulating the interaction of immune cells with the vascular endothelium. Cortactin has been shown to control the integrity of the endothelial barrier and to support neutrophil transendothelial migration in vitro and in vivo in the skin. Here we use cortactin gene-inactivated male and female mice to study the role of this protein in EAE. Inducing EAE by immunization with a myelin oligodendrocyte glycoprotein peptide (MOG35-55) revealed an ameliorated disease course in cortactin gene-deficient female mice compared with WT mice. However, proliferation capacity and expression of IL-17A and IFNγ by cortactin-deficient and WT splenocytes did not differ, suggesting that the lack of cortactin does not affect induction of the immune response. Rather, cortactin deficiency caused decreased vascular permeability and reduced leukocyte infiltration into the brains and spinal cords of EAE mice. Accordingly, cortactin gene-deficient mice had smaller numbers of proinflammatory cuffs, less extensive demyelination, and reduced expression levels of proinflammatory cytokines within the neural tissue compared with WT littermates. Thus, cortactin contributes to the development of neural inflammation by supporting leukocyte transmigration through the blood-brain barrier and, therefore, represents a potential candidate for targeting CNS autoimmunity.SIGNIFICANCE STATEMENT Multiple sclerosis is an autoimmune neuroinflammatory disorder, based on the entry of inflammatory leukocytes into the CNS where these cells cause demyelination and neurodegeneration. Here, we use a mouse model for multiple sclerosis, experimental autoimmune encephalomyelitis, and show that gene inactivation of cortactin, an actin binding protein that modulates actin dynamics and branching, protects against neuroinflammation in experimental autoimmune encephalomyelitis. Leukocyte infiltration into the CNS was inhibited in cortactin-deficient mice, and lack of cortactin in cultured primary brain endothelial cells inhibited leukocyte transmigration. Expression levels of proinflammatory cytokines in the CNS and induction of vascular permeability were reduced. We conclude that cortactin represents a novel potential target for the treatment of multiple sclerosis.

The Role of Forced and Voluntary Training on Accumulation of Neural Cell Adhesion Molecule and Polysialic Acid in Muscle of Mice with Experimental Autoimmune Encephalomyelitis.

It has been suggested that depletion of adhesion molecules is one of the factors associated with or possibly responsible for multiple sclerosis (MS) progression. The aim of this study was to investigate the effect of forced and voluntary training before and after induction of experimental autoimmune encephalomyelitis (EAE) on accumulation of neural cell adhesion molecule (NCAM) and polysialic acid (PSA) in neuromuscular junction denervation in plantaris and soleus muscles in C57BL/6 female mice. A total of 40 female C57BL/6 mice, 10-week-old, were randomly divided into four groups, including induced control groups without EAE induction, induced EAE without training, and forced and voluntary training groups. Myelin oligodendrocyte glycoprotein peptide 35–55 (300 μg in saline; MOG 35–55; KJ Ross-Petersen ApS, Denmark) was injected subcutaneously at the base of the tail of each mouse. Clinical assessment of EAE was performed daily using a 15-point scoring system following immunization. Training groups performed the swimming program for 30 min/day, 5 times/week, for 4 weeks. Mice had access to a treadmill for one hour per day, 5times/week, for 4 weeks in individual cage. The mice were scarified, and the plantaris and soleus muscles were then isolated for investigation of proteins expression using IHC. An analysis of the preventive exercise (before) and recovery exercise (after) of the EAE was performed. Images of the stained sections were taken using a fluorescent microscope. Quantitative image analysis was performed using ImageJ software package. The obtained data from the mean percentage expression of PSA and NCAM in pre- and post-soleus and plantaris muscles showed that the highest and lowest expression levels of PSA and NCAM belonged to control and swim EAE (SE) groups, respectively. The low expression levels of PSA and NCAM were detected in rat with MS without intervention. In conclusion, the relationship between increasing levels of NCAM and PSA protein expression and voluntary and compulsory activity were detectable both in pre and post-soleus and plantaris. However, voluntary activity resulted in more expression levels of NCAM and PSA than that of compulsory. In conclusion, since it has been suggested that depletion of NCAM is one of the factors associated with or possibly responsible for MS progression, these findings show exercise MS progression may be reduced by increasing expression of exercise-related adhesion molecule such as NCAM and PSA (a glycan modification of the NCAM).

