cbd oil for autoimmune encephalitis

Article

Katz D 1,2 , Katz I 1,2 , Shoenfeld Y 1,3*

1 The Zabludowicz Center for Autoimmune Diseases, Chaim Sheba Medical Center, Tel-Hashomer, Israel
2 Faculty of Medicine, The Hebrew University of Jerusalem, Israel
3 Incumbent of the Laura Schwarz-kipp chair for research of autoimmune diseases, Sackler Faculty of Medicine, Tel-Aviv University, Israel

Introduction

The tale of Cannabis sativa is as old as time. Through its first days as an herbal remedy, ranging back to 4000 BC and to Emperor Shen Nung’s Rule (2700 BC), to cannabis low point of being banned internationally at 1925 to its recent re-emergence by prof. Mechoulam isolation of the Tetrahydrocannabinol (THC, 1963), Cannabis is slowly gaining its place in medicine 1,2 .

Cannabis sativa, also known as Marijuana has been called many names, yet the variety of names given to Cannabis does not encompass the vast medical opportunities that lie within the cannabis. As of now, 545 ingredients have been identified, of which over 100 classified as unique to Cannabis 3 . The two main and most researched active ingredients are – Tetrahydrocannabinol (THC) which holds a psychoactive properties and on the other hand, Cannabidiol (CBD) which is considered non psychoactive. The components are joined by the two main known endocannabinoids – Ananamide (AEA) and 2-Arachidonoylglycerol (2-AG) (also discovered by prof. Mechoulam and colleagues) 4,5 . The other half of the cannabinoid system (as we know thus far) comprises of CB1 and CB2 receptors, G-protein coupled receptors. The two receptors differ in distribution and function. While the major psychoactive effect of cannabis is attributed to the CB1 receptor and accordingly widely distributed in neurons, while the CB2 receptor has been linked to maintaining homeostasis and commonly appears in cells of the immune system 6,7 .

Cannabis and the Brain Immune System

It is well established that murine microglial cells express both CB1 and CB2 receptors, yet the pattern of receptors expression differs in location as well as in levels of expression. While CB1 receptor is consistently expressed in microglial cells in low levels, CB2 receptor is indetectable in resting state cells and highly expressed in activated microglia 8,9 . The pattern of expression and distribution of CB2 receptor in microglial cell suggest a role in microglial migration, CB2 receptor was found to be expressed heterogeneously throughout murine microglial cells with particularly high density at the leading edges of lamellipodia and microspkies (cellular protrusions that mediate cell migration). Moreover, 2-AG, AEA and abnormal-cannabidiol increase microglial cell migration 10 .

Another aspect of the endocannabinoid system effect on microglial cell is the attenuation of the immune response induced by LPS (Lipopolysaccharide) stimulation, AEA attenuates the immediate release of IL-6 and NO by microglial cell by induction of MPK-1 11 .

A different mechanism of action is suggested by the inhibition of the IL-1 signaling pathway following administration of the synthetic cannabinoid R(+)WIN 55,212-2. Appling R(+)WIN 55,212-2 to astrocytoma cells priori stimulated by IL-1 resulted in dose dependent inhibition of ICAM-1 and VCAM-1 adhesion molecules induction, as well as IL-8 and NFκB. The effect aforementioned is independent from the cannabinoids receptors CB1 and CB2 as suggested by the lack of regulation of CB1 and CB2 antagonist on the immunomodulating effects mentioned above, implying that there is still much to learn in the field of Cannabis and immunomodulation 12 .

Cannabis and the Blood-Brain-Barrier

The blood-brain-barrier (BBB) as well as the blood-spinal cord-barrier (BSCS) and their disturbance is often postulated as a possible mechanism of pathogenesis in neurological autoimmune disease. A possible link of pathogenesis has been suggested in Multiple Sclerosis 13 , Neuromyelitis Optica1 14 , Guillain-Barré Syndrome 15 , Chronic Inflammatory Demyelinating Polyneuropathy 16 and Antiphospholipid Syndrome with neurological involvement 17 .

In murine model of LPS induced vascular and inflammatory changes CBD counteracts the effect of LPS. Mice which received LPS+CBD showed no cerebral vasodilation, no leukocyte migration, reduced TNF-α and COX-2 levels compared to LPS treated mice and more over exhibited reduced dextran extravasation (dextran extravasation is used as a quantification instrument of BBB integrity) 18 .

Similar effect is obtained by administration of Anandamide to TMEV-infected endothelial brain cell. AEA inhibits VCAM-1 induction in vitro, and thus limit leukocyte migration through a transwell filter (coated with collagen type I and fibronectin) model of the BBB. Accordingly, in vivo experiment correlated the result of the in vitro experiments. AEA increased tone (by UCM-707, an AEA uptake inhibitor) inhibited VCAM-1 induced expression, as well as attenuated microglial cell activation 19 .

A role for CB2 receptor was also exemplified by in vivo murine model. Ex vivo CB2-activted leukocytes were injected to LPS treated mice resulting in adhesion reduction of up to 96% using GP1a (CB2 receptor agonist) in comparison with to non GP1a treated mice 20 .

The beneficial effect of cannabinoid also extends to human brain endothelial cells (BMVEC). Using human cells from HIV-1 CNS infected patients and from seronegative controls, a group of researches demonstrated enhanced CB2 receptor expression in HIV infected cells compared to controls. Further investigation of naive human BMVEC revealed that the increased expression of CB2 receptor can also be accomplished separately by IL-1β, TNF-α and LPS. Once induced and activated, CB2 receptor decreased leukocyte adhesion, prevented up regulation of adhesion molecules, promoted 2.2-2.7 increase in tight junction proteins (occludin and claudin-5) and significantly reduced BBB resistance drop induced by LPS 21 .

The coherence of the above mentioned experiments is also exemplified at the genetic level. Human BMVEC isolated from eleptogenic patients were activated using TNF-α to evaluate consequent gene expression. Out of 33 genes that were up regulated by TNF-α, 31 and 32 genes were suppressed using CB2 agonist O-1966 or JWH-133 respectively 22 .

Cannabinoids protective effect goes beyond the BBB and also extends to the BSCB. Pretreatment by JHW-015, a CB2 receptor agonist prevents down regulation of occludin and ZO-1 induced by spinal cord ischemia reperfusion injury (SCII) in murine in vivo model. Moreover, JWH-015 pretreatment reduces BBB leakage (measured by Evans blue) compared to SCII only group 23 .

Cannabis potential ability to protect BBB integrity is of possible great importance, not only in autoimmune neurologic disorders, but in a vast verity of neurological fields as in Alzheimer’s disease and ischemia injury.

Cannabis and Autoimmune Demyelinating Disease

Multiple Sclerosis (MS) is known as the hallmarks of neurological autoimmune disease with prevalence as high as 200:100,000 in some countries in northern Europe 24 .

MS Patients are characterized by high CSF levels of AEA compared to healthy control. In accordance high levels of AEA were also measured in autoimmune encephalomyelitis (EAE), a murine model of MS. Moreover, increased NAPE-PLD (part of AEA production) activity and reduced FAAH (degrades AEA) activity 25 . CB1 receptor deficient mice exhibit substantial neurodegeneration following EAE induction including higher prevalence of residual paresis and axonal pathology in relation to wild type mice 26 .

CBD treatment of TMEV infected mice induces a wide range of immunomodulatory outcomes. CBD reduce the infiltrate of immune cell to the brain parenchyma and decreased microglial activation. Moreover, CBD treatment has a long lasting effect, an 80 days follow up of the treatment group revealed restoration of both horizontal and vertical motor activities to that of the healthy mice and a correlating reduction in the expression of TNF-α and IL-β1 27 .

MS is positively influenced by a variety of cannabinoids, both natural and synthetic, each demonstrating a different mechanism of action to our knowledge. Among the different cannabinoids we can find Cannabidiol which holds the ability to attenuate a range of neuronal apoptotic pathways 28 , Cannabigerol Quinone which its application on murine neuronal culture results in inhibition of IL-1β, IL-6 and PGE2 release 29 . Also Gp1a, a selective CB2 receptor agonist that modulates EAE development by reducing Th17 differentiation 30 , HU-446 and HU-465 (CBD derivatives) and many more which we won’t elaborate on 31 .

There is scarce evidence regarding clinical use of Cannabis in MS patients. A recent Meta-analysis concluded that cannabinoids (nabilone and nabiximols) were associated with a greater average improvement in spasticity assessed by using numerical rating scale (mean difference, -0.76 [95% CI, -1.38 to -0.14]). Also, the average number of patients who reported an improvement on a global impression of change score was greater using nabiximols rather placebo (OR, 1.44 [95% CI, 1.07-1.94]) 32 . Notably, a new large multi centered blinded study was recently published, in which 489 MS patients participated and received either oral dronabinol (THC) or placebo. The study failed to prove the beneficial outcome of dronabinol use in two main outcomes (time to confirmed EDSS [Extended Disability Status Scale] score progression and change in MSIS-29 [Multiple Sclerosis Impact Scale-29] score). However, while taking into consideration the results of this trail, it is worth mentioning a possible weakness in the trail inclusion criteria. The disease progression in MS as measured by the EDSS scale is not linear, and progression through EDSS 4-5.5 is faster the in EDSS 6-6.6. hus making the EDSS 6+ patient’s population insensitive to treatment during the study period of time, leaving the question of Cannabis medical use in MS patients in need of further research 33,34 . Currently, evidenced based recommendation published in 2014 by the American academy of neurology are: oral cannabis extracts (CBD/THC or CBD alone) are the only products with an A – effective rating, next in line is THC (dronabinol/nabilone) with B rating- probably effective 35 .

