Stages Of Experimental Autoimmune Encephalomyelitis Research Articles

Menstrual blood-derived mesenchymal stromal cells efficiently ameliorate experimental autoimmune encephalomyelitis by inhibiting T cell activation in mice

BackgroundImmunosuppressive properties grant mesenchymal stromal cells (MSCs) promising potential for treating autoimmune diseases. As autologous MSCs suffer from limited availability, the readily available allogeneic MSCs isolated from menstrual blood (MB-MSCs) donated by young, healthy individuals offer great potential. Here, we evaluate the therapeutic potential of MB-MSCs as ready-to-use allo-MSCs in multiple sclerosis, an autoimmune disease developed by the activation of myelin sheath-reactive Th1 and Th17 cells, by application in its animal model experimental autoimmune encephalomyelitis (EAE).MethodsWe assessed the therapeutic effect of MB-MSCs transplanted via either intravenous (i.v.) or intraperitoneal (i.p.) route in EAE in comparison with umbilical cord-derived MSCs (UC-MSCs). We used histology to assess myelin sheath integrity and infiltrated immune cells in CNS and flow cytometry to evaluate EAE-associated inflammatory T cells and antigen-presenting cells in lymphoid organs.ResultsWe observed disease-ameliorating effects of MB-MSCs when transplanted at various stages of EAE (day − 1, 6, 10, and 19), via either i.v. or i.p. route, with a potency comparable to UC-MSCs. We observed reduced Th1 and Th17 cell responses in mice that had received MB-MSCs via either i.v. or i.p. injection. The repressed Th1 and Th17 cell responses were associated with a reduced frequency of plasmacytoid dendritic cells (pDCs) and a suppressed co-stimulatory capacity of pDCs, cDCs, and B cells.ConclusionsOur data demonstrate that the readily available MB-MSCs significantly reduced the disease severity of EAE upon transplantation. Thus, they have the potential to be developed as ready-to-use allo-MSCs in MS-related inflammation.Graphical abstract

ASK1 signaling regulates phase-specific glial interactions during neuroinflammation

Neuroinflammation is well known to be associated with neurodegenerative diseases. Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase that has been implicated in neuroinflammation, but its precise cellular and molecular mechanisms remain unknown. In this study, we generated conditional knockout (CKO) mice that lack ASK1 in T cells, dendritic cells, microglia/macrophages, microglia, or astrocytes, to assess the roles of ASK1 during experimental autoimmune encephalomyelitis (EAE). We found that neuroinflammation was reduced in both the early and later stages of EAE in microglia/macrophage-specific ASK1 knockout mice, whereas only the later-stage neuroinflammation was ameliorated in astrocyte-specific ASK1 knockout mice. ASK1 deficiency in T cells and dendritic cells had no significant effects on EAE severity. Further, we found that ASK1 in microglia/macrophages induces a proinflammatory environment, which subsequently activates astrocytes to exacerbate neuroinflammation. Microglia-specific ASK1 deletion was achieved using a CX3CR1CreER system, and we found that ASK1 signaling in microglia played a major role in generating and maintaining disease. Activated astrocytes produce key inflammatory mediators, including CCL2, that further activated and recruited microglia/macrophages, in an astrocytic ASK1-dependent manner. Astrocyte-specific analysis revealed CCL2 expression was higher in the later stage compared with the early stage, suggesting a greater proinflammatory role of astrocytes in the later stage. Our findings demonstrate cell-type-specific roles of ASK1 and suggest phase-specific ASK1-dependent glial cell interactions in EAE pathophysiology. We propose glial ASK1 as a promising therapeutic target for reducing neuroinflammation.

