Histological features of brain and spinal cord sections from passively induced EAE (WT in WT, WT in <i>Was^−/−</i> and <i>Was^−/−</i> in WT).

<p>Samples were obtained 3 weeks after transfer and stained with H&E (<i>upper panels</i>; CB, cerebellum and SC, spinal cords), anti-CD3 and anti-Iba1 (<i>lower panels</i>). Insets show in detail representative microglial cells within spinal cords. Upper panels are from 10×, lower panels from 20× and insets from 40× original magnification.</p

Development of Central Nervous System Autoimmunity Is Impaired in the Absence of Wiskott-Aldrich Syndrome Protein

<div><p>Wiskott-Aldrich Syndrome protein (WASP) is a key regulator of the actin cytoskeleton in hematopoietic cells. Defective expression of WASP leads to multiple abnormalities in different hematopoietic cells. Despite severe impairment of T cell function, WAS patients exhibit a high prevalence of autoimmune disorders. We attempted to induce EAE, an animal model of organ-specific autoimmunity affecting the CNS that mimics human MS, in <i>Was^−/−</i> mice. We describe here that <i>Was^−/−</i> mice are markedly resistant against EAE, showing lower incidence and milder score, reduced CNS inflammation and demyelination as compared to WT mice. Microglia was only poorly activated in <i>Was^−/−</i> mice. Antigen-induced T-cell proliferation, Th-1 and -17 cytokine production and integrin-dependent adhesion were increased in <i>Was^−/−</i> mice. However, adoptive transfer of MOG-activated T cells from <i>Was^−/−</i> mice in WT mice failed to induce EAE. <i>Was^−/−</i> mice were resistant against EAE also when induced by adoptive transfer of MOG-activated T cells from WT mice. <i>Was^+/−</i> heterozygous mice developed an intermediate clinical phenotype between WT and <i>Was^−/−</i> mice, and they displayed a mixed population of WASP-positive and -negative T cells in the periphery but not in their CNS parenchyma, where the large majority of inflammatory cells expressed WASP. In conclusion, in absence of WASP, T-cell responses against a CNS autoantigen are increased, but the ability of autoreactive T cells to induce CNS autoimmunity is impaired, most probably because of an inefficient T-cell transmigration into the CNS and defective CNS resident microglial function.</p></div

<i>Was</i>⁻^<i>/</i>− mice are resistant against EAE.

<p><b>A</b>, EAE was induced in <i>Was</i>^{−<i>/</i>−} (<i>white circles</i>) and WT mice (<i>black circles</i>) with MOG_35–55 peptide. Data represent mean clinical scores of 10 mice/group ± SEM. The experiment is representative of two independent experiments. Statistical analysis was performed with Sum Rank Test. ^*<i>P</i><0.05. <b>B</b>, Histopathological analyses were performed on spinal cords and brains obtained from <i>Was</i>^{−<i>/</i>−} and WT mice two weeks after the induction of EAE. Representative 5–8 mice per group were analyzed. H&E staining (<i>upper panels</i>) and anti-MBP immunostain (<i>lower panels</i>) highlight inflammatory changes and myelin damage, respectively. Insets report in detail inflammatory foci and demyelinated areas within spinal cords (<i>upper insets</i>) and brain parenchyma (<i>lower insets</i>), respectively. Graphs show percentage of inflammatory cell infiltration and demyelination in spinal cords of WT and <i>Was</i>^{−<i>/</i>−} mice. ^*<i>P</i><0.05; ^**<i>P</i><0.01. <b>C</b>, CD3 (<i>upper panels</i>) and Iba-1 (<i>lower panels</i>) immunostains highlight T-cell infiltration and both macrophages and microglial activation, respectively. Details of the staining as for the previous panels are reported in the insets. In all panels serial sections of representative CNS sample are reported. Panels are 4× and insets 20× original magnification.</p

Peripheral T-cell and antibody response against myelin antigen in <i>Was</i>⁻^<i>/</i>− mice.

<p><b>A</b>, LNCs and splenocytes were harvested from <i>Was</i>^{−<i>/</i>−} and WT mice (5 mice/group) two weeks after EAE induction and pooled cells were cultured <i>in vitro</i> with increasing doses of MOG_35–55 peptide or medium alone. [³H] thymidine was added to LNCs cultures 72 h after stimulation (<i>upper left corner</i>). Cytokine levels were measured on supernatants from splenocytes 48 h after stimulation. <b>B</b>, Antigen-specific IgG antibodies (<i>left panel</i>) and total IgE antibodies (<i>right panel</i>) were measured in duplicate in sera obtained from mice (10 mice/group) 6 weeks after EAE induction. Data are representative of one of the two independent experiments that gave similar results. ^*<i>P</i><0.05; ^**<i>P</i><0.01.</p

Adhesive properties of MOG_35–55-primed WT and <i>Was^−/−</i> T-cell blasts.