Role of SOCS2 in the Regulation of Immune Response and Development of the Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS). Experimental Autoimmune Encephalomyelitis (EAE) is the most widely used animal model for the study of MS. The Suppressor of Cytokine Signaling (SOCS) 2 protein plays a critical role in regulating the immune responses. The role of SOCS2 during EAE has not been explored. EAE was induced in WT and SOCS2−/− mice using myelin oligodendrocyte glycoprotein (MOG35-55) peptide. Brain and spinal cord were examined during the peak (day 14) and recovery phase (day 28) of the disease. SOCS2 was upregulated in the brain of WT mice at the peak and recovery phase of EAE. The development of the acute phase was slower in onset in SOCS2−/− mice and was associated with reduced number of Th1 (CD3+CD4+IFN-γ+) cells in the spinal cord and brain. However, while in WT mice, maximal clinical EAE score was followed by a progressive recovery; the SOCS2−/− mice were unable to recover from locomotor impairment that occurred during the acute phase. There was a prolonged inflammatory response (increased Th1 and decreased Th2 and T regulatory cells) in the late phase of EAE in the CNS of SOCS2−/− mice. Transplantation of bone marrow cells from SOCS2−/− into irradiated WT mice resulted in higher lethality at the early phase of EAE. Altogether, these results suggest that SOCS2 plays a dual role in the immune response during EAE. It is necessary for damage during the acute phase damage but plays a beneficial role in the recovery stage of the disease.

Peptide-Conjugated Silver Nanoparticle for Autoantibody Recognition.

In this work, we considered the autoantibodies proposed as putative biomarkers of demyelination taking into account their reactivity towards myelin oligodendrocyte glycoprotein (MOG) and myelin basic protein (MBP). These myelin proteins are among the most commonly researched targets in the immunopathology of demyelinating diseases. In this context, the development of assays for autoantibody detection can contribute as a predictive value for the early diagnosis of demyelinating diseases. Hence, we aimed to address the application of silver nanoparticles (AgNPs) as a sensing device of autoantibodies. AgNPs were synthesized via a chemical reduction method and characterized using atomic force microscopy (AFM), X-ray diffractometry, dynamic light scattering, and UV-visible spectrophotometry. The process of peptide conjugation on the nanoparticles was also analyzed. The autoantibody recognition by the peptide-conjugated AgNPs was evaluated with UV-visible spectrophotometry, atomic force spectroscopy (AFS), and color changing. AgNPs exhibited spherical morphology, low polydispersity, face-centered cubic crystal structure, and an average size of 29.3±3.0 nm. The hydrodynamic diameter variation and AFM showed that the MBP peptide induced greater agglomeration, compared to MOG peptide. The AFS measurements indicated the efficient binding of peptides to the AgNPs maintaining their activity, revealed by typical adhesion force and shapes of curves. The absorption spectrum features were more affected by the interaction with the specific autoantibodies, which also caused a visible color change in suspension providing a qualitative response. We described a preliminary study of MOG- and MBP-conjugated AgNPs which showed to be applicable in the autoantibody recognition. These have promising implication in the searching for biological markers for diagnostic purposes in the demyelination context, in which the nanoscale sensing exploitation is recent.

The contribution of cyclophilin A to immune-mediated central nervous system inflammation

Cyclophilin A has multiple known functions in inflammation. Intracellular cyclophilin A modulates T helper 2 response (Th2) and extracellular cyclophilin A functions as a leukocyte chemotactic factor. The contribution of cyclophilin A to central nervous system (CNS) inflammation has not been reported. To test the hypothesis that inhibition of cyclophilin A would ameliorate immune-mediated CNS inflammation, we compared the course and neuroimmunology of experimental allergic encephalomyelitis (EAE) in cyclophilin A knockout mice and wild type littermates. There was a trend towards lower incidence of EAE in cyclophilin A knockout mice, but the clinical course of EAE among animals that manifested clinical signs of EAE was similar in cyclophilin A knockout and wild type littermates. Antigen recall response to myelin oligodendrocyte glycoprotein (MOG) peptide showed that interferon-γ release was lower in cyclophilin A knockout mice. Analysis of CNS inflammatory cells showed that CD3+ T cell infiltration into the CNS was lower in cyclophilin A knockout mice. These results showed that the loss of cyclophilin A results in altered peripheral immune activation and CNS leukocyte infiltration, but these changes did not result in a substantial change in the clinical course of EAE.

