Peptide-induced Experimental Autoimmune Encephalomyelitis Research Articles

Matrine inhibits the Wnt3a/β-catenin/TCF7L2 signaling pathway in experimental autoimmune encephalomyelitis

Oligodendrocyte (OL) death and remyelination failure lead to progressive neurological deficits in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Matrine (MAT), a quinolizidine alkaloid component derived from the root of Sophora flavescens, has the capacity to effectively inhibit central nervous system (CNS) inflammation and to promote neuroregeneration. In the present study we explored its regulatory mechanism on the Wnt/β-catenin/TCF7L2 pathway, a negative modulator for myelination, in MOG35‐–55 peptide-induced EAE. Our results clearly indicate that MAT treatment reduced the activation of Wnt3a and β-catenin in the CNS of EAE mice, accompanied by the activation of GSK3β and decreased expression of cyclin D1 and Axin2, two target genes of the Wnt3a/β-catenin pathway. In addition, MAT increased OL maturation and myelination, as evidenced by the decreased number of NG2+Olig2+ cells and the increased numbers of MBP+ and CC1+Olig2+ cells. Taken together, these findings indicate that MAT treatment promoted the maturation of OLs and myelin repair, which is closely related to the modulation of the Wnt/β-catenin/TCF7L2 signaling pathway.

Experimental Autoimmune Encephalomyelitis Animal Models Induced by Different Myelin Antigens Exhibit Differential Pharmacologic Responses to Anti-Inflammatory Drugs

Background and objective Experimental autoimmune encephalomyelitis (EAE) is the most commonly used model for studying autoimmune-mediated myelin degradation in multiple sclerosis (MS). Here, we evaluated the pharmacologic responses of several anti-inflammatory drugs with varying mechanisms of actions (MOAs) using EAE models induced by different MOG immunogens to reveal differential pharmacologic characteristics of the disease models and provide a general guidance in animal model selection for MS research. Methods The pharmacologic responses of anti-inflammatory drugs with different mechanisms of actions (MOAs) were evaluated using EAE models induced by either myelin oligodendrocyte glycoprotein p35-55 （MOG35-55）or p1-128 (MOG1-128). EAE animal models were developed in mice with C57BL/6 background. The animals were treated with different anti-MS medications, including 3 B cell-mediated agents and 2 T cell-mediated agents, respectively. Clinical symptoms were monitored and scored, and pharmacodynamic markers including cytokine secretion, inflammatory cell infiltration, and demyelination in spinal cord were analyzed. Results In MOG35-55 peptide-induced EAE model, T cell modulating agents Secukinumab and Fingolimod significantly alleviated clinical symptoms, while B cell-depleting agents, BTK inhibitors PRN2246 and Telitacicept, displayed minimal therapeutic effects or even exacerbated disease progression. In contrast, both T cell-modulating agents and B cell-depleting agents ameliorated disease severity in MOG1-128-induced EAE model. T cell and B cell infiltration in spinal cord increased with disease progression in MOG1-128-induced EAE model. Conclusions Our results demonstrated that induction of EAE by different myelin antigens resulted in differential pharmacologic responses to drugs with specific MOAs. The MOG35-55 peptide-induced EAE model only responded to T cell-modulating drugs, whereas the MOG1-128 protein-induced EAE model exhibited therapeutic sensitivity to both T cell- and B cell-modulating agents. These data suggest the MOG35-55 peptide-induced EAE model is suitable for assessing T cell-modulating agents while MOG1-128 protein-induced model can be employed to evaluate both T cell- and B cell-modulating agents.

PI3-Kinase p110α Deficiency Modulates T Cell Homeostasis and Function and Attenuates Experimental Allergic Encephalitis in Mature Mice.