Another demyelinating autoimmune disease that shows promise for cannabis treatment is Neuromyelitis Optica (NMO). Plasma levels of 2-AG were found to be elevated in NMO patients compared to healthy patients. Moreover, 2-AG levels were negatively correlated with pain sensitivity, while AEA correlated positively with pain sensitivity 36 .

Multiple Sclerosis and Neuromyelitis Optica are the milestones of medical cannabis implantation in neurologic autoimmune disease, yet only the foundation has been accomplished up to now and further clinical investigation is the core of establishing Cannabis Sativa and its products as a new therapeutic solution.

Cannabis Adverse Effects

Cannabis addiction is one of the main adverse effects of chronic cannabis use, though once considered as only “psychological addiction”, recent evidence revels a physiological ingredient to the addiction 37 . Epidemiological studies indicate that about 9% of adult marijuana users will develop cannabis addiction, while adolescent’s percentages of addiction is as high as 17% 38 .

Another adverse effect of great importance lurking chronic cannabis users is the consequence anatomical changes, a 2013 meta-analysis concluded that chronic cannabis consumption results in reduction of hippocampal grey mater 39 . Accordingly a new research conducted at 2015 demonstrated reduced hippocampus and amygdala volumes 40 .

Acute adverse effects (some may be found beneficial in some indications) include anxiety, dysphoria, psychosis/hallucinations, tachycardia, and stimulation of appetite 39 . Further side effects are listed in table 1.

System Adverse Effect Statistics a References
Neurologic ↓hippocampus & amygdala volumes
↑ Incidence of acute ischemic stroke
Age 15-54
Age 25 -34
Age 45 – 54
Drowsiness
Dizziness
↓Educational performance (adolescents)
Lower IQ
RR 1.13 (1.11-1.15)*
RR 2.26 (2.14 – 2.38)*
RR 1.45 (1.42 – 1.54)*
OR 3.68 (2.24-6.01)*
OR 5.09 (4.10-6.32)*
↓11% (% GCSE† points)*
Linear trend, t test
t: -3.36***
32,40-43
Psychiatric Psychosis Schizophrenia
Anxiety
Depression
OR 1.41 (1.20–1.65)*
OR 1.9 (1.1–3.1)*
OR 1.98 (0.73-5.35)*
OR 1.49 (1.15–1.94)*
32,44,45
Cardiovascular Tachyarrhythmia
Palpitation
Angina
RR 1.5 (1.1–2.1)*
↓ 48% (↓time to, during exercise)**
38,46,47
Pulmonary (cannabis smoking) Chronic Bronchitis symptoms
↑health services for respiratory infections
25%-33% of smokers 46,48
Gastrointestinal Nausea
Diarrhea
Vomiting
Abdominal pain
Constipation
OR 2.08 (1.63-2.65)*
OR 1.65 (1.04-2.62)*
OR 1.67 (1.13-2.47)*
32, 49,50
General Dry mouth OR 3.50 (2.58-4.75)* 38
Cannabis dependence
Withdrawal syndrome
Adults, Adolescents
Anxiety
Insomnia
Appetite disturbance
Depression
Irritability
9%, 17% (percentage of users who will
become addicted)
38
51
Pregnancy Maternal anemia
Decrease birth weight
↑Intensive care unit
pOR 1.36 (1.1 – 1.69)*
pOR 1.77 (1.04 – 3.01)*
pOR 2.02 (1.27 – 3.21)*
52

Table 3: Perfusion analysis from 1H-NMR in 12-month-old female mdx mice.

Reperfusion mean has been calculated during the first 25 minutes after release of ischemia. Wild-type mice, n=6; mdx mice, n=7. **p < 0.01.

(a)The set-up of the tourniquet was sufficient to induce an absence of perfusion in both groups. After release of tourniquet, a rapid and important increase of perfusion was detected and the reperfusion of mdx mice was greater than wild-type mice. A single peak of reperfusion was however observed in mdxmice, when a first rapid and strong peak followed by a second attenuated peak of reperfusion was observed in wild-type animals.
Because the release of ischemia induced movements of the leg, images affected by these movement artifacts, at the moment of ischemia release, were removed from analysis of muscle perfusion.

(b) In the first 5 minutes after ischemic stress release, below a threshold of 250 mL/100 g reperfusion (concerning mostly wt mice), PCr resynthesis rate was dependent on perfusion, an increase in reperfusion leading to a decrease of τPCr. In contrast, above the threshold of 250 mL/100 g reperfusion (concerning mostly mdx mice), PCr resynthesis rate was poorly affected by the increase of post-ischemia reperfusion.

a: Perfusion analysis was performed through from 1H-NMR as described in the Material and Methods section; b: Correlation between reperfusion and PCr resynthesis rate (from 31P-spectroscopy analysis) during the first 300 seconds (5 minutes) after ischemia; n = 6 (wt, a, b); n = 7 (mdx, a, b); τPCr: time of creatine rephosphorylation.

Conclusion

Nowadays Cannabis tends to be considered as a “buzz word”, with global recognition of the potential embodied in medical Cannabis, more and more countries legalize the use of medical cannabis, leaving many physicians overwhelmed due to the rapid changes. In this article we aimed to review the laboratory and clinical evidence regarding Medical Cannabis and neurological autoimmunity diseases.

Unfortunately, lack of clinical data prevents a definitive conclusion. Nonetheless, clinical trials conducted upon MS and NMO patients suggests a future role for medical cannabis in MS and NMO treatment by obtaining relief in patient symptoms. Yet, the trails aforementioned only paves the beginning, much research is yet to be done in order to evaluate the therapeutic effects of cannabis in treating autoimmune neurologic diseases versus.

Unfortunately, lack of clinical data prevents a definitive conclusion. Nonetheless, clinical trials conducted upon MS and NMO patients suggests a future role for medical cannabis in MS and NMO treatment by obtaining relief in patient symptoms. Yet, the trails aforementioned only paves the beginning, much research is yet to be done in order to evaluate the therapeutic effects of cannabis in treating autoimmune neurologic diseases versus.

Another promising aspect is cannabis protective effect on the BBB, having great potential not only in the field of autoimmunity but also in a variety of other pathologies with attributed BBB damage pathogenesis. The field of cannabis immunomodulation and BBB protection is an exciting new medical pathway, but only further research is to say what will be Cannabis sativa place in medical history.

Abbreviations

THC : Tetrahydrocannabiol; CBD : Cannabidiol; AEA : Ananamide; 2-AG : 2-Arachidonoylglcerol; LPS : Lipopolysaccharide; IL-6 : Interleukin 6; NO : Nitric oxide; MPK-1 : Mitogen-activated protein kinase 1; IL-1 : Interleukin 1; ICAM-1 : Intercellular Adhesion Molecule 1; VCAM-1 : vascular cell adhesion molecule 1; IL-8 : Interleukin 8; NFκB : Nuclear factor kappa-light-chain-enhancer of activated B cells; BBB : Blood – brain – barrier; BSCB : Blood – spinal cord – barrier; TNF-α : Tumor necrosis factor α; COX-2 : Cyclooxygenase-2; TMEV : Theiler's Murine Encephalomyelitis Virus.

Cannabidiol Regulates Gene Expression in Encephalitogenic T cells Using Histone Methylation and noncoding RNA during Experimental Autoimmune Encephalomyelitis

Cannabidiol (CBD) has been shown by our laboratory to attenuate experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). In this study, we used microarray and next generation sequencing (NGS)-based approaches to determine whether CBD would alter genome-wide histone modification and gene expression in MOG sensitized lymphocytes. We compared H3K4me3 and H3K27me3 marks in CD4+ T cells from naïve, EAE and CBD treated EAE mice by ChIP-seq. Although the overall methylation level of these two histone marks did not change significantly, the signal intensity and coverage differed in individual genes, suggesting that CBD may modulate gene expression by altering histone methylation. Further analysis showed that these histone methylation signals were differentially enriched in the binding sites of certain transcription factors, such as ZNF143 and FoxA1, suggesting that these transcription factors may play important roles in CBD mediated immune modulation. Using microarray analysis, we found that the expression pattern of many EAE-induced genes was reversed by CBD treatment which was consistent with its effect on attenuating the clinical symptoms of EAE. A unique finding of this study was that the expression of many miRNAs and lncRNAs was dramatically affected by CBD. In summary, this study demonstrates that CBD suppresses inflammation through multiple mechanisms, from histone methylation to miRNA to lncRNA.

Introduction

Marijuana (Cannabis sativa) has many biologically active compounds and its medicinal value has been known for centuries. Two main active ingredients in marijuana are delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD). THC, the psychotropic component, is used for pain relief, stimulating appetite, and as an anti-emetic in cancer and HIV/AIDS patients 1 . CBD is a non-psychoactive compound in marijuana with potential efficacy against epilepsy and autoimmune diseases 2 . THC exerts its function through cannabinoid receptors, CB1 and CB2. CB1 is predominantly expressed in the brain while CB2 is primarily found on immune cells 3 . However, CBD does not bind to or activate cannabinoid receptors directly. In neuronal cells, CBD demonstrates agonist activity on 5-HT1A receptor and regulates 5-HT1A receptor-mediated neurotransmission 4,5 . In immune system, studies from our lab as well as those from others have shown that both THC and CBD have anti-inflammatory properties 2,6,7,8,9 . CBD has been shown to attenuate many autoimmune diseases including experimental autoimmune encephalomyelitis (EAE), autoimmune hepatitis, colitis and collagen-induced arthritis 6,8,10,11 . CBD may exert some of its effect through several receptors, including vanilloid receptor 1 (TRPV1), G-coupled receptor 55 (GPR55), and 5-HT1A receptors depending on the disease model.

