Progression Of Experimental Autoimmune Encephalomyelitis Research Articles

The combined treatment of NAD+ and atorvastatin ameliorates the development of experimental autoimmune encephalomyelitis in C57BL/6 mice

Multiple sclerosis (MS) is a demyelinating and degenerating disorder of the central nervous system impacting many patients worldwide. Due to the complex pathogenesis of MS, drugs to treat MS often show partial effectiveness. Earlier experiments have demonstrated that both atorvastatin and nicotinamide adenine dinucleotide (NAD+) may ameliorate experimental autoimmune encephalomyelitis (EAE), which is known as a classical model of MS, via different protective mechanisms. Since combination therapy can exhibit more beneficial effects than monotherapy, we observed the protective effects of combined treatment of atorvastatin and NAD+ in EAE mice. Our results showed that the combined treatment of these two drugs at half of their optimal dosages had synergistic effect to alleviate the severity of EAE in mice than treatment with each alone. The combined treatment of EAE mice with atorvastatin plus NAD+ relieved the clinical signs and histologic changes, increased the proportion of Treg cells, attenuated IL-17 production, reduced proinflammatory cytokine secretion of Th17 cells, and increased cytokine secretion of Treg cells. In addition, the combined treatment also diminished phosphorylation of both P38 MAPK and ERK, while elevated SIRT1 expression. Taken together, these data suggested that combined treatment with NAD+ and atorvastatin could attenuate the progression of EAE by synergistic immune regulation.

Blockade of Kv1.3 potassium channel inhibits CD8+ T cell-mediated neuroinflammation via PD-1/Blimp-1 signaling.

Kv1.3 potassium channel is considered as a target for the treatment of autoimmune diseases such as multiple sclerosis (MS), since Kv1.3 blockade suppresses memory T cell activation including cytotoxic CD8+ T cells. However, the underlying signaling pathway related to autoimmune CD8+ T cell inhibition by Kv1.3 channel in neuroinflammatory diseases remains unclear. We found that ImK, a selective Kv1.3 blocker, reduced auto-reactive CD8+ T cell infiltration in the spinal cords of experimental autoimmune encephalomyelitis (EAE) rats, an animal model of MS. ImK suppressed transcriptional factor Blimp-1 expression and reduced the cytotoxicity of CD8+ T cells on neuronal cells. Furthermore, ImK upregulated co-inhibitory molecule PD-1 to inhibit B lymphocyte-induced maturation protein (Blimp-1) in an IL-2 independent way. In addition, PD-1 inhibitor impaired the suppression of ImK on CD8+ T cells and accelerated EAE progression. Our study demonstrated a novel regulatory mechanism of Kv1.3 blockade on modulating CD8+ T cell differentiation through PD-1/Blimp-1 signaling. This work expands the understanding of Kv1.3 channel for modulating neuroinflammation.

Vitamin B6 prevents excessive inflammation by reducing accumulation of sphingosine-1-phosphate in a sphingosine-1-phosphate lyase-dependent manner.

Vitamin B6 is necessary to maintain normal metabolism and immune response, especially the anti‐inflammatory immune response. However, the exact mechanism by which vitamin B6 plays the anti‐inflammatory role is still unclear. Here, we report a novel mechanism of preventing excessive inflammation by vitamin B6 via reduction in the accumulation of sphingosine‐1‐phosphate (S1P) in a S1P lyase (SPL)‐dependent manner in macrophages. Vitamin B6 supplementation decreased the expression of pro‐inflammatory cytokines by suppressing nuclear factor‐κB and mitogen‐activated protein kinases signalling pathways. Furthermore, vitamin B6–reduced accumulation of S1P by promoting SPL activity. The anti‐inflammatory effects of vitamin B6 were inhibited by S1P supplementation or SPL deficiency. Importantly, vitamin B6 supplementation protected mice from lethal endotoxic shock and attenuated experimental autoimmune encephalomyelitis progression. Collectively, these findings revealed a novel anti‐inflammatory mechanism of vitamin B6 and provided guidance on its clinical use.

