Testosterone Inhibits Secretion of the Pro-Inflammatory Chemokine CXCL1 from Astrocytes.

Astrocytes play an important role in the regulation of the inflammatory response in the CNS, e.g., in demyelinating diseases. Since the chemokine CXCL1 is known to be secreted by astrocytes and to have a pro-inflammatory effect on immune cells in the CNS, we verified the effect of testosterone on its secretion in vitro (in the astrocytic cell line DI TNC1). Testosterone reduced the increase in CXCL1 production caused by the pro-inflammatory agent lysophosphatidylcholine and restored the basal production level of CXCL1. The androgen receptor (present and functional in the studied cell line) was strongly suggested to mediate this effect-its non-steroid ligand flutamide exerted an agonist-like effect, mimicking the activity of testosterone itself on CXCL1 secretion. This novel mechanism has important implications for the known immunomodulatory effect of testosterone and potentially other androgenic hormones. It provides a potential explanation on the molecular level and shows that astrocytes are important players in inflammatory homeostasis in the CNS and its hormonal regulation. Therefore, it suggests new directions for the development of the therapeutic intervention.

Unexpected central role of the androgen receptor in the spontaneous regeneration of myelin.

Lost myelin can be replaced after injury or during demyelinating diseases in a regenerative process called remyelination. In the central nervous system (CNS), the myelin sheaths, which protect axons and allow the fast propagation of electrical impulses, are produced by oligodendrocytes. The abundance and widespread distribution of oligodendrocyte progenitors (OPs) within the adult CNS account for this remarkable regenerative potential. Here, we report a key role for the male gonad, testosterone, and androgen receptor (AR) in CNS remyelination. After lysolecithin-induced demyelination of the male mouse ventral spinal cord white matter, the recruitment of glial fibrillary acidic protein-expressing astrocytes was compromised in the absence of testes and testosterone signaling via AR. Concomitantly, the differentiation of OPs into oligodendrocytes forming myelin basic protein (MBP)+ and proteolipid protein-positive myelin was impaired. Instead, in the absence of astrocytes, axons were remyelinated by protein zero (P0)+ and peripheral myelin protein 22-kDa (PMP22)+ myelin, normally only produced by Schwann cells in the peripheral nervous system. Thus, testosterone favors astrocyte recruitment and spontaneous oligodendrocyte-mediated remyelination. This finding may have important implications for demyelinating diseases, psychiatric disorders, and cognitive aging. The testosterone dependency of CNS oligodendrocyte remyelination may have roots in the evolutionary history of the AR, because the receptor has evolved from an ancestral 3-ketosteroid receptor through gene duplication at the time when myelin appeared in jawed vertebrates.

The neural androgen receptor: a therapeutic target for myelin repair in chronic demyelination.

Myelin regeneration is a major therapeutic goal in demyelinating diseases, and the failure to remyelinate rapidly has profound consequences for the health of axons and for brain function. However, there is no efficient treatment for stimulating myelin repair, and current therapies are limited to anti-inflammatory agents. Males are less likely to develop multiple sclerosis than females, but often have a more severe disease course and reach disability milestones at an earlier age than females, and these observations have spurred interest in the potential protective effects of androgens. Here, we demonstrate that testosterone treatment efficiently stimulates the formation of new myelin and reverses myelin damage in chronic demyelinated brain lesions, resulting from the long-term administration of cuprizone, which is toxic for oligodendrocytes. In addition to the strong effect of testosterone on myelin repair, the number of activated astrocytes and microglial cells returned to low control levels, indicating a reduction of neuroinflammatory responses. We also identify the neural androgen receptor as a novel therapeutic target for myelin recovery. After the acute demyelination of cerebellar slices in organotypic culture, the remyelinating actions of testosterone could be mimicked by 5α-dihydrotestosterone, a metabolite that is not converted to oestrogens, and blocked by the androgen receptor antagonist flutamide. Testosterone treatment also failed to promote remyelination after chronic cuprizone-induced demyelination in mice with a non-functional androgen receptor. Importantly, testosterone did not stimulate the formation of new myelin sheaths after specific knockout of the androgen receptor in neurons and macroglial cells. Thus, the neural brain androgen receptor is required for the remyelination effect of testosterone, whereas the presence of the receptor in microglia and in peripheral tissues is not sufficient to enhance remyelination. The potent synthetic testosterone analogue 7α-methyl-19-nortestosterone, which has been developed for long-term male contraception and androgen replacement therapy in hypogonadal males and does not stimulate prostate growth, also efficiently promoted myelin repair. These data establish the efficacy of androgens as remyelinating agents and qualify the brain androgen receptor as a promising drug target for remyelination therapy, thus providing the preclinical rationale for a novel therapeutic use of androgens in males with multiple sclerosis.

