Fig 1.
Lack of endogenous Gal-8 expression exacerbates EAE.
Lgals8+/+ (WT) and Lgals8-/- (Gal-8 KO) immunized with MOGp. (A) Clinical scores monitored daily for 25 days show an exacerbated EAE in Gal-8 KO mice (***p<0.001; Mann Whitney; day by day clinical Score comparisons; n = 20 WT; n = 22 Gal-8 KO). (B) Spinal cord histopathological analysis after 10 days of immunization show enhanced immune cell infiltration and demyelination in Gal-8 KO mice, shown by H&E staining (I and II) and luxol fast blue staining (III and IV). Scale bar = 50 μm.
Table 1.
Clinical parameters of EAE progression.
Fig 2.
Gal-8 deficit favors selective Th17 cell differentiation upon polyclonal activation.
Splenocytes isolated from Lgals8+/+ (WT) and Lgals8-/- (KO) mice were analyzed by FACS: (A) Dendritic cells (CD11c+), B cells (CD19+), CD8+ T cells and different CD4+ T cells subsets, naïve (CD44-CD62L+), effector (CD44+CD62L+), memory (CD44+CD62L-) and total cells analyzed in the subset of viable CD4+ CD25- T-cells show no differences between WT and KO mice. Graphics of frequency +/-SD (n = 5). (B) T cell activation by 72 h incubation with anti-CD3 (anti-CD3) and anti-CD28 (anti-CD28) antibodies show Th17 increased frequency in KO mice while Th1 and Th2 cells are similar in WT and KO mice. Graph shows frequency +/-SD (*p<0.05; ANOVA; n = 4).
Fig 3.
Gal-8 deficit favors Th17 polarization during MOGp-induced EAE and ex-vivo re-stimulation.
Th17 and Th1 subpopulations in splenocytes from Lgals8-/- (KO) and Lgals8+/+ (WT) mice obtained after 10 days of EAE induction were analyzed either immediately or after 72 h of ex vivo MOGp re-stimulation, in the absence or presence of Gal-8. Gal-8 KO mice show higher frequency of Th17 cells both at steady state and after MOGp re-stimulation. Incubation with Gal-8 reduced Th17 cells only in Gal-8 KO. Graph shows frequency +/-SD (*p<0.05; ANOVA; n = 4).
Fig 4.
Galectin-8 deficit increases the frequency of total Tregs and CXCR3+ Tregs.
Splenocytes isolated from Lgals8+/+ (WT) and Lgals8-/- (KO) mice were analyzed at steady state for total Tregs (Foxp3+), CXCR3+ and CCR6+ frequency in the Treg (Foxp3+ CD4+) population. Graphs of frequency +/-SEM show increased total Tregs (Foxp3+) and CXCR3+Tregs in KO mice (*p<0.05; **p<0.01; p***<0.001; Student’s t-test; n = 4).
Fig 5.
Gal-8 ameliorates EAE and induces Th17 cell death in vitro.
(A) Gal-8 treatment ameliorates MOGp-induced EAE in C57BL/6 mice. The mice were injected daily by intraperitoneal injection of either PBS (Control) or Gal-8 100 μg/ml. Gal-8-treated animals tend to start the disease later and show lower EAE scores during the acute and chronic phases of the disease (*p<0.05; Control, n = 7; Gal-8 treated, n = 5). (B) Gal-8-induced cell death in Th17 lymphocytes differentiated and activated in vitro. Naive (CD62L+ CD44-) CD4+ T cells were purified by cell sorting and differentiated to a Th17 phenotype. The differentiated Th17 cells were isolated with a commercial kit based on the cell surface expression of IL-17 and activated with anti-CD3/anti-CD28 in the presence or absence of Gal-8 (20 μg/ml) for 72 h. Cell death was determined by cell staining with Annexin V and 7-AAD and analyzed by FACS. Representative dot plots show the frequency of Th17 cells before and after purification (upper panels), the selected gate in the forward scatter versus side scatter analysis (middle panels) and the associated contour-plots show the Annexin V versus 7-AAD analysis (lower panels). Numbers in quadrants indicate the percentage of cells in the respective quadrant.