Experimental autoimmune encephalomyelitis is associated with changes of the microbiota composition in the gastrointestinal tract

The gut microbiome is known to be sensitive to changes in the immune system, especially during autoimmune diseases such as Multiple Sclerosis (MS). Our study examines the changes to the gut microbiome that occur during experimental autoimmune encephalomyelitis (EAE), an animal model for MS. We collected fecal samples at key stages of EAE progression and quantified microbial abundances with 16S V3–V4 amplicon sequencing. Our analysis of the data suggests that the abundance of commensal Lactobacillaceae decreases during EAE while other commensal populations belonging to the Clostridiaceae, Ruminococcaceae, and Peptostreptococcaceae families expand. Community analysis with microbial co-occurrence networks points to these three expanding taxa as potential mediators of gut microbiome dysbiosis. We also employed PICRUSt2 to impute MetaCyc Enzyme Consortium (EC) pathway abundances from the original microbial abundance data. From this analysis, we found that a number of imputed EC pathways responsible for the production of immunomodulatory compounds appear to be enriched in mice undergoing EAE. Our analysis and interpretation of results provides a detailed picture of the changes to the gut microbiome that are occurring throughout the course of EAE disease progression and helps to evaluate EAE as a viable model for gut dysbiosis in MS patients.

Icariin ameliorates the progression of experimental autoimmune encephalomyelitis by down-regulating the major inflammatory signal pathways in a mouse relapse-remission model of multiple sclerosis

This study aimed at investigating whether treatment with icariin (ICA) could modulate the progression of experimental autoimmune encephalomyelitis (EAE) and its potential mechanisms in SJL/J mice. Female SJL/J mice were immunized with PLP139-151 peptide to induce relapse-remitting EAE and the immunized mice were treated with vehicle alone (EAE) or ICA (12.5 or 25 mg/body weights) by gavage daily for 42 days. Compared with the control, the EAE mice developed relapse-remitting EAE and reduced body weights (15.76 ± 0.61 vs. 17.60 ± 0.98 g on day 13; 17.35 ± 0.44 vs. 18.46 ± 0.66 g on day 26), accompanied by severe inflammation with many microglia infiltrates and obvious demyelination in the spinal cord tissues. Conversely, ICA treatment significantly reduced the clinical scores (on day 13, 1.00 ± 0.16 and 1.10 ± 0.33 for ICA 12.5 and 25 mg/kg group, respectively vs. 1.62 ± 0.41 in the EAE group; on day 26, 0.50 ± 0.23 and 0.40 ± 0.24 for ICA 12.5 and 25 mg/kg group, respectively, vs. 1.56 ± 0.29 in the EAE group), mitigated the body weight reduction, spinal cord inflammation and demyelination in EAE mice (pathological scores of 2.33 ± 0.82 and 1.11 ± 0.57 for ICA 12.5 and 25 mg/kg, respectively; vs. 4.78 ± 1.13, P < 0.0001). Furthermore, ICA treatment significantly mitigated the EAE-increased iNOS, TNF-α, CD206 and TGF-β1, but further reduced IL-10 mRNA transcripts in the brain mice. More importantly, ICA treatment significantly mitigated the inflammation-related NF-κB, AKT, ERK1/2, p38, c-Jun and MEK phosphorylation in the brain of EAE mice. ICA treatment ameliorates the progression of EAE by down-regulating the major inflammation-related signal pathways in mice.

Gene Expression Analysis of Astrocyte and Microglia Endocannabinoid Signaling during Autoimmune Demyelination.

