Experimental Autoimmune Encephalomyelitis Mouse Model Research Articles

Proteomic Identification of Immunoproteasome Accumulation in Formalin-Fixed Rodent Spinal Cords with Experimental Autoimmune Encephalomyelitis

Clinically relevant formalin-fixed and paraffin-embedded (FFPE) tissues have not been widely used in neuroproteomic studies because many proteins are presumed to be degraded during tissue preservation. Recent improvements in proteomics technologies, from the 2D gel analysis of intact proteins to the "shotgun" quantification of peptides and the use of isobaric tags for absolute and relative quantification (iTRAQ) method, have made the analysis of FFPE tissues possible. In recent years, iTRAQ has been one of the main methods of choice for high throughput quantitative proteomics analysis, which enables simultaneous comparison of up to eight samples in one experiment. Our objective was to assess the relative merits of iTRAQ analysis of fresh frozen versus FFPE nervous tissues by comparing experimental autoimmune encephalomyelitis (EAE)-induced proteomic changes in FFPE rat spinal cords and frozen tissues. EAE-induced proteomic changes in FFPE tissues were positively correlated with those found in the frozen tissues, albeit with ∼50% less proteome coverage. Subsequent validation of the enrichment of immunoproteasome (IP) activator 1 in EAE spinal cords led us to evaluate other proteasome and IP-specific proteins. We discovered that many IP-specific (as opposed to constitutive) proteasomal proteins were enriched in EAE rat spinal cords, and EAE-induced IP accumulation also occurred in the spinal cords of an independent mouse EAE model in a disability score-dependent manner. Therefore, we conclude that it is feasible to generate useful information from iTRAQ-based neuroproteomics analysis of archived FFPE tissues for studying neurological disease tissues.

Anti-Inflammatory Effect of Erythropoietin Therapy on Experimental Autoimmune Encephalomyelitis

ABSTRACTPrevious studies report that erythropoietin (EPO) has a neuroprotective role in some neurodegenerative diseases, but the mechanisms are not completely elucidated. The aim of this study was to investigate whether EPO exerts neuroprotective role in experimental autoimmune encephalomyelitis (EAE) via the routes of anti-inflammation. We established an EAE mice model treated intraperitoneally with EPO at the dose of 5,000 IU/kg on schedule, and recorded the clinical score and weight fluctuation. The infiltration of inflammatory cells in the spinal cord of EAE mice was observed with hemotoxylin and eosin (HE) staining, and the levels of IL-10, IFN-γ, IL-17, and MHC-II in central nervous system (CNS)-infiltrating cells and peripheral mononuclear cells were detected by flow cytometry or ELISA. EPO therapy ameliorates clinical signs of EAE mice, inhibits the body weight loss, and decreases the infiltration of inflammatory cells in spinal cords. IL-17 and IFN-γ are reduced, while IL-10 is not increased significantly, in both CNS-infiltrating cells and peripheral mononuclear cells of EPO-treated EAE mice, as compared with EAE control group. EPO also reduces the expression of MHC-II on peripheral antigen presentation cells. Our results indicate that EPO exerts a beneficial role in EAE by inhibiting the levels of IL-17 and IFN-γ in peripheral splenic cells and CNS-infiltrating cells.

Amelioration of Experimental Autoimmune Encephalomyelitis in C57BL/6 Mice by Photobiomodulation Induced by 670 nm Light

