A cannabigerol derivative suppresses immune responses and protects mice from experimental autoimmune encephalomyelitis

Abstract

Phytocannabinoids that do not produce psychotropic effects are considered of special interest as novel therapeutic agents in CNS diseases. A cannabigerol quinone, the compound VCE-003, has been shown to alleviate symptoms in a viral model of multiple sclerosis (MS). Hence, we studied T cells and macrophages as targets for VCE-003 and its efficacy in an autoimmune model of MS. Proliferation, cell cycle, expression of activation markers was assessed by FACs in human primary T cells, and cytokine and chemokine production was evaluated. Transcription was studied in Jurkat cells and RAW264.7 cells were used to study the effects of VCE-003 on IL-17-induced macrophage polarization to a M1 phenotype. Experimental autoimmune encephalomyelitis (EAE) was induced by myelin oligodendrocyte glycoprotein (MOG₃₅₋₅₅) immunization and spinal cord pathology was assessed by immunohistochemistry. Neurological impairment was evaluated using disease scores. We show here that VCE-003 inhibits CD3/CD28-induced proliferation, cell cycle progression and the expression of the IL-2Rα and ICAM-1 activation markers in human primary T cells. VCE-003 inhibits the secretion of Th1/Th17 cytokines and chemokines in primary murine T cells, and it reduces the transcriptional activity of the IL-2, IL-17 and TNFα promoters induced by CD3/CD28. In addition, VCE-003 and JWH-133, a selective CB2 agonist, dampened the IL-17-induced polarization of macrophages to a pro-inflammatory M1 profile. VCE-003 also prevented LPS-induced iNOS expression in microglia. VCE-003 ameliorates the neurological defects and the severity of MOG-induced EAE in mice through CB2 and PPARγ receptor activation. A reduction in cell infiltrates, mainly CD4+ T cells, was observed, and Th1 and Th17 responses were inhibited in the spinal cord of VCE-003-treated mice, accompanied by weaker microglial activation, structural preservation of myelin sheets and reduced axonal damage. This study highlights the therapeutic potential of VCE-003 as an agent for the treatment of human immune diseases with both inflammatory and autoimmune components.

Figure 1. Effects of VCE-003 on T-cell proliferation.

(A) Human peripheral T cells were stimulated with the OKT3 (1 µg/ml) and anti-CD28 (0.5 µg/ml) mAbs for 72 h in the presence or absence of increasing concentrations of VCE-003. Thymidine [³H] incorporation was measured by liquid scintillation and represented as the mean d.p.m ± SEM. Three independent experiments were performed. **p≤0.01 and ***p≤0.005 in an unpaired two-tailed Student's t test. (B) T cells were stained with CFSE, pre-treated with VCE-003 and stimulated with the CD3/CD28 mAbs for 6 days. Cell division was identified by flow cytometry. (C) T-cells were pre-treated with VCE-003 and stimulated with the CD3/CD28 mAbs for 72 h. The percentage of subdiploid cells (sub-G₀), and cells entering the S and G₂/M phases of the cell cycle are indicated. The results are representative of three independent experiments.

Figure 2. Effects of VCE-003 on T-cell activation.

(A) Human peripheral T cells were stimulated for 72 h with the OKT3 (1 µg/ml) and anti-CD28 (0.5 µg/ml) mAbs in the presence or absence of increasing concentrations of VCE-003, and the culture supernatants were collected and assayed using a R&D Systems Human Cytokine Array system (A, cytokines; B, chemokines and sICAM-1). (C) Human peripheral T cells were stimulated as indicated in 2A for 72 h, and the expression of CD25 and CD54 was detected by flow cytometry. The numbers represent the percentage of CD25⁺ and CD54⁺ cells and they are representative of three different experiments. (D) Jurkat T cells transfected with TNFα, IL-17 or IL-2 promoter luciferase reporter plasmids were treated for 30 min with increasing concentrations of VCE-003, and then stimulated with OKT3 (1 µg/ml) and anti-CD28 (0.5 µg/ml) mAbs for 6 h before measuring luciferase activity in the cell lysates. The results are the means ± SEM of three measurements and they are expressed as the percentage of inhibition, considering CD3/CD28 stimulation as 100% activation: **p≤0.01 indicates significant changes between CD3+CD28 and VCE-003 treatment.

Figure 3. VCE-003 and JWH-133 reduces IL-17-induced M1 polarization in macrophages.

