EAE Disease Research Articles

Astrocytic MHC-I but Not MHC-II Regulates EAE Disease Severity (S17.009)

Astrocytic MHC-I but Not MHC-II Regulates EAE Disease Severity (S17.009)

TCR signaling induces STAT3 phosphorylation to promote TH17 cell differentiation.

TH17 differentiation is critically controlled by "signal 3" of cytokines (IL-6/IL-23) through STAT3. However, cytokines alone induced only a moderate level of STAT3 phosphorylation. Surprisingly, TCR stimulation alone induced STAT3 phosphorylation through Lck/Fyn, and synergistically with IL-6/IL-23 induced robust and optimal STAT3 phosphorylation at Y705. Inhibition of Lck/Fyn kinase activity by Srci1 or disrupting the interaction between Lck/Fyn and STAT3 by disease-causing STAT3 mutations selectively impaired TCR stimulation, but not cytokine-induced STAT3 phosphorylation, which consequently abolished TH17 differentiation and converted them to FOXP3+ Treg cells. Srci1 administration or disrupting the interaction between Lck/Fyn and STAT3 significantly ameliorated TH17 cell-mediated EAE disease. These findings uncover an unexpected deterministic role of TCR signaling in fate determination between TH17 and Treg cells through Lck/Fyn-dependent phosphorylation of STAT3, which can be exploited to develop therapeutics selectively against TH17-related autoimmune diseases. Our study thus provides insight into how TCR signaling could integrate with cytokine signal to direct T cell differentiation.

Genetic deletion of c-Jun amino-terminal kinase 3 (JNK3) modestly increases disease severity in a mouse model of multiple sclerosis

The c-Jun amino terminal kinases (JNKs) regulate transcription, and studies suggest they contribute to neuropathology in the EAE model of MS. To examine the role of the JNK3 isoform, we compared EAE in JNK3 null mice to wild type (WT) littermates. Although disease severity was similar in female mice, in male JNK3 null mice the day of onset and time to reach 100% incidence occurred sooner, and disease severity was increased. While glial activation in spinal cord was similar, white matter lesions were increased in JNK3 null mice. These results suggest JNK3 normally limits EAE disease in a sex-dependent manner.

Infection with LCMV delays the progression of demyelinating disease in EAE.

Abstract Multiple sclerosis is a chronic autoimmune demyelinating disease that affects approximately 2.8 million people worldwide. While several preventative treatments for multiple sclerosis, including Natalizumab and Mitoxantrone exist, there currently is no cure. It is also unknown what provides the initial trigger of disease episodes or exacerbation of the ongoing disease although viral infections might play a role. In contrast to their role as a driver of demyelination, here we investigate whether viral infections play a potential protective role. Using a well-established animal model of chronic-progressive autoimmune disease known as Experimental Autoimmune Encephalomyelitis, we found that infection with lymphocytic choriomeningitis virus (LCMV) infection delayed onset of paralysis as compared to uninfected controls. Consistent with the reduction in EAE disease scores, we identified reduced numbers of myelin-specific CD4 T cells in the CNS. The myelin-specific CD4 T cells were found in the periphery suggesting that the virus impeded their trafficking to the CNS. Additionally, we analyzed that virus-specific CD8 T cells were found in the CNS in preference to the myelin-specific CD4 T cells. Thus, in contrast to the prevailing paradigm of infections driving autoimmune disease progression, we find that viral infection can lessen acute demyelinating disease. R01NS071518 R01AI147641

Gut microbiome-derived isoflavone metabolites may ameliorate experimental autoimmune encephalomyelitis (EAE) through estrogen receptor signaling in CD8 T-cells