EAE is an animal model of multiple sclerosis (MS). MS is a neurodegenerative autoimmune disease. The disease often begins with inflammatory attacks against the white matter of the brain, producing neurological disorders including loss of sensation, lack of coordination, bowel and bladder incontinence, difficulty in walking and paralysis 12 . EAE partially mimics the immunopathological process of MS by immunizing mice with myelin-derived antigens such as myelin basic protein (MBP), proteolipid protein (PLP), myelin associated glycoprotein (MAG) and myelin oligodendrocyte glycoprotein (MOG) 13 . These immunodominant antigenic epitopes trigger acute and chronic autoimmune disease. It has been shown that in EAE models and MS patients, naïve CD4+ T cells can differentiate into pro-inflammatory Th1 and Th17 cells which cross blood-brain barrier and contribute to neuro inflammation 14,15,16 . On the other hand, FoxP3 + regulatory T cells (Tregs) play a critical role in the downregulation of EAE 17 . In addition, previous studies from our lab have demonstrated that CBD can also attenuate clinical symptoms of EAE by inducing immunosuppressive Myeloid-derived Suppressor Cells (MDSC) 6 .

Although THC and CBD have shown anti-inflammatory property in various disease models, the underlying mechanism is not clear. Our studies have suggested the THC and CBD may modulate histone modification and miRNA expression in immune cells. For example, THC regulates gene expression in lymphocytes by altering histone methylation and acetylation 18,19 . In this study, we investigated whether activation of T cells with MOG antigen would alter gene expression and histone methylation leading to differentiation of T cells into proinflammatory phenotype and whether CBD treatment would reverse these effects. To that end, we used microarray and next generation sequencing (NGS)-based approaches to determine whether CBD could alter histone methylation (H3K4me3 and H3K27me3), and expression of non-coding RNA (miRNA and long non coding RNA (lncRNA)) in CD4+ T lymphocytes from naïve, MOG + vehicle and MOG + CBD treated mice. Our results suggested that histone methylation as well as non coding RNAs may play important roles in inflammatory T cell development and that CBD-mediated immune modulation may result from restoration of such alterations.

Results

Effect of CBD on genome-wide histone H3K4me3 and H3K27me3 methylation in splenic CD4+ T cells

MOG-induced EAE is a well-established mouse model of MS. Recently, we demonstrated that CBD was highly effective in attenuating EAE 6 . To determine whether CBD would affect histone modification in CBD-mediated regulation of T cell functions in this model, we examined two histone marks, H3K4me3 and H3K27me3. While many types of histone methylation have been shown to regulate gene expression, these two histone marks are the most well studied ones. H3K4me3 in the promoter region is associated with transcription activation and H3K27me3 is associated with transcription repression. The global histone methylation profile was examined by ChIP-seq in splenic CD4+ T cells from naïve, MOG + vehicle and MOG + CBD treated mice. The overall genome-wide H3K4me3 and H3K27me3 levels did not differ significantly among those 3 groups (Fig. 1a). However, the intensity and/or coverage of these histone marks in certain genomic regions was significantly altered by CBD in EAE mice. For example, the histone marks differed significantly in the genes of Th2 related cytokines such as IL-4, IL-5 and IL13 (Fig. 1b). Both marks showed lower levels of coverage in naïve cells when compared to MOG-activated cells, suggesting that histone methylation might be important for the expression of these cytokines in MOG activated T cells. Compared to vehicle treatment, CBD treatment led to a lower coverage of H3K27me3 but a higher level coverage of H3K4me3 in these genes, suggesting that CBD might cause an increase in the expression of these anti-inflammatory cytokines. The expression of IL-4, IL-5 and IL-13 in those 3 groups was further determined by real time PCR (Fig. 1c). When T cells were activated by MOG, the expression of IL-5 and IL-13 was significantly increased. CBD treatment tended to further increase their expressions although the difference was only significant for IL-4. This result indicated that besides H3K4me3 and H3K27me3, other histone/DNA modifications could also play important roles in the expression of these cytokines in EAE.

Histone methyaltion in splenic CD4+ T cells. CD4+ T cells from spleens of naïve, MOG + vehicle and MOG + CBD treated mice were isolated as described in the Materials and Methods. Histone methyaltion was determined by ChIP-seq. (a) Genome-wide histone methyaltion marks, H3K4me3 (outward histograms) and H3K27me3 (inward histograms) are presented using Circos plot. Blue: Naïve; Green: MOG + vehicle; Red: MOG + CBD. (b) H3K4me3 and H3K27me3 methylation levels in the genomic region of IL-4, IL-13 and IL-5. (c) Relavite expression level of IL-4, IL-13 and IL-5 determined by real time qPCR (The level in the naïve mice was set as 1).

Because many transcription factors are involved in immune regulation, we further determined whether these 2 histone marks were differentially enriched in certain transcription factor binding motifs. By performing signal enrichment assay, we identified top 10 transcription factor binding motifs with enriched H3K4me3 or H3K27me3 signal after T cell activation by MOG (Fig. 2a,c). We also identified signal enriched motifs in CBD treated vs. vehicle treated cells (Fig. 2b,d). Among them were binding motifs of ZNF143, c-Myc, RUN family and FoxA1. ZNF143 and RUN family members are known to regulate DNA replication and cell cycle 20,21 , while FoxA1 is the factor for the development of a subset of Treg population 22 . Since histone modification affects transcription factor accessibility, the results suggest that genes whose expression is regulated by these transcription factors may be important for MOG-induced inflammation as well as CBD-induced anti-inflammation.

Enriched motifs with H3K4me3 and H3K27me3 marks in T cells. CD4+ T cells from spleens of naïve, MOG + vehicle and MOG + CBD treated mice were isolated as described in the Fig. 1 legend. Enrichment of motifs was identifed by HOMER sofwar using ChIP-seq data. Top 10 known transcription binding motifs are presented here. (a) Enriched motifs with H3K4me3 mark in MOG + vehich sample compared to Naïve sample; (b) Enriched motifs with H3K4me3 mark in MOG-sensitized T cells EAE mice treated with CBD compared to vehicle treatment; (c) Enriched motifs with H3K27me3 mark in MOG-sensitized T cells from EAE mice compared to Naïve; (d) Enriched motifs with H3K27me3 mark in MOG-sensitized T cells from EAE mice treated with CBD compared to vehicle treatment.

CBD-mediated alteration in miRNA expression

Inasmuch as miRNAs are known to play important role in the regulation of immune response, we compared the expression of mature miRNAs in splenic CD4+ T cells from naïve, MOG + vehicle and MOG + CBD groups by microarray. Among 1910 total mature miRNAs, 232 were significantly induced in MOG-activated T cells when compared to naïve T cells. Within those 232 miRNAs, 74 of them were suppressed after CBD treatment. On the other hand, MOG-treatment led to decreased expression of 118 miRNAs compared to naïve and only 23 of them whose expression was reversed by CBD treatment (see Supplementary Data). Figure 3 is the heat map of miRNA expression in these 3 groups.

Expression of microRNA in splenic CD4+ T cells. CD4+ T cells from spleens of naïve, MOG + vehicle and MOG + CBD treated mice were isolated as described in the Fig. 1 legend. Expression of mature miRNAs in splenic CD4+ T cells from naïve mice and MOG-induced EAE mice treated with vehicle (MOG + Veh) or CBD (MOG + CBD) was determined by microarray. Linear fold change > 1.5 was considered as significant. (a) Number of MOG induced miRNAs (topper panel) and suppressed miRNAs (lower panel). CBD suppressed the expression of 74 MOG-induced miRNAs, and induced the expression of 23 MOG-suppressed miRNAs. (b) Heat map showing the expression levels of 1910 miRNAs in 3 groups of sample.

CBD-mediated alteration in transcriptome expression

We also used microarray to compare the transcriptome expression profile in CD4+ T cells isolated from these 3 groups of mice. MOG stimulation changed the expression of many transcripts. Among those altered transcripts, the expression pattern of 1272 transcripts was reversed by CBD treatment. CBD suppressed 876 MOG-induced transcripts, and induced 396 MOG-suppressed transcripts. Interestingly, majority of those altered transcripts were non-coding transcripts. CBD reversed the expression of 556 MOG altered protein coding transcripts and 716 non-coding transcripts (Fig. 4) (see Supplementary Data). Pathway analysis revealed that most altered protein coding transcripts were involved in cell cycle and immune response, which is consistent with the results that MOG increases while CBD inhibits T cell proliferation. The immune related genes as well as their cellular locations are presented in Fig. 5. We also paired altered miRNA from miRNA array with their potential targets from transcriptome array through the use of IPA (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis) 23 . Because miRNA usually suppress the expression of their targets, we only paired the protein coding genes and miRNAs that had opposite expression patterns after CBD treatment (Fig. 6).

Transcriptome expression in splenic CD4+ T cells. CD4+ T cells from spleens of naïve, MOG + vehicle and MOG + CBD treated mice were isolated as described in the Fig. 1 legend. Gene expression in in splenic CD4+ T cells from maive, MOG + vehicle and MOG + CBD mice was determined by transcriptome microarray. (a) Number of protein coding and non-coding transcripts whose expression patterns were reversed by CBD treatment in EAE mice. (b) Heat map showing the expresseion levels of total transcripts as well as protein coding and non-coding transcripts.