The endocannabinoid system is associated with protective effects in multiple sclerosis (MS) that involve attenuated innate immune cell responses. Astrocytes and microglia are modulated by endocannabinoids and participate in the biosynthesis and metabolism of these compounds. However, the role of neuroglial cells as targets and mediators of endocannabinoid signaling in MS is poorly understood. Here we used a microfluidic RT-qPCR screen to assess changes in the expression of the main endocannabinoid signaling genes in astrocytes and microglia purified from female mice during the time-course of experimental autoimmune encephalomyelitis (EAE). We show that astrocytes and microglia upregulate the expression of genes encoding neurotoxic A1 and pro-inflammatory molecules at the acute disease with many of these transcripts remaining elevated during the recovery phase. Both cell populations exhibited an early onset decrease in the gene expression levels of 2-arachidonoylglycerol (2-AG) hydrolytic enzymes that persisted during EAE progression as well as cell-type-specific changes in the transcript levels for genes encoding cannabinoid receptors and molecules involved in anandamide (AEA) signaling. Our results demonstrate that astrocytes and microglia responses to autoimmune demyelination involve alterations in the expression of multiple endocannabinoid signaling-associated genes and suggest that this system may regulate the induction of neurotoxic and pro-inflammatory transcriptional programs in both cell types during MS.

Co-treatments to Boost IDO Activity and Inhibit Production of Downstream Catabolites Induce Durable Suppression of Experimental Autoimmune Encephalomyelitis.

Reinforcing defective tolerogenic processes slows progression of autoimmune (AI) diseases and has potential to promote drug-free disease remission. Previously, we reported that DNA nanoparticles (DNPs) and cyclic dinucleotides (CDNs) slow progression of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, by activating the Stimulator of Interferon Genes (STING) signaling adaptor to stimulate interferon type 1 (IFN-I) production, which induced dendritic cells to express indoleamine 2,3 dioxygenase (IDO) and acquire immune regulatory phenotypes. Here, we show that therapeutic responses to DNPs depend on DNA sensing via cyclic GAMP synthase (cGAS) and interactions between Programmed Death-1 (PD-1) and PD-1 ligands. To investigate how increased tryptophan (Trp) metabolism by IDO promotes therapeutic responses mice were co-treated at EAE onset with DNPs and drugs that inhibit kynurenine aminotransferase-II (KatII) or 3-hydroxyanthranilic acid dioxygenase (HAAO) activity downstream of IDO in the kynurenine (Kyn) pathway. DNP and KatII or HAAO inhibitor co-treatments suppressed EAE progression more effectively than DNPs, while KatII inhibition had no significant therapeutic benefit and HAAO inhibition attenuated but did not prevent EAE progression. Moreover, therapeutic responses to co-treatments were durable as EAE progression did not resume after co-treatment. Thus, using STING agonists to boost IDO activity and manipulating the Kyn pathway downstream of IDO is an effective strategy to enhance tolerogenic responses that overcome autoimmunity to suppress EAE progression.

Arsenic trioxide ameliorates experimental autoimmune encephalomyelitis in C57BL/6 mice by inducing CD4+ T cell apoptosis

BackgroundMultiple sclerosis (MS) is an immune-mediated disease of the central nervous system characterized by severe white matter demyelination. Because of its complex pathogenesis, there is no definite cure for MS. Experimental autoimmune encephalomyelitis (EAE) is an ideal animal model for the study of MS. Arsenic trioxide (ATO) is an ancient Chinese medicine used for its therapeutic properties with several autoimmune diseases. It is also used to inhibit acute immune rejection due to its anti-inflammatory and immunosuppressive properties. However, it is unclear whether ATO has a therapeutic effect on EAE, and the underlying mechanisms have not yet been clearly elucidated. In this study, we attempted to assess whether ATO could be used to ameliorate EAE in mice.MethodsATO (0.5 mg/kg/day) was administered intraperitoneally to EAE mice 10 days post-immunization for 8 days. On day 22 post-immunization, the spinal cord, spleen, and blood were collected to analyze demyelination, inflammation, microglia activation, and the proportion of CD4+ T cells. In vitro, for mechanistic studies, CD4+ T cells were sorted from the spleen of naïve C57BL/6 mice and treated with ATO and then used for an apoptosis assay, JC-1 staining, imaging under a transmission electron microscope, and western blotting.ResultsATO delayed the onset of EAE and alleviated the severity of EAE in mice. Treatment with ATO also attenuated demyelination, alleviated inflammation, reduced microglia activation, and decreased the expression levels of IL-2, IFN-γ, IL-1β, IL-6, and TNF-α in EAE mice. Moreover, the number and proportion of CD4+ T cells in the spinal cord, spleen, and peripheral blood were reduced in ATO-treated EAE mice. Finally, ATO induced CD4+ T cell apoptosis via the mitochondrial pathway both in vitro and in vivo. Additionally, the administration of ATO had no adverse effect on the heart, liver, or kidney function, nor did it induce apoptosis in the spinal cord.ConclusionsOverall, our findings indicated that ATO plays a protective role in the initiation and progression of EAE and has the potential to be a novel drug in the treatment of MS.

