Combination of Cannabinoids, Δ9- Tetrahydrocannabinol and Cannabidiol, Ameliorates Experimental Multiple Sclerosis by Suppressing Neuroinflammation Through Regulation of miRNA-Mediated Signaling Pathways

Abstract

Multiple sclerosis (MS) is a chronic and disabling disorder of the central nervous system (CNS) characterized by neuroinflammation leading to demyelination. Recently a combination of Δ9-tetrahydrocannabinol (THC) and Cannabidiol (CBD) extracted from Cannabis has been approved in many parts of the world to treat MS-related spasticity. THC+CBD combination was also shown to suppresses neuroinflammation, although the mechanisms remain to be further elucidated. In the current study, we demonstrate that THC+CBD combination therapy (10 mg/kg each) but not THC or CBD alone, attenuates murine experimental autoimmune encephalomyelitis (EAE) by reducing neuroinflammation and suppression of Th17 and Th1 cells. These effects were mediated through CB1 and CB2 receptors inasmuch as, THC+CBD failed to ameliorate EAE in mice deficient in CB1 and CB2. THC+CBD treatment also caused a decrease in the levels of brain infiltrating CD4+ T cells and pro-inflammatory molecules (IL-17, INF-γ, TNF-α, IL-1β, IL-6, and TBX21), while increasing anti-inflammatory phenotype such as FoxP3, STAT5b, IL-4, IL-10, and TGF-β. Also, the brain-derived cells showed increased apoptosis along with decreased percentage in G0/G1 phase with increased percentage in G2/M phase of cell cycle. miRNA microarray analysis of brain-derived CD4+ T cells revealed that THC+CBD treatment significantly down-regulated miR-21a-5p, miR-31-5p, miR-122-5p, miR-146a-5p, miR-150-5p, miR-155-5p, and miR-27b-5p while upregulating miR-706-5p and miR-7116. Pathway analysis showed that majority of the down-regulated miRs targeted molecules involved in cycle arrest and apoptosis such as CDKN2A, BCL2L11, and CCNG1, as well as anti-inflammatory molecules such as SOCS1 and FoxP3. Additionally, transfection studies involving miR-21 and use of Mir21^-/- mice suggested that while this miR plays a critical role in EAE, additional miRs may also be involved in THC+CBD-mediated attenuation of EAE. Collectively, this study suggests that combination of THC+CBD suppresses neuroinflammation and attenuates clinical EAE development and that this effect is associated with changes in miRNA profile in brain-infiltrating cells.

Keywords: CB1; CB2; CBD; EAE; THC; miR-21a-5p; multiple sclerosis.

Figure 1

Combination of THC+CBD attenuates EAE by suppressing neuroinflammation. EAE was induced in C57BL/6 mice using CFA+PTX+MOG, as described in Methods. These mice were treated with cannabinoids and the mice were studied for clinical signs of paralysis and neuroinflammation. (A) Experimental timeline. EAE mice were treated daily with THC (10 mg/kg), CBD (10 mg/kg), or a combination of THC+CBD (10 mg/kg each) starting at 8–10 days after MOG immunization. (B) Controls consisting of mice that received CFA+PTX only that received cannabinoids. (C,D) Clinical scoring of EAE symptoms in mice treated with Veh, THC, CBD, or THC+CBD. (E,F) EAE scoring in an extended experiment until day 27 in Veh vs. THC+CBD treated EAE mice. (G) Representative H&E images and LFB staining in spinal cord tissues to detect cellular infiltration and demyelination, respectively. (H,I) EAE scoring in WT or CB1^−/−CB2^−/− double knockout mice treated with either Veh or THC+CBD. Data presented are mean ± SEM. For (B,C,H), significance was determined by two-way ANOVA with a Dunnett post hoc test. For (D,I), one-way ANOVA with a Dunnett post hoc test was used. For (E) Mann-Whitney U test was performed excluding scoring values before initiation of treatment. For (F), an unpaired two-tailed T-test was performed. For (J), one-way ANOVA with a Sidak correction was used. ^****p < 0.0001, ^***p < 0.001, ^**p < 0.01, ^*p < 0.05.

Figure 2

T cell population and phenotypic changes of WT and CB1^−/−CB2 ^−/− EAE mice treated with vehicle or THC+CBD. As shown in Figure 1, EAE was induced in naïve mice then mice were treated with Veh or THC+CBD when symptoms appeared. Inguinal lymph nodes and brain MNCs were collected at the peak of disease (Day 15 post immunization). (A) IL-17A and IFNγ concentration in inguinal lymph node 24 h culture supernatant measured by ELISA. (B) Representative flow cytometry contour plots of encephalitogenic T cells. (C) Quantification of total MNCs, CD3+ T cells, and CD3+CD4+ Th cells per brain. Data presented are mean ± SEM. ^***p < 0.001, ^*p < 0.05 by unpaired two-tailed T-test (A), or Kruskal-Wallis test (C).

