In summary, we have shown that CBD administered to MOG-immunized C57BL/6 mice, at the onset of EAE disease, reduced the severity of the clinical signs of EAE. CBD treatment was accompanied by diminished axonal loss and inflammation (infiltration of T cells and microglial activation). Moreover, CBD prevented proliferation of myelin-specific T cells in vitro. These observations suggest that CBD may have potential for alleviating MS-like pathology.
Cannabidiol (CBD) treatment of control, naïve C57BL/6 mice does not affect spinal cord integrity, basal microglial and T-cell number. The Figure shows representative stainings of spinal cord sections prepared from control healthy mice receiving complete Freund adjuvant either without CBD (Ctrl) or with CBD (Ctrl + CBD) treatment. (A) Haematoxylin and eosin (H&E) staining; (B) Iba-1 staining for the presence of microglial/macrophages; and (C) CD3+ staining for presence of T-cells. Scale bars represent 100 µm. Statistical analysis of the CD3+ and Iba-1 expression is presented in the bar graphs of Figures 5C (CD3+) and and6C 6C (Iba-1).
Several cannabinoids including THC and CBD exert anti-oxidative and neuroprotective properties (Mechoulam et al., 2007). Most of the current MS therapies are directed against various immune cells to achieve immunosuppressive effects. However, increasing evidence shows that immunosuppression alone was not sufficient for therapeutic effect especially in late, secondary progressive MS (Bennett and Stüve, 2009; Jones and Coles, 2010). In these cases, the neurodegenerative processes become resistant to immunomodulation. Indeed, neurodegeneration that consists of neuronal and axonal loss can result from oxidative stress and excitotoxicity and is driven by activated microglial cells and macrophagic/monocytic infiltrates (Hanisch and Kettenmann, 2007). It appears that the cannabinoid system could provide a rescue mechanism in such conditions. Accordingly, the ameliorating activity of THC-like cannabinoids combines CB2 receptor-mediated inhibition of autoreactive T cells and CB1 receptor-mediated neuroprotective activity on neurons (Maresz et al., 2007). Similarly, Croxford et al. (2008) pointed out that cannabinoid-mediated neuroprotection rather than immunosuppression, was relevant for the recovery process at the later, remissive stages of MS.
Cannabidiol (CBD) ameliorates the clinical signs and disease progression of EAE induced by myelin oligodendrocyte glycoprotein (MOG). EAE was induced in C57BL/6 mice by flank s.c. immunization with MOG35-55. Clinical disease scores were recorded daily until day 30 after induction. CBD (CBD + EAE) or its vehicle (EAE group), was injected i.p. during the onset of the disease for 3 consecutive days (days 19–21; indicated by arrows). The mean clinical scores ± SD are shown. Each group consisted of 15 mice. Repeated measure anova showed significant differences between the EAE and CBD + EAE groups (P < 0.001).
Anti-oxidant and neuroprotective properties of CBD
Among the many types of neurodegenerative diseases in which inflammation is involved, multiple sclerosis (MS) is one of those clearly induced and driven by dysfunctional immune system activity. In MS, myelin autoreactive peripheral T cells migrate into the CNS and initiate cytotoxic, degenerative processes that include demyelination, oligodendrocyte cell death and axonal degeneration. These effects lead to neurological deficits such as visual and sensory disturbances, motor weakness, tremor, ataxia and progressive disability as the main clinical symptoms (Compston and Coles, 2008). Infiltrating T cells are constantly reactivated within the CNS parenchyma by microglia. Microglial cells via chemokine and cytokine release and constant antigen presentation potentiate T-cell recruitment to the CNS and facilitate their polarization into cytotoxic phenotypes (Th1 or Th17). Moreover, via released chemokines such as CCL-2, microglial cells recruit other immune cells of myeloid origin, specialized in epitope spreading and phagocytosis of myelin including monocytes, macrophages, B cells and dendritic cells (Jack et al., 2005; Koning et al., 2009). Depletion of microglia or impairment of their function can attenuate disease progression in experimental animal models of MS supporting their role in initiation and development of this disease (Huitinga et al., 1990; Heppner et al., 2005). Thus, suppression of microglia will potentially reduce inflammatory lesions and limit demyelination within the CNS.