Table 2.
Clinical parameters of EAE disease progression (day 0–20).
Fig 6.
Gal-8 expression in mouse brain and presence in human CSF.
(A) Histochemistry of β-gal staining reveals Gal-8 expression in several regions of the mouse brain (S1 Table). Brain slices depict high Gal-8 expression levels in the choroid plexus (CP) of the lateral ventricle (LV) and the dorsal 3rd ventricle (D3V), as well as in the ventrolateral thalamic nucleus. (B) Immunoblot with rabbit anti-Gal-8 antibody show Gal-8 reactivity in the CSF of individuals without MS. Samples C3-11 correspond to non-inflammatory CSF from individuals studied for diplopia (C3), vertiginous syndrome (C7), cephalea (C8 and C11) and febrile syndrome (C9), whereas C6 is an inflammatory CSF from a patient with meningitis. All samples show anti-Gal-8 reactivity, though with variable intensity. *Bands of unknown origin might include Gal-8 dimers or complexes with other proteins, not separable under SDS-PAGE conditions.
Fig 7.
Detection of Gal-8 autoantibodies in sera and CSF from MS patients.
Immunoblot of sera and CSF from different MS patients (Pn) against Gal-8 indicating its (-) or (+) anti-Gal-8 reactivity compared with a negative control (C) from a healthy individual. In some patients (e.g. P2 and P10) anti-Gal-8 reactivity was detected in both sera and CSF, while in others (e.g. P3) was only detected in CSF. P14 is shown only in CSF but is also positive in serum (analyzed in other immunoblot), while P15 and P17 are negative both in CSF and serum (not shown). In most patients (e.g. P4-6) only sera could be analyzed. P9 was only analyzed in CSF.
Fig 8.
Function-blocking activity of anti-Gal-8 autoantibodies.
(A) Anti-Gal-8(+) sera block the adhesion of PBMC to Gal-8-coated coverslips. Graph shows number of adhered cells (Average ± SE of three anti-Gal-8(-) and three anti-Gal-8(+) sera tested in triplicate) (***p<0.001; Student’s t-test). (B) Anti-Gal-8 autoantibodies inhibit Gal-8-induced apoptosis of Th17 cells. In vitro differentiated Th17 cells from IL-17A-GFP reporter mice were purified based on IL-17A expression (GFP+) and incubated with Gal-8 (20 μg/ml) in the presence of lactose, sucrose or anti-Gal-8 antibodies affinity purified from pooled serum of MS patients. The extent of apoptosis was quantified as the frequency of Annexin V+ 7AAD+ cells of the sample relative to the frequency of Annexin V+ 7AAD+ cells of the untreated control. Representative contour plots are shown in upper panels. Quantification of a representative experiment is shown in the lower panel. Values represent mean + SEM of triplicates. Data from a representative from four independent experiments is shown. **, p<0.01; ***, p < .001 by one-way ANOVA followed by Tukey’s post-hoc test.
Table 3.
Frequency of anti-Gal-8 autoantibodies in 58 patients with multiple sclerosis according to relapsing-remitting (RRMS) or progressive forms.
Fig 9.
Anti-Gal-8 autoantibodies correlate with worse disability scores in RRMS patients.
(A) RRMS patients with and without autoantibodies were followed during an average of 12 months. Patients (n = 17) with anti-Gal-8(+) sera have worse EDSS at the end of follow-up than patients (n = 19) without anti-Gal-8 autoantibodies (mean EDSS 1.5 vs 0, *p = 0.002, nonparametric Mann-Whitney U test), independent of the treatment received or number of relapses during this period. (B) At the end of follow-up, 5/17 patients with anti-Gal-8 autoantibodies developed confirmed EDSS worsening vs 0/19 of patients without anti-Gal-8 autoantibodies (*p = 0.016 by Fisher test).
Table 4.
Different EDSS outcome in RRMS patients according to the presence of anti-Gal-8 autoantibodies.