Disease Progression In Experimental Autoimmune Encephalomyelitis Research Articles

Immunoresponsive Gene 1: an important regulator of autoreactive CD4 +T Cells in mouse model of multiple sclerosis

Abstract Th17 and Th1 CD4 T cells play important roles in the pathophysiology of human MS and the mouse model of MS, experimental autoimmune encephalomyelitis (EAE). As per our findings, metabolic changes in MS alter the phenotypic and functionality of immune cells, which may influence their effector activities as disease progresses. IRG1 (Immuno response gene 1), encodes for cis-aconitic acid decarboxylase in TCA cycle & enhances the manufacture of mitochondria-selective metabolite, itaconic acid from cis-aconitate, is one such gene linking metabolism to immune effector function. It modulates inflammation in myeloid cells in addition to its antibacterial activity. The relevance of IRG1 in CNS autoimmunity, however, is still unknown. We used IRG1 KO mice to know what role it plays in EAE. We observed that global IRG1 KO showed significantly worsened clinical disease severity compared to their WT at the peak of the disease (3.3+0.50 vs. 2.1+0.50, p< 0.05). We found an evident early onset of disease induction & severity in the IRG1 KO as compared to the Wt (D9 vs. D13). Furthermore, to examine the mechanism of EAE pathogenesis, found a significantly higher infiltration of CD45 hicells (mainly Th17/Th1 cells) in the CNS of IRG1 KO mice compared to Wt mice. Adoptive transfer of CD45+ cells from immunized IRG1 KO augmented the disease progression in RAG1 deficient mice suggesting a possible role of IRG1 in a hyperactivated Th17 response. Also, itaconate treatment at the dose of 15mg/kg bw attenuated EAE disease relapse in SJL mice. Our findings suggest that IRG1/itaconate negatively regulates the Th17 immune response & moDCs’ function to attenuate the EAE disease progression, which may open a novel therapeutic avenue in the development of MS-related therapies.

Therapeutic Potential of Combined Therapy of Vitamin A and Vitamin C in the Experimental Autoimmune Encephalomyelitis (EAE) in Lewis Rats.

Demyelination, inflammation, oxidative injury, and glial activation are the main pathological hallmarks of multiple sclerosis (MS). Vitamins, as essential micronutrients, seem to be crucial in the pathogenesis of MS, and particularly vitamins A and C were found to have a protective role in MS development or progression. In this study, the therapeutic potential of combined therapy of vitamins A and C on progression of experimental autoimmune encephalomyelitis (EAE) and myelin repair mechanisms was examined. EAE, an animal model of MS, was induced in female Lewis rats. The rats were treated with daily intraperitoneal injections of vitamins A and C and their combination. We found that co-supplementation of vitamins A and C mitigated neurological severity and EAE disease progression. Histological study confirmed a significant reduction in demyelination size, inflammation and immune cell infiltration as well as microglia and astrocyte activation following co-administration of vitamins A and C. Co-administration of vitamins A and C also decreased the levels of pro-inflammatory cytokines (TNF-α, IL1β) and iNOS and increased gene expressions of IL-10, Nrf-2, HO-1, and MBP. Combination therapy of vitamins A and C also increased the total antioxidant capacity and decreased levels of oxidative stress markers. Finally, we proved that co-administration of vitamins A and C has anti-apoptotic and neuroprotective impacts in EAE via decreasing caspase-3 and increasing BDNF and NeuN expressing cells. The present study suggests that combined therapy of vitamins A and C may be an effective strategy for development of alternative medicine in boosting myelin repair in demyelinating diseases.

Prophylactic and Therapeutic Effect of Kynurenine for Experimental Autoimmune Encephalomyelitis (EAE) Disease.