Monitoring retinal changes with optical coherence tomography predicts neuronal loss in experimental autoimmune encephalomyelitis

BackgroundRetinal optical coherence tomography (OCT) is a clinical and research tool in multiple sclerosis, where it has shown significant retinal nerve fiber (RNFL) and ganglion cell (RGC) layer thinning, while postmortem studies have reported RGC loss. Although retinal pathology in experimental autoimmune encephalomyelitis (EAE) has been described, comparative OCT studies among EAE models are scarce. Furthermore, the best practices for the implementation of OCT in the EAE lab, especially with afoveate animals like rodents, remain undefined. We aimed to describe the dynamics of retinal injury in different mouse EAE models and outline the optimal experimental conditions, scan protocols, and analysis methods, comparing these to histology to confirm the pathological underpinnings.MethodsUsing spectral-domain OCT, we analyzed the test-retest and the inter-rater reliability of volume, peripapillary, and combined horizontal and vertical line scans. We then monitored the thickness of the retinal layers in different EAE models: in wild-type (WT) C57Bl/6J mice immunized with myelin oligodendrocyte glycoprotein peptide (MOG35–55) or with bovine myelin basic protein (MBP), in TCR2D2 mice immunized with MOG35–55, and in SJL/J mice immunized with myelin proteolipid lipoprotein (PLP139–151). Strain-matched control mice were sham-immunized. RGC density was counted on retinal flatmounts at the end of each experiment.ResultsVolume scans centered on the optic disc showed the best reliability. Retinal changes during EAE were localized in the inner retinal layers (IRLs, the combination of the RNFL and the ganglion cell plus the inner plexiform layers). In WT, MOG35–55 EAE, progressive thinning of IRL started rapidly after EAE onset, with 1/3 of total loss occurring during the initial 2 months. IRL thinning was associated with the degree of RGC loss and the severity of EAE. Sham-immunized SJL/J mice showed progressive IRL atrophy, which was accentuated in PLP-immunized mice. MOG35–55-immunized TCR2D2 mice showed severe EAE and retinal thinning. MBP immunization led to very mild disease without significant retinopathy.ConclusionsRetinal neuroaxonal damage develops quickly during EAE. Changes in retinal thickness mirror neuronal loss and clinical severity. Monitoring of the IRL thickness after immunization against MOG35–55 in C57Bl/6J mice seems the most convenient model to study retinal neurodegeneration in EAE.

LFA-1 Controls Th1 and Th17 Motility Behavior in the Inflamed Central Nervous System

Leukocyte trafficking is a key event during autoimmune and inflammatory responses. The subarachnoid space (SAS) and cerebrospinal fluid are major routes for the migration of encephalitogenic T cells into the central nervous system (CNS) during experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis, and are sites of T cell activation before the invasion of CNS parenchyma. In particular, autoreactive Th1 and Th17 cell trafficking and reactivation in the CNS are required for the pathogenesis of EAE. However, the molecular mechanisms controlling T cell dynamics during EAE are unclear. We used two-photon laser microscopy to show that autoreactive Th1 and Th17 cells display distinct motility behavior within the SAS in the spinal cords of mice immunized with the myelin oligodendrocyte glycoprotein peptide MOG35−55. Th1 cells showed a strong directional bias at the disease peak, moving in a straight line and covering long distances, whereas Th17 cells exhibited more constrained motility. The dynamics of both Th1 and Th17 cells were strongly affected by blocking the integrin LFA-1, which interfered with the deformability and biomechanics of Th1 but not Th17 cells. The intrathecal injection of a blocking anti-LFA-1 antibody at the onset of disease significantly inhibited EAE progression and also strongly reduced neuro-inflammation in the immunized mice. Our results show that LFA-1 plays a pivotal role in T cell motility during EAE and suggest that interfering with the molecular mechanisms controlling T cell motility can help to reduce the pathogenic potential of autoreactive lymphocytes.