Memantine Modulates Oxidative Stress in the Rat Brain following Experimental Autoimmune Encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an animal model most commonly used in research on the pathomechanisms of multiple sclerosis (MS). The inflammatory processes, glutamate excitotoxicity, and oxidative stress have been proposed as determinants accompanying demyelination and neuronal degeneration during the course of MS/EAE. The aim of the current study was to characterize the role of NMDA receptors in the induction of oxidative stress during the course of EAE. The effect of memantine, the uncompetitive NMDA receptor antagonist, on modulation of neurological deficits and oxidative stress in EAE rats was analyzed using several experimental approaches. We demonstrated that the expression of antioxidative enzymes (superoxide dismutases SOD1 and SOD2) were elevated in EAE rat brains. Under the same experimental conditions, we observed alterations in oxidative stress markers such as increased levels of malondialdehyde (MDA) and decreased levels of sulfhydryl (-SH) groups, both protein and non-protein (indicating protein damage), and a decline in reduced glutathione. Importantly, pharmacological inhibition of ionotropic NMDA glutamate receptors by their antagonist memantine improved the physical activity of EAE rats, alleviated neurological deficits such as paralysis of tail and hind limbs, and modulated oxidative stress parameters (MDA, -SH groups, SOD’s). Furthermore, the current therapy aiming to suppress NMDAR-induced oxidative stress was partially effective when NMDAR’s antagonist was administered at an early (asymptomatic) stage of EAE.

The circular RNA circINPP4B acts as a sponge of miR-30a to regulate Th17 cell differentiation during progression of experimental autoimmune encephalomyelitis

Circular RNAs (circRNAs) regulate gene expression and participate in various biological and pathological processes. However, little is known about the effects of specific circRNAs on T helper cell 17 (Th17) differentiation and related autoimmune diseases, such as multiple sclerosis (MS). Here, using transcriptome microarray analysis at different stages of experimental autoimmune encephalomyelitis (EAE), we identified the EAE progression-related circINPP4B, which showed upregulated expression in Th17 cells from mice with EAE and during Th17 differentiation in vitro. Silencing of circINPP4B inhibited Th17 differentiation and alleviated EAE, characterized by less demyelination and Th17 infiltration in the spinal cord. Mechanistically, circINPP4B served as a sponge that directly targeted miR-30a to regulate Th17 differentiation. Furthermore, circINPP4B levels were associated with the developing phases of clinical relapsing-remitting MS patients. Our results indicate that circINPP4B plays an important role in promoting Th17 differentiation and progression of EAE by targeting miR-30a, which provides a potential diagnostic and therapeutic target for Th17-mediated MS.

Characterization of microglial transcriptomes in the brain and spinal cord of mice in early and late experimental autoimmune encephalomyelitis using a RiboTag strategy

Microglia play an important role in the pathogenesis of multiple sclerosis and the mouse model of MS, experimental autoimmune encephalomyelitis (EAE). To more fully understand the role of microglia in EAE we characterized microglial transcriptomes before the onset of motor symptoms (pre-onset) and during symptomatic EAE. We compared the transcriptome in brain, where behavioral changes are initiated, and spinal cord, where damage is revealed as motor and sensory deficits. We used a RiboTag strategy to characterize ribosome-bound mRNA only in microglia without incurring possible transcriptional changes after cell isolation. Brain and spinal cord samples clustered separately at both stages of EAE, indicating regional heterogeneity. Differences in gene expression were observed in the brain and spinal cord of pre-onset and symptomatic animals with most profound effects in the spinal cord of symptomatic animals. Canonical pathway analysis revealed changes in neuroinflammatory pathways, immune functions and enhanced cell division in both pre-onset and symptomatic brain and spinal cord. We also observed a continuum of many pathways at pre-onset stage that continue into the symptomatic stage of EAE. Our results provide additional evidence of regional and temporal heterogeneity in microglial gene expression patterns that may help in understanding mechanisms underlying various symptomology in MS.

MANF Is Neuroprotective in Early Stages of EAE, and Elevated in Spinal White Matter by Treatment With Dexamethasone

Multiple sclerosis (MS) is a progressive autoimmune disease characterized by T-cell mediated demyelination in central nervous system (CNS). Experimental autoimmune encephalomyelitis (EAE) is a widely used in vivo disease model of MS. Glucocorticoids such as dexamethasone (dex) function as immunosuppressants and are commonly used to treat acute exacerbations of MS. Dex is also often used as a positive control in EAE studies, as it has been shown to promote motor behavior, inhibit immune cell infiltration into the CNS and regulate the activation of glial cell in EAE. This study further validated the effects of intravenously administrated dex by time-dependent fashion in EAE. Dex postponed clinical signs and motor defects in early stages of EAE. Histological analysis revealed that the degeneration of myelin and axons, as well as the infiltration of peripheral immune cells into the white matter of spinal cord was inhibited by dex in early stages of EAE. Additionally, dex-treatment delayed the neuroinflammatory activation of microglia and astrocytes. Furthermore, this study analyzed the expression of the neurotrophic factor mesencephalic astrocyte-derived neurotrophic factor (MANF) in EAE, and the effect of treatment with dex on MANF-expression. We show that in dex-treated EAE mice expression MANF increased within myelinated areas of spinal cord white matter. We also show that intravenous administration with hMANF in EAE mice improved clinical signs and motor behavior in the early stage of EAE. Our report gives insight to the progression of EAE by providing a time-dependent analysis. Moreover, this study investigates the link between MANF and the EAE model, and shows that MANF is a potential drug candidate for MS.