BackgroundThe approved immunomodulatory agents for the treatment of multiple sclerosis (MS) are only partially effective. It is thought that the combination of immunomodulatory and neuroprotective strategies is necessary to prevent or reverse disease progression. Irradiation with far red/near infrared light, termed photobiomodulation, is a therapeutic approach for inflammatory and neurodegenerative diseases. Data suggests that near-infrared light functions through neuroprotective and anti-inflammatory mechanisms. We sought to investigate the clinical effect of photobiomodulation in the Experimental Autoimmune Encephalomyelitis (EAE) model of multiple sclerosis.Methodology/Principal FindingsThe clinical effect of photobiomodulation induced by 670 nm light was investigated in the C57BL/6 mouse model of EAE. Disease was induced with myelin oligodendrocyte glycoprotein (MOG) according to standard laboratory protocol. Mice received 670 nm light or no light treatment (sham) administered as suppression and treatment protocols. 670 nm light reduced disease severity with both protocols compared to sham treated mice. Disease amelioration was associated with down-regulation of proinflammatory cytokines (interferon-γ, tumor necrosis factor-α) and up-regulation of anti-inflammatory cytokines (IL-4, IL-10) in vitro and in vivo.Conclusion/SignificanceThese studies document the therapeutic potential of photobiomodulation with 670 nm light in the EAE model, in part through modulation of the immune response.

Lipocalin 2 is present in the EAE brain and is modulated by natalizumab

Multiple sclerosis (MS) is a demyelinating disease that causes major neurological disability in young adults. A definitive diagnosis at the time of the first episode is still lacking, but since early treatment leads to better prognosis, the search for early biomarkers is needed. Here we characterized the transcriptome of the choroid plexus (CP), which is part of the blood-brain barriers (BBBs) and the major site of cerebrospinal fluid production, in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. In addition, cerebrospinal fluid samples from two cohorts of patients with MS and with optic neuritis (ON) were analyzed to confirm the clinical relevance of the findings. Genes encoding for adhesion molecules, chemokines and cytokines displayed the most altered expression, supporting the role of CP as a site of immune-brain interaction in MS. The gene encoding for lipocalin 2 was the most up-regulated; notably, the cerebrospinal fluid lipocalin 2 levels coincided with the active phases of the disease. Immunostaining revealed that neutrophils infiltrating the CP were the source of the increased lipocalin 2 expression in this structure. However, within the brain, lipocalin 2 was also detected in astrocytes, particularly in regions typically affected in patients with MS. The increase of lipocalin 2 in the cerebrospinal fluid and in astrocytes was reverted by natalizumab treatment. Most importantly, the results obtained in the murine model were translatable into humans since patients from two different cohorts presented increased cerebrospinal fluid lipocalin 2 levels. The findings support lipocalin 2 as a valuable molecule for the diagnostic/monitoring panel of MS.

BBB-endothelial cell response to cerebral cortex demyelination in a mouse model of chronic EAE

Changes in blood-brain barrier (BBB) function have been implicated in demyelinating diseases. This study aimed to investigate the response of cerebral cortex microvessels to nerve fibre demyelination in a chronic model of murine experimental autoimmune encephalomyelitis (EAE) characterized by areas of extensive subpial demyelination along with well-demarcated lesions extended to deeper cortex layers. These cortices showed activation of microglia and astrogliosis with absence of typical perivascular inflammatory infiltrates. On the basis of these data, we have analyzed the expression of two integral proteins of endothelial tight junctions, claudin-5 and occludin, a structural protein of caveolae, caveolin-1, as well as the BBB-specific endothelial transporter, Glut1 in the cerebral cortex of EAE-affected mice by immunofluorescence confocal microscopy. Microvascular endothelial cells showed an increased expression of caveolin-1 and a coincident decrease of both claudin-5 and occludin junctional staining pattern. At a very early disease stage, claudin-5 molecules formed aggregates and vacuoles that also stained for Glut 1, whereas occludin pattern became diffusely cytoplasmic at advanced stages of the disease. Internalization/dismantling and loss of tight junction proteins and impairment of BBB function were confirmed by coexpression of claudin-5 whit the autophagosomal marker MAP1LC3A and by FITC-dextran experiments that showed leakage of the tracer into the perivascular neuropil. Overall, these observations indicate that in the cerebral cortex of EAE mice, during demyelination and independently from the inflammatory involvement of the cortex, a ‘microvascular disease’ characterized by a differential involvement of claudin-5 and occludin occurs, thereby possibly contributing to demyelinating disease progression.