The preventive and therapeutic effects of astragaloside IV (AST-IV) on experimental autoimmune encephalomyelitis in mice

TCM has some advantages over western medicine in delaying the progress of MS/EAE due to its characteristics of multiple targets, overall regulation and less toxic side effects. Qi deficiency and Blood stasis syndrome is one of typical phenomenon in MS/EAE. AST-IV can significantly relieve clinical symptoms of EAE by supplementing qi and promoting blood flow. To investigate the efficacy and mechanism of AST-IV in the treatment of EAE. Female C57BL/6 mice were induced to establish EAE model by myelin oligodendrocyte glycoprotein peptides (MOG35–55), which were randomly divided into EAE group, high-dose and low-dose AST-IV group. After 3 days of modeling, the mice were injected with drug till day 28. High-dose and low-dose AST-IV group were intraperitoneally injected with AST-IV 30 mg/kg/day and 15 mg/kg/day respectively. The injection of PBS was set up as control in a similar manner. High-dose and low-dose AST-IV could significantly reduce the average highest clinical score and alleviate the clinical symptoms of EAE. AST-IV inhibited the inflammatory cells in spinal cord. Flow cytometry analysis showed that AST-IV significantly increased the proportion of CD4+ CD25+ and CD4+ TGF-β+, decrease the proportion of CD4+IL17+, inhibiting the expression of TLR4, Myd88 and NF-kappa B/p65 in spinal cord. AST-IV is effective on EAE by regulating the inflammatory pathway and maintains the balance between Th17 cells and Treg cells. (NNSF of China 81473577, Collaborative Innovation Center Project of Astragalus Resource Industrialization and Industrial Internationalization-hqxtcxzx2016-020. Ma and Xiao are corresponding authors).

Hydroxyfasudil ameliorates experimental autoimmune encephalomyelitis possibly through immunomodulation and anti-inflammatory effects

Background Rho kinase inhibitor Fasudil is effective in the treatment of EAE, but its narrow safety window, lower bioavailability limit the clinical use. Hydroxyfasudil, active metabolite of Fasudil in vivo, exhibits higher biological activity and longer half-period. Objective To observe the therapeutic potential of Hydroxyfasudil in EAE, and explore its possible mechanisms. Material and methods The effects of Hydroxyfasudil on cell viability. We further explored therapeutic effect in EAE model. Female C57BL/6 mice were immunized with myelin oligodendrocyte glycoprotein peptides (MOG35-55), which were divided into EAE group, Hydroxyfasudil group. Hydroxyfasudil was injected intraperitoneally (40 mg/kg/day) on day 3 post immunization. The injection of saline was set up as control in a similar manner. Results Hydroxyfasudil was well tolerant in vitro. Hydroxyfasudil delayed the clinical onset and suppressed the severity of EAE. Demyelination and cell infiltration in spinal cords of Hydroxyfasudil group were significantly decreased. Flow cytometry analysis showed that Hydroxyfasudil reduced CD4+IFN-γ+ and CD4+IL-17+ but increased CD4+CD25+ T cells. Hydroxyfasudil decreased inflammatory cytokine, chemokine and inhibited NF-κB, COX-2, iNOS. According to RT-PCR analysis, Hydroxyfasudil suppressed iNOS, CD68 and TNF-α, while enhanced Arg-1, IL-4 and TGF-β. Conclusion Hydroxyfasudil should be a potential approach for EAE through immunomodulation, anti-inflammatory effects, the transformation of M1 type macrophages to M2, preventing the infiltration of inflammatory cells into CNS. (NNSF of China 81473577, Shanxi Scholarship Council of China 2014-7, 2011 Cultivation Project of Shanxi University of TCM 2011PY-1. Ma and Xiao are corresponding authors).

Potential of Adult Endogenous Neural Stem/Progenitor Cells in the Spinal Cord to Contribute to Remyelination in Experimental Autoimmune Encephalomyelitis.