The main source of reactive oxygen and nitrogen species within the CNS under neurodegenerative conditions are reactive microglia and astrocytes (Hanisch and Kettenmann, 2007). Oxidative signalling negatively affects neuronal and axonal survival, especially of axons lacking a myelin sheath, and results in irreversible damage. Accordingly, deletion of nuclear factor-erythroid 2-related 2 (Nrf2), a redox-sensitive transcription factor that regulates expression of many protective antioxidant and detoxication enzymes and transporters (Kobayashi and Yamamoto, 2005), resulted in exacerbation of EAE (Johnson et al., 2010). CBD has been frequently described as a potent neuroprotective and anti-oxidant agent. CBD reduced glutamate excitotoxicity and hydroperoxide-induced oxidative neuronal damage (Hampson et al., 1998) and provided neuroprotection against 6-hydroxy-dopamine (Lastres-Becker et al., 2005), β-amyloid (Iuvone et al., 2004) and prion toxicities to neurons (Dirikoc et al., 2007) and against neuroinflammation (Esposito et al., 2007; Zuardi, 2008). In this regard, we have shown that exposure of microglial cells in culture to CBD up-regulates a number of anti-oxidative genes including genes that are involved in glutathione synthesis. Many of these genes are under the control of the Nrf2 transcription factor (Juknat et al., 2011). Thus, the anti-oxidative properties of CBD may significantly contribute to the alleviation of EAE pathology and accompany its anti-inflammatory beneficial properties.
Neither mice that received CFA alone nor those that received CFA with 5 mg·kg −1 of CBD exhibited any clinical signs of EAE (data not shown). Next, we evaluated if CBD injections in these healthy mice affected the spinal cords. As shown on Figure 2A , CBD injections did not affect the pattern of H&E histological staining, compared with the group of healthy mice, treated only with CFA. Moreover, Iba-1 microglial expression in Ctrl + CBD group treated with CBD and CFA did not differ from that in the mice given only CFA ( Figure 2B ). CD3+ T cells were absent in spinal cords of mice given CFA, with or without CBD ( Figure 2C ). The amount of staining is presented in Figures 5C and and6C 6C .
Indeed, CBD exerts a wide range of anti-inflammatory properties and regulates cell cycle and function of various immune cells. These effects include suppression of humoral responses, such as release of cytokines, chemokines, growth factors, as well as suppression of immune cell proliferation, activation, maturation, migration and antigen presentation (Mechoulam et al., 2007). In an earlier publication, we showed that CBD inhibited production of the cytokine IL-6 and the chemokine CCL-2 by activated microglial cells and in parallel activated intracellular anti-inflammatory pathways (Kozela et al., 2010).
Consequently, CBD, a compound active on non-CB1 or CB2 receptors (Showalter et al., 1996), has been shown to possess a wide anti-inflammatory profile. CBD was shown to decrease TNFα, IL-2 and IFNγ release from activated splenocytes and macrophages (Malfait et al., 2000; Jan et al., 2007; Kaplan et al., 2008). It also suppresses concavalin A and collagen induced T-cell proliferation (Malfait et al., 2000; Jan et al., 2007), microglial migration (Walter et al., 2003) and cytokine release (Kozela et al., 2010). It suppresses antigen-specific antibody production in splenocytes (Jan et al., 2007), as well as attenuates endothelial inflammation and barrier disruption (Rajesh et al., 2007). These activities of CBD may contribute to its beneficial effects in EAE, as observed in our hands, because many of these immune processes were reported to be involved in EAE pathology at different stages of the disease model.
The effect of CBD treatment on EGR2, STAT5, LAG3, and IL-10 mRNA levels in purified T MOG previously co-cultured with APC. TMOG cells were co-cultured with adherent APC and stimulated with MOG35-55 in the presence or absence of CBD. TMOG cells were then purified using CD4 + microbeads, lysed, and subjected for mRNA extraction and qPCR analysis using gene specific primers. The bar graphs show the levels of the indicated mRNAs as percentage of the amounts observed following stimulation with MOG35-55. (A) EGR2 mRNA (ANOVA F(3,4) = 211.5, P < 0.001); (B) STAT5 mRNA (ANOVA F(3,12) = 6.1, P < 0.01); (C) LAG3 mRNA (ANOVA F(3,8) = 116.5, P < 0.001); (D) IL-10 mRNA (ANOVA F(3,11) = 9.9, P < 0.01); (n = 2 to 4). Symbols: *P < 0.05, **P < 0.01, ***P < 0.001 vs non-stimulated cells; # P < 0.05, ## P < 0.001, ### P < 0.001 vs MOG35-55-stimulated cells.
We would like to note that 8 h MOG35-55 stimulation resulted in a lower yield of encephalitogenic T cells following the use of the CD4 microbead system. We assume that this change in CD4 yield reflects transient downregulation (internalization and turnover) in CD4 receptor expression following antigen stimulation [35,36]. Indeed, immunoblotting of STAT3 and β-actin proteins confirmed a decrease in total level of both of these proteins by about 35% in all MOG-treated samples. This observation corresponds well with these previous reports on post-stimulation CD4 receptor downregulation [35,36]. Addition of 5 μM CBD had no further effect.
