Citations

Abstract Multiple sclerosis is a chronic inflammatory and demyelinating disease of the CNS. Although, MS is well characterized in terms of the role played by immune cells, cytokines and CNS pathology, nothing is known about the metabolic alterations that occur during the disease process in circulation. Here, we profiled the plasma metabolites of B6 with EAE, a known model of chronic MS using untargeted global metabolomics. Using a combination of high-throughput liquid-and-gas chromatography with mass spectrometry, a total of 326 metabolites were identified, with significant changes observed in 105 metabolites (13 up- and 92 down-regulated), that mapped to lipid and amino acid pathways, followed by the peptide, xenobiotic, carbohydrate, nucleotide and energy pathways (p<0.05). To understand the functional role of these alterations, the KEGG metabolic library was analyzed using Metaboanalyst. The top nine most significant pathways in terms of GlobeTest p-value and impact are 1) fatty acid biosynthesis, 2) lysine degradation, 3) histidine metabolism, 4) beta-alanine metabolism, 5) pentose phosphate pathway, 6) arachidonic acid metabolism, 7) linoleic acid, 8) D-arginine and ornithine. Overall, these metabolic changes could be exploited as biomarkers for EAE/MS disease and to design new treatment paradigms where metabolic interventions could be combined with present and experimental therapeutics to achieve better treatment.

Abstract In search of metabolites signature as biomarkers during EAE disease, we profiled urine from B6 EAE using global untargeted metabolomics. Evaluation of metabolomic profiling of urine from EAE and healthy B6 group by using a combination of high-throughput liquid-and-gas chromatography with mass spectrometry, we found that 132 out of 322 (41%) metabolites were differentially altered (P<0.05) indicating robust alteration in the urine metabolomics profile during disease. Among the perturbed metabolites, 11 were up regulated in EAE urine whereas 121 were down regulated. We conducted pathway analysis of the biochemical pathways of the KEGG and considered both concerted changes in metabolite intensity within the pathway (GlobalTest) and alterations of high impact, and found that a number of pathways were significantly altered including glyoxylate and dicarboxylate, phenylanine metabolism, porphyrin & chlorophyll metabolism, primary bile acid biosynthesis, cysteine & methione metabolism, taurine and hypotaurine metabolism, glycine, serine & threonine metabolism, and beta-alanine metabolism. Alteration in these pathways during EAE disease suggesting that perturbation of certain central metabolites could have impact on multiple metabolic pathways. While some of these metabolite changes could easily be developed as biomarkers, the key to translating metabolomics into therapeutics would require figuring out the central altered metabolic pathway(s), once studied in detail.

Abstract AMPK is an energy-sensing metabolic switch implicating in various metabolic disorders. Here we report its new role in controlling the immunopathology of experimental autoimmune encephalitis by polarizing pro-inflammatory phenotype of Macrophage. AMPK alpha1 knockout (alpha1KO) developed severe EAE due to higher expression of IL17 with reduced IFNy and IL4. AMPK alpha1KO also exhibited increased number of infiltrated mononuclear cells in CNS due to impaired blood brain barrier integrity. Macrophage from alpha1KO mice exhibited M1 type phenotype (IL10lowIL12/23high) with stronger capacity to induce allogenic and antigen-specific T-cell response and also heightened the encephalitogenic property of MOG35-55-primed CD4 T cells in B6 mice. Collectively, our data shows AMPK controls the clinical outcome of disease by regulating M1 phenotype-Th17 axis in animal model of MS.

Abstract AMPK is an energy sensing metabolic switch in mammalian cells and regulates diverse metabolic pathway in cells. Recently, we observed that AMPK is lost in all immune cells of Experimental autoimmune encephalomyelitis (EAE), widely used animal model for MS. AMPK is lost at protein level in spleen macrophages, total T cells and their subsets (CD4, CD8 and regulatory T cells) isolated from EAE afflicted animals compared to control. To examine the role of AMPK, EAE was induced in Wt (+/+) and AMPKalpha1 null mice (-/-) and found that AMPKalpha1 (-/-) mice exhibited severe EAE disease. AMPKalpha2 is not involved in enhancing the severity of the disease. Moreover, AMPKalpha1 null mice significantly exhibited severe inflammation and demyelination in CNS. Loss of AMPKalpha1 leads to higher production of IL17 cytokine and reduced production of IFNgamma and IL4 under recall response with MOG35-55 as antigen. AMPKalpha1 KO mice exhibited higher number of infiltrated mononuclear cells in CNS and produced higher amount of IL17 significantly compared to Wt mice. Moreover, adoptive transfer of MOG35-55 specific T cells from AMPKalpha1 KO in Wt mice (KO to Wt) induced significantly higher degree of clinical disease compared to adoptive transfer of MOG specific T cells from Wt to Wt mice suggesting loss of AMPK negatively regulate clinical disease of EAE and identify AMPK as a novel therapeutic target for MS.

