(−)-β-Caryophyllene, a CB2 Receptor-Selective Phytocannabinoid, Suppresses Motor Paralysis and Neuroinflammation in a Murine Model of Multiple Sclerosis

(−)-β-caryophyllene (BCP), a cannabinoid receptor type 2 (CB2)-selective phytocannabinoid, has already been shown in precedent literature to exhibit both anti-inflammatory and analgesic effects in mouse models of inflammatory and neuropathic pain. Herein, we endeavored to investigate the therapeutic potential of BCP on experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS). Furthermore, we sought to demonstrate some of the mechanisms that underlie the modulation BCP exerts on autoimmune activated T cells, the pro-inflammatory scenery of the central nervous system (CNS), and demyelination. Our findings demonstrate that BCP significantly ameliorates both the clinical and pathological parameters of EAE. In addition, data hereby presented indicates that mechanisms underlying BCP immunomodulatory effect seems to be linked to its ability to inhibit microglial cells, CD4+ and CD8+ T lymphocytes, as well as protein expression of pro-inflammatory cytokines. Furthermore, it diminished axonal demyelination and modulated Th1/Treg immune balance through the activation of CB2 receptor. Altogether, our study represents significant implications for clinical research and strongly supports the effectiveness of BCP as a novel molecule to target in the development of effective therapeutic agents for MS.


Introduction
Multiple sclerosis (MS) is a severe inflammatory demyelinating disease of the central nervous system (CNS), and is the most common cause of non-traumatic neurologic disability in young adults, affecting over two million people worldwide. The etiology of MS is heterogeneous in nature and has not been completely elucidated. However, studies with MS patients suggest that the demyelination observed in the CNS results from a T cell-mediated autoimmune response, especially encephalitogenic Th17 and Th1 cells [1]. Moreover, experimental autoimmune encephalomyelitis (EAE) is an experimental model extensively employed for studying clinical, immunological, and neuropathological features of MS [2]. The onset and development of clinical signs in EAE are
The selective agonism of CB2 receptors expressed by immune cells modulates cell migration, and decreases cytokine production [47] and antigen presentation [48]. The rank order of CB2 mRNA expression in immune cells is as follows: lymphocyte B cells > natural killer (NK) cells > monocytes > polymorphonuclear neutrophil cells > CD8+ T cells > CD4+ T cells [49]. Moreover, even though the expression of CB2 mRNA is the least significant in T lymphocytes in comparison to other major leukocyte cells, these receptors are intrinsically connected to T cell migration. In fact, the activation of CB2 receptors by multiple ligands has been shown to inhibit lymphocyte T migration responding to C-X-C motif chemokine 12 (CXCL12), a vastly characterized and effective chemokine, fundamental for T cell recruitment [50]. Taken together, these data indicate that BCP may have suppressed the encephalitogenic effects of T cells by inhibiting their recall responses (Th1 vs. Treg) and, consequently, production of pro-and anti-inflammatory cytokines, respectively. Previous data showed that BCP binds selectively to the CB2 receptor as a full agonist [12][13][14]. Additionally, CB2 receptor activation has been shown to prevent cisplatin-induced nephrotoxicity in experimental colitis, through inhibition of pro-inflammatory chemokines and infiltration of macrophage and neutrophil [31,46].

