Glatiramer Acetate (Copaxone) Modulates Platelet Activation and Inhibits Thrombin-Induced Calcium Influx: Possible Role of Copaxone in Targeting Platelets during Autoimmune Neuroinflammation

Background: Glatiramer acetate (GA, Copaxone, Copolymer-1) is an FDA approved drug for the treatment of MS and it is very effective in suppressing neuroinflammation in experimental autoimmune encephalitis (EAE), an animal model of MS. Although this drug was designed to inhibit pathogenic T cells, the exact mechanism of EAE/MS suppression by GA is still not well understood. Previously we presented evidence that platelets become activated and promote neuroinflammation in EAE, suggesting a possible pathogenic role of platelets in MS and EAE. We hypothesized that GA could inhibit neuroinflammation by affecting not only immune cells but also platelets. Methodology/Principal Findings We investigated the effect of GA on the activation of human platelets in vitro: calcium influx, platelet aggregation and expression of activation markers. Our results in human platelets were confirmed by in-vitro and in-vivo studies of modulation of functions of platelets in mouse model. We found that GA inhibited thrombin-induced calcium influx in human and mouse platelets. GA also decreased thrombin-induced CD31, CD62P, CD63, and active form of αIIbβ3 integrin surface expression and formation of platelet aggregates for both mouse and human platelets, and prolonged the bleeding time in mice by 2.7-fold. In addition, we found that GA decreased the extent of macrophage activation induced by co-culture of macrophages with platelets. Conclusions: GA inhibited the activation of platelets, which suggests a new mechanism of GA action in suppression of EAE/MS by targeting platelets and possibly preventing their interaction with immune cells such as macrophages. Furthermore, the reduction in platelet activation by GA may have additional cardiovascular benefits to prevent thrombosis

In Vivo Induction of Tr1 Cells via Mucosal Dendritic Cells and AHR Signaling

Background: Type 1 regulatory T (Tr1) cells, characterized by the secretion of high levels of the anti-inflammatory cytokine interleukin-10 (IL-10), play an important role in the regulation of autoimmune diseases and transplantation. However, effective strategies that specifically induce Tr1 cells in vivo are limited. Furthermore, the pathways controlling the induction of these cells in vivo are not well understood. Methodology/Principal Findings: Here we report that nasal administration of anti-CD3 antibody induces suppressive Tr1 cells in mice. The in vivo induction of Tr1 cells by nasal anti-CD3 is dependent on IL-27 produced by upper airway resident dendritic cells (DCs), and is controlled by the transcription factors aryl hydrocarbon receptor (AHR) and c-Maf. Subsequently, IL-21 acts in an autocrine fashion to expand and maintain the Tr1 cells induced in vivo by nasally administered anti-CD3. Conclusions/Significance: Our findings identify a unique approach to generate Tr1 cells in vivo and provide insights into the mechanisms by which these cells are induced

Galectin-1 Deactivates Classically Activated Microglia and Protects from Inflammation-Induced Neurodegeneration

SummaryInflammation-mediated neurodegeneration occurs in the acute and the chronic phases of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Classically activated (M1) microglia are key players mediating this process. Here, we identified Galectin-1 (Gal1), an endogenous glycan-binding protein, as a pivotal regulator of M1 microglial activation that targets the activation of p38MAPK-, CREB-, and NF-κB-dependent signaling pathways and hierarchically suppresses downstream proinflammatory mediators, such as iNOS, TNF, and CCL2. Gal1 bound to core 2 O-glycans on CD45, favoring retention of this glycoprotein on the microglial cell surface and augmenting its phosphatase activity and inhibitory function. Gal1 was highly expressed in the acute phase of EAE, and its targeted deletion resulted in pronounced inflammation-induced neurodegeneration. Adoptive transfer of Gal1-secreting astrocytes or administration of recombinant Gal1 suppressed EAE through mechanisms involving microglial deactivation. Thus, Gal1-glycan interactions are essential in tempering microglial activation, brain inflammation, and neurodegeneration, with critical therapeutic implications for MS

Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition)

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer‐reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state‐of‐the‐art handbook for basic and clinical researchers.DFG, 389687267, Kompartimentalisierung, Aufrechterhaltung und Reaktivierung humaner Gedächtnis-T-Lymphozyten aus Knochenmark und peripherem BlutDFG, 80750187, SFB 841: Leberentzündungen: Infektion, Immunregulation und KonsequenzenEC/H2020/800924/EU/International Cancer Research Fellowships - 2/iCARE-2DFG, 252623821, Die Rolle von follikulären T-Helferzellen in T-Helferzell-Differenzierung, Funktion und PlastizitätDFG, 390873048, EXC 2151: ImmunoSensation2 - the immune sensory syste

Effect of glatiramer acetate (GA) on thrombin-induced expression of activation marker CD31 and active form of αIIbβ3 integrin on the surface of mouse platelets.

