Cannabidiol as a Novel Therapeutic for Immune Modulation

Abstract The immune-suppressive effects of cannabidiol (CBD) are attributed to the modulation of essential immunological signaling pathways and receptors. Mechanistic understanding of the pharmacological effects of CBD emphasizes the therapeutic potential of CBD as a novel immune modulator. Studies have observed that the antagonists of CB1 and CB2 receptors and transient receptor potential vanilloid 1 reverse the immunomodulatory effects of CBD. CBD also inhibits critical activators of the Janus kinase/signal transducer and activator of transcription signaling pathway, as well as the nucleotide-binding oligomerization domain-like receptor signaling pathway, in turn decreasing pro-inflammatory cytokine production. Furthermore, CBD protects against cellular damage incurred during immune responses by modulating adenosine signaling. Ultimately, the data overwhelmingly support the immunosuppressive effects of CBD and this timely review draws attention to the prospective development of CBD as an effective immune modulatory therapeutic.

cells. [13][14][15] CB 1 receptor activation initiates down-stream modulation of three main signaling pathways ( Figure 2). 12 Activated CB 1 receptor coupled to G i/o inhibits adenyl cyclase (AC) activity, in turn inhibiting cyclic adenosine monophosphate (cAMP) formation. As a result, intracellular signal transduction, and protein kinase A (PKA) activity is hindered. [16][17][18] The cAMP-PKA signal transduction is crucial for many cellular responses including enzyme activation and gene expression. 19 Studies have also shown that activation of the CB 1 receptor also leads to regulation of mitogen-activated protein kinases (MAPKs) that are vital to inflammatory and immune cellular responses, including extracellular signalregulated protein kinase 1/2 (ERK1/2), p38, and c-jun terminal kinase (JNK). 20,21 The phosphoinositide 3-kinase (PI3K)/ protein kinase B (Akt) is a third pathway involved in cell growth and survival that may also be activated by CB 1 receptor. 22,23 Similarly, CB 2 receptor agonism modulates and inhibits AC and cAMP leading to the inactivation of PKA by G i/o protein coupling. 24,25 CBD and CB 1

and CB 2 Receptors
The low affinity of CBD for both CB 1 and CB 2 receptors (K i > 2000 nM) suggests the pharmacodynamics of CBD to be independent of these receptors. [26][27][28][29] For example, CBD suppressed cytokine production in CB 1 and CB 2 receptor knockout (CNR1 −/− /CNR2 −/− ) mice, suggesting the effects of CBD are independent of CB 1 and CB 2 receptors. 30 Specifically, Interleukin-2 (IL-2), a T-cell-derived cytokine, was significantly reduced in splenocytes of CNR1 −/− /CNR2 −/− mice upon treatment with CBD (1 μM). CBD (10 μM) also significantly decreased adaptive immune response cytokine production of Interferon gamma (IFN-γ). However, CBD has been shown to function as an inverse agonist of CB 1 and CB 2 receptors and can antagonize the effects of CB 1 and CB 2 receptor agonists. 4 The use of CB 1 and CB 2 receptor antagonists reverses the anti-inflammatory and immune-suppressive effects of CBD. 31,32 For instance, while treatment with CBD (10 mg/kg) was shown to reduce gastrointestinal motility in a bacterial lipopolysaccharide (LPS) sepsis mouse model, the effects were reversed upon treatment with CB 1 receptor antagonist, AM251 (1 mg/kg). 31 Similar reversal of CBDinduced neuroprotective effects was observed in a hypoxicischemic brain injury model in newborn pigs. 32 In this model, CBD (1 mg/kg) inhibited inflammatory cytokine interleukin-1 (IL-1) production in hypoxic-ischemic pigs, and coadministration with CB 2 receptor antagonist, AM630 (1 mg/ kg), reversed this CBD-mediated neuroprotective effect. Conclusively, the immune-protective effects of CBD are pharmacologically supported to be independent of CB 1 and CB 2 receptors as well as by inverse agonism of CB 1 and CB 2 .

