Resveratrol (3, 5, 4'-Trihydroxy-trans-Stilbene) Attenuates a Mouse Model of Multiple Sclerosis by Altering the miR-124/Sphingosine Kinase 1 Axis in Encephalitogenic T Cells in the Brain

Abstract

Resveratrol (3,5,4'-trihydroxy-trans-stilbene) (RES) is a naturally-derived phytoestrogen found in the skins of red grapes and berries and has potential as a novel and effective therapeutic agent. In the current study, we investigated the role of microRNA (miRNA) in RES-mediated attenuation of experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis. Administration of RES effectively decreased disease severity, including inflammation and central nervous system immune cell infiltration. miRNA microarray analysis revealed an altered miRNA profile in encephalitogenic CD4+ T cells from EAE mice exposed to RES treatment. Additionally, bioinformatics and in silico pathway analysis suggested the involvement of RES-induced miRNA in pathways and processes that regulated cellular proliferation. Additional studies confirmed that RES affected cell cycle progression and apoptosis in activated T cells, specifically in the brain. RES treatment significantly upregulated miR-124 during EAE, while suppressing associated target gene, sphingosine kinase 1 (SK1), and this too was specific to mononuclear cells in the brains of treated mice, as peripheral immune cells remained unaltered upon RES treatment. Collectively, these studies demonstrate that RES treatment leads to amelioration of EAE development through mechanism(s) potentially involving suppression of neuroinflammation via alteration of the miR-124/SK1 axis, thereby halting cell-cycle progression and promoting apoptosis in activated encephalitogenic T cells. Graphical Abstract Resveratrol alters the miR-124/sphingosine kinase 1 (SK1) axis in encephalitogenic T cells, promotes cell-cycle arrest and apoptosis, and decreases neuroinflammation in experiemental autoimmune encephalomyelitis (EAE).

Keywords: CD4+ T cells; Experimental autoimmune encephalitis (EAE); Multiple sclerosis (MS); Resveratrol (RES); Sphingosine kinase 1 (SK1); microRNA 124 (miR-124).

Fig. 1. Resveratrol diminishes EAE severity and pro-inflammatory cytokines.

MOG_35–55-immunized mice were treated daily, starting on day 2, with vehicle (VEH) or resveratrol (RES) at 100 mg/kg by oral gavage. A) EAE clinical scores of VEH- and RES-treated mice (n > 9). B) Absolute number of brain-infiltrating mononuclear cells (n = 4). Data are representative (n > 9). (C) TNF-α and D) IFNγ serum cytokine levels detected by ELISA on days 7 & 15 post immunization. Brain mononuclear cells were isolated from VEH- or RES-treated mice and stimulated ex vivo with 30μg/mL MOG for 72 h. Supernatants were analyzed for E) IL-17A and F) IL-6. Data presented as the mean ± S.E.M. of triplicates. Significance assessed using two-tailed student’s T test: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. BDL, below detection level

Fig. 2. Resveratrol alters immune cell distribution and activation in the brains of EAE mice.

Infiltrating mononuclear cells were isolated from brains of VEH- or RES-treated EAE mice and stained for the indicated cell surface markers and analyzed by flow cytometry. Representative histograms and bar graphs of combined experiments (n > 3) displayed as percent positive cells of A) CD4 and B) CD11b. C) Absolute cell numbers of T cell co-stimulatory molecule CD28 and activation marker CD69 and D) co-stimulatory molecules CD80 and CD86. E) Representative dot plots of CD11b+/CD45hi and CDllb+/CD45lo mononuclear cells and bar graphs of combined experiments (n > 3). Data is presented as the mean ± S.E.M. Statistical significance (A-D) evaluated using Student’s T test or (E) One-way ANOVA with Tukey’s multiple comparisons: *, p < 0.05; ** p < 0.01; ***, p < 0.001; ****, p < 0.0001

Fig. 3. Resveratrol induces apoptosis in brain-infiltrating cells.

Brain-derived mononuclear cells from VEH- or RES-treated EAE mice were stained with a PI/Rnase solution and cell cycle analysis evaluated using flow cytometry. A) Flow cytometry data from a representative experiment was analyzed and stages of the cell cycle modeled using MODFit LT software. Inset, percentage of cells in stages of cell cycle displayed as pie charts, data are representative. C) Percentage of cells in stages of cell cycle and D) apoptotic cells as indicated by ModFit LT analysis from combined experiments (n > 3). Data represented as the mean ± S.E.M. Significance evaluated using Student’s T test: **, p < 0.01; ***, p < 0.001; ****, p < 0.0001

Fig. 4. Resveratrol alters miRNA expression in brain-derived CD4+ T cells.

CD4+ T cells were purified from the brain as described in Fig. 2 and analyzed for miRNA expression. A) Heatmap representing miRNA fold change in enecphalitogenic CD4+ T cells from VEH- or RES- treated EAE mice. B) Array analysis indicated 13 down- and 52 up-regulated miRNAs in RES- vs VEH-treated EAE mice, changes greater than ± 1.5 fold-change, visualized on an MA plot, where the data was transformed onto M (log ratio) and A (mean average) scales. C) The top 5 up-and down-regulated miRNAs determined by array analysis

Fig. 5. Gene Ontology and Pathway Analysis.

Gene ontology enrichment analysis of target genes was performed and mapped for Biological Process: Immune System Process using Cytoscape bioinformatics software. A) Global analysis revealed significant overlap between target genes and pathways involved in Hematopoiesis or Lymphoid Organ Development, Immune Response and Lymphocyte-mediated Immunity. B) A more detailed analysis using Cytoscape revealed overlap between target genes and Regulation of Activated T cell Proliferation. Statistical significance assessed using Enrichment/Depletion (Two-sided Hypergeometric test) with Benjamini-Hochberg correction. C) Ingenuity Pathway Analysis (IPA) software was used to evaluate pathway analysis of the top 5 up-regulated miRNA target genes; Top Network: Cell Morphology, Development and Growth & Proliferation (p = 4.3 × 10–5–2.0 × 10–2); Functional and Canonical Pathway Analysis statistical significance assessed using Fisher’s exact Test. (SPHK1, sphingosine kinase 1; p38, p38 MAP kinase; PDE10A, phosphodiesterase 10A; CDK6, cyclin-dependent kinase 6; E2F, E2F transcription factor; RB1, retinoblastoma protein; FADD, FAS-associated protein with death domain; CCND1, cyclin-D1)

Fig. 6. Resveratrol alters the miR-124/SK1 axis specifically in the brain.

Spleen- or brain-derived CD4+ cells from VEH- or RES-treated EAE mice were evaluated for levels of A) miR-124 and B) target gene SK1 by qRT-PCR (n > 6). Splenocytes were isolated from VEH- or RES-treated EAE mice and analyzed for C) absolute cell number (n = 3), D) percent CD4+ T cells (and F) percent CD11b + monocytes/macrophages. Data representative of >3 independent experiments. Splenocytes were also evaluated for cell-cycle and apoptosis using PI/Rnase staining and ModFit LT software (n > 4). MNE, mean normalized expression. Statistical significance was evaluated using Student’s T test: p < 0.0001