Immunoregulation of a viral model of multiple sclerosis using the synthetic cannabinoid R+WIN55,212

Abstract

Theiler murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) is a mouse model of chronic-progressive multiple sclerosis (MS) characterized by Th1-mediated CNS demyelination and spastic hindlimb paralysis. Existing MS therapies reduce relapse rates in 30% of relapsing-remitting MS patients, but are ineffective in chronic-progressive disease, and their effects on disability progression are unclear. Experimental studies demonstrate cannabinoids are useful for symptomatic treatment of spasticity and tremor in chronic-relapsing experimental autoimmune encephalomyelitis. Cannabinoids, however, have reported immunosuppressive properties. We show that the cannabinoid receptor agonist, R+WIN55,212, ameliorates progression of clinical disease symptoms in mice with preexisting TMEV-IDD. Amelioration of clinical disease is associated with downregulation of both virus and myelin epitope-specific Th1 effector functions (delayed-type hypersensitivity and IFN-gamma production) and the inhibition of CNS mRNA expression coding for the proinflammatory cytokines, TNF-alpha, IL1-beta, and IL-6. Clinical trials investigating the therapeutic potential of cannabinoids for the symptomatic treatment of MS are ongoing, and this study demonstrates that they may also have potent immunoregulatory properties.

Figure 1

Cannabinoid receptor agonism ameliorates the clinical severity of established TMEV-induced demyelinating disease. Treatment with R(+)WIN55,212 (filled circles) inhibits TMEV-induced clinical disease when administered, at the time of infection (a), at the onset of clinical disease (b), or during established disease (c). Rx, duration of administration of WIN55,212 to mice. *Significant inhibition (P < 0.05) of clinical disease score in R(+)WIN55,212-treated mice compared with S(–)WIN55,212 (open circles) or untreated TMEV mice (filled triangles). Data are representative of three separate experiments.

Figure 2

Cannabinoid treatment increases the susceptibility of mice to TMEV infection and is not cytotoxic to splenocytes. R(+)WIN55,212 treatment of mice at the time of infection (a) (black bars) showed a significant increase in CNS virus titers compared with either S(–)WIN55,212 (gray bars) or untreated (white bars) TMEV-infected mice. Virus titers were not different from controls in mice treated with R(+)WIN55,212 at the onset of disease (b). *Significant increase in viral load in R(+)WIN55,212-treated mice compared with either S(–)WIN55,212 or untreated TMEV mice (P < 0.05). No PFUs were observed in naive mice. R(+)WIN55,212 treatment has no cytotoxic effect, as measured by FACS analysis of splenic CD4⁺, CD8⁺, B220⁺, and CD4⁺CD25⁺ lymphocytes, and F4/80⁺ macrophage populations compared with S(–)WIN55,212 or naive mice (c). Results are expressed as the mean total number of cells (n = 3 per group) ± SEM.

Figure 3

Effects of cannabinoid receptor agonism on T cell proliferation depends upon the cellular activation state. Early R(+)WIN55,212 treatment (filled circles) (postinfection day 0–5) significantly inhibited the proliferation of T lymphocytes compared with S(–)WIN55,212 (open circles) and untreated TMEV-infected mice (filled triangles) upon rechallenge with the TMEV VP2_70-86 peptide (postinfection day 8) (a), but not PLP_139-151 (d). When measured at postinfection day 40, R(+)WIN55,212 treatment did not inhibit T lymphocyte proliferation to either VP2_70-86 or PLP_139-151 peptides during established disease (postinfection day 26–31 or 50–55) (b, c, e, and f). *P < 0.05, compared with either S(–)WIN55,212 or untreated mice.

Figure 4

Cannabinoid treatment inhibits both virus and myelin autoimmune inflammatory DTH responses. Treatment of mice with R(+)WIN55,212 (black bars) at the time of TMEV infection (a), at the onset of clinical disease (b), or during established disease (c), significantly reduces DTH responses to rechallenge with either the immunodominant TMEV peptide VP2_70-86 or the autoreactive myelin PLP_139-151 peptide. *Significant reduced DTH responses compared with either S(–)WIN55,212 (dark gray bars) or untreated TMEV-infected (white bars) mice (P < 0.05). Naive mice (light gray bars) show a low background response to either peptide.

Figure 5

Cannabinoid receptor agonism inhibits Th1-mediated IFN-γ cell secretion. Treatment with R(+)WIN55,212 at the time of infection (a) or at the onset of clinical disease (b) significantly inhibited IFN-γ secretion from T lymphocytes rechallenged with either VP2_70-86 or PLP_139-151 compared with either S(–)WIN55,212 (gray bars) or untreated TMEV-infected mice (white bars). R(+)WIN55,212 treatment during established disease (c) had no significant effect on IFN-γ secretion. *Cytokine response was significantly inhibited compared with either S(–)WIN55,212 or untreated TMEV-infected mice. P < 0.05.

Figure 6

Cannabinoid treatment inhibits mRNA coding for both antiviral and Th1- and Th2-type inflammatory mediators in the CNS. R(+)WIN55,212 treatment at the time of infection (day 0–5) inhibited spinal cord mRNA coding for the innate antiviral mediators, IFN-α, and IFN-β (a). R(+)WIN55,212 treatment at the onset of disease inhibited spinal cord mRNA coding for the proinflammatory Th1 mediators, IL-1β, IL-6, TNF-α, and IFN-γ, but not the IL-12 p40 subunit (b). Treatment at disease onset also inhibited mRNA coding for the Th2 cytokine IL-4, but not IL-10 (c). The mRNA levels are presented as the percentage of intensity of the HPRT control for that sample.