ABSTRACT
Epstein-Barr virus (EBV) is a ubiquitous herpesvirus strongly associated with multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system (CNS). However, the mechanisms linking EBV infection to MS pathology are uncertain. Neuropathological and immunological studies suggest that a persistent EBV infection in the CNS can stimulate a CD8 T-cell response aimed at clearing the virus but inadvertently causing CNS injury. Inasmuch as in situ demonstration of EBV-specific CD8 T cells and their effector function is missing, we searched for EBV-specific CD8 T cells in MS brain tissue using the pentamer technique. Postmortem brain samples from 12 donors with progressive MS and known HLA class I genotype were analyzed. Brain sections were stained with HLA-matched pentamers coupled with immunogenic peptides from EBV-encoded proteins, control virus (cytomegalovirus and influenza A virus) proteins, and myelin basic protein. CD8 T cells recognizing proteins expressed in the latent and lytic phases of the EBV life cycle were visualized in white matter lesions and/or meninges of 11/12 MS donors. The fraction (median value) of CD8 T cells recognizing individual EBV epitopes ranged from 0.5 to 2.5% of CNS-infiltrating CD8 T cells. Cytomegalovirus-specific CD8 T cells were detected at a lower frequency (≤0.3%) in brain sections from 4/12 MS donors. CNS-infiltrating EBV-specific CD8 T cells were CD107a positive, suggesting a cytotoxic phenotype, and stuck to EBV-infected cells. Together with local EBV dysregulation, selective enrichment of EBV-specific CD8 T cells in the MS brain supports the notion that skewed immune responses toward EBV contribute to inflammation causing CNS injury.IMPORTANCE EBV establishes a lifelong and asymptomatic infection in most individuals and more rarely causes infectious mononucleosis and malignancies, like lymphomas. The virus is also strongly associated with MS, a chronic neuroinflammatory disease with unknown etiology. Infectious mononucleosis increases the risk of developing MS, and immune reactivity toward EBV is higher in persons with MS, indicating inadequate control of the virus. Previous studies have suggested that persistent EBV infection in the CNS stimulates an immunopathological response, causing bystander neural cell damage. To verify this, we need to identify the immune culprits responsible for the detrimental antiviral response in the CNS. In this study, we analyzed postmortem brains donated by persons with MS and show that CD8 cytotoxic T cells recognizing EBV enter the brain and interact locally with the virus-infected cells. This antiviral CD8 T cell-mediated immune response likely contributes to MS pathology.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Epstein-Barr Virus Infections/immunology , Epstein-Barr Virus Infections/pathology , Herpesvirus 4, Human/immunology , Multiple Sclerosis/immunology , Multiple Sclerosis/pathology , Adult , Brain/virology , Cytomegalovirus , Female , Genes, MHC Class I , HLA-B Antigens , Humans , Infectious Mononucleosis , Influenza A virus/immunology , Male , Middle AgedABSTRACT
BACKGROUND: It is debated whether multiple sclerosis (MS) might result from an immunopathological response toward an active Epstein-Barr virus (EBV) infection brought into the central nervous system (CNS) by immigrating B cells. Based on this model, a relationship should exist between the local immune milieu and EBV infection status in the MS brain. To test this hypothesis, we analyzed expression of viral and cellular genes in brain-infiltrating immune cells. METHODS: Twenty-three postmortem snap-frozen brain tissue blocks from 11 patients with progressive MS were selected based on good RNA quality and prominent immune cell infiltration. White matter perivascular and intrameningeal immune infiltrates, including B cell follicle-like structures, were isolated from brain sections using laser capture microdissection. Enhanced PCR-based methods were used to investigate expression of 75 immune-related genes and 6 EBV genes associated with latent and lytic infection. Data were analyzed using univariate and multivariate statistical methods. RESULTS: Genes related to T cell activation, cytotoxic cell-mediated (or type 1) immunity, B cell growth and differentiation, pathogen recognition, myeloid cell function, type I interferon pathway activation, and leukocyte recruitment were found expressed at different levels in most or all MS brain immune infiltrates. EBV genes were detected in brain samples from 9 of 11 MS patients with expression patterns suggestive of in situ activation of latent infection and, less frequently, entry into the lytic cycle. Comparison of data obtained in meningeal and white matter infiltrates revealed higher expression of genes related to interferonγ production, B cell differentiation, cell proliferation, lipid antigen presentation, and T cell and myeloid cell recruitment, as well as more widespread EBV infection in the meningeal samples. Multivariate analysis grouped genes expressed in meningeal and white matter immune infiltrates into artificial factors that were characterized primarily by genes involved in type 1 immunity effector mechanisms and type I interferon pathway activation. CONCLUSION: These results confirm profound in situ EBV deregulation and suggest orchestration of local antiviral function in the MS brain, lending support to a model of MS pathogenesis that involves EBV as possible antigenic stimulus of the persistent immune response in the central nervous system.