Transient Receptor Potential Vanilloid 1 (TRPV1)
The transient receptor potential vanilloid 1 (TRPV1) is a homotetrameric membrane protein and is the most wellstudied nonselective ion channel of the transient receptor potential cationic family. 33 TRPV1 is expressed in various immune cells including lymphocytes, microglia, and astrocytes and is involved in pathophysiological responses of the immune system. 34 Numerous exogenous and endogenous stimuli activate TRPV1 with capsaicin as the most notable

Figure 2
Signaling pathway activity downstream of CB 1. Activated CB 1 receptor couples with G i/o to inhibit adenyl cyclase (AC) activity, thus inhibiting cyclic adenosine monophosphate (cAMP) production and protein kinase A (PKA) activity. CB 1 receptor activation also regulates mitogen-activated protein kinases (MAPK) and phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathways. agonist. 35 TRPV1 is responsible for detecting stimuli to maintain and regulate cellular homeostasis by promoting Ca 2+ influx as a secondary messenger for the induction of proinflammatory cytokines and chemokines. 36 Similar to CB 1 and CB 2 receptors described above, activation of TRPV1 may lead to varying downstream signaling such as PI3K/Akt and ERK1/ 2 signaling pathways ( Figure 3). 37 TRPV1 may also lead to transforming growth factor-activated kinase 1 (TAK-1) dependent JNK/MAPK cascade that leads to pro-inflammatory nuclear factor-κB (NF-κB) activation. 38 Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway may also be activated downstream of TRPV1 activation leading to increased expression of pro-inflammatory cytokines. 39

CBD and TRPV1
As TRPV1 activation induces pro-inflammatory and immune mediators, TRPV1 channels are a potential target for novel immune modulatory therapeutics. [40][41][42] Vanilloid receptor knockout (TRPV1−/−) mice treated with CBD dose-dependently (5 mg/kg -50 mg/kg) inhibited induction of myeloid-derived suppressor cells (MDSCs), suggesting that TRPV1 receptors are critical for induction of MDSCs by CBD. 43 CBD (10 mg/kg) was also shown to be an effective therapeutic in a rodent model of thermal hyperalgesia which is associated with immune activation. 44,45 In contrast, coadministration of CBD (10 mg/kg) with TRPV1 antagonist, capsazepine (CPZ) (2 mg/kg), partially reversed the anti-hyperalgesia effects of CBD. Treatment with CPZ (10 mg/kg) fully reversed the anti-hyperalgesia effects, signifying that CBD modulates TRPV1 for immune suppression. 45 Similarly, CBD (10 μM) increased phagocytosis of CNS immune response microglial cells and treatment with CPZ (10 μM) abolished this enhanced phagocytosis, attributing the increased phagocytic effects to modulation of TRPV1. 46 The role of TRPV1 in the immunomodulatory effects of CBD was also apparent in CBD-induced endometrial cancer cell apoptosis. The apoptotic effects of CBD (5 μM) on the endometrial cancer cell line were dependent on TRPV1 modulation as these effects were reversed upon coadministration with TRPV1 antagonist, iRTX (20 nM), hence signifying that CBD may be a potential candidate for immune modulation of TRPV1. 47

Janus Kinase (JAK) and Signal Transducer and Activator of Transcription 3 (STAT3)
Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway is an evolutionary conserved cascade fundamental to T cellmediated immunity and adaptive immune responses. [48][49][50] Broadly, cytokines or growth factors activate their respective transmembrane receptors, facilitating activation of receptor-bound JAKS 51,52 ( Figure 4). Activated JAKs (JAK1, JAK2, JAK3, TYK2) proceed to phosphorylate latent STAT monomers (STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, STAT6) leading to STAT dimerization, translocation to the nucleus, binding to specific DNA promoter sequences, and ultimately transcription of the target gene. 53,54 Signal transducer and activator of transcription 3 (STAT3) is one of the seven STAT proteins involved in the regulation of critical cellular gene transcriptions including cell differentiation, proliferation, metastasis, as well as immune responses. 55 Constitutive activation of JAK-STAT3 signaling is the basis for the development and progression of many cancers; thus, it is not surprising that JAK-STAT3 remains a principal anticancer target. 56 However, as STAT3 is a transcription factor that remains generally confined to the nucleus, the drug-ability of STAT3