Class I phosphoinositide 3-kinases (PI3K) are involved in the development of normal and autoimmune responses, including Experimental Autoimmune Encephalomyelitis (EAE), a mouse model for human multiple sclerosis (MS). Here, the role of the ubiquitously expressed class IA PI3K p110α catalytic subunits in EAE has been analyzed using a model of Cre/flox mediated T cell specific deletion of p110α catalytic chain (p110αΔT). Comparison of two month-old (young) and six month-old (mature) p110αΔT mice and their wild type (WT) counterparts indicated loss of spleen CD4+ T cells that increased with age, indicating a role of p110α in their homeostasis. In contrast, CD4+ T regulatory (Treg) cells were enhanced in mature p110αΔT mice when compared to WT mice. Since Myelin Oligodendrocyte Glycoprotein (MOG) peptide-induced EAE is dependent on, or mediated by CD4+ T cells and CD4+ T cell-derived cytokines and controlled by Treg cells, development of EAE in young and mature WT or p110αΔT mice was analyzed. EAE clinical symptoms and disease scores in six month p110αΔT mice were significantly lower than those of mature WT, or young WT and p110αΔT mice. Furthermore, ex vivo antigen activation of lymph node cells from MOG immunized mature p110αΔT mice induced significantly lower levels of IFN-γ and IL-17A than young p110αΔT or young and mature WT mice. Other cytokines including IL-2, IL-10 or TNF-α showed no significant differences between p110αΔT and WT mature mice. Our data show a lower incidence of MOG-induced EAE in mature p110αΔT mice linked to altered T cell homeostasis and lower secretion of inflammatory cytokines.

Roles of the DOCK-D family proteins in a mouse model of neuroinflammation

The DOCK-D (dedicator of cytokinesis D) family proteins are atypical guanine nucleotide exchange factors that regulate Rho GTPase activity. The family consists of Zizimin1 (DOCK9), Zizimin2 (DOCK11), and Zizimin3 (DOCK10). Functions of the DOCK-D family proteins are presently not well-explored, and the role of the DOCK-D family in neuroinflammation is unknown. In this study, we generated three mouse lines in which DOCK9 (DOCK9-/-), DOCK10 (DOCK10-/-), or DOCK11 (DOCK11-/-) had been deleted and examined the phenotypic effects of these gene deletions in MOG35-55 peptide-induced experimental autoimmune encephalomyelitis, an animal model of the neuroinflammatory disorder multiple sclerosis. We found that all the gene knockout lines were healthy and viable. The only phenotype observed under normal conditions was a slightly smaller proportion of B cells in splenocytes in DOCK10-/- mice than in the other mouse lines. We also found that the migration ability of macrophages is impaired in DOCK10-/- and DOCK11-/- mice and that the severity of experimental autoimmune encephalomyelitis was ameliorated only in DOCK10-/- mice. No apparent phenotype was observed for DOCK9-/- mice. Further investigations indicated that lipopolysaccharide stimulation up-regulates DOCK10 expression in microglia and that microglial migration is decreased in DOCK10-/- mice. Up-regulation of C-C motif chemokine ligand 2 (CCL2) expression induced by activation of Toll-like receptor 4 or 9 signaling was reduced in DOCK10-/- astrocytes compared with WT astrocytes. Taken together, our findings suggest that DOCK10 plays a role in innate immunity and neuroinflammation and might represent a potential therapeutic target for managing multiple sclerosis.

Independent and inter-dependent immunoregulatory effects of NCF1 and NOS2 in experimental autoimmune encephalomyelitis

BackgroundIncreasing evidence has suggested that a single nucleotide polymorphism in the Ncf1 gene is associated with experimental autoimmune encephalomyelitis (EAE). However, the mechanisms of NCF1-induced immunoregulatory effects remain poorly understood. In this study, we focus on NCF1 deficiency-mediated effects on EAE in NOS2 dependent and independent ways.MethodsTo determine the effects of NCF1 and NOS2 during EAE development, we have established recombinant mouse strains deficient at NCF1 and/or NOS2 in a crossbreeding system. Different strains allow us to examine the entire course of the disease in the Nos2-null mice bearing a Ncf1 gene that encodes a mutated NCF1, deficient in triggering oxidative burst, after immunization with recombinant myelin oligodendrocyte glycoprotein (MOG)79-96 peptides. The peptide-induced innate and adaptive immune responses were analyzed by flow cytometry.ResultsNCF1-deficient mice developed a reduced susceptibility to EAE, whereas NCF1-NOS2 double-deficient mice developed an enhanced EAE, as compared with NOS2-deficient mice. Flow cytometry analyses show that double deficiencies resulted in an increase of neutrophils in the spleen, accompanied with higher release of interleukin-1β in neutrophils prior to EAE onset. The additional deficiency in NCF1 had no added effect on either interleukin-17 or interferon-γ secretion of T cells during the priming phase.ConclusionsThese studies show that NCF1 and NOS2 interact to regulate peptide-induced EAE.