Abstract Multiple sclerosis (MS) is an autoimmune demyelinating disease of the CNS which may be influenced by diet- and gut bacteria-produced metabolites. We have shown that phytoestrogen-metabolizing bacteria are reduced in MS patients compared to healthy individuals. The importance of phytoestrogen-metabolizing bacteria was validated in EAE, an animal model of MS, where lack of isoflavones (a type of phytoestrogen) in the diet and/or lack of phytoestrogen-metabolizing bacteria lead to increased disease severity. However, the cellular and molecular pathways through which isoflavones/gut microbiota modulate EAE is unknown. Cellular analysis of the gut and periphery showed that isoflavone-fed mice have an increased number of CD8 T-cell in the colon compared to mice on an isoflavone-free diet. Moreover, CD8 T-cell knockout mice lose isoflavone-mediated disease protection, suggesting an important role of CD8 T-cells in this process. Due to the structural similarity between isoflavone metabolites and endogenous estrogen, we hypothesize that isoflavone metabolites may induce regulatory CD8 T-cells via engagement of estrogen receptors (ERs). Flow cytometric analysis of colonic CD8 T-cells showed that isoflavone-fed mice had higher expression of both ERα and ERβ and produced lower levels of IFNγ, perforin, and IL10. We are utilizing conditional KO mice lacking ERα or ERβ exclusively in CD8 cells to determine the importance of ERs in the generation of diet-induced CD8 T-cells. In summary, our data suggest that bacterial metabolism of isoflavones may lead to EAE disease suppression through immunomodulation of CD8 T-cells via estrogen receptor signaling. Supported by grants from NIH (1R01AI137075-01) and VA (1I01CX002212)

Obesity induced gut dysbiosis contributes to disease severity in an animal model of multiple sclerosis.

BackgroundMultiple sclerosis (MS) is an inflammatory and demyelinating disease of the CNS. The etiology of MS is complex, and results from the interaction of multiple environmental and genetic factors. Although human leukocyte antigen-HLA alleles such as HLA-DR2 and –DR3 are considered the strongest genetic factors, the environmental factors responsible for disease predisposition are not well understood. Recently, diet and gut microbiota have emerged as an important environmental factors linked to the increased incidence of MS. Especially, western diets rich in protein and fat have been linked to the increased incidence of obesity. Numerous clinical data indicate a role of obesity and gut microbiota in MS; however, the mechanistic link between gut microbiota and obesity in the pathobiology of MS remains unclear. The present study determines the mechanisms driving MS severity in the context of obesity utilizing a high-fat diet (HFD) induced obese HLA-DR3 class-II transgenic mouse model of MS.MethodsHLA-DR3 transgenic mice were kept on a standard HFD diet or Normal Chow (NC) for eight weeks. Gut microbiota composition and functional analysis were performed from the fecal DNA of mice. Experimental autoimmune encephalomyelitis-EAE (an animal model of MS) was induced by immunization with the proteolipid protein-PLP91-110 peptide in complete Freud’s Adjuvant (CFA) and pertussis toxin.ResultsWe observed that HFD-induced obesity caused gut dysbiosis and severe disease compared to mice on NC. Amelioration of disease severity in mice depleted of gut microbiota suggested an important role of gut bacteria in severe EAE in obese mice. Fecal microbiota analysis in HFD mice shows gut microbiota alterations with an increase in the abundance of Proteobacteria and Desulfovibrionaceae bacteria and modulation of various bacterial metabolic pathways including bacterial hydrogen sulfide biosynthetic pathways. Finally, mice on HFD showed increased gut permeability and systemic inflammation suggesting a role gut barrier modulation in obesity induced disease severity.ConclusionsThis study provides evidence for the involvement of the gut microbiome and associated metabolic pathways plus gut permeability in obesity-induced modulation of EAE disease severity. A better understanding of the same will be helpful to identify novel therapeutic targets to reduce disease severity in obese MS patients.

Abstract 5 Human Umbilical Cord Blood-Derived Cell Therapy Product, DUOC-01, Promotes Remyelination by Driving the Differentiation of Oligodendrocyte Progenitor Cells