Subject(s)
Brain/immunology , Disease Progression , Epstein-Barr Virus Infections/immunology , Laser Capture Microdissection/methods , Multiple Sclerosis/immunology , Transcription, Genetic/immunology , Adult , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Brain/pathology , Epstein-Barr Virus Infections/pathology , Female , Humans , Male , Middle Aged , Multiple Sclerosis/pathologyABSTRACT
It has long been known that multiple sclerosis (MS) is associated with an increased Epstein-Barr virus (EBV) seroprevalence and high immune reactivity to EBV and that infectious mononucleosis increases MS risk. This evidence led to postulate that EBV infection plays a role in MS etiopathogenesis, although the mechanisms are debated. This study was designed to assess the prevalence and magnitude of CD8+ T-cell responses to EBV latent (EBNA-3A, LMP-2A) and lytic (BZLF-1, BMLF-1) antigens in relapsing-remitting MS patients (nâ=â113) and healthy donors (HD) (nâ=â43) and to investigate whether the EBV-specific CD8+ T cell response correlates with disease activity, as defined by clinical evaluation and gadolinium-enhanced magnetic resonance imaging. Using HLA class I pentamers, lytic antigen-specific CD8+ T cell responses were detected in fewer untreated inactive MS patients than in active MS patients and HD while the frequency of CD8+ T cells specific for EBV lytic and latent antigens was higher in active and inactive MS patients, respectively. In contrast, the CD8+ T cell response to cytomegalovirus did not differ between HD and MS patients, irrespective of the disease phase. Marked differences in the prevalence of EBV-specific CD8+ T cell responses were observed in patients treated with interferon-ß and natalizumab, two licensed drugs for relapsing-remitting MS. Longitudinal studies revealed expansion of CD8+ T cells specific for EBV lytic antigens during active disease in untreated MS patients but not in relapse-free, natalizumab-treated patients. Analysis of post-mortem MS brain samples showed expression of the EBV lytic protein BZLF-1 and interactions between cytotoxic CD8+ T cells and EBV lytically infected plasma cells in inflammatory white matter lesions and meninges. We therefore propose that inability to control EBV infection during inactive MS could set the stage for intracerebral viral reactivation and disease relapse.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Herpesvirus 4, Human/immunology , Multiple Sclerosis, Relapsing-Remitting/immunology , Multiple Sclerosis, Relapsing-Remitting/virology , Trans-Activators/metabolism , Adult , Antibodies, Monoclonal, Humanized/therapeutic use , Brain/virology , Epstein-Barr Virus Infections/complications , Epstein-Barr Virus Infections/immunology , Epstein-Barr Virus Infections/virology , Female , Humans , Interferon-beta/therapeutic use , Male , Middle Aged , Multiple Sclerosis, Relapsing-Remitting/drug therapy , Natalizumab , Seroepidemiologic Studies , Young AdultABSTRACT
The presence of EBV infected B cells in postmortem multiple sclerosis (MS) brain tissue suggests immune evasion strategies. Using immunohistochemical techniques we analysed the expression of the immune checkpoint molecule PD-L1 and its receptor PD-1 in MS brains containing B cell-enriched perivascular infiltrates and meningeal follicles, a major EBV reservoir. PD-1 and PD-L1 immunoreactivities were restricted to CNS-infiltrating immune cells. PD-L1 was expressed on B cells, including EBV infected B cells, while PD-1 was expressed on many CD8+ T cells, including EBV-specific CD8+ T-cells, and fewer CD4+ T cells. PD-L1+ cells and EBV infected cells were in close contact with PD-1+ T cells. PD-L1 expressed by EBV infected B cells could favour local immune evasion leading to EBV persistence and immunopathology in the MS brain.
Subject(s)
B7-H1 Antigen , Epstein-Barr Virus Infections , Multiple Sclerosis , Humans , B7-H1 Antigen/metabolism , Brain/immunology , Brain/pathology , Brain/virology , Epstein-Barr Virus Infections/immunology , Herpesvirus 4, Human/metabolism , Multiple Sclerosis/immunology , Multiple Sclerosis/virology , Programmed Cell Death 1 Receptor/metabolismABSTRACT
OBJECTIVES: Rheumatoid arthritis (RA) is associated with an increased Epstein-Barr virus (EBV) blood DNA load, a robust immune response to EBV and cross-reactive circulating antibodies to viral and self-antigens. However, the role of EBV in RA pathogenesis remains elusive. Here, we investigated the relationship between synovial EBV infection, ectopic lymphoid structures (ELS) and immunity to citrullinated self and EBV proteins. METHODS: Latent and lytic EBV infection was investigated in 43 RA synovial tissues characterised for presence/absence of ELS and in 11 control osteoarthritis synovia using RT-PCR, in situ hybridisation and immunohistochemistry. Synovial production of anti-citrullinated protein (ACPA) and anti-citrullinated EBV peptide (VCP1/VCP2) antibodies was investigated in situ and in vivo in the severe combined immunodeficiency (SCID)/RA chimeric model. RESULTS: EBV dysregulation was observed exclusively in ELS+ RA but not osteoarthritis (OA) synovia, as revealed by presence of EBV latent (LMP2A, EBV-encoded small RNA (EBER)) transcripts, EBER+ cells and immunoreactivity for EBV latent (LMP1, LMP2A) and lytic (BFRF1) antigens in ELS-associated B cells and plasma cells, respectively. Importantly, a large proportion of ACPA-producing plasma cells surrounding synovial germinal centres were infected with EBV. Furthermore, ELS-containing RA synovia transplanted into SCID mice supported production of ACPA and anti-VCP1/VCP2 antibodies. Analysis of CD4+ and CD8+ T-cell localisation and granzyme B expression suggests that EBV persistence in ELS-containing synovia may be favoured by exclusion of CD8+ T cells from B-cell follicles and impaired CD8-mediated cytotoxicity. CONCLUSIONS: We demonstrated active EBV infection within ELS in the RA synovium in association with local differentiation of ACPA-reactive B cells.