IC100: a novel anti-ASC monoclonal antibody improves functional outcomes in an animal model of multiple sclerosis

BackgroundThe inflammasome adaptor apoptosis-associated speck-like protein containing a CARD (ASC) is involved in immune signaling by bridging the interactions between inflammasome sensors and caspase-1. Strong experimental evidence has shown that ASC−/− mice are protected from disease progression in animal models of multiple sclerosis (MS), suggesting that targeting inflammasome activation via ASC inhibition may be a promising therapeutic strategy in MS. Thus, the goal of our study is to test the efficacy of IC100, a novel humanized antibody targeting ASC, in preventing and/or suppressing disease in the experimental autoimmune encephalomyelitis (EAE) model of MS.MethodsWe employed the EAE model of MS where disease was induced by immunization of C57BL/6 mice with myelin oligodendrocyte glycoprotein peptide 35–55 (MOG35–55). Mice were treated with vehicle or increasing doses of IC100 (10, 30, and 45 mg/kg) and clinical disease course was evaluated up to 35 days post EAE induction. Immune cell infiltration into the spinal cord and microglia responses were assessed.ResultsWe show that IC100 treatment reduced the severity of EAE when compared to vehicle-treated controls. At a dose of 30 mg/kg, IC100 significantly reduced the number of CD4+ and CD8+ T cells and CD11b+MHCII+ activated myeloid cells entering the spinal cord from the periphery, and reduced the number of total and activated microglia.ConclusionsThese data indicate that IC100 suppresses the immune-inflammatory response that drives EAE development and progression, thereby identifying ASC as a promising target for the treatment of MS as well as other neurological diseases with a neuroinflammatory component.

Functions and mechanisms of chondroitin disaccharide-induced miRNAs in the regulation of CD44 expression involved in the development of experimental autoimmune encephalomyelitis

Abstract Multiple sclerosis (MS) is a chronic demyelinating neurodegenerative disease of the central nervous system (CNS). In this study, we investigated the effects of chondroitin disaccharide and its induced miR-29b and miR-199b on CD44 expression in mouse lymphocytes, as well as the effects of miR-29b and miR-199b on the development and progression of experimental autoimmune encephalomyelitis (EAE) in mice, an animal model of MS. The effects of chondroitin sulfate disaccharide on the changes in cellular immunity were investigated in EAE induced in mice by MOG and PTX. Real-time PCR and flow cytometry were used to investigate the effects of the anti-miRs and mimics of miR-29b and miR-199b on CD44 expression in mouse splenocytes after electroporation. After the transduction of antai-miRs and mimics of miR-29b and miR-199b into splenocytes, they were adoptively transferred into mice intravenously and the changes in immune cells and cytokines were analyzed by flow cytometry and ELISA. Our studies revealed that chondroitin disaccharide significantly up-regulated several specific miRNAs such as miR-29b and miR-199b, and thus dramatically inhibited EAE in mice. In vitro and in vivo studies demonstrated that miR-29b and miR-199b could down-regulate the expression of CD44 and modulate the expression of IL-17 and IL-4 in the lymphocytes from EAE mice. Therefore, miR-29b and miR-199b could inhibit the expression of CD44, modulate the expression of IL-4 and IL-17, and thus suppress lymphocyte proliferation and migration, leading to significant inhibition of EAE development. Thus, miR-29b and miR-199b could also be used as therapeutic targets for EAE and MS.