CBD regulated expression of immune related genes in splenic CD4+ T cells in EAE mice. Immune related genes whose expression pattern was reversed by CBD treatment in MOG-induced EAE mice are presented. Genes shown in green were induced in EAE mice (compared to naïve) but their expression was decreased by CBD treatment in EAE mice (compared to vehicle treatment). Genes shown in red were suppressed in EAE mice (compared to naïve) but their expressions were induced by CBD treatment in EAE mice (compared to vehicle treatment). The celluar localization of these gene products is also shown.

Expression pairing of differentially expressed genes and miRNAs in splenic CD4+ T cells in CBD treated EAE mice. Genes and miRNAs whose expression was significantly altered in MOG-induced EAE mice compared to naïve mice were used for expression pairing b IPA analysis (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuitypathway-analysis). Those induced by CBD treatment in EAE mice are shown red and those suppressed by CBD are shown green.

CBD mediated change in long non-coding RNA expression

Transcriptome array results showed that CBD altered the expression of many lncRNAs in splenic T cells in MOG-induced EAE mice. However, most of these lncRNAs are predicted ones and have not been validated experimentally. We selected some validated lncRNAs (AW112010 (AW), Mirt2, Neat1, 170071M16Rik (M16), 201011I01Rik (I01), 4931406H21Rik (H21), 6330407A03Rik (A03) and 9310230L23Rik (L23)), and their expressions in CD4+ T cells after MOG + vehicle and MOG + CBD treatment, were further confirmed by real time PCR (Fig. 7a). Although the functions of those lncRNAs are not known at the present time, the results indicated that those lncRNAs might play an important role in MOG-induced EAE development as well as CBD-mediated immune modulation. To determine whether their expressions correlated with histone/DNA modifications, we examined H3K4me3, H3K27me3 and 5mC in their genomic regions. The result suggested that the activation mark H3K4me3 was important for the expression of lncRNA A03, H21, M16 and Mirt2 (Fig. 7b). The suppressive marks might play an important role in the expression of Neat1, because in naïve T cells which had a low expression level, showed increased signals of H3K27me3 in its promoter and 5mC in its promoter CpG island (Fig. 7c). Overall, the patterns of histone H3K4me3 and H3K27me3 methylation as well as 5mC were consistent with the expression patterns of these lncRNAs.

Expression and histone/DNA methylation of lncRNAs in splenic CD4+ T cells. The exprsession of select lncRNA in splenic CD4+ T cells was determined by real time PCR. The amount in cells from naïve mice was set as 1. (a) Relative exprssion level of lncRNAs in MOG-induced EAE mice treated with vehicle (MOG + veh) or CBD (MOG + CBD). (b) Histone H3K4me3 methylation in the selected lncRNA genes as determined by ChIP-seq. (c) Histone H3K27me3 methylation and 5mC in Neat1 gene.

Discussion

CBD has been shown to have an anti-inflammatory effect in several animal models. While our previous studies demonstrated that CBD can induce MDSCs that are highly immunosuppressive and which upon adoptive transfer can attenuate EAE, the direct effect of CBD on T cells was not investigated. In this study, we examined the effects of CBD on global histone H3K4me3 and H3K27me3 methylation and gene expression in splenic CD4+ T cells from EAE mice. Despite the fact that MOG treatment dramatically increases transcription activity in T cells, the overall histone methylation levels do not differ significantly when compared to those in T cells from naïve mice. These results are similar to those from our previous study regarding the effect of THC on histone methylation in SEB activated lymphocytes 19 . These results indicate that MOG stimulation and CBD treatment may not alter global histone methylation/de-methylation activity. However, signal enrichment assay indicates that serval transcription binding motifs are differentially associated with histone methylation marks after CBD treatment in EAE mice. Among them is ZNF143 binding motifs. ZNF143 is a zinc finger protein which is critical for chromatin interaction at promoter area to regulate gene expression 24 . ZNF143 binds to promoters and is required for cell type specific chromatin interactions connecting promoters and regulatory elements 24 . Histone methylation such as H3K4me1 and H3K27ac in the ZNF143 binding sites has been linked to transcription activation 24 . In this study, we have found that ZNF143 binding motif is the top enriched motif associated H3K4me3 mark after MOG treatment, which suggests an enhanced activity of ZNF143 mediated transcription. Interestingly, ZNF143 binding motif is also the top enriched motif associated with the suppressive mark, H3K27me3, after CBD treatment in MOG stimulated cells, suggesting that both active and suppressive histone marks can modulate ZNF143 binding, which in turn, regulate gene expression.

FoxA1 binding motif is the top enriched motif with H3K4me3 mark after CBD treatment compared to the vehicle treatment in MOG stimulated cells. FoxA1 is a member of fork head family transcription factors and regulates the lineage-specific transcription 25 . It has been shown that FoxA1 binds to distinct regions within the genome with specific epigenetic modifications. FoxA1 binding sites have significant enrichment for transcription active marks, H3K4me1 and me2 25 . Our results suggest that CBD can also lead to the enrichment of H3K4me3 in the FoxA1 binding motif. Although FoxP3-expressing Treg cells have been shown to control inflammation in EAE model, studies have also suggested that other immunosuppressive T cells, such as FoxA1+ Treg cells, are involved in EAE and MS 22,26,27,28 . FoxA1+ Treg cells have been identified as IFN-β induced, linage-specific immunosuppressive cells, and IFN-β deletion leads to augmented symptoms of EAE 29 .

In our previous studies, we used RNA-seq to examine effect of cannabinoids such as THC on pri/pre-miRNA expression in SEB challenged lymphocytes 18 . Because the expression profile of precursor miRNAs may not correlate with their mature forms, we used microarray to determine mature miRNA expression in this study. Among those identified miRNA, some are known to regulate immune response. For example, miR-155 has been shown by us as well as by others to have pro-inflammatory property 30 . Some miRNAs such as miR-19, -210 and -223 that are up- regulated in T cells in MOG-induced EAE mice are also up-regulated in T cells from patients with MS 31 . On the other hand, few miRNAs such as miR-181c are down-regulated in EAE compared to naïve. miR-181c is also down-regulated in MS patients 31 . Furthermore, decreased expression of miR-181c in peripheral blood mononuclear cells from patients with myasthenia gravis correlates with elevated serum IL-7 and IL-17 32 . Knockdown of miR-181 has also been shown to enhance LPS-induced production of pro-inflammatory cytokines 33 , suggesting that miR-181c has an anti-inflammatory property. More importantly, the expression patterns of these miRNAs in EAE are reversed by CBD, indicating that modulating the expression of miRNA may constitute one of the mechanisms by which CBD exerts its anti-inflammatory roles in EAE and MS.

Long non coding RNA is another layer of gene expression regulation. We have found that many lncRNAs are induced by MOG treatment. However, the only lncRNA with known function is Neat1 (Nuclear-Enriched Autosomal Transcript 1). Neat1 is essential for the formation of paraspeckes in the nucleus and is a potent regulator in cell proliferation and differentiation 34,35 . Several studies have shown that aberrant expression of Neat1 is associated with various types of cancer, and its overexpression correlates with poor prognosis 36,37 . Overall, Neat1 seems to promote cell proliferation which is consistent with our results that MOG significantly induces Neat1 expression in T cells. Neat1 has also been shown to have a pro-inflammatory property. Neat1 expression is increased in Systemic Lupus Erythematosus and knockdown of Neat1 reduces cytokines such as IL-6 and CXCL10 38 . The expression of Neat1 is also induced by certain viral infections and facilitates the expression of antiviral cytokines such as IL-8 39 . Another MOG-induced lncRNA which is suppressed by CBD is Mirt2 (myocardial infarction-associated transcript 2). It is one of 2 lncRNAs that is up regulated in mice with myocardial infarction 40 . Further study has shown that Mirt2 may be involved in left ventricular remodeling 40 . A recent study shows that the expression of Mirt2 in macrophages is induced by LPS 41 . Furthermore, Mirt2 inhibits the activation of NF-kB and MAPK pathways to limit the expression of proinflammatory cytokines and thus it may serve as a negative regulator of inflammation 41 . Another lncRNA, AW112010, was initially identified as a transcript that might encode a small peptide but was classified as a non-coding RNA 42 . The role of this lncRNA is not known. One study shows that AW112010 is induced in microglia and astrocyte in CNS after virus infection, suggesting it may be involved in inflammation 43 .

In summary, the current study demonstrates that CBD modulates immune response through various mechanisms, from histone and DNA methylation to miRNA and lncRNA expression. The main purpose of this study is to identify potential regulatory elements in CBD-mediated immune modulation. Many candidates identified in this study have no known function and thus the current study provides new avenues to investigate their roles in regulating inflammation.

Materials and Methods

Induction of EAE and Cell Isolation

EAE was induced in female C57BL/6 J mice (8–10 weeks old) by subcutaneous injection of MOG35–55 (NeoMPS, San Diego, CA), as described 6,44,45 . Each mouse received 150 μg of MOG in 100 μl of complete Freund’s adjuvant with 100 μg of inactivated Mycobacterium tuberculosis. Mice also received 200 ng of pertussis toxin (Sigma) in 100 μl of PBS by intraperitoneal injection. Pertussis toxin was injected again 2 days after MOG injection. After MOG injection, mice received CBD (10 mg/kg of body weight) or the vehicle daily by intraperitoneal injection. All animal experiments were carried out in accordance with NIH guideline and approved by University of South Carolina IACUC (protocol #2363; approval data: May 31, 2017). After 7 days, spleens from the naïve control, MOG + vehicle and MOG + CBD (4–7 mice in each group) were collected and single cell suspension was prepared. CD4+ T cells were isolated using EasySep selection kit (StemCell Technologies, Vancouver, BC) according to the provided instructions. The antibody used was FITC-anti-mouse CD4 (BioLegend, San Diego, CA). Isolated cells were cultured in complete RPMI1640 medium. For the MOG + vehicle group, 30 μg/ml of MOG was added to the medium. For the MOG + CBD group, 30 μg/ml of MOG and 10 μM of CBD were added to the medium. Cells were harvested after 48 hr of culturing.