Disruption of hepatocyte Sialylation drives a T cell-dependent pro-inflammatory immune tone.

Through the catalysis of α2,6-linked sialylation, the enzyme ST6Gal1 is thought to play key roles in immune cell communication and homeostasis. Of particular importance, glycans with terminal α2,6-sialic acids are known to negatively regulate B cell receptor signaling and are associated with an immunosuppressive tumor microenvironment that promotes T cell anergy, suggesting that α2,6-sialic acids are a key immune inhibitory signal. Consistent with this model, mice harboring a hepatocyte-specific ablation of ST6Gal1 (H-cKO) develop a progressive and severe non-alcoholic fatty liver disease characterized by steatohepatitis. Using this H-cKO mouse, we have further discovered that loss of hepatocyte α2,6-sialylation not only increases the inflammatory state of the local tissue microenvironment, but also systemic T cell-dependent immune responses. H-cKO mice responded normally to innate and passively induced inflammation, but showed significantly increased morbidity in T cell-dependent house dust mite-antigen (HDM)-induced asthma and myelin oligodendrocyte glycoprotein (MOG) peptide-induced experimental autoimmune encephalomyelitis (EAE). We further discovered that H-cKO mice have a profound shift toward effector/memory T cells even among unchallenged mice, and that macrophages from both the liver and spleen expressed the inhibitory and α2,6-sialic acid-specific glycan binding molecule CD22. These findings align with previously reported pro-inflammatory changes in liver macrophages, and support a model in which the liver microenvironment sets a systemic immune tone that is regulated by tissue α2,6-sialylation and mediated by liver macrophages and systemic T cells.

Reduced expression of the ferroptosis inhibitor glutathione peroxidase-4 in multiple sclerosis and experimental autoimmune encephalomyelitis.

Glutathione peroxidase 4 (GPx4) is the only enzyme capable of reducing toxic lipid hydroperoxides in biological membranes to the corresponding alcohols using glutathione as the electron donor. GPx4 is the major inhibitor of ferroptosis, a non-apoptotic and iron-dependent programmed cell death pathway, which has been shown to occur in various neurological disorders with severe oxidative stress. In this study, we investigate whether GPx4 expression is altered in multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). The results clearly show that mRNA expression for all three GPx4 isoforms (cytoplasmic, mitochondrial and nuclear) decline in multiple sclerosis gray matter and in the spinal cord of MOG35-55 peptide-induced EAE. The amount of GPx4 protein is also reduced in EAE, albeit not in all cells. Neuronal GPx4 immunostaining, mostly cytoplasmic, is lower in EAE spinal cords than in control spinal cords, while oligodendrocyte GPx4 immunostaining, mainly nuclear, is unaltered. Neither control nor EAE astrocytes and microglia cells show GPx4 labeling. In addition to GPx4, two other negative modulators of ferroptosis (γ-glutamylcysteine ligase and cysteine/glutamate antiporter), which are critical to maintain physiological levels of glutathione, are diminished in EAE. The decrease in the ability to eliminate hydroperoxides was also evidenced by the accumulation of lipid peroxidation products and the reduction in the proportion of the docosahexaenoic acid in non-myelin lipids. These findings, along with presence of abnormal neuronal mitochondria morphology, which includes an irregular matrix, disrupted outer membrane and reduced/absent cristae, are consistent with the occurrence of ferroptotic damage in inflammatory demyelinating disorders.