Therapeutic Potential of a Novel Glucagon-like Peptide-1 Receptor Agonist, NLY01, in Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), characterized by demyelination, gliosis, and neurodegeneration. While the currently available disease-modifying therapies effectively suppress the immune attack on the CNS, there are no therapies to date that directly mitigate neurodegeneration. Glucagon-like peptide-1 (GLP-1) is a small peptide hormone that maintains glucose homeostasis. A novel GLP-1 receptor (GLP-1R) agonist, NLY01, was recently shown to have neuroprotective effects in the animal models of Parkinson's disease and is now in a phase 2 clinical trial. In this study, we investigated the therapeutic potential of NLY01 in a mouse model of MS, experimental autoimmune encephalomyelitis (EAE). Our data show that NLY01 delays the onset and attenuates the severity of EAE in a prevention paradigm, when given before disease onset. NLY01 inhibits the activation of immune cells in the spleen and reduces their trafficking into the CNS. In addition, we show that NLY01 suppresses the production of chemokines that are involved in leukocyte recruitment to the site of inflammation. The anti-inflammatory effect of NLY01 at the early stage of EAE may block the expression of the genes associated with neurotoxic astrocytes in the optic nerves, thereby preventing retinal ganglion cell (RGC) loss in the progressive stage of EAE. In the therapeutic paradigm, NLY01 significantly decreases the clinical score and second attack in a model of relapsing-remitting EAE. GLP-1R agonists may have dual efficacy in MS by suppressing peripheral and CNS inflammation, thereby limiting neuronal loss.

STEAP4 expression in CNS resident cells promotes Th17 cell-induced autoimmune encephalomyelitis

BackgroundMultiple sclerosis (MS) is a debilitating neurological disease caused by autoimmune destruction of the myelin sheath. Experimental autoimmune encephalomyelitis (EAE) is a widely used animal model for the pathogenesis of MS. We and others have previously demonstrated that IL-17 is critical for the pathogenesis of EAE. The concentration of IL-17 is significantly higher in the sera of MS patients than in healthy controls and correlates with disease activity. Moreover, anti-IL-17 neutralizing antibody demonstrated promising efficacy in a phase II trial in MS patients, further substantiating a key pathogenic role for IL-17 in MS. While Th17 and IL-17 are emerging as a bona fide drivers for neuroinflammation, it remains unclear what effector molecule executes the inflammatory tissue destruction in Th17-driven EAE.MethodsBy microarray analysis, we found STEAP4 is a downstream molecule of IL-17 signaling in EAE. We then used STEAP4 global knockout mice and STEAP4 conditional knockout mice to test its role in the pathogenesis of EAE.ResultsHere, we report that the metalloreductase, STEAP4, is a key effector molecule that participates and contributes to the pathogenesis of Th17-mediated neuroinflammation in experimental autoimmune encephalomyelitis. STEAP4 knockout mice displayed delayed onset and reduced severity of EAE induced by active immunization. The reduced disease phenotype was not due to any impact of STEAP4 deficiency on myelin reactive T cells. In contrast, STEAP4 knockout mice were resistant to passively induced EAE, pointing to a role for STEAP4 in the effector stage of EAE. Notably, STEAP4 was only induced the spinal cord of EAE mice that received Th17 cells but not Th1 cells. Consistently, STEAP4 deficiency protected from only Th17 but not Th1-induced EAE. Finally, using Nestin-Cre STEAP4fl/fl mice, we showed that ablation of STEAP4 expression in the resident cells in the central nervous system attenuated disease severity in both active immunization and passive Th17 transfer-induced EAE.ConclusionIn this study, we identified STEAP4 as a Th17-specific effector molecule that participates and contributes to the pathogenesis of neuroinflammation, thus potentially provide a novel target for MS therapy.