Intravenous tolerance induces T cell anergy/apoptosis via IFN-γ/IL-27/PD-L1 axis

Abstract The induction of peripheral tolerance by intravenous injection (i.v.) of auto-Ags has shown to suppress experimental autoimmune encephalomyelitis (EAE), a mouse model of Multiple Sclerosis, by eliciting tolerogenic APCs, Tregs, and suppressing self-reactive T cells. The programmed cell death protein (PD)-1 and its ligands, PD-L1 and PD-L2, regulate the balance between T cell activation and immune tolerance. Although PD-L1 and PD-L2 are expressed on a variety of cell types, however, the mechanisms related to peripheral tolerance that induce these molecules are still unclear. Here we show that PD-L1, but not PD-L2, is critical for inducing peripheral tolerance in EAE, and that PD-L1-dependent tolerance relies on both IFN-γ and IL-27. I.v. administration of auto-Ag(s) attenuates the progression of EAE, by inducing PD-L1 expression in monocyte-derived DCs (moDCs) in the CNS, via IFN-γ/ IL-27-dependent mechanism. Indeed, when PD-L1 is blocked, autoantigen(s)/i.v treatment becomes ineffective. Following IFN-γ−/− CD4+ T cell transfer into Rag1−/− mice, i.v. tolerance treatment failed to suppress EAE. Moreover, loss of IFN-γR signaling in classical DCs type I (cDCs I) compromised IL-27 production and consequent PD-L1 expression by moDCs. We also show that IL-27 stimulates moDCs to over-express PD-L1, in a STAT-3-dependent way. Indeed, in CCR2−/−/WSX−/− (CD45.2) → CD45.1 radiation-induced bone marrow chimera mice, auto-Ag(s)/i.v. treatment failed to suppress EAE. Similarly, EAE mice that expressed mutant STAT-3 (muSTAT-3) in CCR2+ cells were also insensitive to i.v. tolerance induction. Our data reveal a mechanism whereby IFN-γ and IL-27 are the key factors in PD-L1 induction and subsequent peripheral tolerance restoration.

Farnesol reduces T cell infiltration in murine CNS inflammatory demyelination

Abstract Farnesol (FOL) is a naturally-produced 15-carbon organic acyclic sesquiterpene alcohol (isoprenol) that acts as a potent blocker of neuronal voltage-gated Ca2+ channels (L- and N-type) and is found in the human brain. FOL has potent anti-oxidant and anti-inflammatory effects in vitro and is neuroprotective in a murine model of neurotoxicity. Because inflammation and neurodegeneration are mechanisms associated with CNS demyelinating diseases, we sought to determine whether FOL treatment would result in protection against experimental autoimmune encephalomyelitis (EAE). We compared the progression of EAE in MOG35–55 immunized female C57BL/6 mice treated orally with FOL (100 mg/kg/daily) emulsified in corn oil, versus vehicle-treated and untreated EAE mice. FOL significantly reduced the average clinical scores of EAE mice when compared to untreated mice and vehicle-treated mice. The protective effect was associated with a significant reduction of CD4+ T cell spinal cord infiltration in FOL-treated mice as assessed by flow cytometry. Although FOL’s mechanism of action remains to be known, we propose that FOL promotes protection against CNS inflammatory demyelination by promoting an anti-inflammatory effect. We compared pro-inflammatory and anti-inflammatory cytokine transcriptional levels in brain tissues of EAE mice and histological analysis of CD4+ T cell CNS infiltration and demyelination. The potential benefit of FOL nanoencapsulation in neuroprotection was explored. We demonstrate that there is a correlation between the reduced neuroinflammation and EAE severity observed in the context of FOL protection. Understanding the neuroprotective effects of FOL may provide insight to novel therapeutic approaches for MS.