BCP Attenuates Disease Progression in Chronic EAE Mice Model
In order to assess the therapeutic potential of BCP, EAE was induced in C57BL/6 mice with myelin MOG35-55 peptide. The onset of EAE clinical symptoms was on day 11 and reached a maximum mean clinical score of 3.5 on day 19 after immunization ( Figure 2). Moreover, weight loss progressed alongside EAE severity, related to both level of motor impairment and reduced food intake, as well as peak of pro-inflammatory cytokines production on the acute phase of disease. Herein, EAE mice reached their lowest weight at the peak of the disease, in comparison to the control group. These same symptoms were not observed in the naïve group ( Figure 2). To evaluate the prophylactic effect of BCP treatment in the same experimental model, since day 0 of immunization, mice were treated orally with 25 and 50 mg/kg BCP (twice/day). In comparison to the untreated EAE group, BCP treatment with 25 mg/kg presented promising results, particularly at the chronic stage of disease; additionally, 50 mg/kg BCP significantly prevented motor paralysis and weight loss induced by EAE immunization (Figure 2). to C-X-C motif chemokine 12 (CXCL12), a vastly characterized and effective chemokine, fundamental for T cell recruitment [50]. Taken together, these data indicate that BCP may have suppressed the encephalitogenic effects of T cells by inhibiting their recall responses (Th1 vs. Treg) and, consequently, production of pro-and anti-inflammatory cytokines, respectively. Previous data showed that BCP binds selectively to the CB2 receptor as a full agonist [12][13][14]. Additionally, CB2 receptor activation has been shown to prevent cisplatin-induced nephrotoxicity in experimental colitis, through inhibition of pro-inflammatory chemokines and infiltration of macrophage and neutrophil [31,46].

BCP Attenuates Disease Progression in Chronic EAE Mice Model
In order to assess the therapeutic potential of BCP, EAE was induced in C57BL/6 mice with myelin MOG35-55 peptide. The onset of EAE clinical symptoms was on day 11 and reached a maximum mean clinical score of 3.5 on day 19 after immunization ( Figure 2). Moreover, weight loss progressed alongside EAE severity, related to both level of motor impairment and reduced food intake, as well as peak of pro-inflammatory cytokines production on the acute phase of disease. Herein, EAE mice reached their lowest weight at the peak of the disease, in comparison to the control group. These same symptoms were not observed in the naïve group ( Figure 2). To evaluate the prophylactic effect of BCP treatment in the same experimental model, since day 0 of immunization, mice were treated orally with 25 and 50 mg/kg BCP (twice/day). In comparison to the untreated EAE group, BCP treatment with 25 mg/kg presented promising results, particularly at the chronic stage of disease; additionally, 50 mg/kg BCP significantly prevented motor paralysis and weight loss induced by EAE immunization (Figure 2). It has been estimated that two-thirds of MS patients suffer from pain during the course of the disease at some point, whereas 40% of the patients are never pain free [51]. MS is the source of many pain syndromes; some prevail for a brief time while others are long lasting. Pain syndromes associated with MS include powerful cramps and spasms, stiffened joints, pressure pain, facial (trigeminal) pain, and other painful sensations, such as burning, itching, and shooting pain [51]. Thus, in the next set of experiments, we determined if BCP treatment could inhibit the mechanical hyperalgesia induced by EAE. In response to stimulation with von Frey filaments on the right hind paw, untreated EAE mice showed long lasting and increased frequency response ( Figure 3). Treatment with BCP (50 mg/kg, p.o., twice/day) notably attenuated mechanical hyperalgesia induced by EAE immunization, especially during pre-motor phase of the disease ( Figure 3). It has been estimated that two-thirds of MS patients suffer from pain during the course of the disease at some point, whereas 40% of the patients are never pain free [51]. MS is the source of many pain syndromes; some prevail for a brief time while others are long lasting. Pain syndromes associated with MS include powerful cramps and spasms, stiffened joints, pressure pain, facial (trigeminal) pain, and other painful sensations, such as burning, itching, and shooting pain [51]. Thus, in the next set of experiments, we determined if BCP treatment could inhibit the mechanical hyperalgesia induced by EAE. In response to stimulation with von Frey filaments on the right hind paw, untreated EAE mice showed long lasting and increased frequency response (Figure 3). Treatment with BCP (50 mg/kg, p.o., twice/day) notably attenuated mechanical hyperalgesia induced by EAE immunization, especially during pre-motor phase of the disease (Figure 3). Interestingly, our data corroborates and expands previous data found in literature, which established that BCP reduced inflammatory (late phase) pain responses though the CB2 receptor in the formalin test, although it did not influence acute (early phase) responses. Furthermore, the same authors suggest that administration of BCP via oral route extenuated mechanical allodynia and thermal hyperalgesia during a neuropathic pain model, as well as reduced spinal neuroinflammation and did not induce signs of tolerance after prolonged treatment [29]. Thus, our results suggest that BCP treatment was able to extinguish clinical EAE signs, including motor impairment and symptoms induced by immunization. Interestingly, our data corroborates and expands previous data found in literature, which established that BCP reduced inflammatory (late phase) pain responses though the CB2 receptor in the formalin test, although it did not influence acute (early phase) responses. Furthermore, the same authors suggest that administration of BCP via oral route extenuated mechanical allodynia and thermal hyperalgesia during a neuropathic pain model, as well as reduced spinal neuroinflammation and did not induce signs of tolerance after prolonged treatment [29]. Thus, our results suggest that BCP treatment was able to extinguish clinical EAE signs, including motor impairment and symptoms induced by immunization. These observations are corroborated by a recent report that determined that BCP showed protective effect on brain damage and chemical induced seizure through inhibition of pro-inflammatory cytokines productions, such as TNF-α and IL-1β, as well as restored the activity of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) [52]. Likewise, Cheng and colleagues demonstrated that BCP, given orally, activated the CB2 receptor and the peroxisome proliferator-activated receptor-γ (PPARγ) pathway, preventing the Alzheimer-like phenotype in APP/PS1 mice. Furthermore, this cognitive effect was positively related with decreased β-amyloid load in the cerebral cortex and hippocampus. In addition, BCP treatment inhibited the levels of COX-2 protein as well as glial activation, and the mRNA levels of the pro-inflammatory cytokines in the cerebral cortex [27]. Hence, to establish the status of these cells and inflammatory mediators in BCP-treated EAE mice, we evaluated glial activation, oxidative damage, and demyelination in brain tissues from both untreated and BCP-treated EAE mice.