Subject(s)
Arthritis, Rheumatoid/virology , Autoimmunity , Herpesvirus 4, Human/physiology , Osteoarthritis/virology , Plasma Cells/virology , Synovial Membrane/virology , Adult , Aged , Aged, 80 and over , Animals , Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/pathology , Female , Herpesvirus 4, Human/genetics , Herpesvirus 4, Human/isolation & purification , Humans , Lymphoid Tissue , Male , Mice , Mice, SCID , Middle Aged , Osteoarthritis/immunology , Osteoarthritis/pathology , Plasma Cells/immunology , Plasma Cells/pathology , Synovial Membrane/immunology , Synovial Membrane/pathology , Viral LoadABSTRACT
Presence of EBV infected B cells and EBV-specific CD8 T cells in the multiple sclerosis (MS) brain suggests a role for virus-driven immunopathology in brain inflammation. Tissue-resident memory (Trm) T cells differentiating in MS lesions could provide local protection against EBV reactivation. Using immunohistochemical techniques to analyse canonical tissue residency markers in postmortem brains from control and MS cases, we report that CD103 and/or CD69 are mainly expressed in a subset of CD8+ T cells that intermingle with and contact EBV infected B cells in the infiltrated MS white matter and meninges, including B-cell follicles. Some Trm-like cells were found to express granzyme B and PD-1, mainly in white matter lesions. In the MS brain, Trm cells could fail to constrain EBV infection while contributing to sustain inflammation.
Subject(s)
Epstein-Barr Virus Infections , Multiple Sclerosis , Humans , Herpesvirus 4, Human , Multiple Sclerosis/pathology , Memory T Cells , Brain/pathology , CD8-Positive T-LymphocytesABSTRACT
To identify differentially expressed genes in multiple sclerosis, microarrays were used in a stringent experimental setting-leukapheresis from disease-discordant monozygotic twins and gene expression profiling in CD4(+) and CD8(+) T-cell subsets. Disease-related differences emerged only in the CD8(+) T-cell subset. The five differentially expressed genes identified included killer cell lectin-like receptor subfamily B, member 1, also known as natural killer receptor protein 1a/CD161, presented by the International Multiple Sclerosis Genetics Consortium as one of the non-MHC candidate loci. Flow cytometric analysis on peripheral blood of healthy donors and patients with multiple sclerosis and rheumatoid arthritis confirmed an upregulation of CD161 at the protein level, showing also a significant excess of CD161(high)CD8(+) T cells in multiple sclerosis. This subset prevalently included chemokine (C-C motif) receptor 6(+), cytokine-producing, effector-memory T cells with proinflammatory profiles. It also included all circulating interleukin-17(+)CD8(+) T cells. In the CD161(high)CD8(+) subset, interleukin-12 facilitated proliferation and interferon-γ production, with CD161 acting as a co-stimulatory receptor. CD161(+)CD8(+)CD3(+) T cells producing interferon-γ were part of intralesional immune infiltrates and ectopic B cell follicles in autopsy multiple sclerosis brains. Variations of CD161 expression on CD8(+) T cells identify a subset of lymphocytes with proinflammatory characteristics that have not been previously reported in multiple sclerosis and are likely to contribute to disease immunopathology.
