Normal-appearing white matter in multiple sclerosis is in a subtle balance between inflammation and neuroprotection

Multiple sclerosis is a chronic inflammatory disease of the CNS. Although progressive axonal injury and diffuse inflammatory damage has been shown in the chronic phase of the disease, little is known about the molecular mechanisms underlying these pathological processes. In order to identify these mechanisms, we have studied the gene expression profile in non-lesion containing tissue, the so-called normal-appearing white matter (NAWM). We performed differential gene expression analysis and quantitative RT-PCR on subcortical white matter from 11 multiple sclerosis and 8 control cases. Differentially expressed genes were further analysed in detail by in situ hybridization and immunofluorescence studies. We show that genes known to be involved in anti-inflammatory and protective mechanisms such as STAT6, JAK1, IL-4R, IL-10, Chromogranin C and Hif-1α are consistently upregulated in the multiple sclerosis NAWM. On the other hand, genes involved in pro-inflammatory mechanisms, such as STAT4, IL-1β and MCSF, were also upregulated but less regularly. Immunofluorescence colocalization analysis revealed expression of STAT6, JAK1, IL-4R and IL-13R mainly in oligodendrocytes, whereas STAT4 expression was detected predominantly in microglia. In line with these data, in situ hybridization analysis showed an increased expression in multiple sclerosis NAWM of HIF-1α in oligodendrocytes and HLA-DRα in microglia cells. The consistency of the expression levels of STAT6, JAK1, JAK3 and IL-4R between the multiple sclerosis cases suggests an overall activation of the STAT6-signalling pathway in oligodendrocytes, whereas the expression of STAT4 and HLA-DRα indicates the activation of pro-inflammatory pathways in microglia. The upregulation of genes involved in anti-inflammatory mechanisms driven by oligodendrocytes may protect the CNS environment and thus limit lesion formation, whereas the activation of pro-inflammatory mechanisms in microglia may favour disease progression. Altogether, our data suggests an endogenous inflammatory reaction throughout the whole white matter of multiple sclerosis brain, in which oligodendrocytes actively participate. This reaction might further influence and to some extent facilitate lesion formatio

Claudin-1 induced sealing of blood-brain barrier tight junctions ameliorates chronic experimental autoimmune encephalomyelitis

In experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), loss of the blood-brain barrier (BBB) tight junction (TJ) protein claudin-3 correlates with immune cell infiltration into the CNS and BBB leakiness. Here we show that sealing BBB TJs by ectopic tetracycline-regulated expression of the TJ protein claudin-1 in Tie-2 tTA//TRE-claudin-1 double transgenic C57BL/6 mice had no influence on immune cell trafficking across the BBB during EAE and furthermore did not influence the onset and severity of the first clinical disease episode. However, expression of claudin-1 did significantly reduce BBB leakiness for both blood borne tracers and endogenous plasma proteins specifically around vessels expressing claudin-1. In addition, mice expressing claudin-1 exhibited a reduced disease burden during the chronic phase of EAE as compared to control littermates. Our study identifies BBB TJs as the critical structure regulating BBB permeability but not immune cell trafficking into CNS during EAE, and indicates BBB dysfunction is a potential key event contributing to disease burden in the chronic phase of EAE. Our observations suggest that stabilizing BBB barrier function by therapeutic targeting of TJs may be beneficial in treating MS, especially when anti-inflammatory treatments have faile

Combinatory multifactor treatment effects on primary nanofiber oligodendrocyte cultures

