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. 2016 Jul;46(7):1783-96.
doi: 10.1002/eji.201546212. Epub 2016 May 25.

Galectin-1 is essential for the induction of MOG35-55 -based intravenous tolerance in experimental autoimmune encephalomyelitis

Elisabeth R Mari  1 , Javad Rasouli  1 , Bogoljub Ciric  1 , Jason N Moore  1   2 , José R Conejo-Garcia  3 , Naveen Rajasagi  4 , Guang-Xian Zhang  1 , Gabriel A Rabinovich  5   6 , Abdolmohamad Rostami  1
Affiliations

Galectin-1 is essential for the induction of MOG35-55 -based intravenous tolerance in experimental autoimmune encephalomyelitis

Elisabeth R Mari et al. Eur J Immunol. 2016 Jul.

Abstract

In experimental autoimmune encephalomyelitis (EAE), intravenous (i.v.) injection of the antigen, myelin oligodendrocyte glycoprotein-derived peptide, MOG35-55 , suppresses disease development, a phenomenon called i.v. tolerance. Galectin-1, an endogenous glycan-binding protein, is upregulated during autoimmune neuroinflammation and plays immunoregulatory roles by inducing tolerogenic dendritic cells (DCs) and IL-10 producing regulatory type 1 T (Tr1) cells. To examine the role of galectin-1 in i.v. tolerance, we administered MOG35-55 -i.v. to wild-type (WT) and galectin-1 deficient (Lgals1(-/-) ) mice with ongoing EAE. MOG35-55 suppressed disease in the WT, but not in the Lgals1(-/-) mice. The numbers of Tr1 cells and Treg cells were increased in the CNS and periphery of tolerized WT mice. In contrast, Lgals1(-/-) MOG-i.v. mice had reduced numbers of Tr1 cells and Treg cells in the CNS and periphery, and reduced IL-27, IL-10, and TGF-β1 expression in DCs in the periphery. DCs derived from i.v.-tolerized WT mice suppressed disease when adoptively transferred into mice with ongoing EAE, whereas DCs from Lgals1(-/-) MOG-i.v. mice were not suppressive. These findings demonstrate that galectin-1 is required for i.v. tolerance induction, likely via induction of tolerogenic DCs leading to enhanced development of Tr1 cells, Treg cells, and downregulation of proinflammatory responses.

Keywords: Galectin-1; Tolerance; Tolerogenic DC; Tr1 cell; Treg cell.

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Conflict of interest statement

Conflict of Interest

The authors declare no commercial or financial conflict of interest.

