Sphingosine 1-phosphate receptor modulation suppresses pathogenic astrocyte activation and chronic progressive CNS inflammation

Abstract

Multiple sclerosis (MS) is an autoimmune inflammatory demyelinating disease of the CNS that causes disability in young adults as a result of the irreversible accumulation of neurological deficits. Although there are potent disease-modifying agents for its initial relapsing-remitting phase, these therapies show limited efficacy in secondary progressive MS (SPMS). Thus, there is an unmet clinical need for the identification of disease mechanisms and potential therapeutic approaches for SPMS. Here, we show that the sphingosine 1-phosphate receptor (S1PR) modulator fingolimod (FTY720) ameliorated chronic progressive experimental autoimmune encephalomyelitis in nonobese diabetic mice, an experimental model that resembles several aspects of SPMS, including neurodegeneration and disease progression driven by the innate immune response in the CNS. Indeed, S1PR modulation by FTY720 in murine and human astrocytes suppressed neurodegeneration-promoting mechanisms mediated by astrocytes, microglia, and CNS-infiltrating proinflammatory monocytes. Genome-wide studies showed that FTY720 suppresses transcriptional programs associated with the promotion of disease progression by astrocytes. The study of the molecular mechanisms controlling these transcriptional modules may open new avenues for the development of therapeutic strategies for progressive MS.

Keywords: EAE; astrocytes; multiple sclerosis; secondary progression; sphingolipid metabolism.

Conflict of interest statement

Conflict of interest statement: These studies were funded in part by a Novartis grant (to F.J.Q. and J.A.).

Figures

Fig. 1.

FTY ameliorates chronic progressive EAE in NOD mice. EAE was induced in NOD/ShiLtJ mice, which were treated with daily i.p. injections of FTY720 or vehicle in the secondary progressive phase of the disease starting from day 40 after disease induction. (A) Clinical scores (Left) and linear regression analysis (Right) of mice under treatment with FTY720 or vehicle (n = 10 mice per group; two-way ANOVA). (B) Kaplan–Meier survival analysis of mice in the experiment described in A by two-way ANOVA. (C) Histologic examination of transversal lumbar spinal cord sections isolated from FTY720- or vehicle-treated mice at day 120. (Left) representative sections stained for Luxol fast blue (LFB) for demyelination or Bielschowsky’s Silver stain (silver) for axonal loss. Representative of three sections of three mice. (Right) Quantification of demyelination and axonal loss in FTY720- or vehicle-treated mice (Student’s t test). (D) Proliferation assay from splenocytes isolated on day 120 of the experiment (n = 5; two-way ANOVA). Throughout, data are mean ± SEM and representative of two independent experiments (*P < 0.05 and **P < 0.01; ns, not significant).

Fig. S1.

Quantification of immune cell subtypes in FTY720- or vehicle-treated NOD EAE mice. EAE was induced in NOD/ShiLtJ mice, which were treated with daily i.p. injections of FTY720 or vehicle in the secondary progressive phase of the disease starting from day 40 after disease induction. (A) Absolute numbers of mononuclear cells, CD11b+CD45highLy6Chigh proinflammatory monocytes, CD19+ B cells, and CD3+CD4+ T cells at day 120 after disease induction in lymph nodes (LN), spleen, and CNS as determined by FACS analysis. (B) Absolute numbers of CNS CD3+CD4+ T cells positive for IL-17, IFN-γ, Foxp3, and IL-10. Data are mean ± SEM and representative of two independent experiments. P values were derived by Student’s t test (**P< 0.01; ns, not significant).

Fig. 2.

FTY720 modulates activation of astrocytes, microglia, and proinflammatory monocytes. Astrocytes, microglia, and proinflammatory monocytes were isolated by FACS sorting at day 120 of EAE, and RNA was subjected to custom-made nCounter Nanostring arrays. (A and B) Fold change in mRNA expression of the indicated genes from sorted astrocytes (A) and microglia (B) from FTY720- or vehicle-treated mice at day 120 of EAE as determined by NanoString analysis [fold change in relative expression as determined by log2(FTY720/vehicle)]. Data are representative of two independent experiments of pooled astrocytes and microglia from three mice per group. (B and C) NanoString analysis of proinflammatory gene clusters (Table S2) from sorted microglia (C) and Ly6C1hi proinflammatory monocytes (D). Data are ratio of count numbers of cells from FTY720-treated to vehicle-treated mice, and are representative of two independent experiments of pooled microglia and Ly6Chi proinflammatory monocytes with three mice per group. Data are mean ± SEM (**P < 0.01 and ***P < 0.001, Student’s t test).

Fig. 3.