Demyelination and remyelination play pivotal roles in the pathological process of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), a well-established animal model of MS. Although increasing evidence shows that various stimuli can promote the activation/induction of endogenous neural stem/progenitor cells (NSPCs) in the central nervous system, the potential contributions of these cells to remyelination following inflammatory injury remain to be fully investigated. In the present study, using an adult mouse model of EAE induced by myelin oligodendrocyte glycoprotein (MOG) peptide, we investigated whether adult NSPCs in the spinal cord can lead to remyelination under inflammatory conditions. Immunohistochemistry showed that cells expressing the NSPC marker Nestin appeared after MOG peptide administration, predominantly at the sites of demyelination where abundant inflammatory cells had accumulated, whereas Nestin+ cells were rarely present in the spinal cord of PBS-treated control mice. In vitro, Nestin+ NSPCs obtained from EAE mice spinal cords could differentiate into multiple neural lineages, including neurons, astrocytes, and myelin-producing oligodendrocytes. Using the Cre–LoxP system, we established a mouse strain expressing yellow fluorescent protein (YFP) under the control of the Nestin promoter and investigated the expression patterns of YFP-expressing cells in the spinal cord after EAE induction. At the chronic phase of the disease, immunohistochemistry showed that YFP+ cells in the injured regions expressed markers for various neural lineages, including myelin-forming oligodendrocytes. These results show that adult endogenous NSPCs in the spinal cord can be subject to remyelination under inflammatory conditions, such as after EAE, suggesting that endogenous NSPCs represent a therapeutic target for MS treatment.

Down-regulation of taurine-up-regulated gene 1 attenuates inflammation by sponging miR-9-5p via targeting NF-κB1/p50 in multiple sclerosis

AimsMultiple sclerosis (MS) is an inflammatory disease of the central nervous system characterized by widespread inflammation. LncRNA taurine-up-regulated gene 1 (TUG1) has been reported to be involved in multiple biological processes and human diseases. The aim of this study was to investigate the role of lncRNA TUG1 in MS and the underlying mechanism. Main methodsExperimental autoimmune encephalomyelitis (EAE) was induced in mice by immunization with myelin oligodendrocyte glycoprotein peptide 35–55 (MOG35–55). Lentiviral vectors encoding sh-TUG1 was constructed to silence TUG1 in MOG-EAE mice by intracerebroventricular (ICV) injection. The effect of TUG1 on inflammation in MS was evaluated by real-time PCR, Western blot, ELISA and Hematoxylin-eosin staining. To further study the mechanism of TUG1 in MS, TUG1 knockdown and miR-9-5p overexpression were performed in LPS-induced BV2 cells. Key findingsDown-regulation of TUG1 improved mice behavior, reduced granulocyte-macrophage colony stimulating factor (GM-CSF) level, decreased the levels of pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin (IL)-6 and IL-17, and increased IL-10 in EAE mice. Notably, TUG1 expression was negatively correlated with miR-9-5p expression, while positively correlated with NF-κB1/p50. Knockdown of TUG1 or enforced expression of miR-9-5p inhibited LPS-induced inflammation in BV2 cells, while these effects were abolished by inhibition of miR-9-5p. We further verified that TUG1 negatively regulated miR-9-5p expression and NF-κB1/p50 is a direct target of miR-9-5p. SignificanceDown-regulation of TUG1 attenuates MS through inhibition of inflammation by sponging miR-9-5p via targeting NF-κB1/p50, suggesting that TUG1 is a potential therapeutic target for MS treatment.

Treatment of Experimental Autoimmune Encephalomyelitis by Sustained Delivery of Low-Dose IFN-α.

Multiple sclerosis (MS) is a chronic autoimmune disease with no curative treatment. The immune regulatory properties of type I IFNs have led to the approval of IFN-β for the treatment of relapsing-remitting MS. However, there is still an unmet need to improve the tolerability and efficacy of this therapy. In this work, we evaluated the sustained delivery of IFN-α1, either alone or fused to apolipoprotein A-1 by means of an adeno-associated viral (AAV) system in the mouse model of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis. These in vivo experiments demonstrated the prophylactic and therapeutic efficacy of the AAV-IFN-α or AAV-IFN-α fused to apolipoprotein A-1 vectors in experimental autoimmune encephalomyelitis, even at low doses devoid of hematological or neurologic toxicity. The sustained delivery of such low-dose IFN-α resulted in immunomodulatory effects, consisting of proinflammatory monocyte and T regulatory cell expansion. Moreover, encephalitogenic T lymphocytes from IFN-α-treated mice re-exposed to the myelin oligodendrocyte glycoprotein peptide in vitro showed a reduced proliferative response and cytokine (IL-17A and IFN-γ) production, in addition to upregulation of immunosuppressive molecules, such as IL-10, IDO, or PD-1. In conclusion, the results of the present work support the potential of sustained delivery of low-dose IFN-α for the treatment of MS and likely other T cell-dependent chronic autoimmune disorders.