Figures

Figure 1
Figure 1. Galectin-1 deficient mice are resistant to i.v. tolerance induction
(A) WT (n=7) and Lgals1−/− (n=6) mice were immunized with MOG35-55 to develop EAE. Two hundred μg of MOG35-55 in PBS were injected i.v. on days 14 and 17 p.i. Mice that received PBS i.v. served as controls. Clinical EAE was scored on a 0–5 scale. Linear-regression curves (right panel). Dashed lines indicate 95% confidence intervals. Cumulative clinical scores were pooled from four experiments. Data show mean ± SEM. (B, C) On day 25 p.i. spinal cords were harvested, and 5 μm sections from the lumbar spinal cord were stained with H&E and Luxol fast blue. Shown are examples of (B) H&E or (C) Luxol fast blue staining for WT and Lgals1−/− mice that received PBS or MOG35-55 i.v.; Magnifications, ×10; scale bar = 10 μm. (D) Mean scores of inflammation and demyelination ± SD in MOG-i.v. and PBS-i.v. mice. One representative experiment of four is shown. *, p<0.05; ***, p < 0.001. For EAE, the area under the curve was calculated for each mouse and values for experimental groups were compared for statistical significance using a Mann-Whitney test for non-parametric data. Parametric data sets were analyzed using a two-tailed, unpaired Student’s t test with Welch’s correction. One-way ANOVA was used for comparisons between multiple groups.
Figure 2
Figure 2. I.v. tolerized Lgals1−/− mice have reduced numbers of Tr1 cells and Tregs in the CNS
MNCs were isolated from the CNS of MOG-i.v. and PBS-i.v. EAE WT (n=7) and Lgals1−/− (n=6) mice on day 25 p.i. (A) MNCs were cultured overnight with MOG35-55 (10 μg/ml), activated with PMA + ionomycin + Golgi plug (PMA/iono/GP), then stained and analyzed by flow cytometry. (B) Representative flow cytometry dot plots showing IL-10 expression in CD4+ T cells. (C) Representative flow cytometry dot plots showing FoxP3 expression on CD4+ T cells. (D) Proportions and absolute numbers of infiltrating Tr1 cells and Tregs were determined for WT and Lgals1−/− MOG-i.v. and PBS-i.v. mice. Pooled data from four experiments are shown. Experiments show mean ± SEM. *, p<0.05, unpaired Student’s t test.
Figure 3
Figure 3. Lack of galectin-1 reduced numbers of tolerogenic DCs in the CNS
MNCs were isolated from the CNS of MOG-i.v. and PBS-i.v. EAE WT (n=7) and Lgals1−/− (n=6) mice on day 25 p.i. MNCs were cultured overnight with MOG35-55, then activated with PMA/iono/GP, stained and analyzed by flow cytometry. (A) Representative flow cytometry dot plots showing CD11c and CD11b expression in MNCs. (B) Proportions of CD11c+CD11b+ DCs were determined for WT and Lgals1−/− MOG i.v. and PBS i.v. mice. (C) MFI was measured for MHC class II, CD80, CD86 and PD-1 in CD11c+CD11b+ DCs. (D) IL-10 mRNA was measured using RT-PCR. Pooled data from four experiments are shown. Experiments show mean ± SEM. *, p<0.05, unpaired Student’s t test.
Figure 4
Figure 4. Lack of galectin-1 leads to increased pro-inflammatory responses and reduced numbers of Tregs in the periphery of i.v. tolerized mice
Splenocytes from MOG-i.v. and PBS-i.v. EAE WT and Lgals1−/− mice on day 25 p.i. were stimulated for three days with MOG35-55. (A) Cell culture supernatants were assayed for IFN-γ, IL-17A and GM-CSF by ELISA (B) Proliferation was measured in splenocytes after three days of culture with MOG35-55. Tritiated adenine was added after 54 hours of culture; its incorporation was measured after 18 hours. CD4+AnnexinV+ and CD4+CD44hi T cells from the spleen were analyzed by flow cytometry on day 25 p.i. (C) IL-10, LAG-3, CD49b and FoxP3 mRNAs in splenocytes were measured using RT-PCR. (D) IL-10 concentrations in cell culture supernatants were measured by ELISA. (E) c-maf, Ahr and IL-21 mRNAs in splenocytes were measured using RT-PCR. Data shown are one representative of four experiments. Experiments show mean ± SD. *, p<0.05, **, p < 0.01, ***, p < 0.001, unpaired Student’s t test.
Figure 5
Figure 5. Lack of galectin-1 reduces tolerogenic DC phenotype in the periphery of i.v. tolerized mice
Splenocytes from MOG-i.v. and PBS-i.v. EAE WT and Lgals1−/− mice on day 25 p.i. were stimulated for three days with MOG35-55. (A) Splenocytes were analyzed by flow cytometry and the proportions of CD11c+CD11b+ DCs were determined. MFI was measured for MHC class II, CD80, CD86 and PD-1 in CD11c+CD11b+ DCs. (B) CD11c+ DC were purified from the splenocytes of WT and Lgals1−/− MOG-i.v. and PBS-i.v. mice, and IL-12a, IL-23a, IL-6, IL-27p28, IL-10 and TGF-β1 mRNAs were measured using RT-PCR. Data shown are pooled from three experiments. Experiments show mean ± SEM. *, p<0.05, **, p < 0.01, ***, p < 0.001, unpaired Student’s t test.
Figure 6
Figure 6. Lack of galectin-1 reduces tolerogenic DC function in the periphery of i.v. tolerized mice
CD11c+ splenic DCs were purified from MOG-i.v. and PBS-i.v. EAE WT and Lgals1−/− mice on day 21 p.i. and pulsed with MOG35-55 peptide. DCs were co-cultured (1:1) with CD4+ T cells from MOG35-55-specific 2D2 mice for 72 hours. (A) CD4+ T cells were analyzed by flow cytometry and representative dot plots showing expression of IL-10 and gated CD4+ T cells. (B) Representative flow cytometry dot plots showing expression of FoxP3 in gated CD4+ T cells. (C) The proportions of LAG-3+CD49b+ Tr1 cells were determined from within the IL-10 expressing CD4+ cells and IL-27p28 concentrations in cell culture supernatants measured by ELISA. Data shown are one representative of three experiments. Experiments show mean ± SD. *, p<0.05, **, p < 0.01, ***, p < 0.001, unpaired Student’s t test.
Figure 7
Figure 7. DCs from Lgals1−/− MOG i.v. mice do not suppress EAE
(A) WT (n=6) and Lgals1−/− (n=6) mice were sacrificed 3 weeks after MOG35-55 i.v. tolerization. CD11c+ DCs were isolated from splenocytes and i.v. transferred into WT EAE mice (1×106 mouse; n=3–5 per group) at disease peak. Mice that received PBS-i.v. served as controls. (B) Two weeks after transfer, MNCs from the CNS of DC-transferred mice and EAE controls were isolated. MNCs were cultured overnight with MOG35-55, activated with PMA/iono/GP, stained and analyzed by flow cytometry. Total numbers of infiltrating cells were gated from CD4+ population. CD4+IFNγ+, CD4+IL-17A+, CD4+GM-CSF+, CD4+IL-10+FoxP3+, and CD4+IL-10+FoxP3− percentages in CD4+ T cells were determined by flow cytometry. (C) Splenocytes from MOG-i.v. and PBS-i.v. EAE WT and Lgals1−/− mice on day 25 p.i. were stimulated for three days with MOG35-55. Cell culture supernatants were assayed for IFN-γ, IL-17A and GM-CSF by ELISA. (D) Proliferation was measured after three days of culture with MOG35-55. Tritiated adenine was added after 54 hours of culture; its incorporation was measured after 18 hours. Data shown are one representative of three experiments. Experiments show mean ± SD. *, p<0.05. For EAE, the area under the curve was calculated for each mouse and values for experimental groups were compared for statistical significance using a Mann-Whitney test for non-parametric data. Parametric data were compared for statistical significance using a two-tailed, unpaired Student’s t test.
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