FTY720 modulates proinflammatory activation of astrocytes. Primary cultures of murine astrocytes were activated with LPS and treated with FTY720 or vehicle and analyzed for gene expression, neurotoxic and chemotaxic mediator production, as well as monocyte polarizing properties. (A) RNA expression of indicated genes from astrocytes isolated 24 h after activation and treatment from three biological replicates. (B) Neurotoxicity assay with supernatants of activated astrocyte cultures treated with FTY720 or vehicle. (C) Migration assays with supernatants from activated FTY720- or vehicle-treated astrocytes using CD11b+Ly6C1hi monocytes as migrating cells. (D) qPCR analysis for expression of the indicated genes from sorted CD11b+Ly6C1hi proinflammatory monocytes that were cocultured with activated FTY720- or vehicle-treated astrocytes and reisolated thereafter for RNA analysis. Throughout, data are mean ± SEM and representative of three independent experiments with three biological replicates. P values were derived by Student’s t test (*P < 0.05, **P < 0.01, and ***P < 0.001).

Fig. S2.

Purity control of in vitro astrocyte cultures. (A) FACS staining of mixed glial cultures and highly pure astrocyte cultures for CD11b and CD45. Numbers indicate percentages in respective gates. (B) Intracellular staining of astrocyte cultures for GFAP. Numbers indicate percentages in respective gate. (C) qPCR analysis for indicated genes of astrocyte and microglia cultures. Data are mean ± SEM and are representative for routine purity controls as performed for each astrocyte culture used in the experiments outlined.

Fig. S3.

Exposure of mouse and human microglia to FTY720 does not dampen proinflammatory activation. Mouse and human microglia were activated and treated with FTY720 or vehicle. (A and B, Left) qPCR analysis of indicated genes expressed in activated murine (A) or human (B) microglia under FTY720 or vehicle treatment 24 h after activation. (A and B, Right) Measurement of NO content in supernatant of FTY720- or vehicle-treated microglia (n = 3 biological replicates). Data are mean ± SEM and representative of three independent experiments. (C and D) Assessment of neurotoxicity (C) and migration induction (D) in supernatants from activated murine microglia under treatment with FTY720 or vehicle. Data are mean ± SEM and representative of two independent experiments. P values were derived by Student’s t test (ns, not significant).

Fig. S4.

FTY720 dampens proinflammatory activation of astrocytes. Astrocytes were activated with LPS or vehicle in the presence or absence of FTY720. (A) Western blot analysis for NF-κB (p65; Left) and quantification of the ratio of nuclear to cytoplasmic fraction of NF-κB p65 (Right) of astrocytes stimulated as indicated. (B and C) Quantification of active components in ACM generated from astrocytes treated as indicated by ELISA and colorimetric analysis (NO). (D and E) Migration and neurotoxicity assays using ACM from astrocytes treated as indicated in the presence of individual or combined blocking antibodies. Data are mean ± SEM and are representative of three independent experiments. P values were derived by one-way ANOVA followed by Tukey’s multiple-comparisons test (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, not significant).

Fig. 4.

Unbiased analyses reveal down-modulatory effects of FTY720 on human astrocytes. Primary human fetal astrocytes were activated with IL-1β in the presence or absence of FTY720, and gene expression was assessed by Affymetrix arrays. (A) Heat map of 16,799 expressed (detected at level 0.1 in at least two of three samples) and 1,221 differentially regulated (B) genes (signal:noise ratio) of activated FTY720- or vehicle-treated human primary astrocytes as assessed by Affymetrix assay. Data represent three replicates of three independent astrocyte cultures. Gene expression levels are row-centered and log2-transformed, and saturated at levels −0.5 and +0.5 for visualization satisfying a false discovery rate (FDR) < 0.1. (C) Ingenuity pathway analysis of the transcriptional profile of activated human primary astrocytes under treatment with FTY720 or vehicle. (D) Ingenuity pathway analysis diagram of NF-κB signaling pathways comparing FTY720 vs. vehicle treatment. Colors code for up- and down-regulation of individual members in red (up) and blue (down).

Fig. 5.

FTY720 mitigates proinflammatory and neurotoxic properties of human astrocytes. Human fetal astrocytes were activated and treated with FTY720 or vehicle. (A) qPCR analysis of indicated genes expressed in activated astrocytes under FTY720 or vehicle treatment 24 h after activation (n = 3 biological replicates). (B) Neurotoxicity assay with supernatants of activated human primary astrocytes treated with FTY720 or vehicle (n = 3 biological replicates). (C) Fold change in mRNA expression of the indicated genes in human activated astrocytes treated with FTY720 or vehicle as determined by Affymetrix arrays [fold change in relative expression as determined by log2(FTY720/vehicle)]. (D) Migration assays with supernatants from activated FTY720- or vehicle-treated human primary astrocytes using CD11b+Ly6C1hi proinflammatory monocytes as migrating cells (n = 3 biological replicates). Throughout, data are mean ± SEM and representative of three independent experiments. P values were derived by Student’s t test (*P < 0.05, **P < 0.01, and ***P < 0.001).