Probiotic upregulation of peripheral IL-17 responses does not exacerbate neurological symptoms in experimental autoimmune encephalomyelitis mouse models

Context: It is of great importance to evaluate the safety of probiotics in dysregulated immune conditions, as probiotics can possibly modulate immune functions in the host.Objective: We tried to confirm the safety of using Lactobacillus casei strain Shirota (LcS) to help prevent autoimmunity in the central nervous system.Methods: We used two chronic experimental autoimmune encephalomyelitis (EAE) models, a relapse and remission type EAE model in SJL/J mice and a durable type model in C57BL/6 mice. LcS was administered from 1 week before antigen sensitization until the end of the experiments, and neurological symptoms and histopathological changes of the spinal cord were observed. Immunological parameters were also examined in the SJL/J mouse model.Results: LcS administration did not exacerbate neurological symptoms or histopathological changes of the spinal cord in either model but instead tended to improve neurological symptoms in the SJL/J mouse EAE model. LcS administration transiently upregulated IL-17 production by antigen-stimulated lymphocytes of draining lymph nodes 7 days after sensitization. Enhanced production of IL-10 and an increase in the percentage of CD4+CD25+ T regulatory cells were also observed at the same sites. Strong expression of IL-17 mRNA was detected in the spinal cord of mice that displayed severe neurological symptoms on day 12, but this expression was not enhanced by LcS administration.Conclusion: These results demonstrate that LcS does not exacerbate, but instead may improve EAE depending on the immunization conditions, and that IL-17 responses at peripheral sites may not always result in a worsening of autoimmune diseases.

Human endometrial-derived mesenchymal stem cells suppress inflammation in the central nervous system of experimental autoimmune encephalomyelitis-induced mice

Human endometrial-derived mesenchymal stem cells suppress inflammation in the central nervous system of experimental autoimmune encephalomyelitis-induced mice

Impaired neurosteroid synthesis in multiple sclerosis

High-throughput technologies have led to advances in the recognition of disease pathways and their underlying mechanisms. To investigate the impact of micro-RNAs on the disease process in multiple sclerosis, a prototypic inflammatory neurological disorder, we examined cerebral white matter from patients with or without the disease by micro-RNA profiling, together with confirmatory reverse transcription-polymerase chain reaction analysis, immunoblotting and gas chromatography-mass spectrometry. These observations were verified using the in vivo multiple sclerosis model, experimental autoimmune encephalomyelitis. Brains of patients with or without multiple sclerosis demonstrated differential expression of multiple micro-RNAs, but expression of three neurosteroid synthesis enzyme-specific micro-RNAs (miR-338, miR-155 and miR-491) showed a bias towards induction in patients with multiple sclerosis (P < 0.05). Analysis of the neurosteroidogenic pathways targeted by micro-RNAs revealed suppression of enzyme transcript and protein levels in the white matter of patients with multiple sclerosis (P < 0.05). This was confirmed by firefly/Renilla luciferase micro-RNA target knockdown experiments (P < 0.05) and detection of specific micro-RNAs by in situ hybridization in the brains of patients with or without multiple sclerosis. Levels of important neurosteroids, including allopregnanolone, were suppressed in the white matter of patients with multiple sclerosis (P < 0.05). Induction of the murine micro-RNAs, miR-338 and miR-155, accompanied by diminished expression of neurosteroidogenic enzymes and allopregnanolone, was also observed in the brains of mice with experimental autoimmune encephalomyelitis (P < 0.05). Allopregnanolone treatment of the experimental autoimmune encephalomyelitis mouse model limited the associated neuropathology, including neuroinflammation, myelin and axonal injury and reduced neurobehavioral deficits (P < 0.05). These multi-platform studies point to impaired neurosteroidogenesis in both multiple sclerosis and experimental autoimmune encephalomyelitis. The findings also indicate that allopregnanolone and perhaps other neurosteroid-like compounds might represent potential biomarkers or therapies for multiple sclerosis.