ChIP-seq

ChIP-seq was performed as described previously 19,46 . Briefly, crosslinked chromatin was fragmented by Micrococcal nuclease digestion and immunoprecipitated with anti-H3K4me3 or anti-H3K27me3 antibodies (Abcam, Cambridge, MA). After ChIP, the crosslinking was reversed and DNA fragments were purified. The sequencing library was constructed using Illumina’s TruSeq sample preparation kit according to the provided instruction. The samples before immunoprecipitation served as input controls. Sequencing was performed using Illumina Nextseq 500 sequencer.

miRNA and transcriptome microarray

RNA was purified by RNeasy and miRNeasy kit (Qiagen,). For miRNA quantification by Affymetrix GeneChip miRNA array, mature miRNAs were labeled by FlashTag Biotin HSR RNA labeling kit. For transcriptome expression analysis by GeneChip Whole Transcript Expression Array, samples were amplified and labeled using GeneChip WT PLUS reagent kit. Microarray hybridization, wash and staining were performed using the Fluidics Station 450. All procedures were carried out according to the protocols provided by Affymetrix. Data were analyzed by Affymetrix Expression Console software. Fold changes >1.5 were considered as significant.

Quantitative Real-time PCR

Total RNA including miRNA was reverse transcribed by miScript II RT kit (Qiagen). mRNA expression was measured by SYBR Green PCR kit and mature miRNA was quantified by miScript Primer Assays (Qiagen). Snord96a and Gapdh were used as the internal controls for miRNA and mRNA, respectively. The amount in the naïve CD4+ T cells was set as 1.

Data analysis

ChIP-seq data were analyzed as described previously 19,46 . The signals were visualized in Integrated Genome Browser (www.bioviz.org) using mouse mm9 genome build as the reference. The genome-wide histone methylation map was generated by the Circos plot 47 . The signal enriched motifs were identified using HOMER motif analysis software 48 . The heatmaps were generated by the R program. Ingenuity Pathway Analysis (Qiagen) was used for functional analysis and pairing of miRNA and mRNA. Statistical analysis was performed using T test (For animal experiments, each group had 4 to 7 mice. For qPCR, n = 3). Significance was determined as p < 0.05 (denoted by *).

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Acknowledgements

This study was supported by NIH grants R01ES019313, R01MH094755, R01AI123947, R01 AI129788, P01 AT003961, P20 GM103641, R01 AT006888 awarded to MN and PSN.

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Department of Pathology, Microbiology and Immunology, School of Medicine, University of South Carolina Columbia, South Carolina, 29209, USA

Xiaoming Yang, Marpe Bam, Prakash S. Nagarkatti & Mitzi Nagarkatti

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X.Y., M.B., P.S.N. and M.N. designed study, analyzed data and edited the manuscript. X.Y. and M.B. performed experiments. Final manuscript was reviewed and approved by all the authors.

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Yang, X., Bam, M., Nagarkatti, P.S. et al. Cannabidiol Regulates Gene Expression in Encephalitogenic T cells Using Histone Methylation and noncoding RNA during Experimental Autoimmune Encephalomyelitis. Sci Rep 9, 15780 (2019). https://doi.org/10.1038/s41598-019-52362-8

Received : 31 May 2019

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Effects of Orally Administered Cannabidiol on Neuroinflammation and Intestinal Inflammation in the Attenuation of Experimental Autoimmune Encephalomyelitis

  • Nicholas Dopkins
  • Kathryn Miranda
  • Mitzi Nagarkatti

Journal of Neuroimmune Pharmacology (2021)

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Cannabidiol Attenuates Experimental Autoimmune Encephalomyelitis Model of Multiple Sclerosis Through Induction of Myeloid-Derived Suppressor Cells

Multiple sclerosis (MS) is a chronic debilitating autoimmune disease without a cure. While the use of marijuana cannabinoids for MS has recently been approved in some countries, the precise mechanism of action leading to attenuate neuroinflammation is not clear. We used experimental autoimmune encephalomyelitis (EAE), a murine model of MS, to explore the anti-inflammatory properties of cannabidiol (CBD), a non-psychoactive cannabinoid. Treatment with CBD caused attenuation of EAE disease paradigms as indicated by a significant reduction in clinical scores of paralysis, decreased T cell infiltration in the central nervous system, and reduced levels of IL-17 and IFNγ. Interestingly, CBD treatment led to a profound increase in myeloid-derived suppressor cells (MDSCs) in EAE mice when compared to the vehicle-treated EAE controls. These MDSCs caused robust inhibition of MOG-induced proliferation of T cells in vitro. Moreover, adoptive transfer of CBD-induced MDSCs ameliorated EAE while MDSC depletion reversed the beneficial effects of CBD treatment, thereby conclusively demonstrating that MDSCs played a crucial role in CBD-mediated attenuation of EAE. Together, these studies demonstrate for the first time that CBD treatment may ameliorate EAE through induction of immunosuppressive MDSCs.

Introduction

Multiple sclerosis (MS) is a chronic autoimmune disease in which inflammatory lesions cause damage to the myelin sheath coating the nerve fibers in the central nervous system (CNS) leading to symptoms ranging from numbness in a limb to paralysis (1). While the exact etiology of MS is unknown, studies have identified that myelin antigen-specific Th1 and Th17 cells from the periphery cross the blood–brain barrier and trigger neuroinflammation ultimately leading to destruction of myelinated neuronal cells and producing paralysis (2–9). The debilitating consequences of MS, and the current lack of effective treatment modalities, have warranted continued research into therapeutic interventions of the disease.

Marijuana cannabinoids have been shown to exhibit potent anti-inflammatory properties and have been shown to be effective in the treatment of a number of autoimmune diseases, including MS (10–12). In addition, cannabidiol (CBD), the major non-psychoactive cannabinoid component of marijuana, has also been shown to exert the neuroprotective effects (13, 14). Such studies have led to the introduction of drugs such as Sativex, which consists primarily of THC and CBD, to alleviate neuropathic pain and spasticity against MS. It is likely that ability of CBD to reduce neuropathic pain and spasticity may be independent of its anti-inflammatory effects. In addition, it is critical to identify the mechanisms through which CBD suppresses neuroinflammation in MS.

While it is well established that experimental autoimmune encephalomyelitis (EAE) pathogenesis is regulated by Tregs, recent studies have suggested that myeloid-derived suppressor cells (MDSCs) may also play a critical role in suppressing neuroinflammation (15). MDSCs are suppressor cells of myeloid lineage that were originally identified in tumor-bearing patients and models of cancer (16). In cancer, MDSCs are believed to drive T cell dysfunction resulting in promotion of tumor growth and metastasis (17–19). More recently, MDSCs have also been shown to be induced at sites of inflammation (20–22), thereby suggesting that they may play a regulatory role to temper down the inflammatory response (10, 12, 23). In autoimmune disease, MDSCs serve as attractive targets for suppressing autoreactive T cell activation and function (24). Interestingly, recent studies from our lab demonstrated that cannabinoids, including CBD, when administered into mice, induce massive numbers of MDSCs that are highly immunosuppressive (10, 25). Given that the mechanistic role of CBD-mediated neuroprotection in MS is poorly understood, we investigated whether CBD conferred a suppressed inflammatory response by triggering increased MDSCs and consequent T cell suppression.

Using EAE as a model of MS, we determined the effect of treatment with CBD on neuroinflammation, specifically focusing on the role of MDSCs. We found that CBD attenuated disease progression primarily via induction of MDSCs inasmuch as depletion of MDSCs could partially reverse disease mitigation, and adoptive transfer of CBD-induced MDSCs into naïve mice protected them from developing EAE.

Materials and Methods

Animal Use and Care

Female C57BL/6 mice were purchased from the National Institutes of Health (NIH) (Bethesda, MD, USA). All animals were housed in the University of South Carolina Animal Facility (Columbia, SC, USA). All animal procedures were performed according to the NIH guidelines under protocols approved by the Institute of Animal Care and Use Committee of the University of South Carolina.

Reagents

The reagents used in this study were purchased as described: CBD (NIH, Bethesda, MD, USA), myelin oligodendrocyte glycoprotein (MOG35–55) peptide, H-MEVGWYRSPFSRVVHLYRNGK-OH (PolyPeptide Laboratories, San Diego, CA, USA), RBC lysis buffer, propidium iodide, hematoxylin and eosin (Sigma-Aldrich, St. Louis, MO, USA), RPMI 1640, l -glutamine, HEPES, phosphate-buffered saline (PBS), and fetal bovine serum (VWR, West Chester, PA, USA), Percoll (GE Healthcare Life Sciences, Pittsburgh, PA, USA).

