Peak Of Experimental Autoimmune Encephalomyelitis Research Articles

Oxysterols promote encephalitogenic CD4 + T cell migration during neuroinflammation

Multiple sclerosis (MS) is a chronic demyelinating disorder of the central nervous system (CNS) leading to progressive neurological dysfunction. Interferon beta (IFNbeta) represents a standard treatment for relapsing–remittingMS that decreases the frequency of relapses and slows disease progression. Exogenous administration of IFNbeta was also demonstrated to exert protective effects in theMS animalmodel of experimental autoimmune encephalomyelitis (EAE) while genetic deletion of IFNbeta in mice leads to an aggravation of disease symptoms. This suggests IFNbeta producing cells as a target for new therapy strategies for autoimmune CNS inflammation. However, neither the source of IFNbeta nor its immunomodulatory mechanisms are known. In this report activated residentmicroglia were identified as themajor IFNbeta source in the CNS during peak EAE presentation using a YFP knock-in reportermodel for IFNbeta. IFNbeta-producingmicroglia migrated efficiently towardsmyelin debris in organotypic slice cultures and IFNbeta deficient microglia failed to induce efficient removal of myelin degradation products in this ex vivo model of lysolecithininduced demyelination, underscoring the prominent role for IFNbeta in controlling myelin clearance. Treatment with IFNbeta alone was sufficient to stimulate specific phagocytotic uptake of degraded myelin by cultured microglia indicating that IFNbeta regulates myelin phagocytosis in primary microglia. These data identify activated microglia as the major endogenous producers of protective IFNbeta at the peak of EAE and demonstrate their involvement in the clearance of myelin debris and possibly neuronal regeneration in CNS autoimmunity. doi:10.1016/j.jneuroim.2014.08.454

Chemokine receptor expression by inflammatory T cells in EAE.

Chemokines direct cellular infiltration to tissues, and their receptors and signaling pathways represent targets for therapy in diseases such as multiple sclerosis (MS). The chemokine CCL20 is expressed in choroid plexus, a site of entry of T cells to the central nervous system (CNS). The CCL20 receptor CCR6 has been reported to be selectively expressed by CD4+ T cells that produce the cytokine IL-17 (Th17 cells). Th17 cells and interferon-gamma (IFNγ)-producing Th1 cells are implicated in induction of MS and its animal model experimental autoimmune encephalomyelitis (EAE). We have assessed whether CCR6 identifies specific inflammatory T cell subsets in EAE. Our approach was to induce EAE, and then examine chemokine receptor expression by cytokine-producing T cells sorted from CNS at peak disease. About 7% of CNS-infiltrating CD4+ T cells produced IFNγ in flow cytometric cytokine assays, whereas less than 1% produced IL-17. About 1% of CD4+ T cells produced both cytokines. CCR6 was expressed by Th1, Th1+17 and by Th17 cells, but not by CD8+ T cells. CD8+ T cells expressed CXCR3, which was also expressed by CD4+ T cells, with no correlation to cytokine profile. Messenger RNA for IFNγ, IL-17A, and the Th1 and Th17-associated transcription factors T-bet and RORγt was detected in both CCR6+ and CXCR3+ CD4+ T cells. IFNγ, but not IL-17A mRNA expression was detected in CD8+ T cells in CNS. CCR6 and CD4 were co-localized in spinal cord infiltrates by double immunofluorescence. Consistent with flow cytometry data some but not all CD4+ T cells expressed CCR6 within infiltrates. CD4-negative CCR6+ cells included macrophage/microglial cells. Thus we have for the first time directly studied CD4+ and CD8+ T cells in the CNS of mice with peak EAE, and determined IFNγ and IL17 expression by cells expressing CCR6 and CXCR3. We show that neither CCR6 or CXCR3 align with CD4 T cell subsets, and Th1 or mixed Th1+17 predominate in EAE.