CBD and JAK/STAT
The use of CBD, to modulate of JAK/STAT is one novel approach towards immunosuppression. Transcriptomic profiles of CBD-treated human Gingival Mesenchymal Stem Cells (hGMSCs) revealed down-regulation of JAK/ STAT pathways, particularly complete down-regulation of Interleukin-6 (IL-6), a potent but not exclusive activator of JAK/STAT3 signaling. 58,59 Similarly, treatment with CBD (ranging from 0.5 μM to 20 μM) has been shown to significantly inhibit IL-2 and IFN-γ production in mouse splenocytes. 30,60 Both IL-2 and IFN-γ are activators of JAK/STAT. IL-2 is also a stimulator of PI3K-Akt and MAPK signaling cascades critical in regulating lymphocyte proliferation. CBD also inhibits another antiinflammatory and immune regulatory cytokine involved in the modulation of JAK/STAT, Interleukin-10 (IL-10), in human leukemic T-cells. 61,62 CBD also has been shown to inhibit JAK/STAT activators, including IFN-γ and inflammatory and immune response tumor necrosis factor (TNF) cytokine in a rheumatoid arthritis model. [63][64][65] This study revealed that CBD (5 mg/kg per day intraperitoneally (ip)) reduced TNF and IFN-γ production in arthritic mice. The anti-arthritic and immunosuppressive effects of CBD were further underlined by concentrationdependent (0-10 μg/mL) inhibition of lymphocyte proliferation. 65 CBD (5 mg/kg) is also effective in the suppression of T-cell proliferation and microglial activation, as well as reduction of axon damage within the spinal cord of in an encephalomyelitis murine model of Multiple Sclerosis (MS) by suppression of fundamental JAK/STAT initiators including IL-2, TNF-α, and IFN-γ. 11 Notably, T cells express many IL receptors that can lead to activation of JAK/STAT; therefore, suppression of T cells by CBD may inhibit activation of this signaling pathway. High concentration of CBD (20 μg/mL) also attenuates TNF-α and IFN-γ expression in the intestinal lymphatic system as quantified in rat mesenteric lymph node and spleen isolated T cells and human lymphocytes. 66 The immunomodulatory effects of CBD have also been observed in Type 1 diabetes mellitus. Type 1 diabetes mellitus is an autoimmune disease leading to T lymphocyte-mediated destruction of insulin-producing pancreatic β cell. 67 Administration of CBD (5 mg/kg/day ip) has shown to significantly prevent the onset of autoimmune diabetes in nonobese diabetes (NOD) prone mice in comparison to mice that did not receive CBD. 68,69 These mice displayed significant reduction in Th1 inflammatory cytokine production of IFN-γ and TNF-α, and an increase in Th2 cytokine production of IL-4 and IL-10, suggesting that CBD may exert an immunomodulatory mechanism resulting in a Th1 to Th2 immune response shift. 70 Similarly, CBD (5 mg/kg/day) was shown to significantly reduce pro-inflammatory cytokine IFN-γ and TNF-α in the plasma of NOD mice treated with CBD. 69 These mice also exhibited a decrease in Th1 cytokine production and increase in the production of Th2-associated cytokines. 69 These results suggest that CBD may have an immunomodulatory role, shifting immune responses from Th1 to Th2. 69 Expression of other initiators and activators of JAK/STAT including IL-6, TNF-α, and IL-12 were also reduced in the CBD-treated splenocytes derived from these non-obese diabetes-prone (NOD) female mice.
CBD has been shown to reduce proinflammatory signaling by modulation of IFNβ/STAT pathway 71 such that CBD (10 μM) reduced IL-1β, IL-6, and IFNβ expression in LPS activated microglial cells. IL-1β activates myeloid differentiation factor 88 (MyD88)-adaptor protein-dependent pathway that leads to activation of NF-κB-dependent transcription. The NF-κB pathway is primarily responsible for regulating the expression of many pro-inflammatory genes including cytokines TNF α, IL-1, and IL-6. IFNβ, on the other hand, activates chemokines such as CXCL10, CCL5, and CCL2 by binding to IFN receptor and inducing phosphorylation of JAK leading to STAT pathway activation. CBD also suppresses NF-κB mediated transcription, in turn enhancing the anti-

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ImmunoTargets and Therapy 2020:9 inflammatory effects. This process is mediated via STAT3 by increasing anti-inflammatory STAT3 phosphorylation while reducing pro-inflammatory STAT1 phosphorylation. 71 Collectively, these studies further illuminate the pharmacodynamics of CBD and underline the immunomodulatory properties of CBD by down-regulation of JAK/STAT signaling cascade, thus supporting the development of CBD as a novel immune modulation therapeutic.

Nucleotide-Binding Oligomerization Domain-Like Receptor (NLR) Signaling Pathway
Nucleotide-binding oligomerization domain-like receptors (NLRs) serve as cellular receptors that detect and orchestrate innate immune system protection in response to cellular stress and insult, such as pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs). 72,73 When dangerous cellular stimuli or stress is detected, the NLR Family Pyrin Domain Containing 3 gene (NLRP3) consisting of a central nucleotide-binding and oligomerization (NACHT) domain and leucine-rich repeat (LRR) forms an inflammasome complex with an adaptor protein apoptosis-associated specklike protein (ASC). The ASC consists of an N-terminal pyrin domain (PYD) and C-terminal caspase-recruitment domain (CARD) and cysteine protease caspase 1 ( Figure 5). 74 This activated NLRP3 inflammasome complex leads to activation of caspase 1, in turn, regulating pro-inflammatory interleukin-1β (IL-1β) and interleukin-18 (IL-18) cytokine production that play significant roles in NF-κB, MAPK and IFN pro-inflammatory pathways. 75

