Galectin-1 deactivates classically activated microglia and protects from inflammation-induced neurodegeneration

Abstract

Inflammation-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.

Figure 1. CNS Expression of Gal1 is Dynamically Regulated during EAE

(A-F) Gal1 mRNA and protein expression in mouse spinal cord tissue and confocal microscopy of spinal cord white matter sections from CFA immunized (control), preclinical EAE (preclinical, 10 dpi), acute EAE (acute, 20 dpi) and chronic EAE (chronic, 40 dpi) mice. (A) Relative Gal1 mRNA expression in mouse spinal cord tissue. (B) Mean Fluorescence Intensity (MFI) of Gal1 immunoreactivity in mouse spinal cord white matter. (C) Immunoblot of Gal1 expression in mouse spinal cord tissue. (D-F) Confocal microscopy analysis. Sections were stained with anti-Gal1 antibody (green), the nuclear marker Topro3 (blue) and anti-GFAP (D, red), anti-CD4 (E, red), or anti-CD11b (F, red) antibodies. Scale bars= 20 μm (left panel). Right panels show 3D reconstruction ortho-view micrographs of representative cells. The images of every marker were acquired using the same parameters. (G) Effect of stimulation of astrocytes with LPS (10 ng/ml), IFN-γ (10 ng/ml), IL-17A (10 ng/ml), IL-4 (10 ng/ml), TGF-β₁ (5 ng/ml) and recombinant Gal1 (5 g/ml) on Gal1 mRNA expression and secretion. Data are representative (C-F) or are the mean ± SEM (A,B,C,G) of three independent experiments. *P<0.05; **P<0.01; ***P<0.005 versus control. See also Figure S1.

Figure 2. Gal1 Differentially Modulates Microglia Activation in vitro

(A) Flow cytometry of resting and polarized microglia subsets incubated with increasing concentrations of recombinant Gal1. (B) Expression of cell surface glycans on M1 (LPS, IFN-γ), M2 (IL-4) and resting (unstimulated) microglia, detected with biotinylated SNA, PNA, L-PHA, HPA and MAL II (black filled histograms) or with FITC-conjugated streptavidin alone (dashed open histograms). Red numbers represent the relative median of intensity (median of intensity (Lectin) – median of intensity (Streptavidin control)). Bar diagrams display the lectin binding as fold change relative to unstimulated microglia. (C) Flow cytometry of M1 microglia activated by IFN-γ (24 h). Control (IFN-γ-MG) or Gal1-treated (IFN-γ-MG_Gal1) microglia were stained with antibodies against CD86 and MHC II. Black lines represent specific antibody binding whereas tinted lines represent unspecific fluorescence signal. Percentage of positive cells and relative median fluorescence (rMFI): (median fluorescence intensity of specific marker signal – median fluorescence intensity of unspecific signal) are shown. Bar diagrams display the relative MFI and percentage of positive cells as fold change relative to IFN-γ-treated microglia. (D-H) Effect of Gal1 on the expression of specific activation markers of IFN-γ- or IL-4-stimulated microglia as determined by quantitative RT-PCR for iNOS (D,G) and arginase (H) mRNA or by bead-based Luminex assay for TNF (E) and CCL2 (F). (I-M) Immunoblot blot (I) and densitometric analysis (J-M) of p-p38MAPK, pERK1/2, pCREB and pIκB-α in neonatal microglia obtained from Lgals1^−/− or WT mice, pre-treated or not with Gal1 (5 μg/ml) and stimulated with LPS. Data are representative (B and C upper panel, I) or are the mean ± SEM (A-H, J-M) of three independent experiments. *P<0.05; **P<0.01; ***P<0.005 versus control. See also Figure S2 and S3.