CD4+ T regulatory and effector cells display a similar wide range of affinities for self during autoimmune disease (BA13P.129)

Abstract The affinity of a T cell receptor (TCR) from a regulatory T cell (Treg) for endogenous self peptide MHC complex is unknown. It is thought that thymic derived Tregs (nTregs) are enriched for high affinity, self-reactive TCRs as an alternative to negative selection. During experimental autoimmune encephalomyelitis (EAE), MOG self-antigen pMHC tetramers identified few (<10%) of the infiltrating CD4+ Tregs and Teffs. The micropipette adhesion frequency assay has previously proven more sensitive for assessing TCR specificity, affinity and frequency of antigen specific cells. Using this assay, it was determined that most of the Tregs and Teffs (>80% each) localized in the central nervous system (CNS) at peak EAE are in fact MOG self antigen specific. Tregs and Teffs possessed similar broad 1000 fold ranges in affinities for MOG with geometric means of 1 and 4 X 10-5 μm4. In the CNS, greater than 90% of the Tregs expressed helios which was consistent with frequencies in the thymus (94%), which suggests an enrichment of nTregs in the CNS during peak EAE. Contrary to the hypothesis that nTregs arise as an alternative to negative selection due to a high affinity for self, our data show that TCR affinity of MOG specific Tregs mimics Teffs with no enrichment of high affinity cells.

R-Ras promotes autoimmunity through negative regulation of natural T regulatory cell numbers by inhibiting tolerogenic dendritic cell development (IRC4P.488)

Abstract R-Ras is a member of the Ras family of small GTPases, which regulate various cellular processes including adhesion, survival, proliferation, trafficking, and cytokine production. R-Ras is expressed by immune cells and has been shown to modulate dendritic cell (DC) function in vitro. Although, higher expression of R-Ras is associated with liver autoimmunity in humans, its pathogenic mechanism is unknown. We used Rras-/- mice to identify the mechanism whereby R-Ras contributes to autoimmunity using experimental autoimmune encephalomyelitis (EAE), a mouse model of the CNS autoimmune disease multiple sclerosis. We found that a lack of R-Ras in peripheral immune cells resulted in attenuated EAE disease. In accordance with reduced EAE disease, numbers of inflammatory immune cells in the CNS of Rras-/- mice at the peak of EAE were significantly reduced. Further investigation revealed that during EAE, absence of R-Ras promoted the formation of MHC IIlo tolerogenic DC concomitant with a significant increase in Neuropilin-1hiCD4+Foxp3+ natural T regulatory cell (nTreg) numbers in the periphery. Mechanistically, we found that Rras-/- tolerogenic DCs induced proliferation of nTregs thereby enhancing their numbers and maintaining them in vivo. Our study suggests a novel role for R-Ras in promoting autoimmunity through negative regulation of nTreg numbers by inhibiting tolerogenic DC development.

Epithelial V-Like Antigen Mediates Efficacy of Anti-Alpha4 Integrin Treatment in a Mouse Model of Multiple Sclerosis

Natalizumab inhibits the transmigration of activated T lymphocytes into the brain and is highly efficacious in multiple sclerosis (MS). However, from a pharmacogenomic perspective, its efficacy and safety in specific patients remain unclear. Here our goal was to analyze the effects of epithelial V-like antigen (EVA) on anti-alpha4 integrin (VLA4) efficacy in a mouse model of MS, experimental autoimmune encephalomyelitis (EAE). EVA has been previously characterized in human CD4 T lymphocytes, mouse thymic development, and choroid plexus epithelial cells. Further analysis here demonstrated expression in B lymphocytes and an increase in EVA+ lymphocytes following immunization. Following active induction of EAE using the MOG35–55 active immunization model, EVA deficient mice developed more severe EAE and white matter tissue injury as compared to wild type controls. This severe EAE phenotype did not respond to anti-VLA4 treatment. In both the control antibody and anti-VLA4 conditions, these mice demonstrated persistent CNS invasion of mature B lymphocyte (CD19+, CD21+, sIgG+), increased serum autoantibody levels, and extensive complement and IgG deposition within lesions containing CD5+IgG+ cells. Wild type mice treated with control antibody also demonstrated the presence of CD19+, CD21+, sIgG+ cells within the CNS during peak EAE disease severity and detectable serum autoantibody. In contrast, wild type mice treated with anti-VLA4 demonstrated reduced serum autoantibody levels as compared to wild type controls and EVA-knockout mice. As expected, anti-VLA4 treatment in wild type mice reduced the total numbers of all CNS mononuclear cells and markedly decreased CD4 T lymphocyte invasion. Treatment also reduced the frequency of CD19+, CD21+, sIgG+ cells in the CNS. These results suggest that anti-VLA4 treatment may reduce B lymphocyte associated autoimmunity in some individuals and that EVA expression is necessary for an optimal therapeutic response. We postulate that these findings could optimize the selection of treatment responders.