Functional characterization of reappearing B cells after anti-CD20 treatment of CNS autoimmune disease

The anti-CD20 antibody ocrelizumab, approved for treatment of multiple sclerosis, leads to rapid elimination of B cells from the blood. The extent of B cell depletion and kinetics of their recovery in different immune compartments is largely unknown. Here, we studied how anti-CD20 treatment influences B cells in bone marrow, blood, lymph nodes, and spleen in models of experimental autoimmune encephalomyelitis (EAE). Anti-CD20 reduced mature B cells in all compartments examined, although a subpopulation of antigen-experienced B cells persisted in splenic follicles. Upon treatment cessation, CD20+ B cells simultaneously repopulated in bone marrow and spleen before their reappearance in blood. In EAE induced by native myelin oligodendrocyte glycoprotein (MOG), a model in which B cells are activated, B cell recovery was characterized by expansion of mature, differentiated cells containing a high frequency of myelin-reactive B cells with restricted B cell receptor gene diversity. Those B cells served as efficient antigen-presenting cells (APCs) for activation of myelin-specific T cells. In MOG peptide-induced EAE, a purely T cell-mediated model that does not require B cells, in contrast, reconstituting B cells exhibited a naive phenotype without efficient APC capacity. Our results demonstrate that distinct subpopulations of B cells differ in their sensitivity to anti-CD20 treatment and suggest that differentiated B cells persisting in secondary lymphoid organs contribute to the recovering B cell pool.

Carnosol Modulates Th17 Cell Differentiation and Microglial Switch in Experimental Autoimmune Encephalomyelitis.

Medicinal plants as a rich pool for developing novel small molecule therapeutic medicine have been used for thousands of years. Carnosol as a bioactive diterpene compound originated from Rosmarinus officinalis (Rosemary) and Salvia officinalis, herbs extensively applied in traditional medicine for the treatment of multiple autoimmune diseases (1). In this study, we investigated the therapeutic effects and molecule mechanism of carnosol in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Carnosol treatment significantly alleviated clinical development in the myelin oligodendrocyte glycoprotein (MOG35–55) peptide-induced EAE model, markedly decreased inflammatory cell infiltration into the central nervous system and reduced demyelination. Further, carnosol inhibited Th17 cell differentiation and signal transducer and activator of transcription 3 phosphorylation, and blocked transcription factor NF-κB nuclear translocation. In the passive-EAE model, carnosol treatment also significantly prevented Th17 cell pathogenicity. Moreover, carnosol exerted its therapeutic effects in the chronic stage of EAE, and, remarkably, switched the phenotypes of infiltrated macrophage/microglia. Taken together, our results show that carnosol has enormous potential for development as a therapeutic agent for autoimmune diseases such as MS.

The flavonoid kurarinone inhibits clinical progression of EAE through inhibiting Th1 and Th17 cell differentiation and proliferation

IntroductionThe flavonoid kurarinone suppresses CD4+ T-cell-mediated chronic inflammatory dermatitis. However, kurarinone's effects upon autoimmune central nervous system (CNS) disease remain unknown. We investigated the potential therapeutic effects and molecular mechanism(s) of kurarinone in an experimental autoimmune encephalomyelitis (EAE) murine model of multiple sclerosis (MS). Materials and methodsMyelin oligodendrocyte glycoprotein (MOG35-55) peptide-induced EAE was constructed in wild-type mice. Effects of kurarinone (100 mg/kg/day) upon clinical scores were assessed based on physical traits and signs. Spinal cord sections were extracted to assess inflammation, demyelination, and mRNA expression of key pro-inflammatory cytokines and chemokines. CNS-infiltrating mononuclear cells (MNCs) and splenocytes were harvested; flow cytometry was then applied to determine CD4+ and CD8+ T-cell percentages as well as Th1/Th2/Th17 subset percentages. Purified naïve CD4+ T-cells underwent in vitro T-cell polarization and proliferation to assess kurarinone's effects. ResultsProphylactic and treatment regimens of kurarinone significantly improved clinical scores in the MOG35-55 peptide-induced EAE model (P < 0.05). Kurarinone significantly lowered CNS inflammation and demyelination (61% and 83% decreases, respectively; P < 0.05), significantly decreased MNC infiltration into CNS tissue (42% decrease; P < 0.05), and significantly inhibited levels of several pro-inflammatory cytokines and chemokines (P < 0.05). Kurarinone significantly lowered CD4+ and CD8+ CNS T-cell counts (51% and 80% decreases, respectively; P < 0.05) and significantly reduced CNS Th1 and Th17 cell percentages (24% and 44% decreases, respectively; P < 0.05). Kurarinone significantly inhibited in vitro Th1, Th2, and Th17 cell differentiation and proliferation (P < 0.05). ConclusionsKurarinone significantly inhibits the clinical progression of EAE through the inhibition of Th1 and Th17 cell differentiation and proliferation. Kurarinone may show promise as an immunomodulatory therapeutic agent in treating MS.