Novel B cell-dependent multiple sclerosis model using extracellular domains of myelin proteolipid protein

Abstract Therapeutic success of B cell-targeting approaches in multiple sclerosis (MS) has intensified research into the role B cells play in pathogenesis and regulation of disease. Historical dissection of B cell function in the MS mouse model experimental autoimmune encephalomyelitis (EAE) has largely been confined to disease induced with either the immunodominant epitope of myelin oligodendrocyte glycoprotein (MOG35–55) or full length recombinant human MOG protein (hrMOG), the latter representing the only available B cell-dependent EAE model. We have demonstrated that myelin proteolipid protein (PLP178–191)-specific CD8 T cells possess striking EAE-suppression capacity. However, little is known regarding the role of B cells in these contexts. Interestingly, unlike MOG35–55, where genetic absence or depletion of B cells yields a more severe disease course, PLP178–191 yields identical EAE progression in both WT and μMT mice, suggesting suboptimal B cell engagement. Given the reports that hrMOG drives a B cell-dependent demyelinating disease versus its MOG35–55 counterpart, we hypothesized that a longer PLP antigen consisting of more epitopes may better engage B cells and provide an additional model for the field. To this end, we designed a novel peptide encompassing the extracellular domains (ECD) of PLP and compared its immunogenicity and disease-inducing capacity with PLP178–191 in μMT and WT mice. We demonstrate here that PLPECD-immunized B cell-deficient mice failed to exhibit EAE disease compared to WT counterparts. PLPECD also induced EAE in B cell-sufficient mice incapable of secreting antibodies, suggesting a predominant antigen presentation role. These results establish a novel, efficient B cell-dependent EAE model.

Activated Protein C Attenuates Experimental Autoimmune Encephalomyelitis Progression by Enhancing Vascular Integrity and Suppressing Microglial Activation.

BackgroundActivated protein C (APC), a serine protease with antithrombotic effects, protects in animal models of ischemic stroke by suppressing inflammation and enhancing vascular integrity, angiogenesis, neurogenesis and neuroprotection. A small number of animal studies suggest it might also have therapeutic potential in multiple sclerosis (MS), though results have been mixed. Based on these conflicting data, the goals of this study were to clarify the therapeutic potential of APC in the experimental autoimmune encephalomyelitis (EAE) model of MS and to determine mechanistically how APC mediates this protective effect.MethodsThe protective potential of APC was examined in a chronic progressive model of EAE. Vascular breakdown, tight junction protein expression and vascular expression of fibronectin and α5β1 integrin as well as vascularity and glial activation were analyzed using immunofluorescence (IF) of spinal cord sections taken from mice with established EAE. The direct influence of APC on microglial activation was evaluated in vitro by a combination of morphology and MMP-9 expression.ResultsAPC attenuated the progression of EAE, and this was strongly associated at the histopathological level with reduced levels of leukocyte infiltration and concomitant demyelination. Further analysis revealed that APC reduced vascular breakdown which was associated with maintained endothelial expression of the tight junction protein zonula occludens-1 (ZO-1). In addition, APC suppressed microglial activation in this EAE model and in vitro studies revealed that APC strongly inhibited microglial activation at both the morphological level and by the expression of the pro-inflammatory protease MMP-9.ConclusionThese findings build on the work of others in demonstrating strong therapeutic potential for APC in the treatment of inflammatory demyelinating disease and suggest that enhancement of vascular integrity and suppression of microglial activation may be important mediators of this protection.