<p>Mouse platelets were isolated and pretreated with PBS or GA (50 µg/ml) for 30 min. After pretreatment with PBS or GA, platelets were activated with thrombin and then analyzed for the expression of activation markers by three-color flow cytometry as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096256#pone-0096256-g002" target="_blank">Fig.2</a>. (A, C) Histograms for CD31 (A, solid lines), or active form of αIIbβ3 integrin (B, solid lines), or proper isotype control (dotted lines) expressions are shown for CD41⁺CD61⁺ gated mouse platelets. Mean fluorescent intensity (MFI) of activation marker expression is shown in upper right corner of each histogram. Mean ± S.E. of four separate experiments is shown in (B) and (D) (*, p<0.05).</p

Analysis of bleeding time in mice after administration of glatiramer acetate.

<p>Mice were administrated intravenously or subcutaneously with GA, or IFN-β, or PBS, and 30–60 minutes later, tail bleeding test was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096256#s5" target="_blank"><i>Methods</i></a>. Duration of bleeding (y-axis, seconds) is shown. Mean ± S.E. of total 9–12 individual animal in each group of three separate experiments is shown (***, p<0.001).</p

The role of glatiramer acetate (GA) in platelet-dependent activation of macrophages <i>in vitro</i>.

<p>Mouse peritoneal macrophages were isolated and cultured alone or co-cultured with autologous platelets with or without glatiramer acetate. After 24 hours of single culture or co-culture with platelets, macrophages were stained for CD11b, F4/80, CD86 and MHC class II and analyzed by four-color flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096256#s5" target="_blank"><i>Methods.</i></a> (<b>A</b>) Histograms for CD86 or MHC class II (solid lines) or proper isotype control (dotted lines) expressions are shown for CD11b⁺F/80⁺ gated macrophages. Percentages of positive cells are shown on the top of each histogram (<b>B, C</b>) Mean ± S.E. of three experiments is shown (*, p<0.05 and ***, p<0.001). <i>Abbreviations</i>: MPh, macrophages; GA, glatiramer acetate, Plat, platelets.</p

Statistical analysis of effects of glatiramer acetate (GA) on thrombin-induced CD62P upregulation and platelet aggregate formation for human and mouse platelets¹.

1<p>Human or mouse platelets were stimulated with thrombin in the presence of GA vs. Control (PBS) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096256#s5" target="_blank"><i>Methods</i></a>.</p>2<p>Optimal concentration of GA is 100 µg/ml for human platelets.</p>3<p>Optimal concentration of GA is 50 µg/ml for mouse platelets.</p>4<p>Mean fluorescence intensity (MFI) of CD62P expression on the surface of CD42a⁺CD61⁺ gated human or CD41⁺CD61⁺ gated mouse platelets was analyzed by FACS as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096256#s5" target="_blank"><i>in Methods</i></a>. Mean ± S.E. of five separate experiments is shown.</p>5<p>***, P<0.001.</p>6<p>*, P<0.05.</p>7<p>Percentages (%) of FCS^hiSSC^hi aggregated CD42a⁺CD61⁺ gated human or CD41⁺CD61⁺ mouse platelets was analyzed by FACS as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096256#s5" target="_blank"><i>Methods</i></a>. Mean ± S.E. of five separate experiments is shown.</p>8<p>**, P<0.01.</p

Effect of glatiramer acetate (GA) on platelet adhesion to collagen and aggregation under flow conditions.

<p>Whole mouse blood samples were collected from normal C57BL/6 mice using sodium citrate as anticoagulant and pretreated with PBS or GA (50 or 100 µg/ml) for 30 min as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096256#s5" target="_blank"><i>Methods</i></a>. After pretreatment with PBS or GA, the whole blood samples were labeled with platelet-specific antibodies and then perfused through the camber with collagen coated glace slides as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096256#s5" target="_blank"><i>Methods</i></a>. Collagen coated slides with aggregated pre-labeled platelets were washed, fixed and subsequently analyzed using fluorescence microscopy. Representative immunofluorescent images for samples treated with PBS or GA (50 or 100 µg/ml) are shown in (<b>A</b>). Average size of thrombi ± S.E. is shown in (<b>B</b>).</p

Effect of glatiramer acetate (GA) on thrombin-induced platelet aggregate formation.

<p>Human (<b>A</b>) or mouse (<b>B</b>) platelets were isolated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096256#s5" target="_blank"><i>Methods</i></a> and were pretreated with PBS or various concentrations of GA (20–500 µg/ml) for 30 min. After pretreatment with GA, platelets were activated with thrombin and then analyzed by FACS for aggregate formation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096256#s5" target="_blank"><i>Methods</i></a>. Contour plots for forward scatter (FSC; x-axes) vs. side scatter (SSC; y-axes) parameters are shown for CD42a⁺CD61⁺ gated human (<b>A</b>) or CD41⁺CD61⁺ gated mouse (<b>B</b>) platelets. Percentages of FCS^hiSSC^hi aggregated platelets are shown on a top of square gates of contour plots. Representative experiment of five is shown.</p