Although LAG3 serves as a CD4 negative co-receptor and thus is mainly expressed on CD4 + T cells, its increased levels have been recently reported on B cells as well . In our hands, the basal frequency of CD19 + LAG3 + B cells in APC/TMOG co-cultures was only 6.9% ± 0.4% and was not affected by the presence of CBD (7.9% ± 0.9%; Table 4). Moreover, neither MOG35-55 stimulation nor MOG + CBD combination had any effect on CD19 + LAG3 + cell frequencies in our APC/TMOG co-cultures (6.0% ± 0.4% and 5.7% ± 0.9%, respectively). Thus, in contrary to the situation in T cells, LAG3 expression in B cells is not regulated by either MOG35-55 stimulation or CBD treatment.
As controls, we analyzed the effects of CBD treatment on the expression of CD69 and of LAG3 on resting splenocytes cultured without TMOG and without MOG35-55 as well as in resting TMOG cultured without MOG35-55 and without APC (both in maintenance medium). These control experiments showed that the levels of CD69 (6.9% ± 1.2%) and of LAG3 (0.5% ± 0.3%) in resting spleen-derived CD4 + splenocytes cultured separately were not significantly affected by CBD treatment (reaching 8.7% ± 1.7% and 0.4% ± 0.4%, respectively; Table 2). Similarly, in resting TMOG cells cultured alone, CBD treatment did not affect the basal levels of CD69 (0.9% ± 0.3% in control cells and 1.0% ± 0.2% in CBD-treated) and of LAG3 (0.7% ± 0.2% and 0.5% ± 0.1%, respectively). These experiments demonstrate that the changes in CD69 and LAG3 expression are induced by CBD treatment only in APC/TMOG co-cultures and not in TMOG or in splenocytes cultured separately.
At the second step, we analyzed the CD4 + CD25 − population for the expression of CD69 molecule. In control, non-stimulated APC/TMOG co-cultures 9.3% ± 0.4% of the CD4 + CD25 − cells were found to be positive for CD69 (Figure 2A). Interestingly, 18 h co-incubation with CBD resulted in a very significant increase in CD4 + CD25 − CD69 + cells reaching 22.5% ± 1.3% (P < 0.001 vs 9.3% ± 0.4% observed in the non-stimulated APC/TMOG co-cultures). MOG35-55-stimulation of APC/TMOG co-cultures resulted in doubling the CD69 expression on CD4 + CD25 − cells up to 18.0% ± 1.5% (P < 0.001 vs non-stimulated cells), and CBD addition together with MOG35-55 further increased the frequency of CD4 + CD25 − CD69 + cells up to 36.6% ± 1.9% (P < 0.001 vs MOG-treated cells).
Our data suggests that CBD exerts its immunoregulatory effects via induction of CD4 + CD25 − CD69 + LAG3 + cells in MOG35-55-activated APC/TMOG co-cultures. This is accompanied by EGR2-dependent anergy of stimulated TMOG cells as well as a switch in their intracellular STAT3/STAT5 activation balance leading to the previously observed decrease in Th17 activity.
We performed gene array analysis of mRNA expression in TMOG cells in search for additional transcripts that are involved in anergy and tolerogenic processes. As shown in Table 3, MOG35-55-stimulation of TMOG cells led to a significant upregulation of various anergy promoters representing the following categories: regulators of cell cycle (Ndrg1 by 6.2-fold, Cdkn1a by 2.6-fold, Casp4 by 2.2-fold, and Fas by 2.6-fold), tolerance inducers (Lag3 by 1.7-fold, Icos by 1.6-fold, Nfatc1 by 3.3-fold), and chemokine recruiting regulatory T cells (Ccl4 by 9.1-fold). The addition of CBD treatment significantly potentiated the MOG35-55-upregulated transcript levels of tolerance inducers, that is, of Lag3 (by 305%), Icos (by 43%), and Nfatc1 (by 21%) and of cell cycle regulators such as Cdkn1a (by 19%), Casp4 (by 22%), and Fas (by 27%). It did not affect the MOG35-55-enhanced levels of Ndrg1 and Ccl4. Treatment with CBD alone resulted in more than twofold increase of the levels of Icos, Ndrg1, and Casp4 in non-stimulated TMOG cells (P < 0.005). Slight but significant increases were observed following CBD treatment for Lag3, Nfatc1, and Fas mRNA transcripts in non-stimulated TMOG cells.