Background:The essential amino acid, tryptophan, is predominantly metabolised through the kynurenine pathway (KP) to generate kynurenine, an aryl-hydrocarbon receptor (AhR) pro-ligand that exerts its effects in a ligand-dependent manner. Interaction between kynurenine and the AhR is an effector mechanism of immunosuppression. We previously found that the KP is involved in multiple sclerosis (MS) disease progression. We postulated that AhR activation by kynurenine might be neuroprotective by encouraging differentiation of Tregs. In this study, we assess both the prophylactic and therapeutic efficiency of kynurenine on disease severity and progression in mice with experimental autoimmune encephalomyelitis (EAE), an MS model.Methods:Myelin oligodendrocyte glycoprotein induced EAE mice (n = 6 per group) were treated with 200 mg/kg L-kynurenine once daily for 10 days beginning on either day 1 of EAE induction (prophylactic) or once they demonstrated motor weakness (therapeutic). Clinical disease severity measured by disease score, time on rotarod, and body weight.Results:The prophylactic kynurenine treatment significantly (P < .0001) prevented the development of a more severe disease course with mice demonstrating diminished relapse rate and improved clinical and behavioural outcomes. However, therapeutic kynurenine did not significantly (P = .4463) decrease the clinical signs until 36 days following induction of disease; after 36 days, it also significantly (P = .0479) reduced disease relapse. Mean body weight measurements only correlated with time on rotarod (r = −.6410; P = .0007) but not clinical scores (r = .1925; P = .3674).Conclusions:Kynurenine ameliorates EAE disease progression prophylactically and reduces relapses therapeutically. Further investigations are needed to elucidate the molecular mechanism explaining the therapeutic role of kynurenine for MS.

Irisin levels in the serum and cerebrospinal fluid of patients with multiple sclerosis and the expression and distribution of irisin in experimental autoimmune encephalomyelitis.

Irisin is a novel hormone-like myokine that plays an important role in central nervous system (CNS) diseases, such as cerebral ischaemia and Alzheimer's disease. However, irisin is rarely investigated in multiple sclerosis (MS), a typical inflammatory demyelinating disease of the CNS, and in experimental autoimmune encephalomyelitis (EAE), a typical model of MS. We determined the levels of irisin in the serum and cerebrospinal fluid in patients with MS. The expression and histological distribution of irisin were determined in EAE. Serum irisin levels in patients with MS and in EAE mice were increased, and the levels of FNDC5/irisin mRNA were decreased in the spinal cord and brain regardless of the onset, peak or chronic phase of EAE. Immunofluorescence staining showed co-localization of irisin and neurones. The levels of irisin fluctuated with disease progression in MS and EAE. Irisin may be involved in the pathological process of MS/EAE.

Piperine Improves Experimental Autoimmune Encephalomyelitis (EAE) in Lewis Rats Through its Neuroprotective, Anti-inflammatory, and Antioxidant Effects.

Inflammation, demyelination, glial activation, and oxidative damage are the most pathological hallmarks of multiple sclerosis (MS). Piperine, a main bioactive alkaloid of black pepper, possesses antioxidant, anti-inflammatory, and neuroprotective properties whose therapeutic potential has been less studied in the experimental autoimmune encephalomyelitis (EAE) models. In this study, the efficiency of piperine on progression of EAE model and myelin repair mechanisms was investigated. EAE was induced in female Lewis rats and piperine and its vehicle were daily administrated intraperitoneally from day 8 to 29 post immunization. We found that piperine alleviated neurological deficits and EAE disease progression. Luxol fast blue and H&E staining and immunostaining of lumbar spinal cord cross sections confirmed that piperine significantly reduced the extent of demyelination, inflammation, immune cell infiltration, microglia, and astrocyte activation. Gene expression analysis in lumbar spinal cord showed that piperine treatment decreased the level of pro-inflammatory cytokines (TNF-α, IL-1β) and iNOS and enhanced IL-10, Nrf2, HO-1, and MBP expressions. Piperine supplementation also enhanced the total antioxidant capacity (FRAP) and reduced the level of oxidative stress marker (MDA) in the CNS of EAE rats. Finally, we found that piperine has anti-apoptotic and neuroprotective effect in EAE through reducing caspase-3 (apoptosis marker) and enhancing BDNF and NeuN expressing cells. This study strongly indicates that piperine has a beneficial effect on the EAE progression and could be considered as a potential therapeutic target for MS treatment. Upcoming clinical trials will provide a deeper understanding of piperine's role for the treatment of the MS.

