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

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

<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

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

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

Table_1_Alpha4 beta7 integrin controls Th17 cell trafficking in the spinal cord leptomeninges during experimental autoimmune encephalomyelitis.pdf

Th1 and Th17 cell migration into the central nervous system (CNS) is a fundamental process in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS). Particularly, leptomeningeal vessels of the subarachnoid space (SAS) constitute a central route for T cell entry into the CNS during EAE. Once migrated into the SAS, T cells show an active motility behavior, which is a prerequisite for cell-cell communication, in situ reactivation and neuroinflammation. However, the molecular mechanisms selectively controlling Th1 and Th17 cell trafficking in the inflamed leptomeninges are not well understood. By using epifluorescence intravital microscopy, we obtained results showing that myelin-specific Th1 and Th17 cells have different intravascular adhesion capacity depending on the disease phase, with Th17 cells being more adhesive at disease peak. Inhibition of αLβ2 integrin selectively blocked Th1 cell adhesion, but had no effect on Th17 rolling and arrest capacity during all disease phases, suggesting that distinct adhesion mechanisms control the migration of key T cell populations involved in EAE induction. Blockade of α4 integrins affected myelin-specific Th1 cell rolling and arrest, but only selectively altered intravascular arrest of Th17 cells. Notably, selective α4β7 integrin blockade inhibited Th17 cell arrest without interfering with intravascular Th1 cell adhesion, suggesting that α4β7 integrin is predominantly involved in Th17 cell migration into the inflamed leptomeninges in EAE mice. Two-photon microscopy experiments showed that blockade of α4 integrin chain or α4β7 integrin selectively inhibited the locomotion of extravasated antigen-specific Th17 cells in the SAS, but had no effect on Th1 cell intratissue dynamics, further pointing to α4β7 integrin as key molecule in Th17 cell trafficking during EAE development. Finally, therapeutic inhibition of α4β7 integrin at disease onset by intrathecal injection of a blocking antibody attenuated clinical severity and reduced neuroinflammation, further demonstrating a crucial role for α4β7 integrin in driving Th17 cell-mediated disease pathogenesis. Altogether, our data suggest that a better knowledge of the molecular mechanisms controlling myelin-specific Th1 and Th17 cell trafficking during EAE delevopment may help to identify new therapeutic strategies for CNS inflammatory and demyelinating diseases.</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

Table_4_Alpha4 beta7 integrin controls Th17 cell trafficking in the spinal cord leptomeninges during experimental autoimmune encephalomyelitis.pdf

Th1 and Th17 cell migration into the central nervous system (CNS) is a fundamental process in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS). Particularly, leptomeningeal vessels of the subarachnoid space (SAS) constitute a central route for T cell entry into the CNS during EAE. Once migrated into the SAS, T cells show an active motility behavior, which is a prerequisite for cell-cell communication, in situ reactivation and neuroinflammation. However, the molecular mechanisms selectively controlling Th1 and Th17 cell trafficking in the inflamed leptomeninges are not well understood. By using epifluorescence intravital microscopy, we obtained results showing that myelin-specific Th1 and Th17 cells have different intravascular adhesion capacity depending on the disease phase, with Th17 cells being more adhesive at disease peak. Inhibition of αLβ2 integrin selectively blocked Th1 cell adhesion, but had no effect on Th17 rolling and arrest capacity during all disease phases, suggesting that distinct adhesion mechanisms control the migration of key T cell populations involved in EAE induction. Blockade of α4 integrins affected myelin-specific Th1 cell rolling and arrest, but only selectively altered intravascular arrest of Th17 cells. Notably, selective α4β7 integrin blockade inhibited Th17 cell arrest without interfering with intravascular Th1 cell adhesion, suggesting that α4β7 integrin is predominantly involved in Th17 cell migration into the inflamed leptomeninges in EAE mice. Two-photon microscopy experiments showed that blockade of α4 integrin chain or α4β7 integrin selectively inhibited the locomotion of extravasated antigen-specific Th17 cells in the SAS, but had no effect on Th1 cell intratissue dynamics, further pointing to α4β7 integrin as key molecule in Th17 cell trafficking during EAE development. Finally, therapeutic inhibition of α4β7 integrin at disease onset by intrathecal injection of a blocking antibody attenuated clinical severity and reduced neuroinflammation, further demonstrating a crucial role for α4β7 integrin in driving Th17 cell-mediated disease pathogenesis. Altogether, our data suggest that a better knowledge of the molecular mechanisms controlling myelin-specific Th1 and Th17 cell trafficking during EAE delevopment may help to identify new therapeutic strategies for CNS inflammatory and demyelinating diseases.</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

Table_3_Alpha4 beta7 integrin controls Th17 cell trafficking in the spinal cord leptomeninges during experimental autoimmune encephalomyelitis.pdf

Th1 and Th17 cell migration into the central nervous system (CNS) is a fundamental process in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS). Particularly, leptomeningeal vessels of the subarachnoid space (SAS) constitute a central route for T cell entry into the CNS during EAE. Once migrated into the SAS, T cells show an active motility behavior, which is a prerequisite for cell-cell communication, in situ reactivation and neuroinflammation. However, the molecular mechanisms selectively controlling Th1 and Th17 cell trafficking in the inflamed leptomeninges are not well understood. By using epifluorescence intravital microscopy, we obtained results showing that myelin-specific Th1 and Th17 cells have different intravascular adhesion capacity depending on the disease phase, with Th17 cells being more adhesive at disease peak. Inhibition of αLβ2 integrin selectively blocked Th1 cell adhesion, but had no effect on Th17 rolling and arrest capacity during all disease phases, suggesting that distinct adhesion mechanisms control the migration of key T cell populations involved in EAE induction. Blockade of α4 integrins affected myelin-specific Th1 cell rolling and arrest, but only selectively altered intravascular arrest of Th17 cells. Notably, selective α4β7 integrin blockade inhibited Th17 cell arrest without interfering with intravascular Th1 cell adhesion, suggesting that α4β7 integrin is predominantly involved in Th17 cell migration into the inflamed leptomeninges in EAE mice. Two-photon microscopy experiments showed that blockade of α4 integrin chain or α4β7 integrin selectively inhibited the locomotion of extravasated antigen-specific Th17 cells in the SAS, but had no effect on Th1 cell intratissue dynamics, further pointing to α4β7 integrin as key molecule in Th17 cell trafficking during EAE development. Finally, therapeutic inhibition of α4β7 integrin at disease onset by intrathecal injection of a blocking antibody attenuated clinical severity and reduced neuroinflammation, further demonstrating a crucial role for α4β7 integrin in driving Th17 cell-mediated disease pathogenesis. Altogether, our data suggest that a better knowledge of the molecular mechanisms controlling myelin-specific Th1 and Th17 cell trafficking during EAE delevopment may help to identify new therapeutic strategies for CNS inflammatory and demyelinating diseases.</p