Subject(s)
CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/metabolism , Multiple Sclerosis/immunology , NK Cell Lectin-Like Receptor Subfamily B/biosynthesis , T-Lymphocyte Subsets/metabolism , Adult , Arthritis, Rheumatoid/immunology , Cell Proliferation/drug effects , Female , Gene Expression Profiling/methods , Humans , Interferon-gamma/metabolism , Interleukin-12/pharmacology , Male , Multiple Sclerosis/blood , Multiple Sclerosis/genetics , Twins, Monozygotic/immunology , Up-RegulationABSTRACT
Meningeal inflammation in the form of ectopic lymphoid-like structures has been suggested to play a prominent role in the development of cerebral cortical grey matter pathology in multiple sclerosis. The aim of this study was to analyse the incidence and distribution of B cell follicle-like structures in an extensive collection of cases with secondary progressive multiple sclerosis with a wide age range and to determine their relationship to diffuse meningeal inflammation, white matter perivascular infiltrates and microglial activation. One hundred and twenty three cases with secondary progressive multiple sclerosis were examined for the presence of meningeal and perivascular immune cell infiltrates in tissue blocks and/or whole coronal macrosections encompassing a wide array of brain areas. Large, dense, B cell-rich lymphocytic aggregates were screened for the presence of follicular dendritic cells, proliferating B cells and plasma cells. Ectopic B cell follicle-like structures were found, with variable frequency, in 49 cases (40%) and were distributed throughout the forebrain, where they were most frequently located in the deep sulci of the temporal, cingulate, insula and frontal cortex. Subpial grey matter demyelinated lesions were located both adjacent to, and some distance from such structures. The presence of B cell follicle-like structures was associated with an accompanying quantitative increase in diffuse meningeal inflammation that correlated with the degree of microglial activation and grey matter cortical demyelination. The median age of disease onset, time to disease progression, time to wheelchair dependence and age at death all differed significantly in these cases when compared with those without B cell follicle-like structures. Our findings suggest that meningeal infiltrates may play a contributory role in the underlying subpial grey matter pathology and accelerated clinical course, which is exacerbated in a significant proportion of cases by the presence of B cell follicle-like structures.
Subject(s)
Cerebral Cortex/pathology , Inflammation/immunology , Inflammation/pathology , Meninges/pathology , Multiple Sclerosis/immunology , Multiple Sclerosis/pathology , Adult , Age of Onset , Aged , Aged, 80 and over , Antigens, CD/immunology , B-Lymphocytes/cytology , B-Lymphocytes/immunology , Cerebral Cortex/immunology , Demyelinating Diseases/pathology , Disease Progression , Female , Humans , Kaplan-Meier Estimate , Male , Meninges/immunology , Middle Aged , Multiple Sclerosis/physiopathologyABSTRACT
The EBV as the 'gluten of MS' hypothesis discussed by Drosu et al. in a recent Editorial envisages the existence of similar mechanisms leading to celiac disease and multiple sclerosis, such as induction of immunity against an ubiquitous exogenous antigen - gluten and EBV, respectively - and subsequent development of autoimmunity that is maintained by persistence of the initial trigger. While this hypothesis provides the rationale for treating MS with antivirals to lower EBV load, it can be misleading when trying to translate concepts of T cell-B cell interaction and autoimmunity development in celiac disease to multiple sclerosis. Here, we propose that EBV might act as the driver of multiple sclerosis without involving autoimmunity.
Subject(s)
Celiac Disease , Epstein-Barr Virus Infections , Multiple Sclerosis , Antiviral Agents , Autoimmunity , Epstein-Barr Virus Infections/complications , Glutens , Herpesvirus 4, Human , HumansABSTRACT
The composition of the intestinal microbiota plays a critical role in shaping the immune system. Modern lifestyle, the inappropriate use of antibiotics, and exposure to pollution have significantly affected the composition of commensal microorganisms. The intestinal microbiota has been shown to sustain inappropriate autoimmune responses at distant sites in animal models of disease, and may also have a role in immune-mediated central nervous system (CNS) diseases such as multiple sclerosis (MS). We studied the composition of the gut mycobiota in fecal samples from 27 persons with MS (pwMS) and in 18 healthy donors (HD), including 5 pairs of homozygous twins discordant for MS. We found a tendency towards higher fungal abundance and richness in the MS group, and we observed that MS twins showed a higher rate of food-associated strains, such as Saccharomyces cerevisiae. We then found that in pwMS, a distinct population of cells with antibacterial and antifungal activity is expanded during the remitting phase and markedly decreases during clinically and/or radiologically active disease. These cells, named MAIT (mucosal-associated invariant T cells) lymphocytes, were significantly more activated in pwMS compared to HD in response to S. cerevisiae and Candida albicans strains isolated from fecal samples. This activation was also mediated by fungal-induced IL-23 secretion by innate immune cells. Finally, immunofluorescent stainings of MS post-mortem brain tissues from persons with the secondary progressive form of the disease showed that MAIT cells cross the blood-brain barrier (BBB) and produce pro-inflammatory cytokines in the brain. These results were in agreement with the hypothesis that dysbiosis of the gut microbiota might determine the inappropriate response of a subset of pathogenic mucosal T cells and favor the development of systemic inflammatory and autoimmune diseases.