Mice Lacking Factor XIII Have Significant Protection from the Progression of Neuroinflammatory Disease

Multiple sclerosis (MS) is a chronic autoimmune inflammatory demyelinating disease of the central nervous system (CNS). Motor and sensory neurologic deficits associated with MS are secondary to autoimmune-mediated demyelination of neurons within the CNS. Autoreactive T cells that target myelin and local microglia/macrophage activation are both central to disease pathogenesis. Fibrin(ogen) deposition, the final product of the coagulation cascade, at sites of blood brain barrier (BBB) breakdown is an early key feature of MS. Growing evidence supports that fibrin(ogen) contributes to MS pathogenesis by serving as a local cue for microglial/macrophage activation through αMβ2-CD11/CD18 interactions. However, the precise contributions of different molecular forms of fibrin(ogen) to the inflammation seen in MS have not been defined. In this study, we determined the roles of soluble fibrinogen, insoluble fibrin polymer and crosslinked fibrin matrix in neuroinflammatory disease by use of a standard murine model of MS, experimental autoimmune encephalomyelitis (EAE). We first sought to determine relative contributions of soluble fibrinogen versus fibrin polymer to MS. FibrinogenAEK (FibAEK) mice, which express a form of fibrinogen that is unable to be cleaved by thrombin and thus form a fibrin polymer, were challenged with EAE. Our results demonstrated that FibAEK mice challenged with EAE developed mildly attenuated clinical symptoms compared with wildtype mice. Notably, these FibAEK mice that are unable to form a fibrin polymer, but still express fibrinogen, were not as protected from EAE disease symptoms as mice that are deficient in fibrinogen. These data suggested that fibrinogen may contribute to the progression of EAE disease without polymerization of fibrinogen. To further explore the role of fibrin(ogen) structure in neuroinflammation, we utilized mice lacking factor XIII (fXIII), a transglutaminase that crosslinks fibrin monomers to form a stable fibrin matrix. FXIIIA-/- mice, which are genetically deficient in the active subunit of fXIII, developed significantly ameliorated EAE clinical symptoms, including lower clinical scores, decreased weight loss, and decreased days of paralysis compared with wildtype animals. Similarly, pharmacological inhibition of transglutaminase activity by cystamine, a transglutaminase inhibitor, significantly ameliorated clinical symptoms in EAE mice. On examination of spinal cords from EAE challenged animals, a significantly reduced area of demyelination was present in the white matter of spinal cords from fXIIIA-/- animals compared to wildtype mice. Further, significantly diminished areas of microglia/macrophage activation were observed in EAE-challenged fXIIIA-/-spinal cords versus wildtype. Correspondingly, a low level of diffuse fibrin deposits was observed in fXIIIA-/- spinal cord white matter. This was in marked contrast to the conspicuous, and significantly increased, fibrin deposition in the spinal cords of wildtype animals challenged with EAE. Further examination of the cytokine and chemokine expression profiles from the spinal cords of mice challenged with EAE suggest that the T helper 17 (Th17) cell responses, including expression of interleukin 17a (IL17a), interleukin 6 (IL6), transforming growth factor β (TGFβ) and chemokine (C-C motif) ligand 20 (CCL20), were decreased in animals lacking factor XIIIA. Together, our results suggest that fibrinogen, and not only polymerized fibrin, may contribute to EAE disease progression. Further, our data reveal that factor XIII-mediated crosslinking is a significant determinant of EAE disease severity. Targeting fibrin crosslinking may be a novel therapeutic strategy for MS/EAE. DisclosuresMullins:Shire: Honoraria; Bayer: Honoraria.

VISTA regulates microglia homeostasis and myelin phagocytosis, and is associated with MS lesion pathology

V-type immunoglobulin domain-containing suppressor of T-cell activation (VISTA) is a negative checkpoint regulator (NCR) that is involved in T-cell quiescence, inhibition of T-cell activation, and in myeloid cells regulates cytokine production, chemotaxis, phagocytosis, and tolerance induction. In the central nervous system (CNS), VISTA is expressed by microglia, the resident macrophage of the parenchyma, and expression is decreased during neuroinflammation; however, the function of VISTA in microglia is unknown. Here, we extensively analyzed VISTA expression in different MS lesion stages and characterized the function of VISTA in the CNS by deleting VISTA in microglia. VISTA is differentially expressed in distinct MS lesion stages. In mice, VISTA deletion in Cx3Cr1-expressing cells induced a more amoeboid microglia morphology, indicating an immune-activated phenotype. Expression of genes associated with cell cycle and immune-activation was increased in VISTA KO microglia. In response to LPS and during experimental autoimmune encephalomyelitis (EAE), VISTA KO and WT microglia shared similar transcriptional profiles and VISTA deletion did not affect EAE disease progression or microglia responses. VISTA KO in microglia in vitro decreased the uptake of myelin. This study demonstrates that VISTA is involved in microglia function, which likely affects healthy CNS homeostasis and neuroinflammation.