Real-world effectiveness of fingolimod in Polish group of patients with relapsing-remitting multiple sclerosis.

OBJECTIVES: Fingolimod is indicated for the treatment of relapsing-remitting multiple sclerosis (RRMS) patients with highly aggressive disease characterized by frequent relapses and active magnetic resonance imaging. Its efficacy has been demonstrated in three large phase III trials, used in the regulatory submissions throughout the world. Fingolimod in licensed in Europe since 2011 but with a growing number of disease-modifying drugs (DMD) becoming available for RRMS, it is important to gather real-world evidence data regarding long-term effectiveness in treated patients with MS. The aim of this study was to assess fingolimod effectiveness in a real life Polish group of RRMS patients receiving fingolimod as second line treatment. PATIENTS AND METHODS: The observational study with retrospective data collection was performed at 13 sites that were asked to document eligible patients in consecutive chronological order to avoid selection bias. Demographic and clinical data from 253 adult patients with RRMS treated with fingolimod were analyzed. RESULTS: Mean treatment time with fingolimod was 42 months. Relapses reduction during 3 years treatment period was observed (2.0 v 0.2) and majority of patients were free of relapses. Mean EDSS score was stable during the time of observation. The proportion of patients who were free from any clinical disease activity, i.e. without relapses and disability progression, was over 70%. During the first and second year of observation significant reduction of new MRI lesions was observed. CONCLUSION: In the Polish group of patients with RRMS treated with fingolimod, the majority of them showed freedom from relapses, disability progression and reduction of new MRI lesions. Switching from injectable immunomodulatory drugs to fingolimod is associated with fewer relapses and lower disability progression.

Overcoming failure to repair demyelination in EAE: gamma-secretase inhibition of Notch signaling.

In multiple sclerosis (MS), myelin destroyed by the immune attack is not effectively repaired by oligodendrocytes (OLs) and MS foci eventually undergo glial scarring. Although oligodendrocyte precursor cells (OPCs) are normally recruited to the lesion areas, they fail to mature and remyelinate the damaged fibers. Activation of the Notch pathway has been shown to inhibit OPC differentiation and to hamper their ability to produce myelin during CNS development. We have recently shown that inhibition of gamma-secretase within the CNS of SJL/J mice with experimental autoimmune encephalomyelitis (EAE) blocks Notch pathway activation in OLs, promotes remyelination, reduces axonal damage and significantly enhances clinical recovery from the disease. Our results suggest that inhibiting the non-myelin permissive environment maintained by Notch pathways within the mature CNS offers a new strategy for treating autoimmune demyelination, including MS.

Linkage disequilibrium screening for multiple sclerosis implicates JAG1 and POU2AF1 as susceptibility genes in Europeans.

By combining all the data available from the Genetic Analysis of Multiple sclerosis in EuropeanS (GAMES) project, we have been able to identify 17 microsatellite markers showing consistent evidence for apparent association. As might be expected five of these markers map within the Major Histocompatibility Complex (MHC) and are in LD with HLA-DRB1. Individual genotyping of the 12 non-MHC markers confirmed association for three of them--D11S1986, D19S552 and D20S894. Association mapping across the candidate genes implicated by these markers in 937 UK trio families revealed modestly associated haplotypes in JAG1 (p=0.019) on chromosome 20p12.2 and POU2AF1 (p=0.003) on chromosome 11q23.1.