Subject(s)
Gastrointestinal Microbiome , Mucosal-Associated Invariant T Cells , Multiple Sclerosis , Animals , Brain , CD8-Positive T-Lymphocytes/pathology , Saccharomyces cerevisiaeABSTRACT
We report that B cell-activating factor of the tumor necrosis factor (TNF) family (BAFF) is expressed in the normal human brain at approximately 10% of that in lymphatic tissues (tonsils and adenoids) and is produced by astrocytes. BAFF was regularly detected by enzyme-linked immunosorbent assay in brain tissue lysates and in normal spinal fluid, and in astrocytes by double fluorescence microscopy. Cultured human astrocytes secreted functionally active BAFF after stimulation with interferon-gamma and TNF-alpha via a furin-like protease-dependent pathway. BAFF secretion per cell was manifold higher in activated astrocytes than in monocytes and macrophages. We studied brain lesions with B cell components, and found that in multiple sclerosis plaques, BAFF expression was strongly up-regulated to levels observed in lymphatic tissues. BAFF was localized in astrocytes close to BAFF-R-expressing immune cells. BAFF receptors were strongly expressed in situ in primary central nervous system (CNS) lymphomas. This paper identifies astrocytes as a nonimmune source of BAFF. CNS-produced BAFF may support B cell survival in inflammatory diseases and primary B cell lymphoma.
Subject(s)
Astrocytes/metabolism , Central Nervous System Neoplasms/metabolism , Lymphoma/metabolism , Membrane Proteins/metabolism , Multiple Sclerosis/metabolism , Tumor Necrosis Factor-alpha/metabolism , B-Cell Activating Factor , B-Cell Activation Factor Receptor , B-Lymphocytes , Brain/metabolism , Humans , Membrane Proteins/genetics , Organ Specificity , RNA, Messenger/metabolism , Receptors, Tumor Necrosis Factor/metabolism , Tumor Necrosis Factor-alpha/genetics , Up-RegulationABSTRACT
OBJECTIVE: Prominent inflammation with formation of ectopic B-cell follicle-like structures in the meninges in secondary progressive multiple sclerosis (MS) (SPMS) is associated with extensive cortical pathology and an exacerbated disease course. Our objective was to evaluate the cellular substrates of the cortical damage to understand the role of meningeal inflammation in MS pathology. METHODS: Using >600 tissue blocks from 37 cases of SPMS and 14 non-neurological controls, we carried out a detailed quantitative analysis of cortical atrophy and layer-specific changes in cell populations in SPMS cases with (F(+) SPMS) and without (Fâ» SPMS) B-cell follicle-like structures. RESULTS: B-cell follicle-like structures were detected in the inflamed meninges of 20 of 37 SPMS cases (54%) and were associated with increased subpial cortical demyelination and cortical atrophy. A clear gradient of neuronal loss was observed in grey matter lesions and normal-appearing grey matter in the motor cortex of F(+) SPMS cases. The density of pyramidal neurons was significantly reduced in layers III and V of the motor cortex. Neuronal loss was accompanied by glia limitans damage with astrocyte loss and an opposite gradient of increased density of activated microglia. No gradient of neuronal loss was seen in Fâ» SPMS cases. INTERPRETATION: We demonstrate substantial cortical neurodegeneration and generalized cell loss in progressive MS in association with meningeal inflammation and lymphoid tissue formation, supporting the hypothesis that cytotoxic factors diffusing from the meningeal compartment contribute to grey matter pathology and the consequent increase in clinical disability.
Subject(s)
Inflammation/etiology , Inflammation/pathology , Meninges/pathology , Multiple Sclerosis/complications , Multiple Sclerosis/pathology , Neurons/pathology , 3',5'-Cyclic-AMP Phosphodiesterases/metabolism , Adult , Aged , Aged, 80 and over , Analysis of Variance , Antigens, CD/metabolism , Astrocytes/pathology , B-Lymphocytes/metabolism , B-Lymphocytes/pathology , Caspase 3/metabolism , Cell Death/physiology , Cerebral Cortex/pathology , Female , Humans , In Situ Nick-End Labeling/methods , Inflammation/immunology , Male , Middle Aged , Multiple Sclerosis/immunology , Myelin Proteins/metabolism , Myelin-Associated Glycoprotein/metabolism , Myelin-Oligodendrocyte Glycoprotein , Neuroglia/pathology , Nitric Oxide Synthase Type II/metabolism , Nogo Proteins , Phosphopyruvate Hydratase/metabolismABSTRACT
OBJECTIVE: Increasing evidence supports a link between Epstein-Barr virus (EBV), a ubiquitous B-lymphotropic human herpesvirus, and common B-cell-related autoimmune diseases. We sought evidence of EBV infection in thymuses from patients with myasthenia gravis (MG), an autoimmune disease characterized by intrathymic B-cell activation. METHODS: Seventeen MG thymuses (6 follicular hyperplastic, 6 diffuse hyperplastic, 5 involuted) and 6 control thymuses were analyzed using in situ hybridization for EBV-encoded small RNAs (EBERs), immunohistochemistry for EBV latent and lytic proteins, and polymerase chain reaction for EBV DNA and mRNA. RESULTS: All 17 MG thymuses showed evidence of active EBV infection, whereas none of the control thymuses were infected. Cells expressing EBERs (12 of 17) and EBV latency proteins (EBNA2, LMP1, and LMP2A) (16 of 17) were detected in medullary infiltrates and in germinal centers. Cells expressing early (BFRF1, BMRF1) and late (p160, gp350/220) lytic phase EBV proteins were present in 16 MG thymuses. Latency (EBNA1, LMP2A) or lytic (BZLF1) transcripts (often both) were present in all MG thymuses, and EBV DNA (LMP1 gene) was detected in 13 MG thymuses. We also found CD8+ T cells, CD56 + CD3-natural killer cells, and BDCA-2+ plasmacytoid dendritic cells in immune infiltrates of MG thymuses, but not germinal centers, suggesting an attempt of the immune system to counteract EBV infection. INTERPRETATION: Dysregulated EBV infection in the pathological thymus appears common in MG and may contribute to the immunological alterations initiating and/or perpetuating the disease.