Estrogen receptor β ligand therapy activates PI3K/Akt/mTOR signaling in oligodendrocytes and promotes remyelination in a mouse model of multiple sclerosis

The identification of a drug that stimulates endogenous myelination and spares axon degeneration during multiple sclerosis (MS) could potentially reduce the rate of disease progression. Using experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, we have previously shown that prophylactic administration of the estrogen receptor (ER) β ligand 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) decreases clinical disease, is neuroprotective, stimulates endogenous myelination, and improves axon conduction without altering peripheral cytokine production or reducing central nervous system (CNS) inflammation. Here, we assessed the effects of therapeutic DPN treatment during peak EAE disease, which represents a more clinically relevant treatment paradigm. In addition, we investigated the mechanism of action of DPN treatment-induced recovery during EAE. Given that prophylactic and therapeutic treatments with DPN during EAE improved remyelination-induced axon conduction, and that ER (α and β) and membrane (m)ERs are present on oligodendrocyte lineage cells, a direct effect of treatment on oligodendrocytes is likely. DPN treatment of EAE animals resulted in phosphorylated ERβ and activated the phosphatidylinositol 3-kinase (PI3K)/serine–threonine-specific protein kinase (Akt)/mammalian target of rapamycin (mTOR) signaling pathway, a pathway required for oligodendrocyte survival and axon myelination. These results, along with our previous studies of prophylactic DPN treatment, make DPN and similar ERβ ligands immediate and favorable therapeutic candidates for demyelinating disease.

High interleukin-10 expression within the central nervous system may be important for initiation of recovery of Dark Agouti rats from experimental autoimmune encephalomyelitis

Dark Agouti (DA) rats are highly susceptible to induction of experimental autoimmune encephalomyelitis (EAE), still they completely recover from the disease. Here, we were interested to determine contribution of major anti-inflammatory cytokines transforming growth factor (TGF)-β and interleukin (IL)-10 to the recovery of DA rats from EAE. To that extent we determined CNS expression of these cytokines in DA rats at different phases of EAE and compared data to those obtained in EAE-resistant Albino Oxford (AO) rats. Higher expression of TGF-β was persistently observed in the CNS of AO rats, even if rats were not immunized. This implied that high TGF-β within the CNS is important for resistance of AO rats to EAE induction. On the contrary, IL-10 expression was consistently higher in DA than in AO rats and it culminated at the peak of EAE. Methylprednisolone suppressed EAE and expression of IL-10 in spinal cord homogenates, while IL-10 was increased in CNS-infiltrating immune cells. This implied that IL-10 might have a significant role in recovery of DA rats from the disease. Thus, we next explored effects of IL-10 on astrocytes, glial cells that largely contribute to control of CNS inflammation. IL-10 stimulated astrocytic expression of an important regulator of neuroinflammation, CXCL12. Thus, IL-10 might contribute to recovery of DA rats from EAE through induction of CXCL12 expression in astrocytes.