Inhibition of Notch signaling enhances tissue repair in an animal model of multiple sclerosis.

Oligodendrocytes (OLs) fail to regenerate myelin destroyed by the immune attack in multiple sclerosis (MS) and lesion areas are eventually largely occupied by astrocytic scar tissue. Loss of OLs in MS does not account for the limited myelin repair as lesions contain a considerable number of OL precursor cells (OPC). Activation of the Notch pathway has been shown to provide inhibitory signals for OPC and to hamper their ability to produce myelin during CNS development. Here we show that gamma-secretase inhibition of Notch signaling within OL of CNS of SJL/J mice with experimental autoimmune encephalomyelitis (EAE) significantly enhanced clinical recovery and in the CNS, promoted remyelination and reduced axonal damage. Functional assays confirmed decreased Notch signaling in inhibitor-treated groups. Therefore, gamma-secretase inhibition led to an environment more conducive to myelin repair and axonal survival. Our results suggest that manipulation of the environment associated with Notch activation in the mature CNS provides a promising therapeutic target in MS.

Central Nervous System and Peripheral Expression of CCL19, CCL21 and Their Receptor CCR7 in Experimental Model of Multiple Sclerosis.

It is well documented that inflammatory chemokines play a significant role in the development of multiple sclerosis (MS) and its model, experimental autoimmune encephalomyelitis (EAE). Recently, the involvement of homeostatic (or lymphoid) chemokines in the pathogenesis of autoimmune diseases has become an object of intensive study. In this work, quantitative analysis of CCL19, CCL21 and CCR7 expression in the central nervous system (CNS), as well as in inflammatory mononuclear cells isolated from several organs during the first attack, remission and the second attack of chronic-relapsing EAE (ChREAE), was performed. Using real-time PCR, RNAse Protection Assay and immunohistochemistry, the expression of both chemokines, as well as of their common receptor CCR7, was analyzed in the brain, spleen, lymph nodes and peripheral blood mononuclear cells. Increased expression of CCL19 and CCL21 was observed mostly in mononuclear inflammatory cells isolated from the CNS during active ChREAE. At the same time the expression of CCR7 in blood mononuclear leukocytes was reduced. This observation extends our current knowledge about the possible role of chemokines CCL19, CCL21 and their receptor CCR7 in the pathogenesis of ChREAE and, by extension, MS.

TNF-alpha microinjection upregulates chemokines and chemokine receptors in the central nervous system without inducing leukocyte infiltration.

Chemokines (chemoattractant cytokines) are key players in the initiation of inflammatory cell accumulation in the central nervous system (CNS). Mechanisms leading to upregulation of chemokines in CNS pathologic conditions remain largely unknown. Numerous in vitro studies showed that inflammatory cytokines stimulate cultured CNS cells to produce chemokines. The main goal of this study was to analyze if an individual proinflammatory cytokine is sufficient to upregulate the chemokine system in the adult CNS in vivo. We analyzed CC chemokine ligand and receptor expression in brains from two different strains of mice (SJL and BALB) after stereotaxic, intracerebral injection of tumor necrosis factor-alpha (TNF-alpha). In both strains, we detected similarly increased expression of chemokines RANTES/CCL5, macrophage inflammatory protein-1alpha (MIP-1alpha)/CCL3, MIP-1beta/CCL4, and MIP-2, as well as chemokine receptors CCR1, CCR2, and CCR5. Interestingly, we did not observe parenchymal leukocyte infiltrates after local TNF-alpha delivery. This observation shows that upregulation of chemokines by TNF-alpha is not sufficient to cause accumulation of leukocytes in the CNS parenchyma in both strains of mice.

A whole genome screen for association in Polish multiple sclerosis patients.