CBD and NLR
An alternative pharmacological mechanism by which CBD induces immunomodulatory effects is by inhibiting activation of the NLR signaling cascade. Next-generation sequencing and gene ontology data reveal that inflammasome activation is inhibited in mesenchymal stem cells treated with CBD (5 μM) in comparison to stem cells not treated with CBD. 76 In this study, CBD prevented NLRP3-inflammasome pathway activation by suppressing the expression of key genes belonging to NLRP3-inflammasome pathway including NLRP3 and caspase 1 and inhibited downstream production of IL-18 in Human Gingival Mesenchymal Stem Cells (hGMSCs). 76 Down-regulation of NLRP3 genes in CBD-treated hGMSCs was also confirmed using immunocytochemical and protein extraction. Gene expression profiling also indicated CBD-  76 To further understand the immunosuppressive effects and modulation of NLRP3 inflammasome pathway, CBD has also been compared to known NLRP3 inflammasome inhibitors, oridonin and MCC950, for their respective inhibition of NLRP3-inflammasome activity. 77 Before evaluating anti-inflammasome activity of CBD, in vitro toxicity studies were conducted to ensure cellular viability of human monocytes treated with CBD. No significant cytotoxicity was observed among cells treated with CBD (0.1, 1, 10 and 100 μM), suggesting CBD as a safe and non-toxic potential phyto-therapeutic. LPS was used to significantly increase the production of pro-inflammatory cytokine IL-1β in monocytes, and CBD (0.1, 1, and 10 μM) successfully attenuated this IL-1β production. 77 As the NLRP3 inflammasome complex is responsible for IL-1β production, specific CBD-induced modulation of NLRP3 was evaluated by

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Peyravian et al ImmunoTargets and Therapy 2020:9 submit your manuscript | www.dovepress.com DovePress comparing IL-1β concentration in LPS-stimulated monocytes treated with CBD, oridonin, and MCC950. CBD and these NLRP3 inhibitors exhibited comparable IL-1β cytokine inhibitory effects, thus stressing the immunomodulatory effects of CBD by inhibition of the NLRP3 inflammasome pathway, hence, further emphasizing CBD as a prospective immunomodulatory agent.

Adenosine Signaling
Adenosine is a purine nucleoside that protects against excessive cellular damage during stress and inflammation. 78,79 The protective effects of adenosine are mediated by G proteincoupled A 2A receptor, which attenuates inflammation via activation of cAMP-mediated pathway, and ultimately leading to inhibition of T cell differentiation, downregulation of neutrophil superoxide production, and inhibition of proinflammatory cytokines expression including TNF-α ( Figure 6). 80,81 However, rapid intracellular uptake of adenosine, facilitated by the equilibrative nucleoside transporter (ENT), prevents adenosine interaction with their respective immune cell adenosine receptors, and, consequently, blocking these protective effects. 82,83 One method for hindering adenosine reuptake and promoting immune modulation is to prevent intracellular transport of adenosine by use of an ENT inhibitor. 84