Induction of EAE and CBD Treatment Regimen

Experimental autoimmune encephalomyelitis was induced in groups of 10 female C57BL/6 mice (6–8 weeks old) as described previously (23, 26, 27). Briefly, we injected 100 µL of 150 µg MOG35–55 peptide emulsified in complete Freund’s adjuvant (Difco, Detroit, MI, USA) containing 4 mg/mL killed Mycobacterium tuberculosis (strain H37Ra; Difco), subcutaneously. Following immunization, 200 ng of pertussis toxin (List Labs, Campbell, CA, USA) was injected i.p. into mice on day 0, followed by a 400 ng pertussis toxin intraperitoneally (i.p.) injection on day 2. CBD (20 mg/kg; 16% DMSO:PBS) was administered daily starting at day 9 through day 25 by i.p. route. EAE mice treated with vehicle were depicted as EAE-VEH and those that received CBD as EAE-CBD.

Clinical scores (0, no clinical signs; 1, limp tail; 2, partial paralysis of hind limbs; 3, complete paralysis of hind limbs or partial hind and front limb paralysis; 4, tetraparalysis; 5, moribund; 6, death) were recorded on a daily basis. The mean score was calculated for each group every day. Each experiment was repeated at least twice with consistent results.

Studies Using MDSCs

Myeloid-derived suppressor cells were isolated from the peritoneal cavity of mice injected with CBD, as described (28) and 4 × 10 6 cells were injected i.p. for adoptive transfer. Splenocytes from naïve mice served as controls. To deplete MDSCs in vivo, we used anti-Gr-1 Abs (RB6-8C5) or isotype control Ab given 3 h after CBD injection at 0.1 mg every 48 h.

Cytokine Detection in Serum and Ex Vivo Splenocytes Cultures

Experimental autoimmune encephalomyelitis mice were bled on day 16 after MOG35–55 immunization and serum was separated. Also, supernatants from cultures of splenocytes activated in vitro with MOG were collected after the 72 h culture. Cytokine levels for IFNγ, IL-10, IL-17, and TNFα were determined for serum and culture supernatants. All cytokines were measured using BioLegend ELISA Max kits (San Diego, CA, USA), as described in Busbee et al. (29).

Staining Cells With Antibodies and Use of Flow Cytometry

Cells were stained with fluorescent conjugated antibodies and analyzed using the Beckman Coulter FC500 (Indianapolis, IN, USA) to determine phenotypes of infiltrating cells in the CNS. Antibodies used: fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD4 (L3T4) (clone GK1.5; rat IgG2b), FITC-conjugated anti-mouse Ly-6G/Ly-6C (Gr-1) (clone RB6-8C5; Rat IgG2b), Phycoerythrin (PE)-conjugated anti-mouse/human CD11b (clone M1/70; Rat IgG2b), Allophycocyanin anti-mouse CD8 (Ly-2) (clone 53-6.7; rat IgG2a), and PE anti-mouse CD3ε (clone 145-2C11; hamster IgG).

Cell Culture

Cell cultures were maintained in complete RPMI 1640 media supplemented with 10% heat-inactivated fetal bovine serum, 10 mM HEPES, 10 mM l -glutamine, 50 µM β-mercaptoethanol, and 100 µg/mL penicillin/streptomycin at 37°C and 5% CO2.

Ex Vivo MOG35–55 Restimulation

Splenocytes from naïve, EAE-VEH, or EAE-CBD mice were isolated 16 days after immunization and cultured in a 96-well plate in the presence of 30 µg/mL MOG35–55 for 3 days. Supernatants were collected for cytokine analysis. Prior to harvest, splenocytes were stimulated with ionomycin, phorbol myristate acetate, Golgi-Plug for 4–6 h using Leukocyte Activation Cocktail (BD Biosciences).

Isolation of CNS Infiltrating Cells

Experimental autoimmune encephalomyelitis-induced mice were given vehicle, or CBD as indicated earlier. On day 16, blood was collected and serum was isolated for cytokine/chemokine analysis. Spleen and inguinal lymph nodes were excised prior to perfusion. Mice were then perfused with 10 mL heparinized PBS, and whole brain and spinal cord tissue were isolated. Tissues were homogenized separately into a single-cell suspension and subjected to red blood cell lysis. Mononuclear cells from whole brain and spinal cord homogenates were isolated using 33% Percoll, as described in Rouse et al. (27). Cells were counted and stained with fluorescently tagged Abs as indicated. Absolute cell count was calculated using the following equation: Total cells bearing a specific maker = Percentage of cells with the marker as analyzed by flow cytometry × absolute number of cells/100.

Isolation of MDSCs

Sixteen hours after CBD injection, mice were euthanized, and the peritoneal exudate was collected. In brief, the peritoneal cavity was washed three times with ice-cold 1× PBS (5 ml/wash) for 5 min with agitation to recover cells. The cells were resuspended in 1 mL, treated with Fc block for 10 min, and labeled with PE-conjugated anti-Gr1. The EasySep-positive PE selection kit (STEMCALL Technologies, Vancouver, BC, Canada) procedure was followed to isolate Gr1 + cells, as described previously. After isolation, cells were labeled with FITC-conjugated anti-CD11b and assessed for purity using flow-cytometric analysis (12, 30). We have indicated the use of purified Gr1 + CD11b + MDSCs in such instances in which the cells have been enriched via positive selection.

Statistics

Statistical analysis was performed using GraphPad Prism 5.0 (San Diego, CA, USA). For EAE experiments, we used groups of 10 mice and figures are representative of at least two independent experiments. The clinical scores of EAE mice, at various time points, were compared between various groups using Mann–Whitney test, as described (27). In in vitro experiments, the data used in each figure represent mean ± SEM of at least three experiments. Statistical difference was calculated using ANOVA and Student’s t-test, and where stated post hoc analysis was performed via Tukey’s method. A value of <0.05 was considered statistically significant.

Results

CBD Attenuates EAE

To study the effect of CBD on EAE, we induced EAE using MOG as an antigen and treated the mice with vehicle (EAE-VEH) or CBD (EAE-CBD) as shown in Figure ​ Figure1A. 1 A. CBD (20 mg/kg) treatment of EAE mice was started at the onset of clinical signs (day 9) and given every day until end of study (day 25) (Figure ​ (Figure1A). 1 A). EAE-VEH mice progressively developed EAE disease as seen from clinical scoring (Figure ​ (Figure1B) 1 B) with 100% incidence and maximum mean score of 4.1 ± 0.17. Treatment with CBD delayed the onset of disease and significantly attenuated clinical signs of EAE (maximum score of 2.2 ± 0.16) (Figure ​ (Figure1B). 1 B). Moreover, the significant increase in mononuclear cell infiltrates seen in the spinal cords and brains of EAE-VEH mice was attenuated upon CBD treatment (Figure ​ (Figure1C). 1 C). In addition, CBD treatment significantly reduced the elevated CD3 + CD4 + and CD3 + CD8 + cell numbers seen in the CNS of EAE-VEH mice (Figure ​ (Figure1D). 1 D). Serum collected from EAE-VEH mice displayed elevated levels of IFNγ and IL-17 while CBD treatment significantly reduced these inflammatory cytokine levels (Figure ​ (Figure1 1 E).

Effect of cannabidiol (CBD) treatment on the development of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. (A) Time line schematic of studies. CBD treatment was administered at time points with gray arrows. Data were assessed at peak of disease unless otherwise stated. (B) Clinical scores (n = 10 mice per group); data were presented as mean ± SEM and analyzed for significance using Mann–Whitney U test. Comparisons were considered significant at p ≤ 0.05, denoted as *. Data are representative of at least two independent experiments; in each experiment, disease incidence was 100% for each group. (C) Total mononuclear cell infiltrates in central nervous system. (D) Absolute cell counts for CD3 + CD4 + T cells and CD3 + CD8 + T cells; mononuclear cells stained with corresponding Abs and then enumerated using total cell count and frequency from flow cytometry. (E) Serum expression level of pro-inflammatory cytokines IL-17 and IFNγ analyzed by ELISA. In panels (D–F), all data represented as mean ± SEM. ANOVA, ***p < 0.0001, and **p < 0.001 with Tukey’s post hoc test.

Effect of CBD Treatment on Pro- and Anti-Inflammatory Cytokines and Transcription Factors

Because EAE is triggered primarily by Th1 and Th17 cells, we next studied the effect of CBD on the cytokines related to these cells. In addition, we also studied the effect of CBD on certain critical transcription factors and cytokines: Tbx21 (T-bet), RORγT, and IL-10. To that end, splenic CD4 + T cells were purified from naïve, EAE-VEH, and EAE-CBD mice on day 16, and total RNA was isolated. EAE-VEH mice had significantly elevated T-bet and RORγT compared to naïve mice and treatment with CBD, significantly reduced these levels. Moreover, expression of IL-10 was significantly increased in EAE-CBD mice when compared to EAE-VEH mice (Figure ​ (Figure2A). 2 A). To further elucidate the effect of CBD on MOG-specific T cell activation and function, we cultured splenocytes from naïve, EAE-VEH, and EAE-CBD mice, in vitro, in the presence of MOG35–55 peptide for 3 days. Supernatants from cells of EAE-VEH mice restimulated with MOG displayed increased IFNγ, IL-17, TNFα, and IL-10 cytokine levels, while similar cells from EAE-CBD mice produced significantly less IFNγ and IL-17, and increased IL-10 production, while producing similar levels of TNFα (Figure ​ (Figure2B). 2 B). Together, these data demonstrated that CBD treatment promoted anti-inflammatory cytokines and transcription factors while decreasing the pro-inflammatory.