Metabolite profiling of small cerebrospinal fluid sample volumes with gas chromatography–mass spectrometry: application to a rat model of multiple sclerosis

Analysis of metabolites in biofluids by gas chromatography–mass spectrometry (GC–MS) after oximation and silylation is a key method in metabolomics. The GC–MS method was modified by a modified vial design and sample work-up procedure in order to make the method applicable to small volumes of cerebrospinal fluid (CSF), i.e. 10 μL, with similar coverage compared to the standard procedure using ≥100 μL of CSF. The data quality of the modified GC–MS method was assessed by analyzing a study sample set in an animal model for multiple sclerosis, including repetitively analysed quality control rat CSF samples. Automated normalization and intra- and inter-batch correction significantly improved the data quality with the majority of metabolites showing a relative standard deviation <20 %. The modified GC–MS method was successfully applied in rat model of multiple sclerosis where statistical analysis of 93 metabolites, of which 73 were (tentatively) identified, in 10 μL of rat CSF showed statistically significant differences in metabolite profiles of rats at the onset and peak of experimental autoimmune encephalomyelitis compared to rats in the control group. The modified GC–MS method presented proved to be a valid and valuable metabolomics method when only limited sample volumes are available.

Reduced expression of plasma membrane calcium ATPase 2 and collapsin response mediator protein 1 promotes death of spinal cord neurons

The mechanisms underlying neuronal pathology and death in the spinal cord (SC) during inflammation remain elusive. We previously showed the important role of plasma membrane calcium ATPases (PMCAs) in the survival of SC neurons, in vitro. We also postulated that a decrease in PMCA2 expression could cause neuronal death during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. The current studies were undertaken to define the specific contribution of PMCA2 to degeneration of SC neurons, the effectors downstream to PMCA2 mediating neuronal death and the triggers that reduce PMCA2 expression. We report that knockdown of PMCA2 in SC neurons decreases collapsin response mediator protein 1 (CRMP1) levels. This is followed by cell death. Silencing of CRMP1 expression also leads to neuronal loss. Kainic acid reduces both PMCA2 and CRMP1 levels and induces neuronal death. Administration of an alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA)/kainate receptor antagonist, at onset or peak of EAE, restores the decreased PMCA2 and CRMP1 levels to control values and ameliorates clinical deficits. Thus, our data link the reduction in PMCA2 expression with perturbations in the expression of CRMP1 and the ensuing death of SC neurons. This represents an additional mechanism underlying AMPA/kainate receptor-mediated excitotoxicity with relevance to neurodegeneration in EAE.

Induction of inhibitory central nervous system-derived and stimulatory blood-derived dendritic cells suggests a dual role for granulocyte-macrophage colony-stimulating factor in central nervous system inflammation

The mononuclear phagocyte system, particularly dendritic cells, plays several pivotal roles in the development of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. Here, we demonstrate that functionally distinct dendritic cell subpopulations are present in the central nervous system during experimental autoimmune encephalomyelitis. At peak experimental autoimmune encephalomyelitis, the majority of dendritic cells consisted of a CD11b(+)F4/80(+) inflammatory dendritic cell subtype. Both granulocyte-macrophage colony-stimulating factor and chemokine (C-C motif) ligand 2 were previously suggested to recruit 'inflammatory' monocyte-derived dendritic cells to the central nervous system during experimental autoimmune encephalomyelitis. We show that intra-cerebral production of granulocyte-macrophage colony-stimulating factor leading to chemokine (C-C motif) ligand 2 induction and attraction of chemokine (C-C motif) receptor 2-positive precursors suffices to recruit dendritic cell populations identical to those observed in experimental autoimmune encephalomyelitis into the central nervous system of healthy mice. This does not occur with fms-like tyrosine kinase-3-ligand treatment. Both during experimental autoimmune encephalomyelitis and upon intra-cerebral granulocyte-macrophage colony-stimulating factor production, all myeloid dendritic cells, lymphoid dendritic cells and periphery-derived inflammatory dendritic cells stimulated T cell proliferation, whereas inflammatory dendritic cells that differentiated from central nervous system precursors inhibited T cell activation and pro-inflammatory cytokine production. Despite the capacity of granulocyte-macrophage colony-stimulating factor to induce central nervous system-derived inhibitory inflammatory dendritic cells, the administration of granulocyte-macrophage colony-stimulating factor into mice with experimental autoimmune encephalomyelitis resulted in exacerbated disease. Granulocyte-macrophage colony-stimulating factor thus has a dual role in the central nervous system: it directs both central nervous system-derived dendritic cells towards an inhibitory phenotype and recruits peripheral dendritic cells exhibiting pro-inflammatory functions.