Subject(s)
Epstein-Barr Virus Infections/complications , Myasthenia Gravis , RNA, Viral/metabolism , Thymus Gland/pathology , Thymus Gland/virology , Viral Proteins/metabolism , Adolescent , Adult , Antigens, CD/metabolism , Epstein-Barr Virus Infections/pathology , Female , Gene Expression Regulation, Viral/physiology , Histocompatibility Antigens/metabolism , Humans , Lymphoid Tissue/metabolism , Lymphoid Tissue/pathology , Lymphoid Tissue/virology , Male , Middle Aged , Myasthenia Gravis/complications , Myasthenia Gravis/pathology , Myasthenia Gravis/virology , RNA, Viral/genetics , Thymus Gland/metabolism , Viral Proteins/genetics , Young AdultABSTRACT
Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare congenital leukodystrophy caused by mutations in the MLC1 gene that encodes a membrane protein of unknown function. In the brain MLC1 protein is mainly expressed in astrocyte end-feet, localizes in lipid rafts and associates with the dystrophin glycoprotein complex (DGC). Using pull-down and co-fractionation assays in cultured human and rat astrocytes, we show here that MLC1 intracellular domains pull-down the DGC proteins syntrophin, dystrobrevin, Kir4.1 and caveolin-1, the structural protein of caveolae, thereby supporting a role for DGC and caveolar structures in MLC1 function. By immunostaining and subcellular fractionation of cultured rat or human astrocytes treated with agents modulating caveolin-mediated trafficking, we demonstrate that MLC1 is also expressed in intracellular vesicles and endoplasmic reticulum and undergoes caveolae/raft-mediated endocytosis. Inhibition of endocytosis, cholesterol lowering and protein kinases A- and C-mediated MLC1 phosphorylation favour the expression of membrane-associated MLC1. Because pathological mutations prevent MLC1 membrane expression, the identification of substances regulating MLC1 intracellular trafficking is potentially relevant for the therapy of MLC.
Subject(s)
Astrocytes/metabolism , Brain/metabolism , Caveolae/metabolism , Caveolin 1/metabolism , Leukoencephalopathies/metabolism , Membrane Proteins/metabolism , Animals , Animals, Newborn , Brain/pathology , Brain/physiopathology , Caveolae/ultrastructure , Cell Line, Tumor , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Cells, Cultured , Cholesterol/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Cytoplasmic Vesicles/metabolism , Cytoplasmic Vesicles/ultrastructure , Dystrophin-Associated Protein Complex/metabolism , Endocytosis/physiology , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/ultrastructure , Humans , Leukoencephalopathies/genetics , Leukoencephalopathies/physiopathology , Membrane Microdomains/metabolism , Membrane Microdomains/ultrastructure , Phosphorylation , Protein Kinase C/metabolism , Protein Transport/physiology , RatsABSTRACT
Signaling from central nervous system (CNS)-infiltrating lymphocytes and macrophages is critical to activate microglia and cause tissue damage in multiple sclerosis (MS). We combined laser microdissection with high-throughput real time RT-PCR to investigate separately the CNS exogenous and endogenous inflammatory components in postmortem brain tissue of progressive MS cases. A previous analysis of immune infiltrates isolated from the white matter (WM) and the meninges revealed predominant expression of genes involved in antiviral and cytotoxic immunity, including IFNγ and TNF. Here, we assessed the expression of 71 genes linked to IFN and TNF signaling and microglia/macrophage activation in the parenchyma surrounding perivascular cuffs at different stages of WM lesion evolution and in gray matter (GM) lesions underlying meningeal infiltrates. WM and GM from non-neurological subjects were used as controls. Transcriptional changes in the WM indicate activation of a classical IFNγ-induced macrophage defense response already in the normal-appearing WM, amplification of detrimental (proinflammatory/pro-oxidant) and protective (anti-inflammatory/anti-oxidant) responses in actively demyelinating WM lesions and persistence of these dual features at the border of chronic active WM lesions. Transcriptional changes in chronic subpial GM lesions indicate skewing toward a proinflammatory microglia phenotype. TNF receptor 2 (TNFR2) mediating TNF neuroprotective functions was one of the genes upregulated in the MS WM. Using immunohistochemistry we show that TNFR2 is highly expressed in activated microglia in the normal-appearing WM, at the border of chronic active WM lesions, and in foamy macrophages in actively demyelinating WM and GM lesions. In lysolecithin-treated mouse cerebellar slices, a model of demyelination and remyelination, TNFR2 RNA and soluble protein increased immediately after toxin-induced demyelination along with transcripts for microglia/macrophage-derived pro- and anti-inflammatory cytokines. TNFR2 and IL10 RNA and soluble TNFR2 protein remained elevated during remyelination. Furthermore, myelin basic protein expression was increased after selective activation of TNFR2 with an agonistic antibody. This study highlights the key role of cytotoxic adaptive immunity in driving detrimental microglia activation and the concomitant healing response. It also shows that TNFR2 is an early marker of microglia activation and promotes myelin synthesis, suggesting that microglial TNFR2 activation can be exploited therapeutically to stimulate CNS repair.