IntroductionDUOC-01 is a macrophage-like cell therapy product manufactured by culturing banked human umbilical cord blood cells under GMP conditions. Currently, the safety of DUOC-01 is being tested as a bridging therapy in children with demyelinating leukodystrophies undergoing unrelated donor umbilical cord blood transplantation after myeloablative conditioning. DUOC-01 protects against loss of function in several preclinical models with demyelinating conditions of the central nervous system, making it an attractive therapy for patients with multiple sclerosis (MS) who experience destruction of myelin sheaths as pathology of their disease. The mechanism by which DUOC-01 promotes remyelination and if it directly influences oligodendrocyte lineage cells is untested.ObjectiveUsing multiple systems (primary oligodendrocyte precursor cell [OPC] cultures, in vitro cerebellar slice cultures, and experimental autoimmune encephalomyelitis [EAE], a mouse model of MS), we examined how DUOC-01 influences numerous steps of pathology and recovery.MethodsUsing a brain slice culture, we added DUOC-01 to the lysophosphatidylcholine (LPC)-treated slices. We quantified myelinated axons by assessing percent co-localization of myelin basic protein and neurofilament in the control, LPC, and LPC+DUOC-01 groups. To test the DUOC-01 effect in the EAE model, we immunized C57BL/6 mice with myelin oligodendrocyte glycoprotein peptide (MOG35-55) in complete Freund’s adjuvant. To match clinical protocols, we incubated DUOC-01 in Ringer’s lactate with hydrocortisone (HC) for 2 hours at room temperature. At the onset of EAE disease symptoms, we injected DUOC-01 into the cerebrospinal fluid by a single intra-cisterna magna injection, then recorded clinical scores daily for 2 weeks. To test if DUOC-01 could directly affect OPCs, we set up a primary OPC culture isolated from neonatal mice and added DUOC-01 treatment to the culture.ResultsIn the cerebellar slice model, we demonstrated a higher number of myelinated neuron fibers in the DUOC-treated group compared with the LPC-treated group. In the EAE model, compared with mice injected with Ringer’s or HC+Ringer’s, mice injected with DUOC-01 derived clinical benefit with lower clinical scores. In the primary OPC culture, the DUOC-01 treatment drove the maturation of OPC to become myelin producing oligodendrocytes.DiscussionOur data suggest that DUOC-01 could be beneficial in treating MS and other diverse neurological demyelinating conditions.

Cell-specific roles for miR-155 during neuroinflammation

Abstract The pro-inflammatory microRNA-155 (miR-155) is highly expressed in the serum and CNS lesions of patients with multiple sclerosis (MS). The whole-body deletion of miR-155 in mice confers resistance to a mouse model of MS, EAE, by reducing the encephalogenic potential of CNS-infiltrating Th17 T cells. Here we utilize single-cell RNA sequencing and cell-specific conditional miR-155 knockouts to determine the importance of miR-155 expression in distinct cell populations. Time course single-cell sequencing revealed reductions in T cells, macrophages, and dendritic cells in whole-body miR-155 knockout mice compared with wild-type controls at day 21. Deletion of miR-155 in T cells, driven by CD4 cre, reduced disease severity similar to whole-body miR-155 knockouts. CD11c cre-driven deletion of miR-155 in dendritic cells also significantly reduced EAE disease score, with both T cell and dendritic cell-specific knockouts showing a reduction in Th17 T cell infiltration into the CNS. Although miR-155 is highly expressed in macrophages during EAE, deletion miR-155 by LysM cre in did not affect disease severity.

Antibiotic treatment during pregnancy and lactation in dams exacerbates clinical symptoms and inflammatory responses in offspring with experimental autoimmune encephalomyelitis

A growing body of evidence demonstrates that an imbalance in the intensive communication between gut microbiota and the host might be associated with immune-related disorders such as multiple sclerosis. This study set out to determine whether antibiotic treatment during pregnancy and lactation can affect the onset and severity of clinical symptoms and inflammatory responses in offspring with experimental autoimmune encephalomyelitis (EAE; a mouse model of multiples sclerosis). Female C57BL/6 mice received antibiotics or vehicles during pregnancy and lactation, then their offspring were induced with EAE in adulthood. We also measured interleukin (IL)-6, tumor necrosis factor (TNF)-α, interferon-gamma (IFN-γ), IL-17A, IL-10, and transforming growth factor (TGF)-β in the serum of offspring. The findings indicate that antibiotic treatment in dams significantly exacerbated the severity of EAE clinical symptoms in both male and female offspring. However, antibiotic treatment only accelerated the onset of EAE disease in male but not female offspring. We did not find any significant changes in cytokines in non-EAE male and female offspring treated with antibiotics. Antibiotic treatment significantly enhanced levels of IL-6, TNF-α, IFN-γ, IL-17A, and TGF-β in EAE-induced male offspring, and IFN-γ, IL-17A, and IL-10 levels in EAE-induced female offspring. There were also sex differences in the onset and severity of EAE disease, and inflammatory cytokines (IL-6, IFN-γ, and IL-17A) between EAE-induced male and female offspring treated with antibiotics. Taken together, this study suggests that antibiotic treatment during pregnancy and lactation in dams might affect the development of the immune system in male and female offspring in adulthood.

Oligodendrocyte-Specific Deletion of FGFR1 Reduces Cerebellar Inflammation and Neurodegeneration in MOG35-55-Induced EAE.