Autoimmune encephalomyelitis in NOD mice is not initially a progressive multiple sclerosis model.

ObjectiveDespite progress in treating relapsing multiple sclerosis (MS), effective inhibition of nonrelapsing progressive MS is an urgent, unmet, clinical need. Animal models of MS, such as experimental autoimmune encephalomyelitis (EAE), provide valuable tools to examine the mechanisms contributing to disease and may be important for developing rational therapeutic approaches for treatment of progressive MS. It has been suggested that myelin oligodendrocyte glycoprotein (MOG) peptide residues 35‐55 (MOG 35‐55)‐induced EAE in nonobese diabetic (NOD) mice resembles secondary progressive MS. The objective was to determine whether the published data merits such claims.MethodsInduction and monitoring of EAE in NOD mice and literature review.ResultsIt is evident that the NOD mouse model lacks validity as a progressive MS model as the individual course seems to be an asynchronous, relapsing‐remitting neurodegenerative disease, characterized by increasingly poor recovery from relapse. The seemingly progressive course seen in group means of clinical score is an artifact of data handling and interpretation. InterpretationAlthough MOG 35‐55‐induced EAE in NOD mice may provide some clues about approaches to block neurodegeneration associated with the inflammatory penumbra as lesions form, it should not be used to justify trials in people with nonactive, progressive MS. This adds further support to the view that drug studies in animals should universally adopt transparent raw data deposition as part of the publication process, such that claims can adequately be interrogated. This transparency is important if animal‐based science is to remain a credible part of translational research in MS.

Chitin Micro Particles Regulate Splenocytes Immune Response in Experimental Autoimmune Encephalomyelitis

Contrasting studies are reported on the induction of IL-10 and IFN-γ via chitin microparticles (CMPs) during immune stimulation. Our previous studies have shown marked protection among CMP treated Leishmania-infected mice via regulated IL-10/IFN-γ response, at the present study, once more, examined the inconsistent responses regarding the immunologic response of CMPS. To verify whether CMPs could indeed up-regulate IL-10/IFN-γ axis, isolated spleen cells from the myelin oligodendrocyte glycoprotein (MOG) induced experimental autoimmune encephalomyelitis (EAE) mice were cultured in the presence of MOG peptide and/or CMPs. The effects of CMPs on IL-10, IFN-γ and IL-17 production were evaluated by Enzyme-linked Immunosorbent Assay (ELISA). Moreover, GATA binding protein 3 (Gata3), T-box transcription factor TBX21 (Tbx21), and RAR-related orphan receptor gamma (RORγT) expressions (real-time PCR) were investigated. MOG alone stimulated the production of IFN-γ (p≤0.004) but not, IL-10 (p≤0.140). MOG/chitin stimulation resulted in a significant increase in IFN-γ and IL-10 levels, respectively; (p≤0.004 and p≤0.003) rather than MOG. Additionally, the expression of Tbx21 (p≤0.001), but not Gata3 (p≤0.08), was increased in the MOG/chitin-treated spleen cells. All in all, CMP supports Gata3 independent IL-10 production and promotes Tbx21 dependent IFN-γ induction. These results, alongside our previous data, indicate that CMPs has particular adjuvant effects.