Subject(s)
Brain/metabolism , Epstein-Barr Virus Infections/complications , Herpesvirus 4, Human/genetics , In Situ Hybridization , Multiple Sclerosis , RNA/metabolism , Antigens, CD20/metabolism , Brain/pathology , Brain/virology , Humans , Lymph Nodes/metabolism , Lymph Nodes/pathology , Lymph Nodes/virology , Multiple Sclerosis/etiology , Multiple Sclerosis/pathology , Multiple Sclerosis/virology , RNA/genetics , RNA, Viral/genetics , RNA, Viral/metabolism , RadioactivityABSTRACT
MLC1 gene mutations have been associated with megalencephalic leukoencephalopathy with subcortical cysts (MLC), a rare neurologic disorder in children. The MLC1 gene encodes a membrane protein (MLC1) with unknown function which is mainly expressed in astrocytes. Using a newly developed anti-human MLC1 polyclonal antibody, we have investigated the biochemical properties and localization of MLC1 in cultured astrocytes and brain tissue and searched for evidence of a relationship between MLC1 and proteins of the dystrophin-glycoprotein complex (DGC). Cultured astrocytes express two MLC1 components showing different solubilisation properties and subcellular distribution. Most importantly, we show that the membrane-associated component of MLC1 (60-64 kDa) localizes in astrocytic lipid rafts together with dystroglycan, syntrophin and caveolin-1, and co-fractionates with the DGC in whole rat brain tissue. In the human brain, MLC1 protein is expressed in astrocyte processes and ependymal cells, where it colocalizes with dystroglycan and syntrophin. These data indicate that the DGC may be involved in the organization and function of the MLC1 protein in astrocyte membranes.
Subject(s)
Astrocytes/metabolism , Dystroglycans/metabolism , Neoplasm Proteins/chemistry , Neoplasm Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/chemistry , Proto-Oncogene Proteins c-bcl-2/metabolism , Animals , Animals, Newborn , Astrocytes/cytology , Astrocytoma/pathology , Brain/cytology , Cells, Cultured , Dystrophin-Associated Proteins/metabolism , Humans , Membrane Microdomains/metabolism , Myeloid Cell Leukemia Sequence 1 Protein , Neoplasm Proteins/genetics , Proto-Oncogene Proteins c-bcl-2/genetics , RNA, Messenger/metabolism , Rats , Subcellular Fractions/metabolismABSTRACT
Megalencephalic leukoencephalopathy with subcortical cysts protein-1 (MLC1) is a membrane protein expressed by perivascular astrocytes. MLC1 mutations cause MLC, an incurable leukodystrophy characterized by macrocephaly, brain edema, cysts, myelin vacuolation, and astrocytosis, leading to cognitive/motor impairment and epilepsy. Although its function is unknown, MLC1 favors regulatory volume decrease after astrocyte osmotic swelling and down-regulates intracellular signaling pathways controlling astrocyte activation and proliferation. By combining analysis of human brain tissues with in vitro experiments, here we investigated MLC1 role in astrocyte activation during neuroinflammation, a pathological condition exacerbating patient symptoms. MLC1 upregulation was observed in brain tissues from multiple sclerosis, Alzheimer's, and Creutzfeld-Jacob disease, all pathologies characterized by strong astrocytosis and release of inflammatory cytokines, particularly IL-1ß. Using astrocytoma lines overexpressing wild-type (WT) or mutated MLC1 and astrocytes from control and Mlc1 knock-out (KO) mice, we found that IL-1ß stimulated WT-MLC1 plasma membrane expression in astrocytoma cells and control primary astrocytes. In astrocytoma, WT-MLC1 inhibited the activation of IL-1ß-induced inflammatory signals (pERK, pNF-kB) that, conversely, were constitutively activated in mutant expressing cells or abnormally upregulated in KO astrocytes. WT-MLC1+ cells also expressed reduced levels of the astrogliosis marker pSTAT3. We then monitored MLC1 expression timing in a demyelinating/remyelinating murine cerebellar organotypic culture model where, after the demyelination and release of inflammatory cytokines, recovery processes occur, revealing MLC1 upregulation in these latter phases. Altogether, these findings suggest that by modulating specific pathways, MLC1 contributes to restore astrocyte homeostasis after inflammation, providing the opportunity to identify drug target molecules to slow down disease progression.