We have performed the first systematic search for MS susceptibility genes completed in the Polish population. This screen was performed using 6000 microsatellite markers typed in pooled DNA from cases (n=200), controls (n=200) and trio families (n=129). Five associated markers are identified, one (D6S2444) from the HLA region and four are from novel regions not previously associated with MS, 2p16 (D2S2153), 3p13 (D3S3568), 7p22 (D7S2521) and 15q26 (D15S649).

Treatment with soluble tumor necrosis factor receptor (sTNFR):Fc/p80 fusion protein ameliorates relapsing-remitting experimental autoimmune encephalomyelitis and decreases chemokine expression.

Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease with pathological and clinical similarities to the major human demyelinating disease multiple sclerosis (MS). Multiple lines of evidence in recent years implicate the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) in the pathogenesis of both EAE and MS. TNF-alpha cellular responses are mediated by signaling through receptors, which are expressed in two functional forms, designated according to molecular weight p55/60 and p75/80. We report a treatment trial using the extracellular domain of the p80 TNFR in a bivalent fusion construct designated soluble tumor necrosis factor receptor (sTNFR):Fc to treat EAE. sTNFR:Fc/p80, given after the onset of clinical signs, reduced the clinical deficit of the first attack of relapsing-remitting EAE (RR-EAE) and the exacerbation rate for subsequent attacks. The effect was reversible as mice treated with sTNFR:Fc/p80 reverted to an exacerbation rate and disease severity typical of placebo-treated animals after treatment was discontinued. Treatment of RR-EAE with sTNFR:Fc/p80 decreased expression of chemokines MIP-1alpha (Monocyte Inflammatory Protein)/CCL3, MIP-1beta/CCL4 and MIP-2/CXCL1-2 in the central nervous system. This treatment trial reveals an important function of TNF in the pathogenesis of RR-EAE and propose the mechanism of beneficial action of sTNFR:Fc/p80 in this disease.

Treatment of multiple sclerosis with methylprednisolone and mitoxantrone modulates the expression of CXC chemokine receptors in PBMC.

Chemokines and their receptors are involved in the development of multiple sclerosis (MS). Methylprednisolone (MP) and mitoxantrone (MTX) are commonly used in the treatment of MS. In this study, we analyzed the expression of chemokine receptors CXCR1, CXCR2, CXCR3, CXCR4, and CXCR5 in peripheral blood mononuclear cells (PBMC) from MS patients before and after treatment with MP or MTX. We observed a significant upregulation of expression of CXCR1 and CXCR2 in untreated MS patients. Treatment of MS with MP stimulated further increase of expression of both receptors. Therapy for MS with MTX resulted in decrease of CXCR2 expression. There was a negative correlation between the expression of CXCR1 and CXCR2 and the cumulative dose of MTX received by patients. These results suggest that CXCR1 and CXCR2 may be involved in MS pathogenesis and that treatment of this disease with MP and MTX may influence expression of those receptors.

Experimental autoimmune encephalomyelitis: CC chemokine receptor expression by trafficking cells.

Chemokines are chemotactic cytokines, which stimulate migration of inflammatory cells towards tissue sites of inflammation. The largest chemokine group, termed CC chemokines (CCLs), act primarily on T cells and monocytes, through CC chemokine receptors (CCRs) belonging to the superfamily of G-protein coupled seven transmembrane domain receptors. CCR expression is a critical determinant of cellular responses to CCLs. In this report, we describe the expression pattern of mRNA encoding selected CCRs in the spinal cord and spleen of perfused and non-perfused mice at different stages of chronic-relapsing EAE (ChREAE). We detected increased expression of receptors (CCR1, CCR5) associated with T helper-1 (Th1) but not those (CCR3, CCR4) associated with Th2 T cells in spinal cord during initial attack and relapse of ChREAE. Expression of these CCRs correlated temporally and spatially with reported previously expression of corresponding CCLs. The principal cells expressing CCR5 were inflammatory cells invading the spinal cord. Our results supported the implication of Th1-associated CCRs in the CNS-specific inflammatory reaction of ChREAE.