Impact of acute inflammation on spinal motoneuron synaptic plasticity following ventral root avulsion.

BackgroundVentral root avulsion is a proximal nerve root lesion in which ventral motor nerve rootlets are torn from surface of the spinal cord, resulting in extensive death of motoneurons. It has been previously shown that if such lesioning is performed in an animal with experimental autoimmune encephalomyelitis (EAE), a significant number of motoneurons can be rescued despite an intense inflammatory reaction. This rescue effect has been attributed to production of a number of neurotrophic factors by invading T cells. Synaptological changes may be involved in neuronal degeneration, and a better understanding of the role of these changes may be of importance for developing new strategies to promote neuronal survival. The objective of the present work was to evaluate neuronal survival, astroglial reaction and synaptic input changes in spinal cord anterior horn motor nuclei after ventral root avulsion in animals with EAE, both during peak disease and after remission.MethodsLewis rats were subjected to unilateral avulsion of lumbar ventral roots (VRA) and divided into three groups: VRA control, VRA at peak of EAE, and VRA during EAE remission. The animals were sacrificed and their lumbar spinal cords processed for immunohistochemistry, transmission electron microscopy, and motoneuron counting.ResultsThe results indicate a reduction in astroglial reaction, a maintenance of microglial reactivity, and increases in synaptic covering of, and survival of, motoneurons in the VRA+EAE group as compared to VRA alone.ConclusionThe present findings indicate that CNS inflammation may directly influence synaptic plasticity as well as the stability of neuronal networks, positively influencing the survival of lesioned neurons.

PARP-1 Deficiency Increases the Severity of Disease in a Mouse Model of Multiple Sclerosis

Poly(ADP-ribose) polymerase-1 (PARP-1) has been implicated in the pathogenesis of several central nervous system (CNS) disorders. However, the role of PARP-1 in autoimmune CNS injury remains poorly understood. Therefore, we studied experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis in mice with a targeted deletion of PARP-1. We identified inherent physiological abnormalities in the circulating and splenic immune composition between PARP-1(-/-) and wild type (WT) mice. Upon EAE induction, PARP-1(-/-) mice had an earlier onset and developed a more severe EAE compared with WT cohorts. Splenic response was significantly higher in PARP-1(-/-) mice largely because of B cell expansion. Although formation of Th1 and Th17 effector T lymphocytes was unaffected, PARP-1(-/-) mice had significantly earlier CD4+ T lymphocyte and macrophage infiltration into the CNS during EAE. However, we did not detect significant differences in cytokine profiles between PARP-1(-/-) and WT spinal cords at the peak of EAE. Expression analysis of different PARP isozymes in EAE spinal cords showed that PARP-1 was down-regulated in WT mice and that PARP-3 but not PARP-2 was dramatically up-regulated in both PARP-1(-/-) and WT mice, suggesting that these PARP isozymes could have distinct roles in different CNS pathologies. Together, our results indicate that PARP-1 plays an important role in regulating the physiological immune composition and in immune modulation during EAE; our finding identifies a new aspect of immune regulation by PARPs in autoimmune CNS pathology.

Matrix Metalloproteinase-12 Deficiency Worsens Relapsing-Remitting Experimental Autoimmune Encephalomyelitis in Association with Cytokine and Chemokine Dysregulation

The elevation of several members of the matrix metalloproteinase (MMP) family promotes the pathophysiology of both multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). Nonetheless, given the multiple activities of MMPs, it remains possible that increased levels of a particular MMP may have beneficial functions during disease progression. We reported previously that MMP-12(-/-) mice of the 129/SvEv strain had a poorer EAE outcome than wild-type controls. However, we did not determine further differences in disease profiles between these groups. Using the EAE model in 129/SvEv mice, we report that disease in both wild-type and MMP-12(-/-) mice follows a relapsing-remitting course. Although both mouse groups had similar clinical onsets, subsequent relapses were more severe in MMP-12(-/-) mice; their residual disability at remission was also higher compared with wild-type controls. The worsened relapses and remissions in MMP-12(-/-) mice occurred despite a deficiency of the antigen recall capacity of lymph node-derived cells as well as a reduction in the proportion of macrophages in the spinal cord during the chronic phase of EAE. Significantly, large increases of levels of chemokines and cytokines were found in the spinal cords of MMP-12(-/-) mice during chronic EAE. These results highlight MMP-12 as a beneficial enzyme in EAE and suggest that therapeutic interventions in multiple sclerosis should avoid targeting MMP-12.