Serum starved RAW264.7 cells were exposed to either JWH-133 (5 µM) or VCE-003 (1 and 2.5 µM) for 18 h and then maintained for 24 h in the presence or absence of recombinant mouse IL-17 (50 ng/ml). A) RT-PCR analysis of M1 gene expression in JWH-133-treated RAW264.7 cells. B) RT-PCR analysis of M1 gene expression in VCE-003-treated RAW264.7 cells. The results are expressed as the fold change with respect to untreated cells and they are the mean ± SEM of four independent experiments. C) CHO-CB2 cells were transiently transfected with the CRE-luc plasmid and then exposed to FSK (1 µM) for 6 h in the presence or absence of the indicated concentrations of WIN-55,212-2 (1 or 10 µM) and VCE-003 (5 µM) before luciferase activity was measured in the cell lysates. The results are expressed as the means ± SEM of three determinations and they are expressed as the percentage inhibition considering FSK stimulation as 100% activation: *p<0.05, **p<0.01 and ***p<0.005 in an unpaired two-tailed Student's t test.

Figure 4. VCE-003 alleviates EAE and prevents CD4⁺ cells infiltration.

(A) VCE-003 significantly ameliorates the clinical signs and disease progression of EAE (squares), and these effects are partially blocked by a CB2 antagonist (AM630, triangles) or PPARγ antagonist (T0070907, circles). The results are shown as the means ± SD: *p = 0.015 EAE + VCE-003 vs EAE + VEH; **p = 0.005 EAE + VCE-003 vs EAE + VEH; ***p<0.001 EAE + VCE-003 vs EAE + VEH; #p = 0.016 EAE + VCE-003 + AM630 vs EAE + VCE-003; ##p = 0.008 EAE + VCE-003 + AM630 vs EAE + VCE-003; ###p<0.001 EAE + VCE-003 + AM630 vs EAE + VCE-003; +p = 0,009 EAE + VCE-003 + T0070907 vs EAE + VCE-003; ++p<0.01 EAE + VCE-003 + T0070907 vs EAE + VCE-003. (B) VCE-003 reduces the number of infiltrates and (C) it significantly reduces the number of CD4⁺ T cells in thoracic spinal cord sections. The figure shows the representative staining of spinal cord sections: LFB (B) and CD4 immunohistochemistry (C). Arrows indicate CD4⁺ T cells. The results (C) are shown as the means ± SEM: **p = 0,004 vs Intact; #p = 0,030 vs EAE + VEH.

Figure 5. VCE-003 significantly reduces microglial activation and preserves myelin structure.

(A) VCE-003 notably reduces microglial activation (Iba1⁺ cells). (B) Quantification of Iba1⁺ cells are shown as means ± SEM (***p<0.001 vs Intact; #p = 0.021 vs EAE + VEH). Thoracic spinal cord sections from symptomatic animals show a clear disruption of myelin, whereas exposure to VCE-003 contributes to maintain the myelin structure (C, RIP immunofluorescence staining; D, LFB staining).

Figure 6. VCE-003 reduces axon damage.

Representative images of axon morphology changes: (A) transversal thoracic spinal cord sections, neurofilament staining in red; (B) longitudinal thoracic spinal cord sections, neurofilament staining in green (please note axon swelling, arrows). (C) VCE-003 treatment helps to preserve the axon structure as showed in longitudinal thoracic spinal cord sections by SMI32 staining.

Figure 7. VCE-003 reduces the expression of inflammatory marker mRNAs in the spinal cord and of iNOS protein in BV2 cells.

VCE-003 decreased the mRNA expression of TNFα, IFNγ, IL-1β and IL-17 (A), and the adhesion molecule ICAM-1 and iNOS (B). (C) Immunoblotting of iNOS. Proteins in lysates (30 µg) from BV2 cells stimulated with LPS (50 ng/ml) and IFNγ (100 u/ml), and pre-treated with VCE-003 (1 µM). Both CB2 and PPARγ antagonists (AM630 1 µM and GW9662 0.1 µM, respectively) were administered 10 min before than VCE-003 treatment. The results are presented as the relative ratio of proteins, where the iNOS signal obtained by densitometric analysis was normalized to the tubulin signal. VCE-003 treatment significantly reverses the LPS-IFNγ-induced effects to the control situation, and both CB2 and PPARγ antagonists blocked this effect of VCE-003. The results in (A, B) are shown as the means ± SEM: *p = 0,011 vs Intact (TNFα); *p = 0,003 vs Intact (ICAM-1); **p<0.01 vs Intact; #p = 0.05 vs EAE + VEH (ICAM-1); ##p<0.01 vs EAE + VEH; ###p<0.001 vs EAE + VEH). The results in (C) are shown as the means ± SEM from three independent experiments performed in triplicate: ***p<0.001 vs CTL; ##p<0.01 vs CTL + LPS- IFNγ; +p = 0.034 vs VCE-003 1 µM + LPS- IFNγ; ++p = 0.002 vs VCE-003 1 µM + LPS- IFNγ.