HMGB1 Promotes the Release of Sonic Hedgehog From Astrocytes.

High mobility group box 1 protein (HMGB1) is known to be a trigger of inflammation in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). However, it may play a different role in some way. Here we investigated the effect of HMGB1 on promoting sonic hedgehog (shh) release from astrocytes as well as the possible signal pathway involved in it. Firstly, shh increased in astrocytes after administration of recombinant HMGB1 or decreased after HMGB1 was blocked when stimulated by homogenate of the onset stage of EAE. Moreover, the expression of HMGB1 receptors, toll-like receptor (TLR) 2 and receptor for advanced glycation end products (RAGE) increased after HMGB1 administration in primary astrocytes. However, the enhancing effect of HMGB1 on shh release from astrocytes was suppressed only after RAGE was knocked out or blocked. Mechanistically, HMGB1 functioned by activating RAGE-mediated JNK, p38, stat3 phosphorylation. Moreover, HMGB1 could induce shh release in EAE. Additionally, intracerebroventricular injection of recombinant shh protein on the onset stage of EAE alleviated the progress of disease and decreased demylination, compared to the mice with normal saline treatment. Overall, HMGB1 promoted the release of shh from astrocytes through signal pathway JNK, p38 and stat3 mediated by receptor RAGE, which may provide new insights of HMGB1 function in EAE.

Prolonging the integrated stress response enhances CNS remyelination in an inflammatory environment.

The inflammatory environment of demyelinated lesions in multiple sclerosis (MS) patients contributes to remyelination failure. Inflammation activates a cytoprotective pathway, the integrated stress response (ISR), but it remains unclear whether enhancing the ISR can improve remyelination in an inflammatory environment. To examine this possibility, the remyelination stage of experimental autoimmune encephalomyelitis (EAE), as well as a mouse model that incorporates cuprizone-induced demyelination along with CNS delivery of the proinflammatory cytokine IFN-γ were used here. We demonstrate that either genetic or pharmacological ISR enhancement significantly increased the number of remyelinating oligodendrocytes and remyelinated axons in the inflammatory lesions. Moreover, the combined treatment of the ISR modulator Sephin1 with the oligodendrocyte differentiation enhancing reagent bazedoxifene increased myelin thickness of remyelinated axons to pre-lesion levels. Taken together, our findings indicate that prolonging the ISR protects remyelinating oligodendrocytes and promotes remyelination in the presence of inflammation, suggesting that ISR enhancement may provide reparative benefit to MS patients.

Regionally diverse astrocyte subtypes and their heterogeneous response to EAE.

Astrocytes fulfil many functions in the central nervous system (CNS), including contribution to the blood brain barrier, synapse formation, and trophic support. In addition, they can mount an inflammatory response and are heterogeneous in morphology and function. To extensively characterize astrocyte subtypes, we FACS‐isolated and gene expression profiled distinct astrocyte subtypes from three central nervous system regions; forebrain, hindbrain and spinal cord. Astrocyte subpopulations were separated based on GLAST/SLC1A3 and ACSA‐2/ATP1B2 cell surface expression. The local brain environment proved key in establishing different transcriptional programs in astrocyte subtypes. Transcriptional differences between subtypes were also apparent in experimental autoimmune encephalomyelitis (EAE) mice, where these astrocyte subtypes showed distinct responses. While gene expression signatures associated with blood–brain barrier maintenance were lost, signatures involved in neuroinflammation and neurotoxicity were increased in spinal cord astrocytes, especially during acute disease stages. In chronic stages of EAE, this reactive astrocyte signature was slightly decreased, while obtaining a more proliferative profile, which might be relevant for glia scar formation and tissue regeneration. Morphological heterogeneity of astrocytes previously indicated the presence of astrocyte subtypes, and here we show diversity based on transcriptome variation associated with brain regions and differential responsiveness to a neuroinflammatory insult (EAE).

PD-1 Blockade Reverses Obesity-Mediated T Cell Priming Impairment.