Multiple sclerosis (MS) is a chronic inflammatory and degenerative disease of the central nervous system (CNS). MS commonly affects the cerebellum causing acute and chronic symptoms. Cerebellar signs significantly contribute to clinical disability, and symptoms such as tremor, ataxia, and dysarthria are difficult to treat. Fibroblast growth factors (FGFs) and their receptors (FGFRs) are involved in demyelinating pathologies such as MS. In autopsy tissue from patients with MS, increased expression of FGF1, FGF2, FGF9, and FGFR1 was found in lesion areas. Recent research using mouse models has focused on regions such as the spinal cord, and data on the expression of FGF/FGFR in the cerebellum are not available. In recent EAE studies, we detected that oligodendrocyte-specific deletion of FGFRs results in a milder disease course, less cellular infiltrates, and reduced neurodegeneration in the spinal cord. The objective of this study was to characterize the role of FGFR1 in oligodendrocytes in the cerebellum. Conditional deletion of FGFR1 in oligodendrocytes (Fgfr1ind−/−) was achieved by tamoxifen application, EAE was induced using the MOG35-55 peptide. The cerebellum was analyzed by histology, immunohistochemistry, and western blot. At day 62 p.i., Fgfr1ind−/− mice showed less myelin and axonal degeneration compared to FGFR1-competent mice. Infiltration of CD3(+) T cells, Mac3(+) cells, B220(+) B cells and IgG(+) plasma cells in cerebellar white matter lesions (WML) was less in Fgfr1ind−/−mice. There were no effects on the number of OPC or mature oligodendrocytes in white matter lesion (WML). Expression of FGF2 and FGF9 associated with less myelin and axonal degeneration, and of the pro-inflammatory cytokines IL-1β, IL-6, and CD200 was downregulated in Fgfr1ind−/− mice. The FGF/FGFR signaling protein pAkt, BDNF, and TrkB were increased in Fgfr1ind−/− mice. These data suggest that cell-specific deletion of FGFR1 in oligodendrocytes has anti-inflammatory and neuroprotective effects in the cerebellum in the EAE disease model of MS.

Vascular and immunopathological role of Asymmetric Dimethylarginine (ADMA) in Experimental Autoimmune Encephalomyelitis.

Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide synthase (NOS) inhibitor/uncoupler inducing vascular pathology. Vascular pathology is an important factor for the development and progression of CNS pathology of MS, yet the role of ADMA in MS remains elusive. Patients with multiple sclerosis (MS) are reported to have elevated blood levels of ADMA, and mice with experimental autoimmune encephalomyelitis (EAE, an animal model of MS) generated by auto-immunization of myelin oligodendrocyte glycoprotein (MOG) and blood-brain barrier (BBB) disruption by pertussis toxin also had increased blood ADMA levels in parallel with induction of clinical disease. To explore the role of ADMA in EAE pathogenesis, EAE mice were treated with a daily dose of ADMA. It is of special interest that ADMA treatment enhanced the BBB disruption in EAE mice and exacerbated the clinical and CNS disease of EAE. ADMA treatment also induced the BBB disruption and EAE disease in MOG-immunized mice even without pertussis toxin treatment, suggesting the role of ADMA in BBB dysfunction in EAE. T-cell polarization studies also documented that ADMA treatment promotes TH 1- and TH 17-mediated immune responses but without affecting Treg-mediated immune response in EAE mice as well as in in vitro T-cell culture. Taken together, these data, for the first time, document the vascular and immunopathogenic roles of ADMA in EAE, thus pointing to the potential of ADMA-mediated mechanism as a new target of potential therapy for MS.

Apigenin Modulates Dendritic Cell Activities and Curbs Inflammation Via RelB Inhibition in the Context of Neuroinflammatory Diseases.