Antigen dose affects disease kinetics in a murine model of demyelinating disease

Abstract Multiple sclerosis is an autoimmune disorder caused by immune cell damage to the myelin sheath lining nerve cells, leading to neuronal damage and dysfunction. The murine model of the disease - experimental autoimmune encephalomyelitis (EAE) - is induced by inoculation with MOG (myelin oligodendrocyte glycoprotein) peptide and an adjuvant, leading to recapitulation of disease that is mediated primarily by CD4 T cells. In this study, we look at the effect of MOG dosage on demyelinating disease kinetics in WT and MOG-deficient animals, as MOG-deficient mice have different numbers of MOG specific CD4 Teff and Tregs. The T cells from MOG deficient animals have a higher affinity for MOG, expand faster, and cause exacerbated disease in an adoptive transfer model compared to CD4 T cells from wildtype mice. Our results indicate that the dose of MOG peptide used to induce disease can also modulate disease course. Using tenfold lower doses changes T cell and disease kinetics resulting in disease that resolves compared to controls. Taken together, we can use varying doses of MOG as a tool to probe CD8 and CD4 T cell expansion and monitor migration of immune cells to the CNS. Further understanding of the cellular tides during disease course enables us to gain insight on how cell ratios in different sites contribute to disease pathogenesis. Using this information, we are developing mathematical modeling tools that could be used to analyze and predict disease progression.

GMCSF-neuroantigen vaccines engender FOXP3+ regulatory T cell responses through low-efficiency self-antigen recognition events integrated through diminished CD40L-CD40 signaling

Abstract Neuroantigen (NAg)-specific tolerogenic vaccines that induce myelin-specific FOXP3+ regulatory T cells (Tregs) may be an effective treatment for Multiple Sclerosis. Previous studies showed that fusion proteins comprised of GM-CSF and NAg (GMCSF-NAg) are potent tolerogens that inhibit several rodent models of experimental autoimmune encephalomyelitis. GMCSF-NAg-induced T cell responses were investigated in vivo by use of 2D2-FIG mice, which have a FOXP3 GFP reporter (FIG) and a transgenic TCR (2D2) which recognizes myelin oligodendrocyte glycoprotein peptide 35–55 (MOG) as a low-efficiency antigen and neurofilament medium peptide 13–37 (NFM) as a high-efficiency antigen. A single subcutaneous vaccination of 2D2-FIG mice with GMCSF-MOG resulted in high numbers and percentages of Tregs in the blood, spleen, and lymph nodes, whereas GMCSF-NFM elicited a dominant conventional T cell (Tcon) response. To determine how low and high-efficiency TCR responses regulate cell lineage fate, we investigated TCR-induced CD40L expression. GMCSF-NFM, but not GMCSF-MOG, induced significant CD40L expression when cultured with 2D2 responder T cells. Pretreatment of 2D2-FIG mice with an agonistic anti-CD40 monoclonal antibody prevented GMCSF-MOG induction of Tregs and favored Tcon responses. MOG35–55 was a partial TCR antagonist that blocked NFM stimulation of CD40L expression in 2D2 T cell cultures. Similarly, GMCSF-MOG was functionally dominant and induced Tregs in 2D2-FIG mice when mixed with an equimolar dose of GMCSF-NFM. These data indicated that low-efficiency antigen recognition is a key parameter for the induction of Treg responses and is in part controlled through CD40L-CD40 signaling pathways to determine T cell lineage fate.

High dose of dexamethasone protects against EAE-induced motor deficits but impairs learning/memory in C57BL/6 mice

Multiple sclerosis (MS) is an autoimmune and neuroinflammatory disease characterized by demyelination of the Central Nervous System. Immune cells activation and release of pro-inflammatory cytokines play a crucial role in the disease modulation, decisively contributing to the neurodegeneration observed in MS and the experimental autoimmune encephalomyelitis (EAE), the widely used MS animal model. Synthetic glucocorticoids, commonly used to treat the MS attacks, have controversial effects on neuroinflammation and cognition. We sought to verify the influence of dexamethasone (DEX) on the EAE progression and on EAE-induced cognitive deficits. In myelin oligodendrocyte glycoprotein peptide (MOG35-55)-induced EAE female mice, treated once with DEX (50 mg/kg) or not, on the day of immunization, DEX decreased EAE-induced motor clinical scores, infiltrating cells in the spinal cord and delayed serum corticosterone peak. At the asymptomatic phase (8-day post-immunization), DEX did not protected from the EAE-induced memory consolidation deficits, which were accompanied by increased glucocorticoid receptor (GR) activity and decreased EGR-1 expression in the hippocampus. Blunting hippocampal GR genomic activation with DnGR vectors prevented DEX effects on EAE-induced memory impairment. These data suggest that, although DEX improves clinical signs, it decreases cognitive and memory capacity by diminishing neuronal activity and potentiating some aspects of neuroinflammation in EAE.