CBD and Adenosine Signaling
CBD is a competitive ENT inhibitor (K i < 250 nM) resulting in subsequent anti-inflammatory and immunosuppressive effects by inhibiting adenosine uptake and enhancing adenosine signaling. 85 In vitro data highlight properties of CBD similar to adenosine receptor agonists such as enhancing endogenous adenosine signaling and inhibiting adenosine uptake in murine microglia and macrophages pretreated with CBD. 86 Moreover, in vivo treatment with CBD (1 mg/kg) decreased TNF-α as measured in the serum of LPS-treated mice. 86 However, this effect was eliminated in an A 2A receptor knockout mouse model and annulled in the presence A 2A adenosine receptor antagonist. 86 In a separate study, adenosine uptake inhibition was compared in rat retinal microglial cells treated with CBD and the ENT inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBMPR) to understand if the ENT isinvolved in the pharmacodynamic role of CBD 87 In this study, CBD (0.5 μM) alone was shown to inhibit adenosine uptake in retinal microglial cells and treatment with NBMPR alone inhibited adenosine uptake in a dose-dependent manner. However, adenosine uptake inhibition was not enhanced in CBD-treated cells that were also treated with NBMPR, which lead to the suggestion that the immunosuppressive effects are mediated by competitive binding to ENT. 87 Other studies have reported that TNF-α in LPS-treated mice is reduced in the presence of adenosine uptake inhibitors. 88 As a result, endogenous adenosine is readily available to bind to the A 2A receptor. To study if CBD inhibits TNF-α by A 2A receptor mediation, rats were treated with CBD (1 mg/kg) and A 2A receptor antagonist ZM241385 (10 mg/kg). In these experiments, CBD-treated rats showed significant reduction of TNF-α, while treatment with the ZM241385 alone or treatment with CBD and ZM241385 did not inhibit TNF-α concentration, suggesting that CBD inhibits adenosine uptake and reduces TNF-α production by A 2A receptor modulation. 87 The immunosuppressive effects of CBD by inhibition of adenosine reuptake and regulation of A 2A receptor have also been evaluated in a murine model of Multiple Sclerosis. For that, mice were infected with Theiler's Murine Encephalomyelitis Virus (TMEV) RNA to induce the deleterious inflammatory effects, such as encephalomyelitis, and then treated with CBD. 89 Treatment with CBD (5 mg/kg ip) inhibited VCAM-1 production, a mediator of lymphocyte adhesion and leukocyte infiltration produced in response to inflammation. CBD was also found to reduce CCL2 and CCL5 chemokine expression as well as pro-inflammatory cytokines TNF-α and IL-1β. 89 CCL2 and CCL5 are associated with macrophage,

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ImmunoTargets and Therapy 2020:9 T cell, and leukocyte recruitment during inflammation. 90 These pharmacological effects of CBD were also observed to be dependent on A 2A receptor mediation in vivo. ZM241385 was administered to TMEV-induced mice and then treated with CBD shortly after. CBD attenuated VCAM-1 expression and immune cell infiltration. However, these effects were not observed in mice receiving ZM241385 alone, suggesting that the A 2A receptor is involved in the mechanism of action of CBD. 89 These studies further solidify the immunosuppressive effects of CBD and link the modulatory properties of CBD to inhibition of adenosine uptake and its enhancement of adenosine signaling.

Conclusions
Mechanistic understanding of fundamental pharmacological pathways and receptors followed by a survey and analytical review of promising work involving CBD in the context of the aforementioned receptors and signaling cascades validate CBD as a prospective immune modulatory therapeutic agent. CBD exerts immunomodulatory effects in CB 1 and CB 2 receptor knockout mice, suggesting that the significant reduction of immune response by CBD is independent of CB 1 and CB 2 receptors. However, receptor antagonists of CB 1 and CB 2 receptors have shown to reverse the immune modulatory effects of CBD, indicating that CBD is also an inverse agonist of CB 1 and CB 2 receptors. Co-administration of CBD with a TRPV1 antagonist reversed CBD inhibition of immune activation, further indicating the involvement of TRPV1 in the immunomodulatory effects of CBD. Moreover, CBD also downregulates JAK/STAT signaling by inhibiting immune regulatory cytokines, such as TNF-α and IFN-γ, involved in the modulation of JAK/STAT. CBD also prevents NLRP3-inflammasome pathway activation as well as suppresses gene expression of downstream proteins of the NLRP3-inflammasome pathway. These immunosuppressive effects result in decreased production of proinflammatory cytokines, including IL-1β and IL-18. CBD has also been shown to inhibit cellular adenosine uptake, resulting in enhanced adenosine signaling and protection against tissue injury during inflammation and immune response. Adenosine receptor, A 2A , inhibition with an antagonist or in A 2A receptor knockout mouse, was shown to reverse these immune modulatory effects of CBD. Ultimately, thefindings emphasize the immunesuppressive properties of CBD and its potential use as an attractive therapeutic strategy for immunomodulation.
However, to enhance CBD as a promising therapeutic option, additional mechanistic studies should be conducted in the context of other autoimmune disorders, using various immune system models and stimuli, to generate new knowledge of the immune modulatory effects of CBD in vitro and in vivo. In addition, long-term studies are needed to determine CBD's therapeutic efficacy in chronic immune diseases. Safety and toxicity should be evaluated in these long-term studies to identify any limitations associated with using CBD as a recurring immune modulator. Furthermore, as CBD is a highly hydrophobic molecule and subject to extensive first pass metabolism, its dosages are relatively high. Therefore, there are limitations on how varying dosage affects exhibited immunomodulatory properties. Conclusively, further studies should be conducted to attain a comprehensive understanding of CBD pharmacokinetics and dosages required for therapeutic immune modulation with the ultimate purpose of enhancing CBD as a prospective immune modulatory therapeutic.