Neuroinflammation leads to tissue injury causing many of the clinical symptoms of Multiple Sclerosis, an autoimmune disorder of the central nervous system (CNS). While T cells, specifically Th1 and Th17 cells, are the ultimate effectors of this disease, dendritic cells (DCs) mediate T cell polarization, activation, etc. In our previous study, Apigenin, a natural flavonoid, has been shown to reduce EAE disease severity through amelioration of demyelination in the CNS as well as the sequestering of DCs and other myeloid cells in the periphery. Here, we show that Apigenin exerts its effects possibly through shifting DC modulated T cell responses from Th1 and Th17 type towards Treg directed responses evident through the decrease in T-bet, IFN-γ (Th1), IL-17 (Th17) and increase in IL-10, TGF-β and FoxP3 (Treg) expression in cells from both normal human donors and EAE mice. RelB, an NF-κβ pathway protein is central to DC maturation, its antigen presentation capabilities and DC-mediated T cell activation. Apigenin reduced mRNA and protein levels of RelB and also reduced its nuclear translocation. Additionally, siRNA-mediated silencing of RelB further potentiated the RelB-mediated effects of Apigenin thus confirming its role in Apigenin directed regulation of DC biology. These results provide key information about the molecular events controlled by Apigenin in its regulation of DC activity marking its potential as a therapy for neuroinflammatory disease. Graphical Abstract.

Molecular characteristics of eae-positive clinical Shiga toxin-producing Escherichia coli in Sweden

ABSTRACT Shiga toxin (Stx)-producing Escherichia coli (STEC) can cause a wide range of symptoms from asymptomatic carriage, mild diarrhea to bloody diarrhea (BD) and hemolytic uremic syndrome (HUS). Intimin, encoded by the eae gene, also plays a critical role in STEC pathogenesis. Herein, we investigated the prevalence and genetic diversity of eae among clinical STEC isolates from patients with diarrhea, BD, HUS as well as from asymptomatic STEC-positive individuals in Sweden with whole-genome sequencing. We found that 173 out of 239 (72.4%) of clinical STEC strains were eae positive. Six eae subtypes (ϵ1, γ1, β3, θ, ζ and ρ) were identified eae and its subtype γ1 were significantly overrepresented in O157:H7 strains isolated from BD and HUS patients. ϵ1 was associated with O121:H19 and O103:H2 strains, and β3 to O26:H11 strains. The combination of eae subtype γ1 and stx subtype (stx 2 or stx 1+stx 2) is more likely to cause severe disease, suggesting the possibility of using eae genotypes in risk assessment of STEC infection. In summary, this study demonstrated a high prevalence of eae in clinical STEC strains and considerable genetic diversity of eae in STEC strains in Sweden from 1994 through 2018, and revealed association between eae subtypes and disease severity.

NF-kB/mTOR/MYC axis drives PRMT5 protein induction after T cell activation

Abstract Multiple sclerosis (MS) is a debilitating autoimmune disease of the central nervous system (CNS) mediated by CD4+ T cells. Clinical data has provided evidence that MS patient T cells display an activated or memory phenotype and genome-wide association studies (GWAS) have identified single nucleotide polymorphisms linked to NF-kB complex and MYC genes in MS patients. These data imply a role for TcR (T cell receptor) signaling pathways in MS. PRMT5 is the major type II arginine methyltransferase catalyzing the symmetric dimethylation of histones and other proteins. Our lab has recently reported PRMT5 plays a crucial role in inflammatory T cell expansion and EAE disease. From cancer studies, we are aware of links between NF-kB/MYC signaling and PRMT5 induction. Here, we unravel the impact of NF-kB/MYC pathways in T cell PRMT5 expression and pathogenic T cell responses. We used naïve and memory mouse Th1/Th2 CD4+T cells as models to identify mechanisms controlling PRMT5 protein expression in initial and recall T cell activation. Naïve (initial) mouse T cell activation resulted in NF-kB-dependent transient Prmt5 transcription and NF-kB, mTOR and Myc-dependent PRMT5 protein induction. Recall activation in memory T cells showed rapid induction of PRMT5 protein expression, supported by both transcription and loss of Prmt5-targeting miRNAs. These results highlight the importance of the NF-kB/mTOR/MYC axis in PRMT5-driven pathogenic T cell expansion and may guide targeted therapeutic strategies for MS.