BCP Inhibits Glial Activation, Oxidative Damage, and Demyelination during EAE Development
Therefore, in order to further assay the mechanisms of action of BCP, in a new set of experiments, the expression of ionized calcium binding adaptor molecule 1 (Iba-1), inducible nitric oxide synthase (iNOS), and neurofilament-H was measured in the lumbar spinal cord of EAE-untreated and treated mice. There was a significant increase in the expression of Iba-1 and iNOS in the lumbar spinal cord of the EAE-control group (Figure 4). Moreover, the expression of neurofilament-H, a demyelination marker, was increasingly expressed by EAE immunization in the control group ( Figure 5). Relevantly, BCP (50 mg/kg, p.o.) treatment downregulated the microglial activation, oxidative damage ( Figure 5), and, consequently, demyelination ( Figure 6). This finding validates and expands previous data demonstrating that through activation of BV2 microglia following hypoxic exposure, BCP inhibited cytotoxicity by decreasing the release of pro-inflammatory cytokines, including TNF-α, IL-6, and IL-1β. Additionally, the same authors demonstrated that BCP downregulated the activation of nuclear factor κB (NF-κB), and CB2R small RNA interference completely abolished these actions [28]. Altogether, our research indicates that These observations are corroborated by a recent report that determined that BCP showed protective effect on brain damage and chemical induced seizure through inhibition of pro-inflammatory cytokines productions, such as TNF-α and IL-1β, as well as restored the activity of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) [52]. Likewise, Cheng and colleagues demonstrated that BCP, given orally, activated the CB2 receptor and the peroxisome proliferator-activated receptor-γ (PPARγ) pathway, preventing the Alzheimer-like phenotype in APP/PS1 mice. Furthermore, this cognitive effect was positively related with decreased β-amyloid load in the cerebral cortex and hippocampus. In addition, BCP treatment inhibited the levels of COX-2 protein as well as glial activation, and the mRNA levels of the pro-inflammatory cytokines in the cerebral cortex [27]. Hence, to establish the status of these cells and inflammatory mediators in BCP-treated EAE mice, we evaluated glial activation, oxidative damage, and demyelination in brain tissues from both untreated and BCP-treated EAE mice.