The MAO inhibitor phenelzine improves functional outcomes in mice with experimental autoimmune encephalomyelitis (EAE)

Multiple sclerosis (MS) and the animal model, experimental autoimmune encephalomyelitis (EAE), are both accompanied by motor and non-motor symptoms. Pathological changes in the activities of key neurotransmitters likely underlie many of these symptoms. We have previously described disturbances in the levels of 5-hydroxytryptamine (5-HT/serotonin), noradrenaline (NE) and γ-aminobutyric acid (GABA) in a mouse model of EAE. The potential therapeutic effect of a drug that targets these three neurotransmitters, the antidepressant and anti-panic drug phenelzine (PLZ), was assessed in mice with MOG(35-55) induced EAE. The neurotransmitter content of EAE and control tissue after PLZ administration was first evaluated by HPLC. The ability of PLZ treatment to modulate EAE disease course and clinical signs was then assessed. Daily PLZ treatment, starting seven days after disease induction, delayed EAE onset, reduced disease severity in the chronic phase and was associated with substantial improvements in exploratory behavior and a novel measure of sickness and/or depression. Upon completion of the experiment, PLZ's effects on histopathological markers of the disease were examined. No differences were observed in T cell infiltration, microglia/macrophage reactivity, demyelination or axonal injury in PLZ-treated spinal cords. However, EAE mice treated with PLZ showed a normalization of 5-HT levels in the ventral horn of the spinal cord that might account for the improvements in behavioral outcomes. These results demonstrate the therapeutic potential of MAO inhibitors such as PLZ in MS. Additionally, the behavioral changes observed in EAE mice indicate that alterations in non-motor or 'affective' measures may be valuable to consider in addition to traditional measures of gross locomotor function.

Dopamine Induces IL-6–Dependent IL-17 Production via D1-Like Receptor on CD4 Naive T Cells and D1-Like Receptor Antagonist SCH-23390 Inhibits Cartilage Destruction in a Human Rheumatoid Arthritis/SCID Mouse Chimera Model

A major neurotransmitter dopamine transmits signals via five different seven-transmembrane G protein-coupled receptors termed D1-D5. Several studies have shown that dopamine not only mediates interactions into the nervous system, but can contribute to the modulation of immunity via receptors expressed on immune cells. We have previously shown an autocrine/paracrine release of dopamine by dendritic cells (DCs) during Ag presentation to naive CD4(+) T cells and found efficacious results of a D1-like receptor antagonist SCH-23390 in the experimental autoimmune encephalomyelitis mouse model of multiple sclerosis and in the NOD mouse model of type I diabetes, with inhibition of Th17 response. This study aimed to assess the role of dopaminergic signaling in Th17-mediated immune responses and in the pathogenesis of rheumatoid arthritis (RA). In human naive CD4(+) T cells, dopamine increased IL-6-dependent IL-17 production via D1-like receptors, in response to anti-CD3 plus anti-CD28 mAb. Furthermore, dopamine was localized with DCs in the synovial tissue of RA patients and significantly increased in RA synovial fluid. In the RA synovial/SCID mouse chimera model, although a selective D2-like receptor antagonist haloperidol significantly induced accumulation of IL-6(+) and IL-17(+) T cells with exacerbated cartilage destruction, SCH-23390 strongly suppressed these responses. Taken together, these findings indicate that dopamine released by DCs induces IL-6-Th17 axis and causes aggravation of synovial inflammation of RA, which is the first time, to our knowledge, that actual evidence has shown the pathological relevance of dopaminergic signaling with RA.