Despite obesity reaching pandemic proportions, its impact on antigen-specific T cell responses is still unclear. We have recently demonstrated that obesity results in increased expression of PD-1 on T cells, and checkpoint blockade targeting PD-1/PD-L1 surprisingly resulted in greater clinical efficacy in cancer therapy. Adverse events associated with this therapy center around autoimmune reactions. In this study, we examined the impact of obesity on T cell priming and on autoimmune pathogenesis using the mouse model experimental autoimmune encephalomyelitis (EAE), which is mediated by autoreactive myelin-specific T cells generated after immunization. We observed that diet-induced obese (DIO) mice had a markedly delayed EAE onset and developed milder clinical symptoms compared to mice on control diet (CD). This delay was associated with impaired generation of myelin-specific T cell numbers and concurrently correlated with increased PD-L1 upregulation on antigen-presenting cells in secondary lymphoid organs. PD-1 blockade during the priming stage of EAE restored disease onset and severity and increased numbers of pathogenic CD4+ T cells in the central nervous system (CNS) of DIO mice to similar levels to those of CD mice. Administration of anti–PD-1 after onset of clinical symptoms did not increase EAE pathogenesis demonstrating that initial priming is the critical juncture affected by obesity. These findings demonstrate that obesity impairs antigen-specific T cell priming, but this can be reversed with PD-1 blockade. Our results further suggest that PD-1 blockade may increase the risk of autoimmune toxicities, particularly in obesity.

Oligodendrocyte-specific Argonaute profiling identifies microRNAs associated with experimental autoimmune encephalomyelitis

BackgroundMicroRNAs (miRNAs) belong to a class of evolutionary conserved, non-coding small RNAs with regulatory functions on gene expression. They negatively affect the expression of target genes by promoting either RNA degradation or translational inhibition. In recent years, converging studies have identified miRNAs as key regulators of oligodendrocyte (OL) functions. OLs are the cells responsible for the formation and maintenance of myelin in the central nervous system (CNS) and represent a principal target of the autoimmune injury in multiple sclerosis (MS).MethodsMiRAP is a novel cell-specific miRNA affinity-purification technique which relies on genetically tagging Argonaut 2 (AGO2), an enzyme involved in miRNA processing. Here, we exploited miRAP potentiality to characterize OL-specific miRNA dynamics in the MS model experimental autoimmune encephalomyelitis (EAE).ResultsWe show that 20 miRNAs are differentially regulated in OLs upon transition from pre-symptomatic EAE stages to disease peak. Subsequent in vitro differentiation experiments demonstrated that a sub-group of them affects the OL maturation process, mediating either protective or detrimental signals. Lastly, transcriptome profiling highlighted the endocytosis, ferroptosis, and FoxO cascades as the pathways associated with miRNAs mediating or inhibiting OL maturation.ConclusionsAltogether, our work supports a dual role for miRNAs in autoimmune demyelination. In particular, the enrichment in miRNAs mediating pro-myelinating signals suggests an active involvement of these non-coding RNAs in the homeostatic response toward neuroinflammatory injury.

Regulatory T cells suppress Th17 cell Ca2+ signaling in the spinal cord during murine autoimmune neuroinflammation.

T lymphocyte motility and interaction dynamics with other immune cells are vital determinants of immune responses. Regulatory T (Treg) cells prevent autoimmune disorders by suppressing excessive lymphocyte activity, but how interstitial motility patterns of Treg cells limit neuroinflammation is not well understood. We used two-photon microscopy to elucidate the spatial organization, motility characteristics, and interactions of endogenous Treg and Th17 cells together with antigen-presenting cells (APCs) within the spinal cord leptomeninges in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Th17 cells arrive before the onset of clinical symptoms, distribute uniformly during the peak, and decline in numbers during later stages of EAE. In contrast, Treg cells arrive after Th17 cells and persist during the chronic phase. Th17 cells meander widely, interact with APCs, and exhibit cytosolic Ca2+ transients and elevated basal Ca2+ levels before the arrival of Treg cells. In contrast, Treg cells adopt a confined, repetitive-scanning motility while contacting APCs. These locally confined but highly motile Treg cells limit Th17 cells from accessing APCs and suppress Th17 cell Ca2+ signaling by a mechanism that is upstream of store-operated Ca2+ entry. Finally, Treg cell depletion increases APC numbers in the spinal cord and exaggerates ongoing neuroinflammation. Our results point to fundamental differences in motility characteristics between Th17 and Treg cells in the inflamed spinal cord and reveal three potential cellular mechanisms by which Treg cells regulate Th17 cell effector functions: reduction of APC density, limiting access of Th17 cells to APCs, and suppression of Th17 Ca2+ signaling.