CD40 function in immune development and response: Cell type requirements and cytoplasmic domain function

Abstract CD40 is a TNFR family member and serves as a costimulatory molecule expressed on multiple antigen presenting cell (APC) lineages. The functional significance of CD40 expression is illustrated by human disease and mouse models of life-threatening immune deficiency. CD40-CD40L deficiency affects multiple aspects of immune development and response. However, cell type-specific requirements for CD40 and requirements for CD40 signaling domains have not been comprehensively analyzed. In this study, we use conditional CD40 knockout mice to study cell type requirements and generated CD40 intracellular domain mutants by CRISPR to determine domain-specific functions. CD40 expression on B cells but not DC is required for CD40 dependent superantigen-specific negative selection and for T-cell dependent class-switched antibody responses. TRAF2/3 binding domains of CD40 are critical for these CD40 functions, while deletion of putative TRAF6 binding domain had minimal effect. In contrast, in vivo priming of cytokine-producing T cells depends on CD40 expression on DC but not B cells and requires both TRAF2/3 and TRAF6 domains. CD40 expression on both B cells and DC is required for maximal autoimmune EAE disease induced by recombinant human MOG (rhMOG), and both TRAF2/3 and TRAF6 domains of CD40 are required for this autoimmune response. Initial results indicate that CD40 on thymic epithelial cells and DC contributes to negative selection of tissue restricted antigen-binding T cells. These results identify different CD40-dependent APC requirements and suggest distinct CD40 signaling pathways for specific immune responses. Analysis of the linkage between cell type-specific CD40 function and intracellular signaling is under study.

Immune cell derived BDNF does not mediate neuroprotection of the murine anti-CD52 antibody in a chronic autoimmune mouse model

The murine anti-CD52 antibody, an equivalent of the humanized antibody alemtuzumab, which is successfully used in the treatment of multiple sclerosis, was used to explore a potential neuroprotective effect driven by immune cell derived brain-derived neurotrophic factor (BDNF). Therefore, lineage specific constitutive knock-out mice with a BDNF deficiency in T cells and macrophages were used and compared to treated wildtype mice. Neither therapeutic nor preventive application of the murine anti-CD52 antibody in an animal model of multiple sclerosis, the MOG35–55 EAE, revealed a beneficial contribution of immune cell derived BDNF to the disease outcome. Furthermore, preventive application of the murine anti-CD52 antibody worsened the clinical EAE disease course and could only be overcome by a prolonged recovery phase after treatment and before disease induction.

Isotype-Switched Autoantibodies Are Necessary To Facilitate Central Nervous System Autoimmune Disease in Aicda-/- and Ung-/- Mice.

B cell-depleting therapies have been shown to ameliorate symptoms in multiple sclerosis (MS) patients; however, the mechanism of action remains unclear. Following priming with Ag, B cells undergo secondary diversification of their BCR, including BCR class-switch recombination (CSR) and somatic hypermutation (SHM), with both processes requiring the enzyme activation-induced (cytidine) deaminase. We previously reported that activation-induced (cytidine) deaminase is required for full clinical manifestation of disease in an animal model of MS (experimental autoimmune encephalomyelitis; EAE) provoked by immunization with the extracellular domain of recombinant human myelin oligodendrocyte glycoprotein (hMOG). In this study, we investigated the role of CSR versus SHM in the pathogenesis of EAE. We found that passive transfer of class-switched anti-MOG IgG1 Abs into hMOG-primed Aicda-/- mice is sufficient to fully rescue EAE disease. In addition, we found that the nature of the Ag is an important determinant of EAE severity in Aicda-/- mice because the lack of a diversified BCR does not affect the induction of EAE when immunized with the extracellular domain of rat MOG. To discriminate the effect of either CSR or SHM, we induced EAE in uracil DNA glycosylase-deficient mice (Ung-/-) that exhibit a defect primarily in CSR. We observed that Ung-/- mice exhibit milder clinical disease compared with control mice, concomitant with a reduced amount of anti-MOG IgG1 class-switched Abs that preserved normal affinity. Collectively, these results indicate that CSR plays an important role in governing the incidence and severity of EAE induced with hMOG but not rat MOG.