Analysis of the mitochondrial proteome in multiple sclerosis cortex

Mitochondrial dysfunction has been proposed to play a role in the neuropathology of multiple sclerosis (MS). Previously, we reported significant alterations in the transcription of nuclear-encoded electron transport chain genes in MS and confirmed translational alterations for components of Complexes I and III that resulted in reductions in their activity. To more thoroughly and efficiently elucidate potential alterations in the expression of mitochondrial and related proteins, we have characterized the mitochondrial proteome in postmortem MS and control cortex using Surface-Enhanced Laser Desorption Ionization Time of Flight Mass Spectrometry (SELDI-TOF-MS). Using principal component analysis (PCA) and hierarchical clustering techniques we were able to analyze the differential patterns of SELDI-TOF spectra to reveal clusters of peaks which distinguished MS from control samples. Four proteins in particular were responsible for distinguishing disease from control. Peptide fingerprint mapping unambiguously identified these differentially expressed proteins. Three proteins identified are involved in respiration including cytochrome c oxidase subunit 5b (COX5b), the brain specific isozyme of creatine kinase, and hemoglobin β-chain. The fourth protein identified was myelin basic protein (MBP). We then investigated whether these alterations were consistent in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. We found that MBP was similarly altered in EAE but the respiratory proteins were not. These data indicate that while the EAE mouse model may mimic aspects of MS neuropathology which result from inflammatory demyelinating events, there is another distinct mechanism involved in mitochondrial dysfunction in gray matter in MS which is not modeled in EAE.

Glycogen Synthase Kinase-3 Is an Early Determinant in the Differentiation of Pathogenic Th17 Cells

CD4(+) T cells are critical for host defense but are also major drivers of immune-mediated diseases. The classical view of Th1 and Th2 subtypes of CD4(+) T cells was recently revised by the identification of the Th17 lineage of CD4(+) T cells that produce IL-17, which have been found to be critical in the pathogenesis of autoimmune and other diseases. Mechanisms controlling the differentiation of Th17 cells have been well described, but few feasible targets for therapeutically reducing Th17 cells are known. The generation of Th17 cells requires IL-6 and activation of STAT3. During polarization of CD4(+) T cells to Th17 cells, we found that inhibition of glycogen synthase kinase-3 (GSK3) blocked IL-6 production, STAT3 activation, and polarization to Th17 cells. Polarization of CD4(+) T cells to Th17 cells increased by 10-fold the expression of GSK3β protein levels in Th17 cells, whereas GSK3β was unaltered in regulatory T cells. Diminishing GSK3 activity either pharmacologically or molecularly blocked Th17 cell production, and increasing GSK3 activity promoted polarization to Th17 cells. In vivo inhibition of GSK3 in mice depleted constitutive Th17 cells in intestinal mucosa, blocked Th17 cell generation in the lung after Francisella tularensis infection, and inhibited the increase in spinal cord Th17 cells and disease symptoms in the experimental autoimmune encephalomyelitis mouse model of multiple sclerosis. These findings identify GSK3 as a critical mediator of Th17 cell production and indicate that GSK3 inhibitors provide a potential therapeutic intervention to control Th17-mediated diseases.

Relapse of Experimental Autoimmune Encephalomyelitis after Discontinuation of FTY720 (Fingolimod) Treatment, but Not after Combination of FTY720 and Pathogenic Autoantigen

FTY720 (Fingolimod) is known to have a significant therapeutic effect on experimental autoimmune encephalomyelitis (EAE). Here, we used an EAE mouse model, which had been established by immunizing C57BL/6J mice with a partial peptide of myelin oligodendrocyte glycoprotein (MOG₃₅₋₅₅), to examine the relapse of EAE upon discontinuation of treatment with FTY720 alone or in combination with MOG₃₅₋₅₅. Relapse was confirmed to occur in all animals (n=6) within one week after discontinuation of FTY720, with increase in the number of lymphocytes infiltrating the spinal cord and demyelination. However, in the case of combination therapy with FTY720 and MOG₃₅₋₅₅, relapse following discontinuation of treatment was completely suppressed. The autoantigenic peptide might serve to suppress the clonal selection of relapse-associated autoantigen-specific T cells.

Fibrinogen depleting agent batroxobin has a beneficial effect on experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) was characterized with widespread demyelination and axonal loss of central nervous system (CNS). Fibrinogen (fibrin) deposition was considered as one of the pathogenesis of MS. Therefore, we explored the effects of fibrinogen depleting agent batroxobin in experimental autoimmune encephalomyelitis (EAE) mice model. Our study showed that prevention and suppression with batroxobin significantly ameliorated clinical severity of EAE, reduced inflammatory cells infiltration, and demyelination, and suppressed the activation of astrocytes and macrophages comprising the CD11b(+) population. Batroxobin treatment leads to reduced expression of p-Akt and increased expression of MBP as compared to control. In addition, batroxobin treatment partly reversed the dendric-like formation of macrophages irritated by fibrinogen in vitro. The reduced severity of EAE mice treated with batroxobin suggests that strategy targeting fibrin as a potential therapy for EAE may be beneficial for the treatment of MS patients.

