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Background: MS is a chronic inflammatory disease of the CNS leading to demyelination and neurodegeneration, with a complex and still to be clarified aetiology. Several data, coming from patients' samples and from animal models, show that Oxidative Status (OS) plays an important role in MS pathogenesis. Overproduction of reactive oxidative species by macrophages/microglia can bring about cellular injury and ensuing cell death by oxidizing cardinal cellular components. Oxidized molecules are present in active MS lesions and are associated with neurodegeneration. Methods: We undertook a structured search of bibliographic databases for peer-reviewed research literature focusing on OS in MS. The contents of the selected papers were described in the context of a conceptual framework. A special emphasis was given to the results of our study in the field. Results: The results of our three recent studies were put in the context and discussed taking into account the literature on the topic. Oxidative damage underpinned an imbalance shared by MS and neurodegenerative diseases such as Alzheimer and Parkinson diseases. In people with clinically isolated syndrome (an early phase of MS) oxidative stress proved to contribute to disease pathophysiology and to provide biomarkers that may help predict disease evolution. A drug screening platform based on multiple assays to test the remyelinating potential of library of approved compounds showed two anti-oxidants, edaravone and 5-methyl-7- methoxyisoflavone, as active drugs. Moreover, an analysis of 'structure activity relationship' showed off-targets sites of these compounds that accounted for their remyelinating activity, irrespective of their antioxidant action. Conclusion: Overall, edaravone emerges as a candidate to treat complex disease such as MS, where inflammation, oxidative stress and neurodegeneration contribute to disease progression, together or individually, in different phases and disease types. Furthermore, approaches based on drug repositioning seem to maintain the promise of helping discover novel treatment for complex diseases, where molecular targets are largely unknown.

The cause and the pathogenic mechanisms leading to multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system (CNS), are still under scrutiny. During the last decade, awareness has increased that multiple genetic and environmental factors act in concert to modulate MS risk. Likewise, the landscape of cells of the adaptive immune system that are believed to play a role in MS immunopathogenesis has expanded by including not only CD4 T helper cells but also cytotoxic CD8 T cells and B cells. Once the key cellular players are identified, the main challenge is to define precisely how they act and interact to induce neuroinflammation and the neurodegenerative cascade in MS. CD8 T cells have been implicated in MS pathogenesis since the 80’s when it was shown that CD8 T cells predominate in MS brain lesions. Interest in the role of CD8 T cells in MS was revived in 2000 and the years thereafter by studies showing that CNS-recruited CD8 T cells are clonally expanded and have a memory effector phenotype indicatingin situantigen-driven reactivation. The association of certain MHC class I alleles with MS genetic risk implicates CD8 T cells in disease pathogenesis. Moreover, experimental studies have highlighted the detrimental effects of CD8 T cell activation on neural cells. While the antigens responsible for T cell recruitment and activation in the CNS remain elusive, the high efficacy of B-cell depleting drugs in MS and a growing number of studies implicate B cells and Epstein-Barr virus (EBV), a B-lymphotropic herpesvirus that is strongly associated with MS, in the activation of pathogenic T cells. This article reviews the results of human studies that have contributed to elucidate the role of CD8 T cells in MS immunopathogenesis, and discusses them in light of current understanding of autoreactivity, B-cell and EBV involvement in MS, and mechanism of action of different MS treatments. Based on the available evidences, an immunopathological model of MS is proposed that entails a persistent EBV infection of CNS-infiltrating B cells as the target of a dysregulated cytotoxic CD8 T cell response causing CNS tissue damage.

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-1β. Using astrocytoma lines overexpressing wild-type (WT) or mutated MLC1 and astrocytes from control and Mlc1 knock-out (KO) mice, we found that IL-1β stimulated WT-MLC1 plasma membrane expression in astrocytoma cells and control primary astrocytes. In astrocytoma, WT-MLC1 inhibited the activation of IL-1β–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.

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.

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., 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.

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. Electronic supplementary material The online version of this article (10.1186/s12974-017-1049-5) contains supplementary material, which is available to authorized users.

Ectopic B-cell follicle-like structures (ELS) are found in the meninges of patients with secondary progressive multiple sclerosis (SPMS). Because cells expressing the transcriptional regulator retinoic acid receptor-related orphan receptor-γt (RORγt) and producing interleukin 17 (IL17), e.g. T helper 17 cells and lymphoid tissue inducer (LTi) cells, have been implicated in the formation of ELS, we studied RORγt and IL17 expression in brain tissue from patients with SPMS an assessed their relationships to immune infiltrates and meningeal ELS. By immunohistochemistry, small numbers of RORγt-positive cells were detected in the meninges of 6 of 12 SPMS cases analyzed. RORγt-positive cells were localized in B-cell follicles or aggregates and nearby diffuse meningeal infiltrates, and predominantly co-expressed CD3. Only a few RORγt-positive, CD3-negative cells were observed, suggesting the presence of group 3 innate lymphoid cells, which comprise the LTi cell subset. Some IL17-positive cells, co-expressing in part RORγt and predominantly CD3, were found in meningeal B-cell follicles from 4 SPMS cases. Rare RORγt-positive and IL17-positive cells were detected in white matter. Gene expression analysis of laser dissected meningeal infiltrates and white matter lesions confirmed low frequencies and virtual absence of RORγt and IL17 signals, respectively. Thus, there is selective migration or survival of RORγt-positive cells in MS patient meninges and an association of these cells with ELS.

