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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.

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.

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.

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.