Figure 3

Differentially expressed miRNAs in brain-infiltrating CD4+ T cells upon THC+CBD treatment in EAE mice. Total RNA was isolated and pooled from CD4+ T cells obtained from the brains of EAE mice post-treated with vehicle (n = 5) or THC+CBD (n = 5) on day 15. MicroRNA microarray expression levels of differentially expressed miRNAs were generated. (A) The fold change distribution of all 1,908 miRNAs tested. (B) Proportional Venn diagram illustrating fold change of miRNAs that were >1.5- fold dysregulated following treatment with THC+CBD in EAE mice. (C) Heat map of the 157 dysregulated miRNAs. The color scale denotes those miRNAs that were upregulated (red) and downregulated (green). (D) Pathway analysis of miRs mediating dysregulation in gene expression following THC+CBD treatment. (E–M) Expression levels of selected upregulated and downregulated miRNAs were validated by qRT-PCR using Snord96a as a small RNA endogenous control. Data presented are mean ± SEM. ^***p < 0.001, ^**p < 0.01, ^*p < 0.05 by unpaired two-tailed T-test.

Figure 4

Expression of miRNA target genes involved in Th cell polarization. As described in Figures 2, 3, CD4+ T cells or total MNCs were isolated from brains of EAE mice on day 15 post immunization. For (A–K), RNA was extracted from CD4+ T cells and used for miR target gene validation. For (L), total MNCs were cultured for 24 h and supernatants were collected for cytokine analysis. (A–K) qRT-PCR validation of the target genes (A) Foxp3, (B) STAT5B, (C) IL-10, (D) GATA3, (E) IL-4 (F) STAT3, (G) IL-17A, (H) TBX21, (I) IFN-γ, (J) IL-6, and (K) IL-1β using GAPDH as endogenous control. (L) IL-17A, IFN-γ, TNF-α, IL-6 IL-1β, IL-10, and TGF-β concentration measured in MNC culture supernatants by ELISA. Data are expressed as the mean ± S.E.M. and statistical significance is indicated as ^***p < 0.001, ^**p < 0.01 by unpaired two-tailed T-test.

Figure 5

THC+CBD treatment induces cell cycle arrest / apoptosis in brain MNCs. As shown in Figure 1, EAE was induced in naïve mice and treatment with Veh or THC+CBD was initiated at the onset of symptoms. CD4+ T cells and total MNCs were isolated from the brains of mice on day 15 post immunization for gene and cell cycle analysis. (A–D) qRT-PCR validation of miR target genes involved in cell cycle/apoptosis. (E) Representative flow cytometry histograms of PI staining in brain MNCs using ModFit software. (F) Cell cycle phase quantification. (G) Representative flow cytometry pseudocolor plots of brain MNCs in early apoptosis (AnnexinV⁺ PI⁻) or late apoptosis (AnnexinV⁺PI⁺). (H) Quantification of brain MNCs in early or late apoptosis. Data represented are mean ± SEM. ^***p < 0.001, ^**p < 0.01, ^*p < 0.05 by unpaired two-tailed T-test (A–D), or two-way ANOVA with a Tukey post hoc test (F,H).

Figure 6

Role of miR-21 downregulation on THC+CBD-mediated amelioration of EAE. For (A–D), miR-21a-5p transfection assays were performed in CD4+ T cells purified from naïve WT C57BL6 mice. Cells were cultured for 24 h and transfected with miR-21a-5p mimic, inhibitor, or mock (transfection reagent only). For (E,F) EAE was induced in WT and Mir21^−/− mice as described in Methods then treated with Veh or THC+CBD upon development of EAE symptoms. (A–D) qRT-PCR validation for miR-21 and the target genes. (E) Agarose gel electrophoresis of genotyping for the parents and the first generation of Mir21^−/− mice (“miR-21 KO”). (F) EAE scoring in WT and Mir21^−/− mice. (G) Quantification of the clinical scores. (H) Representative flow cytometry histograms of brain MNCs stained with PI study cell cycle analysis. (I) Quantification of cell cycle phases. Data are expressed as the mean ± S.E.M. and statistical significance is indicated as ^***p < 0.001, ^**p < 0.01, ^*p < 0.05. For (F), statistical significance is vs. WT EAE+Veh. Significance was determined by one- or two-way ANOVA with Tukey post hoc corrections.