4-Ethylguaiacol modulates neuroinflammation and Th1/Th17 differentiation to ameliorate disease severity in experimental autoimmune encephalomyelitis

Abstract Multiple sclerosis (MS) is a progressive autoimmune disease characterized by the accumulation of pathogenic inflammatory immune cells in the central nervous system (CNS) that subsequently causes focal inflammation, demyelination, and neuronal damage. Experimental Autoimmune Encephalomyelitis (EAE) is a well-established murine model of MS. Presently, growing evidences show that phenolic compounds exert anti-inflammatory effects by inhibiting the production of inflammatory molecules. 4-ethylguaiacol (4-EG), a phenolic compound, which was detected in foods, such as green bell peppers, corns, sesames, and coffee has been shown to possess anti-inflammatory effects through attenuating peripheral immune responses. However, whether 4-EG exerts anti-inflammatory effects on modulating the CNS inflammatory immune responses remains unexplored. In this study we assessed the therapeutic effect of 4-EG in EAE using both chronical and relapsing-remitting animal models. Our results showed that 4-EG not only ameliorated disease severity in chronic EAE but also mitigated disease progression in relapsing-remitting EAE. Further investigations revealed that 4-EG suppressed MG activation, inhibited Th1 and Th17 infiltration, and mitigated BBB disruption in the CNS of EAE. Furthermore, 4-EG suppressed Th1 and Th17 differentiation in the periphery of EAE. Finally, 4-EG induced HO-1 expression in the CNS of EAE as well as in MG and macrophages. In summary, our work demonstrates for the first time that 4-EG confers a protection against autoimmune disease EAE through modulating neuroinflammation and inhibiting Th1/Th17 differentiation, suggesting that 4-EG could be developed as a therapeutic agent for the treatment of MS/EAE.

Eomesodermin in CD4+T cells is essential for Ginkgolide K ameliorating disease progression in experimental autoimmune encephalomyelitis.

Eomesodermin (Eomes), a transcription factor, could suppress the Th17 cell differentiation and proliferation through directly binding to the promoter zone of the Rorc and Il17a gene, meanwhile the expression of Eomes is suppressed when c-Jun directly binds to its promoter zone. Ginkgolide K (1,10-dihydroxy-3,14-didehydroginkgolide, GK) is a diterpene lactone isolated from the leaves of Ginkgo biloba. A previous study indicated that GK could decrease the level of phospho JNK (c-Jun N-terminal kinase). Here, we reported the therapeutic potential of Ginkgolide K (GK) treatment to ameliorate experimental autoimmune encephalomyelitis (EAE) disease progression.Methods: EAE was induced in both wildtype and CD4-Eomes conditional knockout mice. GK was injected intraperitoneally. Disease severity, inflammation, and tissue damage were assessed by clinical evaluation, flow cytometry of mononuclear cells (MNCs), and histopathological evaluation. Dual-luciferase reporter assays were performed to measure Eomes transcription activity in vitro. The potency of GK (IC50) was determined using JNK1 Kinase Enzyme System.Results: We revealed that GK could ameliorate EAE disease progression by the inhibition of the Th17 cells. Further mechanism studies demonstrated that the level of phospho JNK was decreased and the level of Eomes in CD4+T cells was dramatically increased. This therapeutic effect of GK was almost completely interrupted in CD4-Eomes conditional knockout mice.Conclusions: These results provided the therapeutic potential of GK treatment in EAE, and further suggested that Eomes expression in CD4+T cells might be essential in this process.

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.

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.

CPT1A plays a key role in the development and treatment of multiple sclerosis and experimental autoimmune encephalomyelitis