Decreased severity of experimental autoimmune encephalomyelitis during resveratrol administration is associated with increased IL-17 +IL-10 + T cells, CD4 − IFN-γ + cells, and decreased macrophage IL-6 expression

Experimental autoimmune encephalomyelitis (EAE), an animal model of Multiple Sclerosis, is induced after injection of PLP 139–151 myelin peptide in complete Freund's adjuvant into SJL/J mice. During EAE, T cells and macrophages infiltrate the brain, produce cytokines IL-17, IFN-γ, TNF-α, or IL-6, and bring about autoimmune neuroinflammation. However, infiltrating T cells which simultaneously produce IL-17 and IL-10 or infiltrating CD4 − NKT cells that produce IFN-γ protect against EAE. Resveratrol, a plant polyphenol, exhibits anti-inflammatory properties. To determine if resveratrol can relieve EAE, SJL/J mice were administered diets enriched in resveratrol at EAE injection. EAE symptoms were significantly less compared with controls in mice fed resveratrol. At day 56 of EAE, splenic T cells from mice fed 0%, 0.04% or 0.08% resveratrol that were restimulated with PLP 139–151 produced similar levels while splenic T cells from mice fed 0.02% resveratrol produced significantly higher levels of IL-17, IFN-γ, and TNF-α. At peak EAE (day 14), mice fed resveratrol had higher numbers of IL-17 + T cells, IL-17 +/IL-10 + T cells, and CD4 −IFN-γ + cells in the brain and spleen compared with controls. Adoptive transfer of day 14 EAE encephalogenic T cells into mice fed resveratrol reduced the severity of EAE. In addition, resveratrol directly suppressed expression of IL-6 and IL-12/23 p40 but increased expression of IL-12 p35 and IL-23 p19 from macrophages. Therefore resveratrol protection against EAE is not associated with declines in IL-17 + T cells but is associated with rises in IL-17 +/IL-10 + T cells and CD4 -IFN-γ + and with repressed macrophage IL-6 and IL-12/23 p40 expression.

Novel role for SLPI in MOG-induced EAE revealed by spinal cord expression analysis

BackgroundExperimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte protein (MOG) in female Dark Agouti (DA) rats is a chronic demyelinating animal model of multiple sclerosis (MS). To identify new candidate molecules involved in the evolution or repair of EAE-lesions we used Affymetrix oligonucleotide microarrays to compare the spinal cord transcriptome at the peak of EAE, during remission and at the first relapse with healthy DA rats.MethodsUntreated DA rats and DA rats immunised with MOG protein were sacrificed at defined time points. Total RNA was isolated from spinal cord tissue and used for hybridization of Affymetrix rat genome arrays RG U34 A-C. Selected expression values were confirmed by RealTime PCR.Adult neural stem cells were incubated with recombinant secretory leukocyte protease inhibitor (SLPI). Proliferation was assessed by BrdU incorporation, cyclin D1 and HES1 expression by RealTime PCR, cell differentiation by immunofluorescence analysis and IkappaBalpha degradation by Western blot.ResultsAmong approximately 26,000 transcripts studied more than 1,100 were differentially regulated. Focussing on functional themes, we noticed a sustained downregulation of most of the transcripts of the cholesterol biosynthesis pathway. Furthermore, we found new candidate genes possibly contributing to regenerative processes in the spinal cord. Twelve transcripts were solely upregulated in the recovery phase, including genes not previously associated with repair processes. Expression of SLPI was upregulated more than hundredfold during EAE attack. Using immunohistochemistry, SLPI was identified in macrophages, activated microglia, neuronal cells and astrocytes. Incubation of adult neural stem cells (NSC) with recombinant SLPI resulted in an increase of cell proliferation and of differentiation towards oligodendrocytes. These processes were paralleled by an upregulation of the cell-cycle promotor cyclin D1 and a suppression of the cell differentiation regulator HES1. Finally, SLPI prevented the degradation of IkappaBalpha, which may explain the suppression of the cell differentiation inhibitor HES1 suggesting a possible mechanism of oligodendroglial differentiation.ConclusionWe identified novel features of gene expression in the CNS during EAE, in particular the suppression of genes of cholesterol biosynthesis and a strong upregulation of SLPI, a gene which is for the first time associated with autoimmune inflammation. The capacity of SLPI to increase proliferation of adult NSC and of oligodendroglial differentiation suggests a novel role for SLPI in the promotion of tissue repair, beyond its known functions in the prevention of tissue damages by protease inhibition damage and modulation of inflammatory reactions.