An Animal Model of Cortical and Callosal Pathology in Multiple Sclerosis

The pathological and radiological hallmarks of multiple sclerosis (MS) include multiple demyelinated lesions disseminated throughout the white matter of the central nervous system (CNS). More recently, the cerebral cortex has been shown to be affected in MS, but the elucidation of events causing cortical demyelination has been hampered by the lack of animal models reflecting such human cortical pathology. In this report, we have described the presence of cortical gray matter and callosal white matter demyelinating lesions in the chronic experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Similar to the pathological lesions of MS patients, EAE lesions have been classified as type I-leukocortical, type II-intracortical and type III-subpial. All of these lesions had varying degrees of demyelination, inflammatory cells and reactive astrocytes. Similar to MS, cortical layers during EAE showed demyelination, microglia activation, synaptic protein alterations and apoptotic cells. In addition, the callosal white matter during EAE had many inflammatory demyelinating lesions and axon degeneration. Functional electrophysiological conduction analysis showed deficits in both myelinated and unmyelinated callosal axons during early and late EAE. The chronic EAE mouse model has features that mimic cortical and callosal pathology of MS, and can be potentially used to screen agents to prevent these features of disease.

Expression and activation of matrix metalloproteinase-9 and NADPH oxidase in tissues and plasma of experimental autoimmune encephalomyelitis in mice

Experimental autoimmune encephalomyelitis (EAE) is a widely used animal model for multiple sclerosis (MS) that can be induced by immunization with myelin antigens such as myelin oligodendrocyte glycoprotein (MOG). The objective of this study was (i) to investigate how matrix metalloproteinase-9 (MMP-9) and NADPH oxidase enzymes are affected in the EAE mouse model and (ii) to know whether peripheral organs also express these enzymes in the EAE model. MOG 33–55 was administered subcutaneously on two sites over the back. Pertussis toxin was administered intraperitoneally immediately after MOG and again two days later. A significant difference was observed in body weights and clinical signs of EAE-induced mice. MMP-9 and NADPH oxidase enzymes were measured in central nervous system (CNS) tissues, peripheral tissues and plasma of EAE-induced mice. The primary findings include the distribution pattern of MMP-9 in CNS and peripheral tissues, and alterations in the enzymatic expression of MMP-9 and NADPH oxidase in the CNS tissues, spleen and plasma of EAE-induced mice. From these results, it can be considered that the spleen as well as the CNS can act as target organs in EAE disease, and plasma MMP-9 and NADPH oxidase may contribute to the pathogenesis of the disease.

Centrally Administered Pertussis Toxin Inhibits Microglia Migration to the Spinal Cord and Prevents Dissemination of Disease in an EAE Mouse Model

BackgroundExperimental autoimmune encephalomyelitis (EAE) models are important vehicles for studying the effect of infectious elements such as Pertussis toxin (PTx) on disease processes related to acute demyelinating encephalomyelitis (ADEM) or multiple sclerosis (MS). PTx has pleotropic effects on the immune system. This study was designed to investigate the effects of PTx administered intracerebroventricularly (icv) in preventing downstream immune cell infiltration and demyelination of the spinal cord.Methods and FindingsEAE was induced in C57BL/6 mice with MOG35–55. PTx icv at seven days post MOG immunization resulted in mitigation of clinical motor symptoms, minimal T cell infiltration, and the marked absence of axonal loss and demyelination of the spinal cord. Integrity of the blood brain barrier was compromised in the brain whereas spinal cord BBB integrity remained intact. PTx icv markedly increased microglia numbers in the brain preventing their migration to the spinal cord. An in vitro transwell study demonstrated that PTx inhibited migration of microglia.ConclusionCentrally administered PTx abrogated migration of microglia in EAE mice, limiting the inflammatory cytokine milieu to the brain and prevented dissemination of demyelination. The effects of PTx icv warrants further investigation and provides an attractive template for further study regarding the pleotropic effects of infectious elements such as PTx in the pathogenesis of autoimmune disorders.