BCP Inhibits Glial Activation, Oxidative Damage, and Demyelination during EAE Development
Therefore, in order to further assay the mechanisms of action of BCP, in a new set of experiments, the expression of ionized calcium binding adaptor molecule 1 (Iba-1), inducible nitric oxide synthase (iNOS), and neurofilament-H was measured in the lumbar spinal cord of EAE-untreated and treated mice. There was a significant increase in the expression of Iba-1 and iNOS in the lumbar spinal cord of the EAE-control group (Figure 4). Moreover, the expression of neurofilament-H, a demyelination marker, was increasingly expressed by EAE immunization in the control group ( Figure 5). Relevantly, BCP (50 mg/kg, p.o.) treatment downregulated the microglial activation, oxidative damage ( Figure 5), and, consequently, demyelination ( Figure 6). This finding validates and expands previous data demonstrating that through activation of BV2 microglia following hypoxic exposure, BCP inhibited cytotoxicity by decreasing the release of pro-inflammatory cytokines, including TNF-α, IL-6, and IL-1β.
Additionally, the same authors demonstrated that BCP downregulated the activation of nuclear factor κB (NF-κB), and CB2R small RNA interference completely abolished these actions [28]. Altogether, our research indicates that BCP selectively increases the infiltration/differentiation of Treg and inhibits Th1 myelin-specific cells in the CNS through activation of the CB2 cannabinoid receptor. These results are in agreement with Bento and colleagues, who demonstrated that AM630, a CB2 antagonist, reverted the inhibitory effect of BCP on pro-inflammatory chemokine CINC-1/CXCL1 levels and mRNA expression of TNF-α in the IEC-6 cell line. In addition, the same authors showed that the therapeutic effects of BCP on experimental colitis were reversed by concomitant administration of CB2 and PPAR-γ antagonists [46]. Thus, further investigation is required to verify whether BCP modulates the course of EAE through direct interaction with CB2 receptor or endogenous CB pathways, such as activation of peroxisome proliferator-activated receptor-γ (PPARγ). BCP selectively increases the infiltration/differentiation of Treg and inhibits Th1 myelin-specific cells in the CNS through activation of the CB2 cannabinoid receptor. These results are in agreement with Bento and colleagues, who demonstrated that AM630, a CB2 antagonist, reverted the inhibitory effect of BCP on pro-inflammatory chemokine CINC-1/CXCL1 levels and mRNA expression of TNF-α in the IEC-6 cell line. In addition, the same authors showed that the therapeutic effects of BCP on experimental colitis were reversed by concomitant administration of CB2 and PPAR-γ antagonists [46]. Thus, further investigation is required to verify whether BCP modulates the course of EAE through direct interaction with CB2 receptor or endogenous CB pathways, such as activation of peroxisome proliferator-activated receptor-γ (PPARγ).   BCP selectively increases the infiltration/differentiation of Treg and inhibits Th1 myelin-specific cells in the CNS through activation of the CB2 cannabinoid receptor. These results are in agreement with Bento and colleagues, who demonstrated that AM630, a CB2 antagonist, reverted the inhibitory effect of BCP on pro-inflammatory chemokine CINC-1/CXCL1 levels and mRNA expression of TNF-α in the IEC-6 cell line. In addition, the same authors showed that the therapeutic effects of BCP on experimental colitis were reversed by concomitant administration of CB2 and PPAR-γ antagonists [46]. Thus, further investigation is required to verify whether BCP modulates the course of EAE through direct interaction with CB2 receptor or endogenous CB pathways, such as activation of peroxisome proliferator-activated receptor-γ (PPARγ).