Abstract MHC class Ia molecules are regulators of antiviral immunity and are thought to function similarly in the presentation of self and antigenic peptides to T cells. However, differences in natural polymorphisms found among class Ia loci in humans and mice indicate these genes may not function equivalently. We used transgenic mice to evaluate the equivalence of MHC Ia gene function in directing cellular immunity against the picornavirus, Theiler’s encephalomyelitis virus, and found that effective antiviral immunity was promoted by H-2D transgenes but not by H-2K in this system. Because transgenes evaluated in our study encoded the peptide binding domain of the Db antigen presenting molecule expressed in the context of the Db or Kb genetic framework, our findings indicate that structural features distinguishing these genes mapping outside of these coding sequences determine ability to direct effective viral immunity. Irrespective of which transgene was expressed, the mice developed robust anti viral T cell responses against the immunodominant TMEV epitope and the T cells were recruited to infected tissue. However, only when the Db antigen binding domain was expressed in the context of the H-2D genetic framework was virus cleared. Our findings suggest that differences in tissue specific class I gene expression at the site of infection is a key factor in this differential function of class Ia genes. Such difference could underlie natural variations in class Ia polymorphism.

Abstract MS is an inflammatory demyelinating disease of CNS with a presumed autoimmune origin. Prevalent data from animal model of disease, experimental autoimmune encephalomyelitis (EAE) suggests that myelin-specific CD4 T-cells play a major role in disease. Role of CD8 T-cells in MS/EAE has been enigmatic as it has been assigned either regulatory or pathogenic role in disease. Previously we have shown that PLPp91-110 peptide can induce inflammatory neurological disease in HLA-DR3 transgenic (Tg) mice. In order to understand the role of CD8 T cells in this humanized model, we generated HLA-DR3 Tg mice lacking CD8 T cells (DR3.CD8-/-). These mice showed earlier onset and more severe disease when compared to CD8 sufficient HLA-DR3 Tg mice indicating that CD8 T-cells plays a regulatory role. However, pathological analysis of DR3.CD8-/- mice with EAE showed less inflammation and demyelination in CNS compared to DR3.CD8 sufficient mice suggesting a second pathogenic role of CD8 T cells. Thus, our data suggests both a regulatory as well as a pathogenic role of CD8 T cells in EAE and possibly in MS. We have characterized both subsets in detail and show that CD8+CD122- T cells are regulatory subset of CD8 T cells and suppress CD4 T cells by inhibiting antigen presentation pathway, while CD8+CD122- T cells are pathogenic subset. Thus ours is the first study to show a dual role of CD8 T cells in the onset, incidence, progression, severity and pathogenesis of EAE/MS.

Abstract Intracranial infection of susceptible mice with Theiler’s virus results in persistent infection and spinal cord demyelination similar to human multiple sclerosis. While central nervous system infiltrating lymphocytes (CNS-ILs) in these mice display no virus-specific CTL activity, the cells were found to be activated killers using a specificity-independent assay. We previously demonstrated that the depletion of T cells in persistently infected mice significantly decreases demyelinating disease. Consequently, we have investigated the killing pathways employed by CNS-ILs that are isolated from persistently infected animals, the relative contribution of CD4 and CD8 cells in the generation of these CTLs, and the reactivity of this cell population to two putative autoantigens in the CNS. In vitro or in vivo manipulation of T cell populations using Abs or genetic knockout strategies demonstrate that the cytotoxic activity is primarily mediated by CD8+ T cells, and that perforin is an important molecule in the effector pathway. Since effector functions in infected mice were not inhibited by the depletion of CD4 cells with mAb but was blocked genetically in CD4 knockout mice, CD4+ T cells appear to play a helper role in the generation of CD8+ CTLs. We found no evidence of autoimmune-mediated demyelination, as the CD8+ CTLs were not reactive to two major myelin autoantigens, myelin basic protein and proteolipid protein. Our finding that CNS-ILs that are isolated from mice susceptible to persistent virus infection are neither specific for virus or myelin autoantigens is consistent with the possibility that CD8+ CTLs mediate CNS damage as a result of nonspecific activation by virus.