Direct modulation of myelin-autoreactive CD4+ and CD8+ T cells in EAE mice by a tolerogenic nanoparticle co-carrying myelin peptide-loaded major histocompatibility complexes, CD47 and multiple regulatory molecules.

PurposeNumerous nanomaterials have been reported in the treatment of multiple sclerosis or experimental autoimmune encephalomyelitis (EAE). But most of these nanoscale therapeutics deliver myelin antigens together with toxins or cytokines and underlay the cellular uptake and induction of tolerogenic antigen-presenting cells by which they indirectly induce T cell tolerance. This study focuses on the on-target and direct modulation of myelin-autoreactive T cells and combined use of multiple regulatory molecules by generating a tolerogenic nanoparticle.Materials and methodsPoly(lactic-co-glycolic acid) nanoparticles (PLGA-NPs) were fabricated by co-coupling MOG40–54/H-2Db-Ig dimer, MOG35–55/I-Ab multimer, anti-Fas, PD-L1-Fc and CD47-Fc and encapsulating transforming growth factor-β1. The resulting 217 nm tolerogenic nanoparticles (tNPs) were administered intravenously into MOG35–55 peptide-induced EAE mice, which was followed by the investigation of therapeutic outcomes and the in vivo mechanism.ResultsFour infusions of the tNPs durably ameliorated EAE with a marked reduction of clinical score, neuroinflammation and demyelination. They were distributed in secondary lymphoid tissues, various organs and brain after intravenous injection, with retention over 36 h, and made contacts with CD4+ and CD8+ T cells. Two injections of the tNPs markedly decreased the MOG35–55-reactive Th1 and Th17 cells and MOG40–55-reactive Tc1 and Tc17 cells, increased regulatory T cells, inhibited T cell proliferation and elevated T cell apoptosis in spleen. Transforming growth factor-β1 and interleukin-10 were upregulated in the homogenates of central nervous system and supernatant of spleen cells.ConclusionOur data suggest a novel therapeutic nanoparticle to directly modulate autoreactive T cells by surface presentation of multiple ligands and paracrine release of cytokine in the antigen-specific combination immunotherapy for T cell-mediated autoimmune diseases.

Effects of C2ta genetic polymorphisms on MHC class II expression and autoimmune diseases.

Antigen presentation by the MHC-II to CD4+ T cells is important in adaptive immune responses. The class II transactivator (CIITA in human and C2TA in mouse) is the master regulator of MHC-II gene expression. It coordinates the transcription factors necessary for the transcription of MHC-II molecules. In humans, genetic variations in CIITA have been associated with differential expression of MHC-II and susceptibility to autoimmune diseases. Here we made use of a C2ta congenic mouse strain (expressing MHC-II haplotype H-2q ) to investigate the effect of the natural genetic polymorphisms in type I promoter of C2ta on MHC-II expression and function. We demonstrate that an allelic variant in the type I promoter of C2ta resulted in an increased expression of MHC-II on macrophages (72-151% higher mean florescence intensity) and conventional dendritic cells (13-65% higher mean florescence intensity) in both spleen and peripheral blood. The increase in MHC-II expression resulted in an increase in antigen presentation to T cells in vitro and increased T-cell activation. The differential MHC-II expression in B6Q.C2ta, however, did not alter the disease development in models of rheumatoid arthritis (collagen-induced arthritis and human glucose-6-phosphate-isomerase325-339 -peptide-induced arthritis), or multiple sclerosis (MOG1-125 protein-induced and MOG79-96 peptide-induced experimental autoimmune encephalomyelitis). This is the first study to address the role of an allelic variant in type I promoter of C2ta in MHC-II expression and autoimmune diseases; and shows that C2ta polymorphisms regulate MHC-II expression and T-cell responses but do not necessarily have a strong impact on autoimmune diseases.