BCP Therapeutic Treatment Inhibits Progression the Clinical Signs of EAE
The observed anti-inflammatory action of BCP on the induction phase of EAE led us to further examine if commencement of therapy after onset of disease, during chronic phase, could also exert therapeutic response. In order to explore this hypothesis, BCP was given to ongoing active EAE in C57BL/6 mice, starting after mice had already presented visible clinical symptoms (score of 1.5 ± 0.5) of disease (on day 15). BCP at 50 mg/kg administered twice daily notably blocked the clinical severity of the disease, indicating maximal inhibition of 62% based on the cumulative score ( Figure  6).

BCP Prophylactic Treatment Downregulated CD4+ and CD8+ T Lymphocytes
Since CD4+ Th-induced inflammation is thought to be responsible for the neurodegenerative component of chronic EAE, we asked whether BCP would interfere with CD4+ and CD8+ T cells. The percentage of CD4+ and CD8+ T cells was significantly lower in EAE, moreover, both CD4+ and CD8+ T cells were significantly decreased in BCP prophylactically treated animals (Figure 7). In order to evaluate activation of T lymphocytes, the co-expression of CD69 cellular marker was measured through the double staining of CD4+CD69+ and CD8+CD69+. In fact, the population of CD4+CD69+ and CD8+CD69+ T lymphocytes were remarkably increased in EAE-untreated animals when assessed 25dpi, when compared with control naive mice. Furthermore, BCP treatment (50 mg/kg, p.o.) inhibited both CD4+ and CD8+ T cells, as well as their activation in the peripheral lymphoid tissue during EAE development (Figure 8).

BCP Therapeutic Treatment Inhibits Progression the Clinical Signs of EAE
The observed anti-inflammatory action of BCP on the induction phase of EAE led us to further examine if commencement of therapy after onset of disease, during chronic phase, could also exert therapeutic response. In order to explore this hypothesis, BCP was given to ongoing active EAE in C57BL/6 mice, starting after mice had already presented visible clinical symptoms (score of 1.5 ± 0.5) of disease (on day 15). BCP at 50 mg/kg administered twice daily notably blocked the clinical severity of the disease, indicating maximal inhibition of 62% based on the cumulative score ( Figure 6).

BCP Prophylactic Treatment Downregulated CD4+ and CD8+ T Lymphocytes
Since CD4+ Th-induced inflammation is thought to be responsible for the neurodegenerative component of chronic EAE, we asked whether BCP would interfere with CD4+ and CD8+ T cells. The percentage of CD4+ and CD8+ T cells was significantly lower in EAE, moreover, both CD4+ and CD8+ T cells were significantly decreased in BCP prophylactically treated animals ( Figure 7). In order to evaluate activation of T lymphocytes, the co-expression of CD69 cellular marker was measured through the double staining of CD4+CD69+ and CD8+CD69+. In fact, the population of CD4+CD69+ and CD8+CD69+ T lymphocytes were remarkably increased in EAE-untreated animals when assessed 25dpi, when compared with control naive mice. Furthermore, BCP treatment (50 mg/kg, p.o.) inhibited both CD4+ and CD8+ T cells, as well as their activation in the peripheral lymphoid tissue during EAE development (Figure 8).

Isolation of Lymphocytes
The 10-week-old EAE C57BL/6 female mice were euthanized and inguinal lymph nodes were removed. The lymph nodes were lysed to prepare as single-cell suspensions. The cells were washed twice and resuspended in RPMI 1640 with the concentration adjusted to 1 × 10 6 cells/mL total lymphocytes. The lymphocytes were pretreated with a range of concentrations of BCP (1-100 µM) for 1 h followed by stimulation with MOG35-55 (10 µg/mL) for 48 h. The production of immune mediators was determined by ELISA.