Expression profile in splenic CD4 + T cells from experimental autoimmune encephalomyelitis (EAE) mice. (A) Splenocytes were isolated from naïve, EAE-VEH, and EAE-cannabidiol (CBD) on day 16 and CD4 + T cells were purified using MACs selection kit. Total RNA was isolated and samples were analyzed for T-bet, IL-10, and RORγT. Data are representative of at least two independent experiments; in each experiment, n = 3–5 mice per group. (B) Splenocytes from naïve (n = 4), EAE-VEH (n = 4), and EAE-CBD (n = 4) were isolated 16 days after disease induction. Cells were restimulated with MOG35–55 peptide (30 μg/mL) for 3 days. Supernatants were collected and analyzed for IFNγ, IL-17, IL-10, and TNFα. Data represented as mean ± SEM. ANOVA, ***p < 0.0001, **p < 0.001, and *p < 0.05 with Tukey’s post hoc test.

Treatment With CBD Leads to Induction of MDSCs and Suppression of MOG-Specific T Cell Proliferation

CD11b + Gr-1 + MDSCs have been shown to play a crucial role in attenuating inflammation and our laboratory has previously reported that CBD treatment induces high levels of CD11b + Gr-1 + MDSCs that suppress autoimmune hepatitis (10). In the current study, we therefore investigated if the ability of CBD to suppress EAE was related to induction of MDSCs. Because CBD was administered by i.p. route, we enumerated the infiltration of MDSCs into the peritoneal cavity and found that there was a dramatic influx of CD11b + Gr-1 + MDSCs in EAE-CBD mice when compared to EAE-VEH mice that was demonstrable on days 10 and 12 and tapered off at day 16 (Figure ​ (Figure3A). 3 A). Next, we determined if CBD treatment led to increase in the number of CD11b + Gr-1 + cells in the CNS. However, the data indicated that CBD treatment failed to increase the numbers of CD11b + Gr-1 + cells in the CNS and in fact, EAE-VEH mice had higher numbers of CD11b + Gr-1 + cells in the spinal cord and brain than EAE-CBD mice (Figure ​ (Figure3B). 3 B). The decreased numbers of CD11b + Gr-1 + cells seen in EAE-CBD mice in the CNS may be due to the fact that there was dramatically decreased numbers of infiltrating cells in the CNS as shown before (Figure ​ (Figure1E). 1 E). These data suggested that CBD induces CD11b + Gr-1 + cells in the periphery but not in the CNS. In addition to MDSCs, neutrophils have also been known to express CD11b + Gr-1 + phenotype. However, we have shown previously that CBD-induced CD11b + Gr-1 + cells are MDSCs and not neutrophils, because they are highly immunosuppressive while neutrophils are not (10). To further determine if CBD-induced CD11b + Gr-1 + cells were indeed MDSCs with immunosuppressive functions, we cultured splenocytes from EAE-VEH mice in vitro with MOG35–55 peptide for 3 days in the presence or absence of CBD-induced MDSCs isolated from intraperitoneal lavage and assessed their ability to suppress MOG-specific T cell proliferation. CBD-induced CD11b + Gr-1 + were found to be highly immunosuppressive and inhibited the proliferation in a dose-dependent manner, thereby confirming that they were MDSCs (Figure ​ (Figure3C). 3 C). When we enumerated the number of viable T cells in such cultures, we found that the T cells were all viable while the total viable cell number decreased significantly (Figure ​ (Figure3D), 3 D), thereby suggesting that MDSCs were not killing the T cells but inhibiting them from proliferating. To determine further the mechanism of suppression of T cell proliferation, we measured the levels of cytokines in these cultures and found that there was decreased production of IFNγ and IL-17, while induction of IL-10 was significantly increased, which was dependent on the number of MDSCs in culture (Figure ​ (Figure4). 4 ). Because MDSCs are well known to produce IL-10, and our data showed dose-dependent response, together these studies suggested that CBD-induced MDSCs may reduce MOG-specific T cell proliferation, at least in part, by producing IL-10 and inhibiting inflammatory cytokines such as IFNγ and IL-17.

Altered expression profile of myeloid-derived suppressor cells (MDSCs) following cannabidiol (CBD) treatment. Absolute cell counts for MDSC: cells were isolated from intraperitoneal lavage (A), spinal cord and brain (B) on indicated day. Cells were stained with CD11b and Gr-1 (MDSC) then enumerated using total cell count and frequency from flow cytometry. Data represented as mean ± SEM (n = 3–5 per sample). Suppressive function of CBD-induced MDSCs was tested using ex vivo restimulation of experimental autoimmune encephalomyelitis (EAE)-VEH splenocytes in the presence of MOG35–55 (30 μg/mL) for 3 days. Cells were pulsed with thymidine and analyzed using BetaScint Counter and were assessed for proliferation (C) and total viable cell number (D). Data represented as mean ± SEM. ANOVA, ***p < 0.0001, **p < 0.001, and *p < 0.05 with Tukey’s post hoc test.

Cannabidiol (CBD)-induced myeloid-derived suppressor cells (MDSCs) alter MOG-stimulated T cell inflammatory cytokine secretion. Regulatory function of CBD-induced MDSCs was tested using ex vivo restimulation of experimental autoimmune encephalomyelitis (EAE)-VEH splenocytes in the presence of MOG35–55 (30 μg/mL) for 3 days, as detailed in Figure ​ Figure3 3 legend. Culture supernatants were collected and analyzed for IFNγ, IL-17, and IL-10. Data represented as mean ± SEM. ANOVA, **p < 0.001, and *p < 0.05 with Tukey’s post hoc test.

Adoptive Transfer of CBD-Induced MDSCs Attenuate EAE Disease Progression

To confirm that CBD-induced MDSCs were in fact attenuating the clinical disease, we performed adoptive transfer experiments. To this end, we transferred purified CBD-induced MDSCs into MOG35–55 immunized mice on days 7 and 9. Four million cells were injected i.p into immunized mice, and similar numbers of splenocytes from naïve mice served as controls (Figure ​ (Figure5A). 5 A). The transferred MDSCs were able to attenuate disease progression, as indicated by significant reduction in clinical scores (Figure ​ (Figure5B) 5 B) and total cellular infiltration in CNS, including the numbers of CD4 + and CD8 + T cells (Figure ​ (Figure5C). 5 C). When we assessed the MDSCs levels in the CNS tissues, we found that there was no significant increase in the percentage and absolute numbers of MDSCs in adoptively transferred mice when compared to controls (Figures ​ (Figures5D,E). 5 D,E). Interestingly, however, the spleens showed a significant increase in their percentage (Figure ​ (Figure5F). 5 F). These data together suggested that the adoptively transferred MDSCs were suppressing MOG-specific T cell activation in the periphery and not by migrating to the CNS and blocking the inflammation there.

Adoptive transfer of cannabidiol (CBD)-induced myeloid-derived suppressor cells (MDSCs) attenuates experimental autoimmune encephalomyelitis (EAE). (A) Time line schematic of studies. CBD-induced MDSCs were administered at time points with gray arrows (d7 and d9) via i.p. injection. Data were assessed at peak of disease unless otherwise stated. CBD-induced MDSCs from IP cavity were collected and 4 × 10 6 MDSCs or splenocytes from naïve C57BL/6 as a control were adoptively transferred. (B) Clinical scores (n = 5 mice per group); data were presented as mean ± SEM and analyzed for significance using Mann–Whitney U test. Comparisons were considered significant at p ≤ 0.05, denoted as *. Data are representative of at least two independent experiments; in each experiment, disease incidence was 100% for each group. (C) Total mononuclear cell infiltrates in central nervous system and absolute cell counts for CD3 + CD4 + and CD3 + CD8 + T cells; mononuclear cells stained with corresponding Abs and then enumerated using total cell count and frequency from flow cytometry. (D) Mononuclear cells stained with CD11b and Gr1 Abs and then analyzed by flow cytometry. Representative histograms shown. (E) Absolute cell counts for MDSC (CD11b + Gr1 + ) enumerated using total cell count and frequency from flow cytometry. (F) Splenocytes were stained with CD11b and Gr1 Ab and then analyzed by flow cytometry. Representative histograms shown. Vertical bars in this figure represent data collected from three to five mice per group expressed as mean ± SEM. ANOVA, ***p < 0.0001, and *p < 0.05 with Tukey’s post hoc test.

Attenuation of EAE by CBD Treatment Can Be Reversed With MDSC Depletion

To further corroborate the role of MDSCs in CBD-mediated attenuation of EAE, we performed MDSC depletion studies using RB6-8C5 (anti Gr-1 Ab), which is highly effective in depleting such cells, as previously shown by us (12). Mice were administered CBD into EAE mice as before, and RB6-8C5 was administered every other day, 3 h after CBD treatment (Figure ​ (Figure6A). 6 A). As an isotype control, similar concentrations of normal IgG2b Ab were used. CBD was able to significantly attenuate disease progression as seen before, and MDSC depletion reversed this effect, as indicated by clinical scores (Figure ​ (Figure6B). 6 B). MDSC depletion was confirmed via peritoneal cavity lavage 18 h after CBD injection on day 9 (sacrificed day 10). MDSCs were significantly reduced in frequency and cell number following treatment with RB6-8C5 when compared to mice that were treated with control Ab (Figures ​ (Figures6C,D). 6 C,D). This effect was also seen at day 16 (data not shown). Moreover, the significant reduction in total cellular infiltration and specific T cell subsets (CD4 + and CD8 + ) mediated by CBD was significantly reversed upon RB6-8C5 treatment (Figures ​ (Figures6E,F). 6 E,F). Together, the MDSC depletion studies corroborated the adoptive transfer experiments suggesting that CBD-mediated attenuation of EAE is mediated primarily through induction of MDSCs.