Human mutations in carnitine palmitoyl transferase 1A (CPT1A) are correlated with a remarkably low prevalence of multiple sclerosis (MS) in Inuits (P479L) and Hutterites (G710E). To elucidate the role of CPT1A, we established a Cpt1a P479L mouse strain and evaluated its sensitivity to experimental autoimmune encephalomyelitis (EAE) induction. Since CPT1a is a key molecule in lipid metabolism, we compared the effects of a high-fat diet (HFD) and normal diet (ND) on disease progression. The disease severity increased significantly in WT mice compared to that in Cpt1 P479L mice. In addition, WT mice receiving HFD showed markedly exacerbated disease course when compared either with Cpt1a P479L mice receiving HFD or WT control group receiving ND. Induction of EAE caused a significant decrease of myelin basic protein expression in the hindbrain of disease affected WT mice in comparison to Cpt1a P479L mice. Further, WT mice showed increased expression of oxidative stress markers like Nox2 and Ho-1, whereas expression of mitochondrial antioxidants regulator Pgc1α was increased in Cpt1a P479L mice. Our results suggest that, lipids metabolism play an important role in EAE, as shown by the higher severity of disease progression in both WT EAE and WT EAF HFD-fed mice in contrast to their counterpart Cpt1a P479L mutant mice. Interestingly, mice with downregulated lipid metabolism due to the Cpt1a P479L mutation showed resistance to EAE induction. These findings support a key role for CPT1A in the development of EAE and could be a promising target in MS treatment.

Re-examining the potential of targeting ABHD6 in multiple sclerosis: Efficacy of systemic and peripherally restricted inhibitors in experimental autoimmune encephalomyelitis

α/β-Hydrolase domain-containing 6 (ABHD6) contributes to the hydrolysis of the major endocannabinoid 2-arachidonoylglycerol (2-AG) in the central nervous system (CNS) and in the periphery. ABHD6 blockade has been proposed as novel strategy to treat multiple sclerosis (MS), based on the observation that the inhibitor WWL70 exerts protective anti-inflammatory effects in experimental autoimmune encephalomyelitis (EAE). According to recent data, WWL70 exhibits off-target anti-inflammatory activity in microglial cells and the potential of ABHD6 as drug target in MS remains controversial. Here we further investigated the role of ABHD6 during autoimmune demyelination by comparing the efficacy of two novel inhibitors with different CNS permeability in vivo. Preventive treatment with the systemically active inhibitor KT182 ameliorated the neurological signs of EAE during the time-course of disease. By contrast, administration of the peripherally restricted compound KT203 was ineffective in attenuating EAE symptomatology. Both inhibitors failed to improve corticospinal tract conduction latency and to attenuate inflammation at EAE recovery phase, despite being equally active at targeting brain ABHD6. Chronic administration of KT182 was associated to a partial loss of brain CB1 receptor coupling ability, suggesting the engagement of CB1 receptor-mediated mechanisms during the EAE disease progression. In cultured neurons, KT182 attenuated NMDA-stimulated excitotoxicity and mitochondrial calcium overload. However, these protective effects were not attributable to ABHD6, as they were not mimicked by the alternative inhibitors KT203, KT195 and WWL70. These results indicate that ABHD6 blockade exerts only modest therapeutic effects against autoimmune demyelination and call into question its utility as novel drug target in MS.

MicroRNA-181c promotes Th17 cell differentiation and mediates experimental autoimmune encephalomyelitis

Among T helper (Th) cell subsets differentiated from naive CD4+ T cells, IL-17-producing Th17 cells are closely associated with the pathogenesis of autoimmune diseases, including multiple sclerosis (MS) and the MS animal model, experimental autoimmune encephalomyelitis (EAE). The modulation of Th17 differentiation offers a potential avenue for treatment. Although a series of microRNAs (miRNAs) that modulate autoimmune disease development have been reported, further studies on miRNA roles in Th17 differentiation and MS pathogenesis are still warranted. Here, we demonstrated that mice with miR-181c knockdown presented with delayed EAE and slowed disease progression, along with a decreased Th17 cell population. We also found that miR-181c was a Th17 cell-associated miRNA and that Smad7, a negative regulator of TGF-β signaling, was a potential target of miR-181c. miR-181c knockdown rendered T cells less sensitive to TGF-β-induced Smad2/3, enhancing the expression of IL-2 which has been reported to inhibit Th17 cell differentiation. Moreover, through the analysis of published miRNA expression profiles from the Gene Expression Omnibus database, increased miR-181c levels were found in peripheral blood from MS patients. Our results identified a novel miRNA that promotes Th17 cell differentiation and autoimmunity, thus miR-181c may serve as a potential treatment target in patients with MS.

BJ-2266 ameliorates experimental autoimmune encephalomyelitis through down-regulation of the JAK/STAT signaling pathway.