Flow Cytometry Assay
Herein, we performed CD4+ and CD8+ T cells quantification by flow cytometry. Inguinal lymph nodes (LN) obtained from each animal group were macerated in RPMI 1640 medium and filtered through a 220 µm filter. The resulting suspension was centrifuged at 1500× g for 7 min, then supernatant was discarded, and the cell pellet was resuspended in RPMI 1640 medium supplemented with 10% fetal bovine serum, 20 mM HEPES, 3 × 10 −5 M 2-mercaptoethanol, 100 U/mL penicillin, and 100 mg/mL streptomycin. The cells were incubated with the following antibodies for 20 min at 4 • C: anti-CD4-PerCP, anti-CD8a-APC, and anti-CD69-PE. The data were collected with FACS Canto II (BD Biosciences, San Jose, CA, USA) and analyzed by means of FlowJo (version 7.5, FlowJo, LLC, Ashland, OR, USA) software.

Histology
To assess the microglial activation, oxidative response and demyelination in the CNS, treated and untreated groups of C57BL/6 mice were euthanized at the chronic stage of EAE disease (day 30) by CO 2 asphyxiation. Their spinal cords of the lumbar region were removed and fixed in 10% buffered formalin. After paraffin embedding, transverse sections of spinal cord of 12 µm-thick (nine sections per animal) were stained with Iba-1, iNOS, and NF-H, respectively. The spinal cord sections from five representative mice (BCP treated and untreated) were processed and stained with Hoechst 33342 dye (for nuclei stain). A Q imaging digital camera connected to an Olympus Bx4 microscope was used. The immunostaining was assessed at four levels of the lumbar spinal cord. Specifically, four alternate 5-µm sections of the lumbar spinal cord with an individual distance of 150 µm were obtained between L4 and L6. A threshold optical density that best discriminated staining from the background was obtained using the NIH ImageJ 1.36b imaging software (NIH, Bethesda, MD, USA). We captured four images of each spinal cord subregion (dorsal, ventral, lateral, central) per section (eight images per section and 32 images per mouse, n = 5 animals/group). Then, the total pixel intensity was determined and the data were expressed as optical density (O.D.).

Mechanical Hyperalgesia
Mice were assessed for mechanical withdrawal thresholds in an individual Plexiglas housing (9 cm × 7 cm × 11 cm) with wire mesh floor, and given time to adjust to the ambiance while exploring and grooming until settling down. A von Frey filament-0.4 g (Stoeling Company, Wood Dale, IL, USA) was handled in an ascending movement in order to pressure the plantar surface of the hind-paw. The bending force of the von Frey filament triggering the withdrawal of the hind-paw was recorded. Hind-paw withdrawal was considered as positive response. Each hind-paw was measured three times and the average values from the three measurements were recorded as the mechanical paw withdrawal threshold. Animals were evaluated until day 10 post-immunization, before onset of motor dysfunctions.

Statistical Analysis
Statistical analysis was performed with the GraphPad Prism 6 software (San Diego, CA, USA) by analysis of variance (ANOVA). Post hoc analyses were executed by the Bonferroni's multiple comparison test or Newman-Keuls test. Results are expressed as mean ± standard error of the mean (SEM); p < 0.05 and p < 0.001 were considered statistically significant.

Conclusions
Altogether, a dietary non-psychoactive CB2 receptor ligand sesquiterpene BCP preventively or therapeutically blocked the development and progression of clinical and neurological signs of EAE, which was correlated with the hindrance of immune cell activation, neuroinflammation, and demyelinating processes in the CNS. Moreover, our findings also demonstrate that the effect of BCP on EAE most likely occurs due to the activation of the CB2 receptor, resulting in a corresponding downregulation of the migration of encephalitogenic T cells into the CNS, which in turn may increase Treg vs. inhibit Th1 polarization and expression. Furthermore, our data establishes that BCP constitutes an alluring therapeutic molecule towards the treatment of MS, along with other autoimmune diseases.