title: Lentivirus-mediated estrogen receptor α overexpression in the central nervous system ameliorates experimental autoimmune encephalomyelitis in mice.
abstract: Multiple sclerosis (MS) is an autoimmune disease characterized by inflammatory cell infiltration of the central nervous system (CNS) and multifocal demyelination. Clinical data and clinical indicators demonstrate that estrogen improves the relapse-remittance of MS patients. This study aimed to investigate the anti-inflammatory effects and the underlying mechanism(s) of action of estrogen and estrogen receptor α (ERα) in an experimental autoimmune encephalomyelitis (EAE) mouse model of MS. An ERα recombinant lentivirus was constructed. Mouse neurons were cultured in serum-free culture medium, and ERα recombinant lentivirus with a multiplicity of infection (MOI) of 5 was used to infect the neurons. Furthermore, neuronal ERα mRNA and protein expression were detected using real-time quantitative PCR and western blot analysis. We sterotaxically injected ERα recombinant lentivirus into the lateral ventricle of mouse brains, and successfully identified infected neurons using Flag immunofluorescence staining to determine the optimal dose. A total of 75 C57BL/6 mice were ovariectomized. After 2 weeks, EAE was induced with myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide. The EAE mice were divided into 5 groups: the estrogen group (treatment with estradiol), the ERα agonist group (treatment with raloxifene), the ERα recombinant lentivirus group (ERα group, treatment with ERα recombinant lentivirus), the empty virus group and the normal saline (NS) group; clinical symptoms and body weight were compared among the groups. We assessed EAE-related parameters, detected pathological changes with immunohistochemistry and quantified the expression of myelin basic protein (MBP), matrix metalloproteinase-9 (MMP-9), and a subset of EAE-related cytokines using enzyme-linked immunosorbent assay (ELISA). We successfully constructed an ERα recombinant lentivirus. C57BL/6 mouse neurons can survive in culture for at least 8 weeks. During that period, the recombinant lentivirus was able to infect the neurons, while sustaining green fluorescence protein (GFP) expression. ERα recombinant lentivirus also infected the neurons at a MOI of 5. The ERα mRNA and protein expression levels were higher in the infected neurons compared to the uninfected ones. We successfully infected the CNS of C57BL/6 mice by stereotaxically injecting ERα recombinant lentivirus into the lateral ventricle of the mouse brains and induced EAE. The lentivirus-mediated overexpression of ERα reduced the incidence of EAE, ameliorated the clinical symptoms, inhibited inflammatory cell CNS infiltration, and reduced nerve fiber demyelination. MMP-9, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin (IL)-17 and IL-23 expression levels were decreased, while those of MBP and IL-4 were increased. These data demonstrate that it is possible to induce the overexpression of ERα using a recombinant lentivirus, and that this novel intervention ameliorates EAE in a mouse model. Mechanistically, estrogen and ERα inhibit inflammatory responses, and ERα alleviates damage to the myelin sheath. Collectively, our findings support the potential use of ERα as a therapeutic target for the treatment of MS.
authors.affiliation: Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, P.R. China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Hu
authors.forename: Xinyue
authors.initials: X
authors.lastname: Qin
mesh_terms: D000818:Animals; D001835:Body Weight; D001921:Brain / Q000378:metabolism / Q000473:pathology; D002490:Central Nervous System / Q000378:metabolism* / Q000473:pathology; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D003711:Demyelinating Diseases / Q000150:complications / Q000473:pathology; D004681:Encephalomyelitis, Autoimmune, Experimental / Q000150:complications / Q000235:genetics* / Q000473:pathology / Q000517:prevention & control*; D047628:Estrogen Receptor alpha / Q000235:genetics* / Q000378:metabolism / Q000627:therapeutic use*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007150:Immunohistochemistry; D007249:Inflammation / Q000150:complications / Q000473:pathology; D016086:Lentivirus / Q000378:metabolism*; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D004676:Myelin Basic Protein; D009412:Nerve Fibers / Q000378:metabolism / Q000473:pathology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics; D013116:Spinal Cord / Q000201:enzymology / Q000473:pathology; D014162:Transfection
chemical_list: D016207:Cytokines; D047628:Estrogen Receptor alpha; D004676:Myelin Basic Protein; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D020780:Matrix Metalloproteinase 9