CD4+ T cells differentiate into distinct effector subsets upon antigenic stimulation. Cytokines, and micro-environmental factors present during T-cell priming, direct differentiation of naïve CD4+ T cells into pro-inflammatory Th1 and Th17 cells. From extensive screening of 2,4,5-trimethylpyridin-3-ol derivatives with various functional groups at C(6)-position, BJ-2266, a 6-thioureido-derivative, showed potent inhibitory activity on in vitro T helper (Th)-cell differentiation. This compound inhibited IFN-γ and IL-17 production from polyclonal CD4+ T cells and ovalbumin (OVA)-specific CD4+ T cells that were activated by T-cell receptor (TCR) engagement. We assessed the inhibitory effect of BJ-2266 in experimental autoimmune encephalomyelitis (EAE). Our results suggest that BJ-2266 treatment significantly suppresses EAE disease progression with reduced generation of Th1 and Th17 cells. Notably, Th-cell differentiation was significantly suppressed by BJ-2266 treatment with no effect on apoptosis, activation and proliferation of activated T cells. Furthermore, adoptive transfer of BJ-2266 treated MOG-reactive Th1 and Th17 cells led to a lower EAE disease score and better clinical recovery from EAE. The underlying mechanism of BJ-2266 effect involved the inhibition of JAK/STAT phosphorylation that is critical for Th-cell differentiation. We conclude that BJ-2266 regulates the JAK/STAT pathway in response to cytokine signals and subsequently suppresses the differentiation of Th-cell responses.

Hyaluronan synthesis is necessary for autoreactive T-cell trafficking, activation, and Th1 polarization.

The extracellular matrix polysaccharide hyaluronan (HA) accumulates at sites of autoimmune inflammation, including white matter lesions in multiple sclerosis (MS), but its functional importance in pathogenesis is unclear. We have evaluated the impact of 4-methylumbelliferone (4-MU), an oral inhibitor of HA synthesis, on disease progression in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Treatment with 4-MU decreases the incidence of EAE, delays its onset, and reduces the severity of established disease. 4-MU inhibits the activation of autoreactive T cells and prevents their polarization toward a Th1 phenotype. Instead, 4-MU promotes polarization toward a Th2 phenotpye and induction of Foxp3(+) regulatory T cells. Further, 4-MU hastens trafficking of T cells through secondary lymphoid organs, impairs the infiltration of T cells into the CNS parenchyma, and limits astrogliosis. Together, these data suggest that HA synthesis is necessary for disease progression in EAE and that treatment with 4-MU may be a potential therapeutic strategy in CNS autoimmunity. Considering that 4-MU is already a therapeutic, called hymecromone, that is approved to treat biliary spasm in humans, we propose that it could be repurposed to treat MS.

B7-H3 Promotes Pathogenesis of Autoimmune Disease and Inflammation by Regulating the Activity of Different T Cell Subsets.

B7-H3 is a cell surface molecule in the immunoglobulin superfamily that is frequently upregulated in response to autoantigens and pathogens during host T cell immune responses. However, B7-H3's role in the differential regulation of T cell subsets remains largely unknown. Therefore, we constructed a new B7-H3 deficient mouse strain (B7-H3 KO) and evaluated the functions of B7-H3 in the regulation of Th1, Th2, and Th17 subsets in experimental autoimmune encephalomyelitis (EAE), experimental asthma, and collagen-induced arthritis (CIA); these mouse models were used to predict human immune responses in multiple sclerosis, asthma, and rheumatoid arthritis, respectively. Here, we demonstrate that B7-H3 KO mice have significantly less inflammation, decreased pathogenesis, and limited disease progression in both EAE and CIA mouse models when compared with littermates; these results were accompanied by a decrease in IFN-γ and IL-17 production. In sharp contrast, B7-H3 KO mice developed severe ovalbumin (OVA)-induced asthma with characteristic infiltrations of eosinophils in the lung, increased IL-5 and IL-13 in lavage fluid, and elevated IgE anti-OVA antibodies in the blood. Our results suggest B7-H3 has a costimulatory function on Th1/Th17 but a coinhibitory function on Th2 responses. Our studies reveal that B7-H3 could affect different T cell subsets which have important implications for regulating pathogenesis and disease progression in human autoimmune disease.