IL-3 promotes the development of experimental autoimmune encephalitis.

Little is known about the role of IL-3 in multiple sclerosis (MS) in humans and in experimental autoimmune encephalomyelitis (EAE). Using myelin oligodendrocyte glycoprotein (MOG) peptide-induced EAE, we show that CD4+ T cells are the main source of IL-3 and that cerebral IL-3 expression correlates with the influx of T cells into the brain. Blockade of IL-3 with monoclonal antibodies, analysis of IL-3 deficient mice, and adoptive transfer of leukocytes demonstrate that IL-3 plays an important role for development of clinical symptoms of EAE, for migration of leukocytes into the brain, and for cerebral expression of adhesion molecules and chemokines. In contrast, injection of recombinant IL-3 exacerbates EAE symptoms and cerebral inflammation. In patients with relapsing-remitting MS (RRMS), IL-3 expression by T cells is markedly upregulated during episodes of relapse. Our data indicate that IL-3 plays an important role in EAE and may represent a new target for treatment of MS.

Nrf2-dysregulation correlates with reduced synthesis and low glutathione levels in experimental autoimmune encephalomyelitis.

This study investigates the possible mechanism(s) underlying glutathione (GSH) deficiency in the mouse spinal cord during the course of myelin oligodendrocyte glycoprotein35-55 peptide-induced experimental autoimmune encephalomyelitis (EAE), a commonly used animal model of multiple sclerosis. Using the classical enzymatic recycling method and a newly developed immunodot assay, we first demonstrated that total GSH levels (i.e. free GSH plus all its adducts) are reduced in EAE, suggesting an impaired synthesis. The decline in the levels of this essential antioxidant tripeptide in EAE coincides temporally and in magnitude with a reduction in the amount of γ-glutamylcysteine ligase, the rate-limiting enzyme in GSH synthesis. Other enzymes involved in GSH biosynthesis, whose genes also contain antioxidant-response elements, including glutathione synthetase, cystine/glutamate antiporter, and γ-glutamyl transpeptidase (γ-GT) are diminished in EAE as well. Low levels of γ-glutamylcysteine ligase, glutathione synthetase, and γ-GT are the consequence of reduced mRNA expression, which correlates with diminished expression of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in both the cytosol and nucleus. Interestingly, the low Nrf2 expression does not seem to be caused by increased degradation via Kelch-like ECH-associated protein 1-dependent or Kelch-like ECH-associated protein 1-independent mechanisms (such as glycogen synthetase kinase-3β activation), or by reduced levels of Nrf2 mRNA. This suggests that translation of this important transcription factor and/or other still unidentified post-translational processes are altered in EAE. These novel findings are central toward understanding how critical antioxidant and protective responses are lost in inflammatory demyelinating disorders.

Antigen-oriented T cell migration contributes to myelin peptide induced-EAE and immune tolerance