Myeloid-derived suppressor cell (MDSC) depletion negates the ameliorative effect of cannabidiol (CBD) on experimental autoimmune encephalomyelitis (EAE). (A) Time line schematic of studies. CBD was administered at time points with gray arrows and RB6-8C5 (anti-Gr-1) antibody was given every other day 3 h after CBD via i.p. injection. (B) Clinical scores (n = 5 mice per group); data were presented as mean ± SEM and analyzed for significance using Mann–Whitney U test. Comparisons were considered significant at p ≤ 0.05, denoted as *. Data were assessed at peak of disease unless otherwise stated and are representative of at least two independent experiments; in each experiment, disease incidence was 100% for each group. (C) On day 10, efficacy of RB6-8C5 was tested on cells recovered from peritoneal cavity. IgG2b isotype Ab was used as control. Cells were stained with CD11b and Gr1 Abs to detect MDSCs and analyzed by flow cytometry. Representative histograms shown. (D) Total number of cells in IP lavage and absolute cell counts for MDSC (CD11b + Gr1 + ) enumerated in top and bottom panels, respectively. (E) Total cell count of mononuclear infiltrating cells in central nervous system (CNS). (F) Mononuclear cells from the CNS were stained for CD3 + CD4 + and CD3 + CD8 + T cells for each group and absolute cell counts of these T cell subsets were enumerated. Vertical bars in this figure represent data collected from three to five mice per group expressed as mean ± SEM. ANOVA, ***p < 0.0001, and **p < 0.001 with Tukey’s post hoc test.

Discussion

Given that Sativex (combination of THC and CBD) is already approved for clinical use for the treatment of MS in Europe and other countries, and the recent legalization of medical marijuana in several states in the US, it is important to understand the underlying mechanism of this therapy for MS and other related inflammatory diseases. Sativex is known to reduce neuropathic pain in patients with MS. However, such effects may be independent of the anti-inflammatory properties exhibited by CBD. The anti-inflammatory benefits of CBD have been studied more recently in EAE models (31, 32). In this study, we further elucidate the effect of CBD in a model of autoimmune neuroinflammation and demonstrate for the first time that observed CBD-induced effects may be mediated by induction of MDSCs.

We chose to use a dose of 20 mg/kg body weight of CBD, which translates to 1.6 mg/kg when converted to human equivalent dose. This adds up to

96 mg for an average human adult (60 kg). During a clinical trial, patients with Huntington disease were given 700 mg of CBD daily, for 6 weeks with no toxic effects relative to placebo (33). Also, in a recent clinical trial in epileptic children, CBD was administered at a daily dose of up to 50 mg/kg/day (34). Furthermore, based on previous studies performed by our lab, the 20 mg/kg is an optimal dose in the mouse model for suppressing inflammation and was deemed suitable and safe (10).

Consistent with other reports (32, 35), our data showed that CBD is effective at attenuating EAE, indicated by a decrease in clinical signs, delay of disease onset, diminished cellular infiltration and tissue damage in the CNS. MOG35–55 induced EAE is primarily driven by the concerted effort of Th1 and Th17 cells, producing inflammatory cytokines and effector cells to indirectly and directly damage the myelin sheath in CNS (36). In the current study, we also found systemic expression of IFNγ and IL-17 to be significantly increased at the peak of EAE. Furthermore, this effect was largely blocked with CBD treatment.

Cannabidiol has been previously shown in vitro to modulate Th17 responses by inhibiting IL-17 and IL-6 secretion and promoting IL-10 (11). These data are consistent with the current study inasmuch as CBD treatment in vivo caused significant suppression of IL-17 and IFNγ in the serum. Moreover, splenocytes from CBD-treated EAE mice that were restimulated in vitro, with MOG35–55, secreted less IFNγ and IL-17, while producing higher levels of IL-10. While our studies showed that MDSCs play a crucial role in inhibiting MOG-specific T cell proliferation both in vitro and in vivo using adoptive transfer, others have also noted that CBD treatment in vitro, may have a direct effect by inhibiting T cell proliferation or induction of apoptosis (32, 37–39). Thus, CBD may mediate its effect on inflammatory T cells both indirectly through MDSCs and directly.

Myeloid-derived suppressor cells, co-express CD11b and Gr-1 antigens and are highly immunosuppressive in nature. While other cells types have also been shown to have similar expression patterns, specifically neutrophils, we have shown previously that cannabinoids induce MDSCs rather than neutrophils (10, 12, 25, 30, 40, 41). We found that cannabinoid induced MDSCs are highly immunosuppressive while the neutrophils from the same animals were not (30). MDSCs were originally discovered in cancer patients; however, recently they have been shown to potently disrupt innate and adaptive immune responses (42). MDSCs utilize a multitude of immunosuppressive functions including; depletion of l -arginine by arginase, resulting in T cell-cycle arrest and inhibition of proliferation, expression of reactive oxygen species, secretion of IL-10, and induction of Tregs (43, 44). The role of MDSCs in EAE has at times been controversial, with some reports suggesting that circulating myeloid precursors act to perpetuate disease (45) and while others have demonstrated a regulatory role (46–48). Moreover, Ioannou et al. (47) demonstrated that patients with active MS have significantly elevated MDSCs in peripheral blood compared to healthy controls. In addition, a recent study highlighted the therapeutic potential of MDSCs in patients with MS, noting that within the MS phenotypes expression and function of MDSCs was altered (49). Because MDSCs are induced at sites of inflammation, it is not surprising that their levels are increased in the CNS during EAE, as seen in the current study as well.

In the current study, we found that following CBD injection in EAE mice, high levels of MDSCs were induced in the peritoneal cavity similar to our previous findings (10, 25). In such mice, we also noted an increase in MDSCs in the spleens but not in the CNS. In fact, in the CNS, we noted that EAE-CBD mice had lower levels of MDSCs than EAE-VEH mice. This can be explained by the fact that MDSCs induced in the periphery may not be able to migrate to the CNS. Thus, the MDSCs may inhibit MOG-specific T cell induction in the secondary lymphoid organs thereby preventing such cells from migrating into the CNS to cause the clinical disease. Therefore, we sought to elucidate this hypothesis. In doing so, we demonstrated in vitro that CBD-induced MDSCs were able to suppress MOG-specific T cell proliferation. Interestingly, alteration of local cytokine milieu may represent a potential mechanism for this decrease in proliferation because in such cultures with CBD-induced MDSCs, there was increased production of IL-10 and decreased induction of pro-inflammatory cytokines (IFNγ and IL-17) that was dose-dependent. IL-10 has also been shown to decrease the levels of B7 co-stimulatory molecule that is expressed on antigen-presenting cells which can bind to CD28 expressed on T cells leading to T cell activation (50–52). Thus, this could be one of the pathways through which CBD may decrease T cell activation. Second, it has been proposed that increased inflammation, particularly chronic type, induces MDSCs (43). Thus, it is also possible that in EAE-VEH mice, due to strong neuroinflammation, MDSCs are induced in higher numbers, whereas in EAE-CBD mice, due to markedly attenuated neuroinflammation, there is decreased MDSC induction. The findings that CBD induces MDSCs in the periphery but not in the CNS of EAE mice, together suggested that CBD may be acting in the periphery to attenuate MOG-specific T cell induction rather than acting directly at the CNS to decrease neuroinflammation. This observation is also supported by our findings that adoptive transfer of MDSCs failed to cause an increase of MDSCs in the CNS.

The current study conclusively demonstrated the critical role of MDSCs in CBD-mediated attenuation of EAE using both adoptive transfer experiments as well as in vivo depletion studies. These studies are consistent with our previous studies showing that CBD-induced MDSCs can attenuate autoimmune hepatitis (10). We also utilized RB6-8C5, an antibody against Gr-1, to deplete MDSCs while treating EAE mice with CBD. Although Gr-1 is expressed on other cells, it has been extensively used to study the effect of MDSC depletion (53–55). Our results showed that treatment with RB6-8C5 led to marked decrease in MDSCs and reversed the ability of CBD to attenuate EAE. These studies, combined with the adoptive transfer experiments conclusively demonstrated the pivotal role played by MDSCs in CBD-mediated amelioration of EAE.

In conclusion, we have demonstrated that the mitigation of EAE with CBD comes from its ability to target a range of anti-inflammatory pathways, including (i) induction of anti-inflammatory MDSCs and (ii) decrease in pro-inflammatory and induction of anti-inflammatory cytokines. Because CBD is non-psychoactive, our studies suggest that CBD may constitute an excellent candidate for the treatment of MS and other autoimmune diseases. Our studies provide further evidence of the importance of MDSCs and that manipulation of such cells may constitute novel therapeutic modality to treat MS and other autoimmune diseases.

Ethics Statement

This study was carried out in accordance with the recommendations of Guide for the Care and Use of Laboratory Animals, National Institute of Health. The protocol was approved by the University of South Carolina Institutional Animal care and Use Committee.

Author Contributions

Participated in research design, performed data analysis, and wrote or contributed to the writing of the manuscript: DE, MN, and PN. Conducted experiments: DE and NS. Contributed new reagents or analytic tools: MN and PN.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer, MR, and handling Editor declared their shared affiliation at the time of the review.

Footnotes

Funding. This work was supported in part by National Institutes of Health grants P01AT003961, R01AT006888, R01AI123947, R01AI129788, R01ES019313, R01MH094755, and P20GM103641.

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