CD11b+Ly-6Chi Suppressive Monocytes in Experimental Autoimmune Encephalomyelitis

Innate immune cells may regulate adaptive immunity by balancing different lineages of T cells and providing negative costimulation. In addition, CD11b(+)Gr-1(+) myeloid-derived suppressor cells have been described in tumor, parasite infection, and severe trauma models. In this study, we observe that splenic CD11b(+) cells markedly increase after experimental autoimmune encephalomyelitis (EAE) immunization, and they suppress T cell proliferation in vitro. Although >80% of CD11b(+) cells express varying levels of Gr-1, only a small population of CD11b(+)Ly-6C(high) inflammatory monocytes (IMC) can efficiently suppress T cell proliferation and induce T cell apoptosis through the production of NO. IFN-gamma produced by activated T cells is essential to induce IMC suppressive function. EAE immunization increases the frequencies of IMC in the bone marrow, spleen, and blood, but not in the lymph nodes. At the peak of EAE, IMC represent approximately 30% of inflammatory cells in the CNS. IMC express F4/80 and CD93 but not CD31, suggesting that they are immature monocytes. Furthermore, IMC have the plasticity to up-regulate NO synthase 2 or arginase 1 expression upon different cytokine treatments. These findings indicate that CD11b(+)Ly-6C(high) IMC induced during EAE priming are powerful suppressors of activated T cells. Further understanding of suppressive monocytes in autoimmune disease models may have important clinical implications for human autoimmune diseases.

Glia Maturation Factor Regulation of STAT Expression: A Novel Mechanism in Experimental Autoimmune Encephalomyelitis

Inflammatory cytokines are implemented in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. We previously demonstrated that glia maturation factor (GMF), a brain protein, isolated, sequenced and cloned in our laboratory, induce expression of proinflammatory cytokine/chemokine in the central nervous system (CNS). We found GMF-deficient (knockout) mice relatively resistant to EAE development after immunization with encephalitogenic MOG peptide 35-55. Consistent with these findings, the expression of proinflammatory cytokines in CNS of mice with EAE differed profoundly between wild type and GMF-knockout mice. In the present study we examined the expressions of six murine signal transducers and activators of transcription (STATs) genes, which are known to regulate the cytokine-dependent signal transduction pathways in autoimmune inflammation. The expressions of STATs genes were evaluated in the brains and spinal cords of wild type and GMF-knockout mice at the peak of EAE by quantitative real-time RT-PCR. Compared to GMF-knockout mice, the expressions of STAT1, STAT2, STAT3, STAT4, STAT5, and STAT6 genes were significantly (P < 0.001) upregulated in the wild type mice exhibiting EAE symptoms. The results are consistent with the diminished development of EAE in the GMF-knockout mice. A significant suppression of STATs expression in GMF-knockout mice suggests GMF as an upstream effector of JAK/STAT signaling.