Suppression of encephalitogenic T-cell responses by cilostazol is associated with upregulation of regulatory T cells

Cilostazol is a specific phosphodiesterase III inhibitor. Recent data show that cilostazol has anti-inflammatory effects and administration of cilostazol ameliorates experimental autoimmune encephalomyelitis (EAE). In this study, we used a mouse EAE model to explore the role of cilostazol in Th1 and Th17 cell-mediated immune responses. We found that cilostazol suppressed mitogen or antigen-induced T-cell responses and Th17 cell differentiation in vitro, which correlated with enhanced Treg-cell responses. Beginning of oral administration of cilostazol at the onset of EAE significantly inhibited encephalitogenic T cells, reduced the levels of inflammatory cytokines in the central nervous system, and ameliorated the severity of EAE. Moreover, administration of cilostazol markedly enhanced Treg-cell response in vivo. Cilostazol, therefore, may exert its therapeutic effects through upregulation of Treg-cell activity.

Notch signaling regulates human and mouse TH17 development. (139.13)

Abstract TH17 immune cells, a recently discovered subset of CD4+ T-helper cells, are known to play important role in pathogenesis of several autoimmune diseases, inflammatory disorders and cancers. Although Notch ligand DLL4 was shown to drive TH17 polarization, downstream pathways conducting those signals are unknown. We demonstrated that the Notch signaling pathway has an important role in TH17 polarization. We found that gamma secretase inhibitors (GSI) that inhibit Notch signaling blocked the in-vitro polarization of mouse or human naïve CD4+ T cells towards the TH17 pathway without affecting cell proliferation. Next, we showed that, among various Notch receptors, Notch-1 is upregulated in TH17 polarizing cells and that knocking down Notch-1 by siRNA inhibited TH17 polarization. We further discovered that, in addition to polarizing naïve T cells towards TH17 pathway, Notch-1 is important for maintenance of this phenotype. Furthermore, reporter luciferase assays and chromatin immunoprecipitation (ChIP) assays suggest that the IL-17 as well as RORC promoters are direct transcriptional targets of Notch-1. Finally, in-vivo administration of GSI inhibits TH17 mediated disease progression in mouse experimental autoimmune encephalomyelitis model of multiple sclerosis. Thus our data suggest a key role for Notch signaling, especially Notch-1, in TH17 polarization.

Suppression of Experimental Autoimmune Encephalomyelitis by Ghrelin

Ghrelin is a recently identified gastric hormone that displays strong growth hormone-releasing activity mediated by the growth hormone secretagogue receptor. While this unique endogenous peptide participates in the regulation of energy homeostasis, increases food intake, and decreases energy expenditure, its ability to inhibit the production of proinflammatory cytokines in vitro indicates its role in the regulation of inflammatory process in vivo. Here we examine the effect of exogenous ghrelin on the development of experimental autoimmune encephalomyelitis (EAE), a representative model of multiple sclerosis. In the C57BL/6 mouse model of EAE induced by sensitization to myelin oligodendrocyte glycoprotein 35-55 peptide, we found that alternate-day s.c. injections of ghrelin (5 mug/kg/day) from day 1 to 35 significantly reduced the clinical severity of EAE. The suppression of EAE was accompanied by reduced mRNA levels of proinflammatory cytokines such as TNF-alpha, IL-1beta, and IL-6 in the spinal cord cellular infiltrates and microglia from ghrelin-treated mice at the peak of disease, suggesting the role of ghrelin as an antiinflammatory hormone. Consistently, ghrelin significantly suppressed the production of proinflammatory cytokines in LPS-stimulated microglia in vitro. These results shed light on the new role of ghrelin in the regulation of inflammation with possible implications for management of human diseases.