Treatment with soluble myelin peptide can efficiently and specifically induce tolerance to demyelination autoimmune diseases including multiple sclerosis, however the mechanism underlying this therapeutic effect remains to be elucidated. In actively induced mouse model of experimental autoimmune encephalomyelitis (EAE) we analyzed T cell and innate immune cell responses in the central nervous system (CNS) and spleen after intraperitoneal (i.p.) infusion of myelin oligodendrocyte glycoprotein (MOG). We found that i.p. MOG infusion blocked effector T cell recruitment to the CNS and protected mice from EAE and lymphoid organ atrophy. Innate immune CD11b+ cells preferentially recruited MOG-specific effector T cells, particularly when activated to become competent antigen presenting cells (APCs). During EAE development, mature APCs were enriched in the CNS rather than in the spleen, attracting effector T cells to the CNS. Increased myelin antigen exposure induced CNS-APC maturation, recruiting additional effector T cells to the CNS, causing symptoms of disease. MOG triggered functional maturation of splenic APCs. MOG presenting APCs interacted with MOG-specific T cells in the spleen, aggregating to cluster around CD11b+ cells, and were trapped in the periphery. This process was MHC II dependent as an MHC II directed antibody blocked CD4+ T cell cluster formation. These findings highlight the role of myelin peptide-loaded APCs in myelin peptide-induced EAE and immune tolerance.

Induction of autoimmune disease by deletion of CTLA-4 in mice in adulthood

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is essential for immunological (self-) tolerance, but due to the early fatality of CTLA-4 KO mice, its specific function in central and peripheral tolerance and in different systemic diseases remains to be determined. Here, we further examined the role of CTLA-4 by abrogating CTLA-4 expression in adult mice and compared the resulting autoimmunity that follows with that produced by congenital CTLA-4 deficiency. We found that conditional deletion of CTLA-4 in adult mice resulted in spontaneous lymphoproliferation, hypergammaglobulinemia, and histologically evident pneumonitis, gastritis, insulitis, and sialadenitis, accompanied by organ-specific autoantibodies. However, in contrast to congenital deficiency, this was not fatal. CTLA-4 deletion induced preferential expansion of CD4(+)Foxp3(+) Treg cells. However, T cells from CTLA-4-deficient inducible KO mice were able to adoptively transfer the diseases into T cell-deficient mice. Notably, cell transfer of thymocytes de novo produced myocarditis, otherwise not observed in donor mice depleted in adulthood. Moreover, CTLA-4 deletion in adult mice had opposing impacts on induced autoimmune models. Thus, although CTLA-4-deficient mice had more severe collagen-induced arthritis (CIA), they were protected against peptide-induced experimental autoimmune encephalomyelitis (EAE); however, onset of protein-induced EAE was only delayed. Collectively, this indicates that CTLA-4 deficiency affects both central and peripheral tolerance and Treg cell-mediated suppression.

Therapeutic effect of baicalin on experimental autoimmune encephalomyelitis is mediated by SOCS3 regulatory pathway.

Natural compounds derived from medicinal plants have long been considered a rich source of novel therapeutic agents. Baicalin (Ba) is a bioactive flavonoid compound derived from the root of Scutellaria baicalensis, an herb widely used in traditional medicine for the treatment of various inflammatory diseases. In this study, we investigate the effects and mechanism of action of Ba in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Ba treatment effectively ameliorated clinical disease severity in myelin oligodendrocyte glycoprotein (MOG)35–55 peptide-induced EAE, and reduced inflammation and demyelination of the central nervous system (CNS). Ba reduced infiltration of immune cells into the CNS, inhibited expression of proinflammatory molecules and chemokines, and prevented Th1 and Th17 cell differentiation via STAT/NFκB signaling pathways. Further, we showed that SOCS3 induction is essential to the effects of Ba, given that the inhibitory effect of Ba on pathogenic Th17 responses was largely abolished when SOCS3 signaling was knocked down. Taken together, our findings demonstrate that Ba has significant potential as a novel anti-inflammatory agent for therapy of autoimmune diseases such as MS.

C-reactive protein directly suppresses Th1 cell differentiation and alleviates experimental autoimmune encephalomyelitis.

Human C-reactive protein (CRP) is a serum-soluble pattern recognition receptor that serves as a marker of inflammation and directly contributes to innate immunity. In this study, we show that human CRP also directly contributes to adaptive immunity, that is, native CRP binds specifically to human Jurkat T cells and to mouse naive CD4(+) T cells and modulates their Th1 and Th2 responses. In vitro both exogenously added (purified) and endogenously expressed (via transfection) human CRP inhibited Th1 differentiation and augmented Th2 differentiation of naive CD4(+) T cells. In vivo for human CRP transgenic compared with wild-type mice, a lesser proportion of the T cells recovered from the spleens of healthy animals were Th1 cells. Moreover, in both CRP transgenic mice and in wild-type mice treated with human CRP, during myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis both the Th1 cell response and disease severity were inhibited. These pattern recognition-independent actions of CRP directly on T cells highlights the potential for this soluble pattern recognition receptor to act as a tonic regulator of immunity, shaping global adaptive immune responses during both homeostasis and disease.