Monocytes and Monocyte-Derived Antigen-Presenting Cells Have Distinct Gene Signatures in Experimental Model of Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory disease mediated by a complex interaction between the autoreactive lymphocytes and the effector myeloid cells within the central nervous system (CNS). In a murine model of MS, experimental autoimmune encephalomyelitis (EAE), Ly6Chi monocytes migrate into the CNS and further differentiate into antigen-presenting cells (APCs) during disease progression. Currently, there is no information about gene signatures that can distinguish between monocytes and the monocyte-derived APCs. We developed a surface marker-based strategy to distinguish between these two cell types during the stage of EAE when the clinical symptoms were most severe, and performed transcriptome analysis to compare their gene expression. We report here that the inflammatory CNS environment substantially alters gene expression of monocytes, compared to the monocyte differentiation process within CNS. Monocytes in the CNS express genes that encode proinflammatory cytokines and chemokines, and their expression is mostly maintained when the cells differentiate. Moreover, monocyte-derived APCs express surface markers associated with both dendritic cells and macrophages, and have a significant up-regulation of genes that are critical for antigen presentation. Furthermore, we found that Ccl17, Ccl22, and Ccr7 are expressed in monocyte-derived APCs but not the Ly6Chi monocytes. These findings may shed light on identifying molecular signals that control monocyte differentiation and functions during EAE.

Upregulation of periostin in MOG-induced experimental autoimmune encephalomyelitis in mice

Periostin has dual roles as a multifunctional cytokine and an extracellular matrix protein that binds to several integrins on a variety of cells. This study evaluated whether the expression and cellular localization of periostin changes in the spinal cord of mice with experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis. Western blot analysis revealed that periostin was significantly upregulated in the spinal cord of EAE-induced mice in the effector stage, and did not significantly decrease in the recovery stage of EAE, compared with normal control. Periostin was constitutively detected in the neurons, ependymal cells, vascular endothelial cells, and astrocytes of normal mice. In the paralytic stage of EAE, periostin immunoreactivity was detected in some macrophages in the subarachnoid space (SAS), as well as in some microglial cells; in addition, periostin immunoreactivity was enhanced in astrocytes and vascular endothelial cells in EAE lesions. Collectively, these results suggest that periostin, either from monocyte-derived macrophages in SAS or glial cells in the parenchyma, partly facilitates the migration of inflammatory cells in the effector stage of EAE; moreover, periostin in the recovery stage of EAE appears to be involved in the plasticity of damaged central nervous system tissues.

The Potassium Channel Kv1.5 Expression Alters During Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) is a chronic, inflammatory, neurodegenerative disease with an autoimmune component. It was suggested that potassium channels, which are involved in crucial biological functions may have a role in different diseases, including MS and its animal model, experimental autoimmune encephalomyelitis (EAE). It was shown that voltage-gated potassium channels Kv1.5 are responsible for fine-tuning in the immune physiology and influence proliferation and differentiation in microglia and astrocytes. Here, we explored the cellular distribution of the Kv1.5 channel, together with its transcript and protein expression in the male rat spinal cord during different stages of EAE. Our results reveal a decrease of Kv1.5 transcript and protein level at the peak of disease, where massive infiltration of myeloid cells occurs,together with reactive astrogliosis and demyelination. Also, we revealed that the presence of this channel is not found in infiltrating macrophages/microglia during EAE. It is interesting to note that Kv1.5 channel is expressed only in resting microglia in the naïve animals. Predominant expression of Kv1.5 channel was found in the astrocytesin all experimental groups, while some vimentin+ cells, resembling macrophages, are devoid of Kv1.5 expression. Our results point to the possible link between Kv1.5 channel and the pathophysiological processes in EAE.