Studying rebound mechanisms in MS patients after interruption of natalizumab may advance our understanding of MS pathogenesis. To verify the role of Epstein-Barr virus (EBV) infection and anti-EBV immunity in post-natalizumab rebound, we analyzed postmortem brain tissue of a case of fatal MS relapse. Using immunohistochemistry, EBV latent, early and late lytic proteins and CD8+ T cells sticking to EBV lytically-infected cells were detected in multiple inflammatory white matter lesions. Using the pentamer technology on brain sections, EBV-specific CD8+ T cells were observed perivascularly. Cell-free EBV DNA was detected in cerebrospinal fluid. These findings confirm and extend the results obtained in another case of post-natalizumab fatal MS relapse, suggesting that this condition represents an EBV-associated immune reconstitution inflammatory syndrome.

There is no treatment for the myelin loss in multiple sclerosis, ultimately resulting in the axonal degeneration that leads to the progressive phase of the disease. We established a multi-tiered platform for the sequential screening of drugs that could be repurposed as remyelinating agents. We screened a library of 2,000 compounds (mainly Food and Drug Administration (FDA)-approved compounds and natural products) for cellular metabolic activity on mouse oligodendrocyte precursors (OPC), identifying 42 molecules with significant stimulating effects. We then characterized the effects of these compounds on OPC proliferation and differentiation in mouse glial cultures, and on myelination and remyelination in organotypic cultures. Three molecules, edaravone, 5-methyl-7-methoxyisoflavone and lovastatin, gave positive results in all screening tiers. We validated the results by retesting independent stocks of the compounds, analyzing their purity, and performing dose-response curves. To identify the chemical features that may be modified to enhance the compounds’ activity, we tested chemical analogs and identified, for edaravone, the functional groups that may be essential for its activity. Among the selected remyelinating candidates, edaravone appears to be of strong interest, also considering that this drug has been approved as a neuroprotective agent for acute ischemic stroke and amyotrophic lateral sclerosis in Japan.

Mutations in the MLC1 gene, which encodes a protein expressed in brain astrocytes, are the leading cause of MLC, a rare leukodystrophy characterized by macrocephaly, brain edema, subcortical cysts, myelin and astrocyte vacuolation. Although recent studies indicate that MLC1 protein is implicated in the regulation of cell volume changes, the exact role of MLC1 in brain physiology and in the pathogenesis of MLC disease remains to be clarified. In preliminary experiments, we observed that MLC1 was poorly expressed in highly proliferating astrocytoma cells when compared with primary astrocytes, and that modulation of MLC1 expression influenced astrocyte growth. Because volume changes are key events in cell proliferation and during brain development MLC1 expression is inversely correlated to astrocyte progenitor proliferation levels, we investigated the possible role for MLC1 in the control of astrocyte proliferation. We found that overexpression of wild type but not mutant MLC1 in human astrocytoma cells hampered cell growth by favoring epidermal growth factor receptor (EGFR) degradation and by inhibiting EGF-induced Ca(+) entry, ERK1/2 and PLCγ1 activation, and calcium-activated KCa3.1 potassium channel function, all molecular pathways involved in astrocyte proliferation stimulation. Interestingly, MLC1 did not influence AKT, an EGFR-stimulated kinase involved in cell survival. Moreover, EGFR expression was higher in macrophages derived from MLC patients than from healthy individuals. Since reactive astrocytes proliferate and re-express EGFR in response to different pathological stimuli, the present findings provide new information on MLC pathogenesis and unravel an important role for MLC1 in other brain pathological conditions where astrocyte activation occurs.