<p><b>A,</b> The expression of LFA-1, α₄-integrin, PSGL-1, CD44 and L-selectin was analyzed by flow cytometry on MOG_35–55-primed T-cell blasts obtained form WT or <i>Was^−/−</i> immunized mice. The mean fluorescence intensity (MFI) of staining is shown. <b>B,</b> WT or <i>Was^−/−</i> MOG_35–55-primed T-cell blasts were left to adhere spontaneously on slides coated with purified ICAM-1, with or without CXCL12. <b>C,</b> WT or <i>Was^−/−</i> MOG_{35<i>–</i>55}-primed T-cell blasts were stimulated with CXCL12 chemokine or control buffer and then labeled with anti-LFA-1 antibody and LFA-1 distribution on cell surface was analyzed with the ImageStream system. <b>D,</b> WT or <i>Was^−/−</i> MOG_35–55-primed T-cell blasts were injected in the right carotid of LPS-treated mice to analyze their interaction with brain pial vessels expressing ICAM-1, VCAM-1 and endothelial selectins <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086942#pone.0086942-Piccio1" target="_blank">[22]</a>. Rolling interactions and stable adhesions were evaluated by analyzing at least 100 cells/venule. In all panels, data are mean ± SD of three independent experiments. <b>E,</b> WT or <i>Was^−/−</i> LN cells isolated from MOG_35–55-immunized mice were seeded in transwells and induced to migrate in the presence or absence of CXCL12 for 3 h. Frequency of cells migrated to the bottom wells of transwell plates are indicated in the graph. Each bar is representative of a pool of 15 mice. Statistical analysis was performed with Fisher's Exact Test. ^**<i>P</i><0.01.</p

<i>Was^+/−</i> heterozygous mice are not resistant to EAE induction.

<p><b>A,</b> EAE was induced in <i>Was^−/−</i> (<i>white circles</i>), <i>Was^+/−</i> (<i>grey circles</i>) and WT mice (<i>black circles</i>) with MOG_35–55 peptide. Data represent mean clinical scores of 5 mice/group ± SEM. <b>B,</b> Histopathological analyses were performed on spinal cords obtained from <i>Was^−/−</i>, <i>Was^+/−</i> and WT mice 14 days after the induction of EAE. WASP (<i>left panels</i>) and WASP-CD3 (<i>right panels</i>) immunostains highlight WASP expression in total CNS infiltrates and in T-cell infiltrates, respectively. Details of the staining are reported in the insets. In all panels serial sections of representative CNS samples are reported. Panels for WASP single stainings are 20×, insets 40× and WASP-CD3 double stainings 60× original magnification.</p

Passive transfer of EAE.

<p>EAE was induced with injection of 5×10⁶ T-cell blasts per mouse derived from WT or <i>Was^−/−</i> mice actively immunized with MOG_35–55 peptide. ^a)Data represent mean ± SEM for those mice that developed the disease.</p

Clinical traits of EAE in <i>Was</i>^{−<i>/</i>−} and wild type mice.

<p>Data are shown as mean ± SEM. ^a)Mice reached a peak of disease severity at day 22 after immunization in Experiment 1 and at day 14 in Experiment 2. ^*<i>P</i><0.05, ^**<i>P</i><0.01, ^***<i>P</i><0.005 <i>vs Was</i>^{−<i>/</i>−} group.</p

Endogenous erythropoietin as part of the cytokine network in the pathogenesis of experimental autoimmune encephalomyelitis

Erythropoietin (EPO) is of great interest as a therapy for many of the central nervous system (CNS) diseases and its administration is protective in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Endogenous EPO is induced by hypoxic/ischemic injury, but little is known about its expression in other CNS diseases. We report here that EPO expression in the spinal cord is induced in mouse models of chronic or relapsing-remitting EAE, and is prominently localized to motoneurons. We found a parallel increase of hypoxia-inducible transcription factor (HIF)-1 alpha, but not HIF-2 alpha, at the mRNA level, suggesting a possible role of non-hypoxic factors in EPO induction. EPO mRNA in the spinal cord was co-expressed with interferon (IFN)-gamma and tumor necrosis factor (TNF), and these cytokines inhibited EPO production in vitro in both neuronal and glial cells. Given the known inhibitory effect of EPO on neuroinflammation, our study indicates that EPO should be viewed as part of the inflammatory/anti-inflammatory network in MS