Figure 3. Gal1-glycan Interactions Promote Retention of CD45 on the Surface of Microglial Cells and Augment its Phosphatase Activity

(A) Co-immunoprecipitation followed by immunoblotting of Gal1 and CD45 expression in lysates from microglia cells incubated with LPS (10 ng/ml) for 18 h and further stimulated or not with recombinant Gal1. Input, whole cell lysate; IB, immunoblot; IP, immunoprecipitation. (B) Confocal microscopy of CD45 and Gal1 co-localization in BV-2 microglia cells incubated with LPS for 18 h and further exposed to FITC-conjugated Gal1. Scale bar = 25 m. Inset scale bar = 10 m. (C,D) Flow cytometry analysis of CD45 and CD80 expression in unpermeabilized BV-2 microglia cells incubated with LPS for 18 h and then stimulated with PBS or Gal1 for the indicated time periods. Nonspecific binding determined with isotype-matched control antibodies is shown for treatment at t = 1 min. Numbers outside parentheses show the percentage of positive cells. Numbers in parentheses represent the relative mean fluorescence intensity (rMFI): (median fluorescence intensity of specific marker signal – median fluorescence intensity of unspecific signal) for each time analyzed. (E) Confocal microscopy of CD45 internalization in LPS-stimulated microglia cells treated with Gal1 or vehicle control for 30 min. Cells were fixed, permeabilized and probed with monoclonal antibodies against CD45 and EEA1. Upper panel, representative images of EEA1 (green) - and CD45 (red)-stained microglia cells. Scale bar = 25 m. Inset scale bar = 10 m. Lower panel, CD45/EEA1 overlay normalized to total EEA1 staining determined by MBF-ImageJ Colocalization Analysis by defining a box of set dimensions and scoring the incidence of superposition in 6 randomly selected areas. (F) CD45-specific phosphatase activity in microglia treated with LPS and further exposed to Gal1 in the absence or presence of a CD45-specific phosphatase inhibitor for the indicated time periods. (G) Co-immunoprecipitation followed by immunoblotting of Gal1 and CD45 expression in lysates from microglia cells transfected with C2GnT1, GnT5 or scrambled (Scr) siRNA, stimulated with LPS (10 ng/ml) and further treated with Gal1. Input, whole cell lysate. Data are representative of two independent experiments. (H) CD45-specific phosphatase activity in microglia cells transfected with GnT5, C2GnT1 or scrambled (Scr) siRNA, treated with LPS and further exposed to Gal1 for 30 min in the absence or presence of a CD45-specific phosphatase inhibitor. Similar results were observed by pre-incubating BV-2 microglia cells with Gal1 before exposure to LPS as in Figure 2. Data are representative (A-E, G) or are the mean ± SEM (E lower panel, F, H) of three independent experiments. *P<0.05 versus LPS; **P<0.01 versus LPS; ^#P<0.05 versus LPS plus Gal1. See also Figure S4.

Figure 4. Endogenous Gal1 Controls Classical Microglia Activation in vivo and Limits EAE Neuropathology

(A-E) Confocal microscopy of spinal cord white matter of WT and Lgals1^−/− mice 35 days after immunization with MOG35-55. (A) Left. Spinal cord sections were stained for Iba1 (green) and MHC II (red). Insert shows low magnification micrograph of representative cells. Middle. 3D reconstruction ortho-view of low magnification migrograph. Right. Quantification of MHC II, Iba1 double positive microglia cells (n=20). (B) Left. Spinal cord sections were stained for Tuj1 (green). Middle. 2.5D intensity analysis of Tuj1 staining. Right. mean fluorescence intensity (MFI) of immunoreactivity against Tuj1. (C) Left. Spinal cord sections were stained for GAP43 (green). Middle. 2.5D intensity analysis of GAP43 staining. Right mean fluorescence intensity (MFI) of immunoreactivity against GAP43. (D) Left. Spinal cord sections were stained for MBP (green). Middle. 2.5D intensity analysis of MBP staining. Right mean fluorescence intensity (MFI) of immunoreactivity against MBP. (E) Left. Spinal cord sections were stained for GFAP (green). Middle. 2.5D intensity analysis of GFAP staining. Right mean fluorescence intensity (MFI) of immunoreactivity against GFAP. Scale bars= 20 μm. (A-E) Data are representative (images) or are the mean (bars) ± SEM of three independent experiments. *P < 0.05; **P < 0.01.