Dual Inhibition of Interleukin-23 and Interleukin-17 Offers Superior Efficacy in Mouse Models of Autoimmunity.

Therapies targeting either interleukin (IL)-23 or IL-17 have shown promise in treating T helper 17 (Th17)-driven autoimmune diseases. Although IL-23 is a critical driver of IL-17, recognition of nonredundant and independent functions of IL-23 and IL-17 has prompted the notion that dual inhibition of both IL-23 and IL-17 could offer even greater efficacy for treating autoimmune diseases relative to targeting either cytokine alone. To test this hypothesis, we generated selective inhibitors of IL-23 and IL-17 and tested the effect of either treatment alone compared with their combination in vitro and in vivo. In vitro, using a novel culture system of murine Th17 cells and NIH/3T3 fibroblasts, we showed that inhibition of both IL-23 and IL-17 completely suppressed IL-23-dependent IL-22 production from Th17 cells and cooperatively blocked IL-17-dependent IL-6 secretion from the NIH/3T3 cells to levels below either inhibitor alone. In vivo, in the imiquimod induced skin inflammation model, and in the myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis model, we demonstrated that dual inhibition of IL-17 and IL-23 was more efficacious in reducing disease than targeting either cytokine alone. Together, these data support the hypothesis that neutralization of both IL-23 and IL-17 may provide enhanced benefit against Th17 mediated autoimmunity and provide a basis for a therapeutic strategy aimed at dual targeting IL-23 and IL-17.

73: Enhanced in vivo efficacy using a novel long-life type I interferon variant in a mouse model of multiple sclerosis

IFN β is a common therapeutic option to treat multiple sclerosis (MS). It is unique amongst the family of type I IFNs in that it binds to the interferon receptors with high affinity, conferring exceptional biological properties. We have previously reported the generation of an interferon superagonist (dubbed YNS α 8) that is built on the backbone of a low affinity IFN, but modified to exhibit higher receptor affinity than IFN β . Here, YNS α 8 was fused with a 600-residue hydrophilic, unstructured N-terminal polypeptide chain, comprising Proline, Alanine and Serine (PAS), in order to prolong its plasma half-life via “PASylation”. PAS-IFN α 8 exhibited a 10-fold increased half-life in both pharmacodynamic and pharmacokinetic assays in a transgenic mouse model harboring the human receptors [1] , notably without any detectable loss in biological potency or bioavailability. This long-life superagonist conferred significantly improved protection from MOG35–55 peptide-induced experimental autoimmune encephalomyelitis (EAE) compared to IFN β , despite being injected with a 4-fold decrease in frequency and at an overall 16-fold lower dosage. These data were corroborated by FACS and immunohistochemistry, showing a decrease of myeloid lineage cells in the central nervous system (CNS) for PAS-YNS α 8 treated animals. Importantly, PAS-IFN α 8 did not induce antibodies upon repeated administration, and its biological efficacy remained unchanged after 21 days of treatment. We next extracted RNA from spleen-derived CD4 + cells and performed high throughput qPCR for ∼ 200 IFN-response genes enriched for immune function. A striking strong correlation of gene expression and EAE clinical response was observed for two genes - CD274 (PD-L1) and CXCR3. An IFN-responsive role for PD-L1 in EAE clinical response has been recently reported elsewhere [2] . This preclinical study thus supports that we have generated a novel, potent and pharmacologically long-acting IFN-variant with potential to treat multiple sclerosis with greater efficacy than current IFN therapies available for use today. It is also provides us with a means to unravel the central IFN-response genes that are providing clinical benefit in EAE and potentially for MS.