Gene Therapy induced Tregs: A treatment for relapsing-remitting MS in mice

Abstract Autoimmune disease is caused by a breakdown of tolerance against endogenous protein(s). New therapies are being developed that utilize the natural abilities of regulatory T (Treg) cells to suppress autoimmune effector cells in an antigen-specific manner. Recently, we demonstrated a clinically relevant in vivo gene immunotherapy approach for restoring Ag-specific tolerance using hepatic adeno-associated virus (AAV) gene transfer. This approach was shown to be remarkably successful at preventing and reversing a monophasic, MOG-induced EAE disease. Additionally, we have shown the dynamic ability of this treatment to prevent and/or reverse disease induced with various MOG epitopes, in genetically diverse mouse strains. However, it is unclear if this approach would be effective in the more complicated, PLP induced relapsing-remitting disease model. Thus, we re-engineered our therapeutic vector to contain the full-coding sequence of PLP. This is critically important as the established RR-EAE (SJL) model is characterized by epitope spreading; a phenomenon that may more closely resemble multiple sclerosis. SJL mice received a single peripheral injection of AAV. PLP vector, or control, 2 weeks prior to PLP 139–151 induced EAE. By day 14, control mice developed significant neurological deficits followed by a relapsing-remitting disease course. In contrast, the treated mice failed to exhibit any neurological symptoms of disease throughout the duration of the experiment, ~75 days. The long-term prevention of PLP induced disease, provides the first evidence of an effective Treg based therapy for combating epitope drift in RR-EAE, and potentially a new paradigm for treating the autoimmune disease multiple sclerosis.

Potential Role of Nitrobiolome in Autoimmune Disease: Efficacy of GSNO in EAE.

Abstract S-nitrosoglutathione (GSNO) is a physiological nitric oxide (NO) carrier molecule important in modulation of NO-signaling. Cellular GSNO homeostasis is maintained by its synthesis by nitric oxide synthase (NOS) as well as its catabolism by GSNO reductase (GSNOR). Treatment of EAE mice (wild-type /WT) with exogenous GSNO or GSNOR inhibitor (N6022) significantly alleviated the clinical disease and associated myelin damage. In addition, more profound alleviation of EAE disease was observed in mice deficient in GSNOR (GSNOR−/−). However, EAE disease was further exacerbated in mice deficient in inducible NOS (iNOS−/−) as compared to WT mice. As expected, the spleen levels of S-nitrosothiol proteins were higher in GSNOR−/− EAE mice but lower in iNOS−/− EAE mice compared to WT EAE mice indicating a negative correlation of spleen GSNO levels with EAE disease severities. Both GSNO and N6022 treated EAE mice had decreased TH17 and increased CD4+/CD25+/FOXP3− Treg differentiations without any significant alterations in other CD4+ T cell subset differentiation. Accordingly, CD4 T+ cells from GSNO or N6022 treated EAE mice produced lower amount of IL-17 and higher amount of IL-10 compared to the cells from untreated WT EAE mice. GSNOR−/− EAE mice also exhibited decreased T 17 and increased CD4+/CD25+/FOXP3− Treg differentiations. On the other hand, iNOS−/− EAE mice exhibited increased TH17, but decreased Tregs differentiations compared to WT EAE mice. In summary, these studies document that nitrobiolome (GSNO) participates in differentiation of autoreactive TH17 and Treg subsets during the course of EAE, and that GSNO as a potential therapeutic drug for multiple sclerosis. Supported in-part by VA(BX002829) and NIH(NS037766).

Correction of mitochondrial and peroxisomal defects by lovastatin and AICAR in EAE.

Abstract In multiple sclerosis, the CNS disease progression continues despite treatment with FDA approved immunomodulatory drugs. Recent studies report that loss of mitochondria and peroxisomes contributes to the CNS disease of MS. In EAE, the loss of myelin proteins paralleled the loss of peroxisomes (catalase and PMP70) and loss of mitochondria (complex I and citrate synthase) as well as levels of ATP. Using EAE animal model, we investigated the efficacy of immune modulatory drugs (lovastatin or AMPK activator AICAR) for protections of mitochondria, peroxisomes, and myelin in relation to the clnical disease. While lovastatin or AICAR attenuated the EAE disease, but individually these drugs provided only partial protection of mitochondria and peroxisomes. However, combination therapy of lovastatin and AICAR was able to protect mitochondria, peroxisomes, and myelin as well as greater efficacy against clinical disease of EAE as compared to individual drug treatments. Lovastatin-mediated inhibition of RhoA induced the expression of transcription factors, PPARα, PPARγ, and catalase and thus biogenesis of peroxisomes and AICAR-mediated activation of AMPK induced expression of transcription factors, PGC-1α, SIRT1, and MnSOD and thus biogenesis of mitochondria in EAE spinal cords. In summary, these studies document that oral medication of combination of immunomodulatory drugs (lovastatin and AICAR) protect against the loss of mitochondria and peroxisomes and energy homeostasis in EAE resulting in greater efficacy in EAE supporting a potential therapeutic approach for progressive MS. Supported in-part by VA:BX002829 and NIH:NS037766.