Figure 5. Gal1 Negatively Regulates Microglia-induced Neurotoxicity

(A-D) Twenty four-h co-culture of pre-treated microglia (untreated MG, LPS-treated MG, LPS plus Gal1-treated MG and Gal1-treated MG) with high-density cortical neuronal cultures. (A) Representative fluorescence photomicrographs of Map2⁺ (green) neurons. (B) Density of Map2⁺ cell bodies per mm² (n=10). (C) High magnification photomicrographs of single Map2⁺ neurons and pixel intensity analysis (below). (D) Percentage of beaded axons per total axons (n=10) (A) Scale bar= 50 μm, (C) Scale bar= 5 μm. Data are representative (A,C) or are the mean ± SEM (B,D) of three independent experiments. ***P < 0.005. See also Figure S5.

Figure 6. Astrocytes Control Microglia Activation and Limit EAE Severity via Gal1

(A) Flow cytometry of MHC II in cultured CD11b⁺ microglia. Microglia were exposed to conditioned media from control or TGF-β₁-stimulated WT or Lgals1^−/− astrocytes. Percentage of CD11b⁺MHC II⁺ cells. (B,C) Lgals1^−/− mice were immunized with 200 μg MOG35-55 and injected with PBS (vehicle) or clodronate-containing liposomes (CLD) into the right lateral ventricle (day 7 and 9 post immunization). When reaching a clinical score of 1, mice were divided into 2 groups and received either WT or Lgals1^−/− (KO) astrocytes into the same injection site. (B) Diagram illustrating the experimental time line (left) and injection site (right). (C) Clinical score (left) and linear regression curves of disease (right) for each group (dashed lines, 95% confidence intervals). Data are representative (A, upper panel) or are the mean ± SEM (A lower panel, C) of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.005. See also Figure S6.

Figure 7. Gal1 Therapy Ameliorates EAE, Limits Microglia Activation, Controls Axonal Loss and Promotes Synaptic Repair

(A) Disease score (left) of vehicle-treated and Gal1-treated (100 μg/ day) mice, immunized with 200 μg MOG35-55 and linear regression curves of disease for each group (right, dashed lines, 95% confidence intervals). (B) Bielschowsky silver (upper panel) and Luxol fast blue (lower panel) staining of spinal cord after 35 days of EAE in Gal1- or vehicle-treated mice. (C-G) Confocal microscopy of spinal cord white matter on day 35 post-immunization in Gal1- or vehicle-treated mice. (C) Left, sections were stained for Iba1 (green) and MHC II (red). Insert shows low magnification micrograph of representative cells. Middle. 3D reconstruction ortho-view of low magnification micrograph. Right, graph represents a quantification of Iba1, MHCII double positive cells in different groups. (D) Left, spinal cord sections were stained for Tuj1 (green). Middle, 2.5D intensity analysis of Tuj1 staining Right, mean fluorescence intensity (MFI) of immunoreactivity against Tuj1. (E) Left, spinal cord sections were stained for GAP43 (green). Middle, 2.5D intensity analysis of GAP43 staining Right, mean fluorescence intensity (MFI) of immunoreactivity against GAP43. (F) Left, spinal cord sections were stained for MBP (green). Middle. 2.5D intensity analysis of MBP staining. Right, mean fluorescence intensity (MFI) of immunoreactivity against MBP. (G) Left, Spinal cord sections were stained for GFAP (green). Middle, 2.5D intensity analysis of GFAP staining. Right, mean fluorescence intensity (MFI) of immunoreactivity against GFAP. (H,I) Neonatal microglia were pre-treated in vitro with vehicle, LPS, Gal1 or LPS plus Gal1 for 24 h before transfer to the right lateral ventricle of Lgals1^−/− EAE mice (day 9 post-immunization; n=6 per group). (H) Diagram illustrating the injection site. (I) Clinical score (left) and linear regression curves of disease for each group (right; dashed lines; 95% confidence intervals). Scale bars = 20 μm. Data are the mean ± SEM (A, C-G, I) or are representative (B-G, images) of four independent experiments. *P<0.05; **P<0.01; ***P<0.005 versus vehicle. See also Figure S7.