Targeted immunomodulation of inflammatory monocytes across the blood-brain barrier by curcumin-loaded nanoparticles delays the progression of experimental autoimmune encephalomyelitis

It is difficult to carry out early diagnosis and treatment of Multiple sclerosis (MS) because of the complex pathogenesis elicited by diversified autoantigens. Monocytes play important roles in the process of MS, especially as most of the amplified inflammatory monocytes cross the BBB to promote neuron injury and recruit more immune cells to infiltrate the central nervous system (CNS). Here, we propose monocytes as an effective immunotherapy target for MS. We used High-density lipoprotein-mimicking peptide-phospholipid scaffold (HPPS) as a carrier to improve the bioavailability of curcumin. Curcumin-loaded HPPS (Cur-HPPS) were taken up specifically and efficiently by monocytes through the scavenger receptor class B type I (SR-B1) receptor. This delivery hindered inflammatory monocytes across the BBB in EAE mice, inhibited the proliferation of microglia, and restricted the infiltration of other effector immune cells, resulting in the reduction of EAE morbidity from 100% to 30%. It attributed to the immunomodulatory effect of Cur-HPPS on inflammatory monocytes, which inhibited NF-κB activation and downregulated the expression of adhesion-and migration-related molecules. Meanwhile, infiltrated monocytes in the CNS of EAE mice characterize early inflammation. Therefore, targeted modulation of monocytes with HPPS carrying therapeutic and/or imaging agents offers a novel strategy for MS diagnosis and treatment.

TNFR2 limits proinflammatory astrocyte functions during EAE induced by pathogenic DR2b-restricted T cells.

Multiple sclerosis (MS) is an autoimmune neuroinflammatory disease where the underlying mechanisms driving disease progression have remained unresolved. HLA-DR2b (DRB1*15:01) is the most common genetic risk factor for MS. Additionally, TNF and its receptors TNFR1 and TNFR2 play key roles in MS and its preclinical animal model, experimental autoimmune encephalomyelitis (EAE). TNFR2 is believed to ameliorate CNS pathology by promoting remyelination and Treg function. Here, we show that transgenic mice expressing the human MHC class II (MHC-II) allele HLA-DR2b and lacking mouse MHC-II and TNFR2 molecules, herein called DR2bΔR2, developed progressive EAE, while disease was not progressive in DR2b littermates. Mechanistically, expression of the HLA-DR2b favored Th17 cell development, whereas T cell-independent TNFR2 expression was critical for restraining of an astrogliosis-induced proinflammatory milieu and Th17 cell responses, while promoting remyelination. Our data suggest the TNFR2 signaling pathway as a potentially novel mechanism for curtailing astrogliosis and promoting remyelination, thus providing new insights into mechanisms limiting progressive MS.

Severity of the autoimmune encephalomyelitis symptoms in mouse model by inhibition of LAT-1 transporters

Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease of the dynamic immune system acting against the myelin sheath of neuronal axons in the abluminal (brain-facing) side of the central nervous system. This immune system is greatly influenced by the free amino acid environment from the luminal (blood-facing) side. Whether the luminal and abluminal free amino acid balance influences EAE disease progression is still unclear. Changes in free amino acid levels on both sides of the blood–brain barrier were observed with or without blocking of the l-amino acid transporter (LAT-1) during EAE disease progression. Brain tissue, plasma, splenocytes samples were used to measure free amino acid by LC–MS/MS. Samples were also used to measure cytokines by ELISA and numbers of immune cells by flow cytometry. In the chronic stage of EAE progression, clinical scores of LAT-1-inhibited EAE mice were higher than those of normal EAE mice. Significantly elevated T-cell counts, MMP-9 activity, and IL-6 levels were found in the LAT-1-inhibited EAE group. Inhibition of LAT-1 with 2-amino-2-norbornanecarboxylic acid (BCH) treatment resulted in decreased leucine concentration in splenocytes and increased leucine levels in plasma. The leucine levels on the abluminal side of LAT-1-inhibited EAE mice were also significantly higher than those of control mice but not those of EAE mice. The increased leucine concentration present at the luminal side crossed the blood brain barrier (BBB) and fueled inflammation with concurrent disease severity in the abluminal side of EAE mice.