TREM2-Transduced Myeloid Precursors Mediate Nervous Tissue Debris Clearance and Facilitate Recovery in an Animal Model of Multiple Sclerosis

BackgroundIn multiple sclerosis, inflammation can successfully be prevented, while promoting repair is still a major challenge. Microglial cells, the resident phagocytes of the central nervous system (CNS), are hematopoietic-derived myeloid cells and express the triggering receptor expressed on myeloid cells 2 (TREM2), an innate immune receptor. Myeloid cells are an accessible source for ex vivo gene therapy. We investigated whether myeloid precursor cells genetically modified to express TREM2 affect the disease course of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis.Methods and FindingsEAE was induced in mice by immunization with a myelin autoantigen. Intravenous application of TREM2-transduced bone marrow–derived myeloid precursor cells at the EAE peak led to an amelioration of clinical symptoms, reduction in axonal damage, and prevention of further demyelination. TREM2-transduced myeloid cells applied intravenously migrated into the inflammatory spinal cord lesions of EAE-diseased mice, showed increased lysosomal and phagocytic activity, cleared degenerated myelin, and created an anti-inflammatory cytokine milieu within the CNS.ConclusionsIntravenously applied bone marrow–derived and TREM2-tranduced myeloid precursor cells limit tissue destruction and facilitate repair within the murine CNS by clearance of cellular debris during EAE. TREM2 is a new attractive target for promotion of repair and resolution of inflammation in multiple sclerosis and other neuroinflammatory diseases.

Diminished cytokine and chemokine expression in the central nervous system of GMF-deficient mice with experimental autoimmune encephalomyelitis

Pro-inflammatory cytokines/chemokines are implemented in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model with clinical and pathological similarities to multiple sclerosis. We have previously shown that over-expression of glia maturation factor (GMF) in glial cells cause excessive production and secretion of pro-inflammatory cytokines/chemokines sufficient to destroy the myelin-forming oligodendroglial cell in vitro. In this present investigation, we evaluate the expression of pro-inflammatory mediators in the central nervous system (CNS) of GMF+/+ (wild type) mice and GMF−/− (GMF-knockout) mice at the peak of EAE induced by immunization with MOG 35–55 peptide. GMF+/+ (Wt) mice developed severe EAE with a maximal mean clinical score of 3.6 ± 0.5 by day 16 post-immunization, whereas GMF-KO mice showed significantly delayed EAE with an average onset on day 26 pi with reduced mean clinical score of 1.3 ± 0.3. Three of fifteen Wt mice as compared to none of GMF-KO mice died of EAE. Encephalitogenic cells from Wt mice transferred to recipient GMF-KO mice caused very mild and with low incidence of EAE. We determined the differences in the expression of cytokines, IFN-γ, TNF-α, IL-1 β, IL-6, IL-4, IL-10, and chemokines, MIP-1, MIP-2, IP-10, MCP-1, GM-CSF mRNA by quantitative real-time RT–PCR in brain and spinal cord. Our results demonstrate significantly low levels of pro-inflammatory cytokines/chemokines in the CNS of GMF-KO mice and increased expression in Wt mice with EAE. Our data suggest that GMF play a critical role in CNS inflammation.

Antigen‐Specific Therapy Promotes Repair of Myelin and Axonal Damage in Established EAE

Inflammation results in CNS damage in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), an animal model of MS. It is uncertain how much repair of injured myelin and axons can occur following highly selective anti-inflammatory therapy in EAE and MS. In this study, SJL/J mice with established EAE were treated successfully with an antigen-specific recombinant T cell receptor ligand (RTL), RTL401, a mouse I-A(s)/PLP-139-151 construct, after the peak of EAE. To define the mechanisms by which late application of RTL401 inhibits EAE, we evaluated mice at different time points to assess the levels of neuroinflammation and myelin and axon damage in their spinal cords. Our results showed that RTL401 administered after the peak of acute EAE induced a marked reduction in inflammation in the CNS, associated with a significant reduction of demyelination, axonal loss and ongoing damage. Electron microscopy showed that RTL-treated mice had reduced pathology compared with mice treated with vehicle and mice at the peak of disease, as demonstrated by a decrease in continued degeneration, increase in remyelinating axons and the presence of an increased number of small, presumably regenerative axonal sprouts. These findings indicate that RTL therapy targeting encephalitogenic T cells may promote CNS neuroregenerative processes.