Abstract We used a murine model of multiple sclerosis (MS) induced by Theiler's murine encephalomyelitis virus (TMEV) to test the effect of IL-6 on central nervous system (CNS) demyelination. Administration of human rIL-6 (2.5 micrograms/dose), beginning one day before infection and then twice daily for 28 days, dramatically reduced demyelination and inflammation in the spinal cord of susceptible SJL/J mice. Benefit also was observed when rIL-6 was used as a therapeutic agent and begun on day 15 after infection, a time in which there is the first evidence of inflammation and demyelination in the spinal cord. Suppression of myelin damage by treatment with rIL-6 was associated with fewer virus Ag-positive cells in the spinal cord. Infectious CNS virus titers, as measured by plaque assay, were reduced in rIL-6-treated animals on day 15 after infection, but not on day 7, 22, or 29 after infection. Total serum Igs and virus-specific Igs, as detected by indirect ELISA, were increased markedly in rIL-6-treated mice, whereas no effect was observed on TMEV-neutralizing Ab titers. In vivo administration of rIL-6 inhibited a murine CNS-demyelinating disease induced by a virus, suggesting that this IL may have application for the treatment of human MS.

Abstract In susceptible mouse strains, the wild-type Daniel’s (wt-DA) strain of Theiler’s murine encephalomyelitis virus induces a persistent central nervous system (CNS) infection with chronic demyelination. The virus is cleared from resistant mice with no resulting demyelination. We characterized the role of the DA L* protein in late demyelination and persistent infection. The DA genome has two alternative reading frames, encoding the virus polyprotein and L*, respectively. The mutant virus DAL*-1 fails to synthesize L* and does not persist in the CNS of wt-DA-susceptible SJL/J or B10.S mice. Since class I-restricted cytotoxicity has been shown to determine resistance to virus persistence and demyelination in this model, virus-specific cytotoxicity in the CNS of DA-resistant (B6 or B10) and -susceptible (SJL/J and B10.S) mice during the acute stage of DA and DAL*-1 infection was characterized. Following intracerebral inoculation with DAL*-1, virus-specific Db- and Kb-restricted CTLs were demonstrated in the CNS of resistant B10 mice, whereas only Db-restricted CTL were found in wt-DA-inoculated mice. CTLs specific to wt-DA or DAL*-1 recognized class I-presented peptides from either of the viruses. Of particular interest, Ks-restricted virus-specific cytotoxicity-restricted CTLs were identified in the CNS of susceptible SJL/J (H-2s) and B10.S (H-2s) mice inoculated with DAL*-1. In contrast, no virus-specific CTLs were identified in the CNS of SJL/J and B10.S mice inoculated with wt-DA. We propose that L* inhibits the generation of H-2K-restricted virus-specific cytotoxicity in the CNS, permitting a persistent infection in susceptible strains, with subsequent inflammatory demyelination in the CNS similar to that in human multiple sclerosis.

Abstract Antibodies directed against self-Ags are frequently considered detrimental, and have been shown to play a pathogenic role in certain autoimmune diseases. However, the presence of autoreactive Abs in normal individuals suggests that some autoantibodies could participate in normal physiology. Our previous studies demonstrated that monoclonal autoantibodies SCH94.03 and SCH94.32, generated from the splenocytes of uninfected SJL/J mice injected with normal homogenized spinal cord, promote central nervous system remyelination when passively transferred into syngeneic mice chronically infected with Theiler's murine encephalomyelitis virus, an established experimental model of multiple sclerosis. In this study we show that these two monoclonal autoantibodies are identical, and have phenotypic characteristics of natural autoantibodies. By using a solid phase assay system, SCH94.03 and SCH94.32 showed reactivity toward several protein Ags and chemical haptens, with prominent reactivity toward spectrin, (4-hydroxy-3-nitrophenyl)acetyl, and fluorescein. Sequence analysis showed that both SCH94.03 and SCH94.32 were encoded by identical germline Ig light chain V kappa 10/J kappa 1 and heavy chain V23/DFL16.1/JH2 genes, with no definitive somatic mutations. These results indicate that a natural autoantibody participates in a beneficial physiologic response to central nervous system injury.