Dendritic cell upregulation of programmed death ligand-1 via DNA demethylation inhibits experimental autoimmune encephalomyelitis

Dendritic cells (DCs) play key roles in regulating T cell proliferation and differentiation, and epigenetic modification involves in this process. In the periphery, programmed death ligand-1 (PD-L1) expressed on antigen-presenting cells interacts with programmed death-1 (PD-1) on T cells to negatively regulate T cell responses. In this study, we investigate whether DNA demethylation in DCs, downmodulates CD4+ T cell activation, to halt progression of experimental autoimmune encephalomyelitis (EAE). These results showed that during the development of bone marrow-derived DCs (BMDCs), DNA hypomethylation by 0.1 μM and 1 μM 5-aza-2′-deoxycytidine (5-aza) upregulated PD-L1, but not CD40, CD80, or CD86, with surprising downregulation of PD-L2. In co-culture, 5-aza-treated BMDCs, as well as CD11c+ cells from 5-aza-treated EAE mice, inhibited EAE CD4+ T cell proliferation and cytokine secretion. Additionally, in vivo 5-aza pretreatment arrested disease progression, inflammatory cell infiltration, and CNS demyelination, in EAE mice. Compared to DCs from vehicle control-treated EAE rodents, DCs from 5-aza-treated EAE mice upregulated PD-L1, in correlation with hypomethylation of the Cd274 promoter. Furthermore, antibody-mediated blockage of PD-L1 rescued EAE progression from 5-aza treatment, in vivo, while also disinhibiting EAE CD4+ T cell proliferation, by 5-aza-treated DCs, in vitro. Consequently, we conclude that PD-L1 is upregulated via DNA hypomethylation in DCs, resulting in downregulation of autoimmune effector T cell functions, thereby halting progression of EAE.

Continuous Immune-Modulatory Effects of Human Olig2+ Precursor Cells Attenuating a Chronic-Active Model of Multiple Sclerosis.

Neuroglial precursor cells (NPC) possess immune-modulatory properties by which they prevent immune-mediated injury in experimental autoimmune encephalomyelitis (EAE). It is unclear whether cell transplantation in a clinical-relevant setup induces ongoing therapeutic effects in a chronic-active model of progressive multiple sclerosis (MS). We examined whether human embryonic stem cell (hESC)-derived NPCs inhibit progressive EAE in Biozzi AB/H mice, manifesting with chronic-active neuroinflammation and demyelinated plaques. hESC-derived NPCs were propagated for 6-8 weeks as spheres enriched for Olig2+ cells to switch from neuronal to glial commitment and to enrich for oligodendrocyte progenitor cells. NPC were transplanted intracerebroventricularly at 30 days post-EAE induction, after the acute relapse. We evaluated effects of cell transplantation on clinical parameters, neuroinflammation, myelination, and axonal loss. Transplanted animals exhibited a significantly milder disease, reduced neuroinflammation, reduced demyelination, and reduced axonal loss as compared to control EAE mice. Toluidine-blue semi-thin staining showed a bystander neuroprotective effect of human precursor cells preventing the loss of myelinated fibers in superficial layer of the cervical dorsal funiculus. Human Olig2+ cells were detected along spinal cord meninges after 65 days of follow-up. In co-cultures in vitro, Olig2+ human precursors inhibited Concanavalin A-induced murine T cell activation and proliferation. To conclude, glial-committed human NPC induce ongoing immune-regulatory and neuroprotective effects, following transplantation into mice with a clinical-relevant model of chronic-active MS and during established disease, entering the chronic phase. These properties highlight the therapeutic potential of human NPC transplantation in chronic MS and their delivery via the cerebrospinal fluid.

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

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