Subject(s)
Astrocytes/pathology , Inflammation/pathology , Membrane Proteins/metabolism , Signal Transduction , Adult , Aged , Alzheimer Disease/pathology , Animals , Astrocytes/metabolism , Cell Membrane/metabolism , Demyelinating Diseases/pathology , Disease Models, Animal , ErbB Receptors/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Humans , Interleukin-1beta/metabolism , Male , Membrane Proteins/deficiency , Mice, Knockout , Middle Aged , Models, Biological , Mutation/genetics , NF-kappa B/metabolism , Phosphorylation , Rats , Up-RegulationABSTRACT
The expression patterns of tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a pleiotropic cytokine with proinflammatory and cell death-inducing activities, and its receptor, fibroblast growth factor-inducible 14 (Fn14), were examined in postmortem brain tissue samples from patients with multiple sclerosis (MS) and controls. Immunohistochemical analysis and real-time reverse transcription-polymerase chain reaction demonstrated that both TWEAK and Fn14 were upregulated in the MS compared with control unaffected brain samples. Perivascular and meningeal macrophages and astrocytes and microglia associated with lesions were identified as the main sources of TWEAK in the MS brains. The highest frequency of TWEAK+ cells was found at edges of chronic active white matter lesions and in subpial cortical lesions inMS cases with abundant meningeal inflammation and ectopic B-cell follicles. Neurons and reactive astrocytes expressing Fn14 were mainly localized in the cerebral cortex in highly infiltrated MS brains. Numerous TWEAK-expressing microglia were associated with the extensive loss of myelin and astrocytosis, neuronal damage, and vascular abnormalities in subpial cortical lesions; this suggests that TWEAK could synergize with other cytotoxic factors diffusing from the inflamed meninges to promote cortical injury. Taken together, these findings indicate that the TWEAK/Fn14 pathway contributes to inflammation and tissue injury and is, therefore, a potential therapeutic target in MS.
Subject(s)
Brain/immunology , Encephalitis/immunology , Microglia/immunology , Multiple Sclerosis/immunology , Receptors, Tumor Necrosis Factor/metabolism , Tumor Necrosis Factors/immunology , Astrocytes/immunology , B-Lymphocytes/immunology , Blood-Brain Barrier/immunology , Blood-Brain Barrier/pathology , Blood-Brain Barrier/physiopathology , Brain/pathology , Brain/physiopathology , Cerebral Arteries/immunology , Cerebral Arteries/pathology , Cerebral Arteries/physiopathology , Cerebral Cortex/immunology , Cerebral Cortex/pathology , Cerebral Cortex/physiopathology , Chemotaxis, Leukocyte/immunology , Cytokine TWEAK , Encephalitis/pathology , Encephalitis/physiopathology , Gliosis/immunology , Gliosis/pathology , Gliosis/physiopathology , Humans , Macrophages/immunology , Meninges/immunology , Meninges/pathology , Multiple Sclerosis/pathology , Multiple Sclerosis/physiopathology , Myelin Sheath/immunology , Myelin Sheath/pathology , Receptors, Tumor Necrosis Factor/analysis , Receptors, Tumor Necrosis Factor/genetics , TWEAK Receptor , Tumor Necrosis Factors/genetics , Tumor Necrosis Factors/metabolism , Up-Regulation/immunologyABSTRACT
The roles of plasmacytoid dendritic cells (pDCs) and their response to interferon (IFN)-beta therapy in multiple sclerosis (MS) patients are poorly understood. We identified pDC accumulation in white matter lesions and leptomeninges of MS brains and abundant expression of the Type I IFN-induced protein MxA, mainly in perivascular CD3+ lymphocytes in lesions, indicating Type I IFN production by activated pDCs. The pDC chemoattractant chemerin was detected in intralesional cerebrovascular endothelial cells, and the chemerin receptor was expressed on infiltrating leukocytes, including pDCs. The effect of IFN-beta on pDC phenotype and function was evaluated in MS patients before and during IFN-beta treatment. Although IFN-beta did not modify the frequency and immature phenotype of circulating pDC, they showed lower expression of major histocompatibility complex Class II and blood-dendritic cell antigen 2 molecules and upregulation of CD38 and B7H1 costimulatory molecules. On exposure to CpG (a site where cytosine [C] lies next to guanine [G] in the DNA sequence [the p indicates that C and G are connected by a phosphodiester bond]) oligodeoxynucleotides in vitro, pDCs from IFN-beta-treated MS patients showed reduced expression of the pDC maturation markers CD83 and CD86 molecules; in vitro IFN-beta treatment of pDCs from healthy donors resulted in lower secretion of proinflammatory cytokines, including IFN-alpha, and a decreased ability to stimulate allogeneic T cells in response to maturative stimuli. These data indicate that IFN-beta modulates the immunologic functions of pDC, thus identifying pDCs as a novel target of IFN-beta therapy in MS patients.