Multiple sclerosis (MS) is a chronic inflammatory demyelinating and neurodegenerative disease of the central nervous system. Neurological deficits are attributed to inflammatory demyelination, which compromises axonal function and survival. These are mitigated in experimental models by rapid and often complete remyelination of affected axons, but in MS this endogenous repair mechanism frequently fails, leaving axons increasingly vulnerable to the detrimental effects of inflammatory and metabolic stress. Understanding the molecular basis of remyelination and remyelination failure is essential to develop improved therapies for this devastating disease. However, recent studies suggest that this is not due to a single dominant mechanism, but rather represents the biological outcome of multiple changes in the lesion microenvironment that combine to disrupt oligodendrocyte differentiation. This identifies a pressing need to develop technical platforms to investigate combinatory and/or synergistic effects of factors differentially expressed in MS lesions on oligodendrocyte proliferation and differentiation. Here we describe protocols using primary oligodendrocyte cultures from Bl6 mice on 384-well nanofiber plates to model changes affecting oligodendrogenesis and differentiation in the complex signaling environment associated with multiple sclerosis lesions. Using platelet-derived growth factor (PDGF–AA), fibroblast growth factor 2 (FGF2), bone morphogenetic protein 2 (BMP2) and bone morphogenetic protein 4 (BMP4) as representative targets, we demonstrate that we can assess their combinatory effects across a wide range of concentrations in a single experiment. This in vitro model is ideal for assessing the combinatory effects of changes in availability of multiple factors, thus more closely modelling the situation in vivo and furthering high-throughput screening possibilities

DARC shuttles inflammatory chemokines across the blood-brain barrier during autoimmune central nervous system inflammation

Trafficking of T cells into the CNS is a pathophysiological hallmark of multiple sclerosis. Using an invitro model of the blood-brain barrier, Minten etal. reveal that the atypical chemokine receptor DARC shuttles inflammatory chemokines across the barrier, where they contribute to immune cell trafficking into the brai

Polarizing receptor activation dissociates Fibroblast Growth Factor 2 mediated inhibition of myelination from its neuroprotective potential

Fibroblast growth factor (FGF) signaling contributes to failure of remyelination in multiple sclerosis, but targeting this therapeutically is complicated by its functional pleiotropy. We now identify FGF2 as a factor up-regulated by astrocytes in active inflammatory lesions that disrupts myelination via FGF receptor 2 (FGFR2) mediated activation of Wingless (Wnt) signaling; pharmacological inhibition of Wnt being sufficient to abrogate inhibition of myelination by FGF2 in tissue culture. Using a novel FGFR1-selective agonist (F2 V2) generated by deleting the N-terminal 26 amino acids of FGF2 we demonstrate polarizing signal transduction to favor FGFR1 abrogates FGF mediated inhibition of myelination but retains its ability to induce expression of pro-myelinating and immunomodulatory factors that include Cd93, Lif, Il11, Hbegf, Cxcl1 and Timp1. Our data provide new insights into the mechanistic basis of remyelination failure in MS and identify selective activation of FGFR1 as a novel strategy to induce a neuroprotective signaling environment in multiple sclerosis and other neurological diseases

Cocapture of cognate and bystander antigens can activate autoreactive B cells

Autoantibodies against myelin oligodendrocyte glycoprotein (MOG) are associated with autoimmune central nervous system diseases like acute disseminated encephalomyelitis (ADEM). For ADEM, it is speculated that a preceding infection is the trigger of the autoimmune response, but the mechanism connecting the infection to the production of MOG antibodies remains a mystery. We reasoned that the ability of B cells to capture cognate antigen from cell membranes, along with small quantities of coexpressed “bystander” antigens, might enable B-cell escape from tolerance. We tested this hypothesis using influenza hemagglutinin as a model viral antigen and transgenic, MOG-specific B cells. Using flow cytometry and live and fixed cell microscopy, we show that MOG-specific B cells take up large amounts of MOG from cell membranes. Uptake of the antigen from the membrane leads to a strong activation of the capturing B cell. When influenza hemagglutinin is also present in the membrane of the target cell, it can be cocaptured with MOG by MOG-specific B cells via the B-cell receptor. Hemagglutinin and MOG are both presented to T cells, which in turn are activated and proliferate. As a consequence, MOG-specific B cells get help from hemagglutinin-specific T cells to produce anti-MOG antibodies. In vivo, the transfer of MOG-specific B cells into recipient mice after the cocapture of MOG and hemagglutinin leads to the production of class-switched anti-MOG antibodies, dependent on the presence of hemagglutinin-specific T cells. This mechanism offers a link between infection and autoimmunity. Keywords: tolerance; autoantibodies; antigen capture; antigen presentation; influenz

Claudin-1 induced sealing of blood-brain barrier tight junctions ameliorates chronic experimental autoimmune encephalomyelitis

In experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), loss of the blood-brain barrier (BBB) tight junction (TJ) protein claudin-3 correlates with immune cell infiltration into the CNS and BBB leakiness. Here we show that sealing BBB TJs by ectopic tetracycline-regulated expression of the TJ protein claudin-1 in Tie-2 tTA//TRE-claudin-1 double transgenic C57BL/6 mice had no influence on immune cell trafficking across the BBB during EAE and furthermore did not influence the onset and severity of the first clinical disease episode. However, expression of claudin-1 did significantly reduce BBB leakiness for both blood borne tracers and endogenous plasma proteins specifically around vessels expressing claudin-1. In addition, mice expressing claudin-1 exhibited a reduced disease burden during the chronic phase of EAE as compared to control littermates. Our study identifies BBB TJs as the critical structure regulating BBB permeability but not immune cell trafficking into CNS during EAE, and indicates BBB dysfunction is a potential key event contributing to disease burden in the chronic phase of EAE. Our observations suggest that stabilizing BBB barrier function by therapeutic targeting of TJs may be beneficial in treating MS, especially when anti-inflammatory treatments have failed

EEF1A1 Deacetylation Enables Transcriptional Activation of Remyelination

Remyelination of the peripheral and central nervous systems (PNS and CNS, respectively) is a prerequisite for functional recovery after lesion. However, this process is not always optimal and becomes inefficient in the course of multiple sclerosis. Here we show that, when acetylated, eukaryotic elongation factor 1A1 (eEF1A1) negatively regulates PNS and CNS remyelination. Acetylated eEF1A1 (Ac- eEF1A1) translocates into the nucleus of myelinating cells where it binds to Sox10, a key transcription factor for PNS and CNS myelination and remyelination, to drag Sox10 out of the nucleus. We show that the lysine acetyltransferase Tip60 acetylates eEF1A1, whereas the histone deacetylase HDAC2 deacetylates eEF1A1. Promoting eEF1A1 deacetylation maintains the activation of Sox10 target genes and increases PNS and CNS remyelination efficiency. Taken together, these data identify a major mechanism of Sox10 regulation, which appears promising for future translational studies on PNS and CNS remyelination

Brain Citrullination Patterns and T Cell Reactivity of Cerebrospinal Fluid-Derived CD4+ T Cells in Multiple Sclerosis

Immune responses to citrullinated peptides have been described in autoimmune diseases like rheumatoid arthritis (RA) and multiple sclerosis (MS). We investigated the post-translational modification (PTM), arginine to citrulline, in brain tissue of MS patients and controls (C) by proteomics and subsequently the cellular immune response of cerebrospinal fluid (CSF)-infiltrating T cells to citrullinated and unmodified peptides of myelin basic protein (MBP). Using specifically adapted tissue extraction- and combined data interpretation protocols we could establish a map of citrullinated proteins by identifying more than 80 proteins with two or more citrullinated peptides in human brain tissue. We report many of them for the first time. For the already described citrullinated proteins MBP, GFAP, and vimentin, we could identify additional citrullinated sites. The number of modified proteins in MS white matter was higher than control tissue. Citrullinated peptides are considered neoepitopes that may trigger autoreactivity. We used newly identified epitopes and previously reported immunodominant myelin peptides in their citrullinated and non-citrullinated form to address the recognition of CSF-infiltrating CD4+ T cells from 22 MS patients by measuring proliferation and IFN-γ secretion. We did not detect marked responses to citrullinated peptides, but slightly more strongly to the non-modified version. Based on these data, we conclude that citrullination does not appear to be an important activating factor of a T cell response, but could be the consequence of an immune- or inflammatory response. Our approach allowed us to perform a deep proteome analysis and opens new technical possibilities to analyze complex PTM patterns on minute quantities of rare tissue samples

Fibroblast growth factor signalling in multiple sclerosis: inhibition of myelination and induction of pro-inflammatory environment by FGF9

The failure of remyelination in multiple sclerosis is largely unexplained. Lindner et al. report that glial cells in demyelinating lesions show increased expression of fibroblast growth factor 9 (FGF9). This induces astrocyte-dependent responses that inhibit remyelination and stimulate expression of pro-inflammatory chemokines, supporting a feedback loop that amplifies disease activit