Background The potential role of the human immunodeficiency virus-1 (HIV-1) accessory protein Nef in the pathogenesis of neuroAIDS is still poorly understood. Nef is a molecular adapter that influences several cellular signal transduction events and membrane trafficking. In human macrophages, Nef expression induces the production of extracellular factors (e.g. pro-inflammatory chemokines and cytokines) and the recruitment of T cells, thus favoring their infection and its own transfer to uninfected cells via exosomes, cellular protrusions or cell-to-cell contacts. Murine cells are normally not permissive for HIV-1 but, in transgenic mice, Nef is a major disease determinant. Both in human and murine macrophages, myristoylated Nef (myr+Nef) treatment has been shown to activate NF-κB, MAP kinases and interferon responsive factor 3 (IRF-3), thereby inducing tyrosine phosphorylation of signal transducers and activator of transcription (STAT)-1, STAT-2 and STAT-3 through the production of proinflammatory factors. Methodology/Principal Findings We report that treatment of BV-2 murine microglial cells with myr+Nef leads to STAT-1, -2 and -3 tyrosine phosphorylation and upregulates the expression of inducible nitric oxide synthase (iNOS) with production of nitric oxide. We provide evidence that extracellular Nef regulates iNOS expression through NF-κB activation and, at least in part, interferon-β (IFNβ) release that acts in concert with Nef. All of these effects require both myristoylation and a highly conserved acidic cluster in the viral protein. Finally, we report that Nef induces the release of neurotoxic factors in the supernatants of microglial cells. Conclusions These results suggest a potential role of extracellular Nef in promoting neuronal injury in the murine model. They also indicate a possible interplay between Nef and host factors in the pathogenesis of neuroAIDS through the production of reactive nitrogen species in microglial cells.

Gray matter lesions are thought to play a key role in the progression of disability and cognitive impairment in multiple sclerosis (MS) patients, but whether gray matter damage is caused by inflammation or secondary to axon loss in the white matter, or both, is not clear. In an analysis of postmortem brain samples from 44 cases of secondary progressive MS, 26 cases were characterized by meningeal inflammation with ectopic B-cell follicles and prominent gray matter pathology; subpial cortical lesions containing dense perivascular lymphocytic infiltrates were present in 11 of these cases. Because intracortical immune infiltrates were enriched in B-lineage cells and because we have shown previously that B cells accumulating in the MS brain support an active Epstein-Barr virus (EBV) infection, we investigated evidence of EBV in the infiltrated cortical lesions. Cells expressing EBV-encoded small RNA and plasma cells expressing EBV early lytic proteins (BZLF1, BFRF1) were present in all and most of the intracortical perivascular cuffs examined, respectively. Immunohistochemistry for CD8-positive cells, granzyme B, perforin, and CD107a indicated cytotoxic activity toward EBV-infected plasma cells that was consistently observed in infiltrated cortical lesions, suggesting active immune surveillance. These findings indicate that both meningeal and intraparenchymal inflammation may contribute to cortical damage during MS progression, and that intracortical inflammation may be sustained by an EBV-driven immunopathologic response, similar to findings in white matter lesions and meninges.

Fine regulation of the innate immune response following brain injury or infection is important to avoid excessive activation of microglia and its detrimental consequences on neural cell viability and function. To get insights on the molecular networks regulating microglia activation, we analyzed expression, regulation and functional relevance of tumor necrosis factor receptors (TNFR) 2 in cultured mouse microglia. We found that microglia upregulate TNFR2 mRNA and protein and shed large amounts of soluble TNFR2, but not TNFR1, in response to pro-inflammatory stimuli and through activation of TNFR2 itself. By microarray analysis, we demonstrate that TNFR2 stimulation in microglia regulates expression of genes involved in immune processes, including molecules with anti-inflammatory and neuroprotective function like granulocyte colony-stimulating factor, adrenomedullin and IL-10. In addition, we identify IFN-γ as a regulator of the balance between pro- and anti-inflammatory/neuroprotective factors induced by TNFR2 stimulation. These data indicate that, through TNFR2, microglia may contribute to the counter-regulatory response activated in neuropathological conditions.

A cardinal feature of multiple sclerosis (MS) is the persistent intrathecal synthesis of antibodies. Our previous finding that a large fraction of B cells infiltrating the MS brain are infected with Epstein-Barr virus (EBV) raises the possibility that this virus, because of its ability to establish a latent infection in B cells and interfere with their differentiation, contributes to B-cell dysregulation in MS. The aim of this study was to gain further insight into EBV latency programs and their relationship to B-cell activation in the MS brain. Immunohistochemical analysis of postmortem MS brain samples harboring large EBV deposits revealed that most B cells in white matter lesions, meninges, and ectopic B-cell follicles are CD27+ antigen-experienced cells and coexpress latent membrane protein 1 and latent membrane protein 2A, 2 EBV-encoded proteins that provide survival and maturation signals to B cells. By combining laser-capture microdissection with preamplification reverse transcription-polymerase chain reaction techniques, EBV latency transcripts (latent membrane protein 2A, EBV nuclear antigen 1) were detected in all MS brain samples analyzed. We also found that B cell-activating factor of the tumor necrosis factor family is expressed in EBV-infected B cells in acute MS lesions and ectopic B-cell follicles. These findings support a role for EBV infection in B-cell activation in the MS brain and suggest that B cell-activating factor of the tumor necrosis factor family produced by EBV-infected B cells may contribute to this process resulting in viral persistence and, possibly, disruption of B-cell tolerance.