Extrapituitary prolactin promotes generation of Eomes-positive helper T cells mediating neuroinflammation

Induction of eomesodermin-positive CD4+ T cells (Eomes+ T helper [Th] cells) has recently been correlated with the transition from an acute stage to a later stage of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Moreover, these cells' pathogenic role has been experimentally proven in EAE. While exploring how the pathogenic Eomes+ Th cells are generated during the course of EAE, we unexpectedly found that B cells and MHC class II+ myeloid cells isolated from the late EAE lesions strikingly up-regulated the expression of prolactin (PRL). We demonstrate that such PRL-producing cells have a unique potential to induce Eomes+ Th cells from naïve T cells ex vivo, and that anti-MHC class II antibody could block this process. Furthermore, PRL levels in the cerebrospinal fluid were significantly increased in the late phase of EAE, and blocking the production of PRL by bromocriptine or Zbtb20-specific siRNA significantly reduced the numbers of Eomes+ Th cells in the central nervous system (CNS) and ameliorated clinical signs in the later phase of EAE. The PRL dependency of Eomes+ Th cells was confirmed in a series of in vitro and ex vivo experiments. Collectively, these results indicate that extrapituitary PRL plays a crucial role in the CNS inflammation mediated by pathogenic Eomes+ Th cells. Cellular interactions involving PRL-producing immune cells could be considered as a therapeutic target for the prevention of chronic neuroinflammation.

Non-invasive visual evoked potentials to assess optic nerve involvement in the dark agouti rat model of experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein.

Experimental autoimmune encephalomyelitis (EAE) is the primary disease model of multiple sclerosis (MS), one of the most diffused neurological diseases characterized by fatigue, muscle weakness, vision loss, anxiety and depression. EAE can be induced through injection of myelin peptides to susceptible mouse or rat strains. In particular, EAE elicited by the autoimmune reaction against myelin oligodendrocyte glycoprotein (MOG) presents the common features of human MS: inflammation, demyelination and axonal loss. Optic neuritis affects visual pathways in both MS and in several EAE models. Neurophysiological evaluation through visual evoked potential (VEP) recording is useful to check visual pathway dysfunctions and to test the efficacy of innovative treatments against optic neuritis. For this purpose, we investigate the extent of VEP abnormalities in the dark agouti (DA) rat immunized with MOG, which develops a relapsing-remitting disease course. Together with the detection of motor signs, we acquired VEPs during both early and late stages of EAE, taking advantage of a non-invasive recording procedure that allows long follow-up studies. The validation of VEP outcomes was determined by comparison with ON histopathology, aimed at revealing inflammation, demyelination and nerve fiber loss. Our results indicate that the first VEP latency delay in MOG-EAE DA rats appeared before motor deficits and were mainly related to an inflammatory state. Subsequent VEP delays, detected during relapsing EAE phases, were associated with a combination of inflammation, demyelination and axonal loss. Moreover, DA rats with atypical EAE clinical course tested at extremely late time points, manifested abnormal VEPs although motor signs were mild. Overall, our data demonstrated that non-invasive VEPs are a powerful tool to detect visual involvement at different stages of EAE, prompting their validation as biomarkers to test novel treatments against MS optic neuritis.

CCL26 and CCR3 are associated with the acute inflammatory response in the CNS in experimental autoimmune encephalomyelitis

Chemokine ligand 26 (CCL26) is a member of the eotaxin family. It works by interacting exclusively with chemokine receptor 3 (CCR3) and acts as an eosinophil-selective chemoattractant. There is an emerging role for eotaxins in autoimmune diseases. Studies have reported that chemokine ligand 11 (CCL11) and CCL26 are upregulated in patients with neuromyelitis optica spectrum disorder (NMOSD) during remission, CCL26 levels appear to be decreased in relapsing-remitting multiple sclerosis (RRMS), whereas CCL26 levels are significantly increased in secondary progressive multiple sclerosis (SPMS), indicating that CCL26 participates in the pathogenesis of multiple sclerosis (MS). We investigated the levels of CCL26, CCR3 and claudin-5 (a marker of changes in BBB (blood-brain barrier) permeability) at different stages of experimental autoimmune encephalomyelitis (EAE) to explore the underlying immune mechanisms of EAE. Our results showed that the levels of CCL26 and CCR3 in EAE rats were significantly increased compared with those in the control group. The levels of CCL26 in the serum and in brain tissues as well as the protein expression of CCR3 in brain tissues were positively correlated with the inflammatory scores of brain tissues from EAE rats and were negatively correlated with the protein expression of claudin-5. We concluded that CCL26, which in turn binds to the receptor CCR3, showed pro-inflammatory effects and aggravated tissue damage involving BBB impairment, especially in the acute stage of EAE. Our study uncovers another possible immunopathological mechanism of MS and provides a possible target for immune therapy.