Voluntary wheel running delays disease onset and reduces pain hypersensitivity in early experimental autoimmune encephalomyelitis (EAE)

Multiple sclerosis (MS) is classically defined by motor deficits, but it is also associated with the secondary symptoms of pain, depression, and anxiety. Up to this point modifying these secondary symptoms has been difficult. There is evidence that both MS and the animal model experimental autoimmune encephalomyelitis (EAE), commonly used to study the pathophysiology of the disease, can be modulated by exercise. To examine whether limited voluntary wheel running could modulate EAE disease progression and the co-morbid symptoms of pain, mice with EAE were allowed access to running wheels for 1h every day. Allowing only 1h every day of voluntary running led to a significant delay in the onset of clinical signs of the disease. The development of mechanical allodynia was assessed using Von Frey hairs and indicated that wheel running had a modest positive effect on the pain hypersensitivity associated with EAE. These behavioral changes were associated with reduced numbers of cFOS and phosphorylated NR1 positive cells in the dorsal horn of the spinal cord compared to no-run EAE controls. In addition, within the dorsal horn, voluntary wheel running reduced the number of infiltrating CD3+ T-cells and reduced the overall levels of Iba1 immunoreactivity. Using high performance liquid chromatography (HPLC), we observed that wheel-running lead to significant changes in the spinal cord levels of the antioxidant glutathione. Oxidative stress has separately been shown to contribute to EAE disease progression and neuropathic pain. Together these results indicate that in mice with EAE, voluntary motor activity can delay the onset of clinical signs and reduce pain symptoms associated with the disease.

Migration of i.c. injected BMDCs from brain to deep cervical lymph nodes is CCR7 dependent but does not promote EAE disease progression (CCR1P.244)

Abstract Cell-mediated antigen (Ag) drainage from the central nervous system (CNS) is incompletely understood. In other organs, Ag-presenting cells—such as dendritic cells (DCs)—migrate from organ tissue through lymphatics to draining lymph nodes (dLNs); however, classical lymphatic vessels are not found in the CNS and few migratory tissue DCs populate neuropil. In the context of neuroinflammation associated with experimental autoimmune encephalomyelitis (EAE), intracerebral (i.c.) injection of myelin peptide-loaded bone marrow (BM)DCs exacerbates disease, suggesting that increasing the number of CNS-DCs promotes EAE progression. However, whether CNS-infiltrating DCs transport Ag to dLNs for T cell priming or restimulate myelin-specific effector T cells in situ within developing CNS inflammatory lesions is unknown. Since migration of i.c. injected BMDCs to dLNs is pertussis toxin sensitive—and thus likely regulated by G proteins/ chemokine receptors—we sought to identify key chemokine receptors that govern DC migration from CNS to dLN. Here we report that 1) CCR7+/+ but not CCR7-/- BMDCs appear in dLNs after i.c. injection, 2) CCR7-/- DCs accumulate in CNS but not dLNs during EAE compared to CCR7+/+ DCs, 3) CCR7 is not necessary for i.c. injected BMDCs to exacerbate EAE, and 4) ablation of CCR7+/+ DCs during onset of EAE leads to increased CNS T cell cytokine production. These data suggest CCR7-dependent DC migration from CNS to dLN might regulate T cell responses in early EAE.

SIRT1 enhances Th17 cell generation by deacetylating RORγt (P1163)

Abstract Although Sirtuin 1 (SIRT1), an NAD-dependent protein deacetylase, has emerged as a critical regulator of the cell cycle, replicative senescence, inflammation, and metabolism, its contribution to immune function remains to be fully investigated. Here we report that SIRT1 regulates the function of IL-17 producing CD4 T helper cells (Th17 cells) by modulating the activity of the Th17 master transcription factor RORγt. We found that Sirt1 interacts with RORγt and increases its transcriptional activity by deacetylating lysine residues K16 and K81. In mixed hematopoietic chimeras, cells from donors in which Sirt1 is deleted in ROR γ+ cells (SIRT1-cKO) exhibited compromised Th17 differentiation. Moreover, SIRT1-cKO mice were resistant to developing experimental autoimmune encephalomyelitis (EAE) upon MOG peptide immunization. Importantly, the SIRT1 specific inhibitor Ex-527 substantially inhibited trans-differentiation to Th17 cells in vitro, and injection of Ex-527 into mice strongly blocked EAE disease progression in vivo. These findings reveal a previously unappreciated proinflammatory role of SIRT1 in CD4 T cells, and provide significant evidence that pharmacological SIRT1 inhibitors might constitute a novel approach in the treatment of Th17-mediated autoimmune diseases such as multiple sclerosis.