Immune Responses Regulated by Cannabidiol

Abstract

Introduction: Cannabidiol (CBD) as Epidiolex^® (GW Pharmaceuticals) was recently approved by the U.S. Food and Drug Administration (FDA) to treat rare forms of epilepsy in patients 2 years of age and older. Together with the increased societal acceptance of recreational cannabis and CBD oil for putative medical use in many states, the exposure to CBD is increasing, even though all of its biological effects are not understood. Once such example is the ability of CBD to be anti-inflammatory and immune suppressive, so the purpose of this review is to summarize effects and mechanisms of CBD in the immune system. It includes a consideration of reports identifying receptors through which CBD acts, since the “CBD receptor,” if a single one exists, has not been definitively identified for the myriad immune system effects. The review then provides a summary of in vivo and in vitro effects in the immune system, in autoimmune models, with a focus on experimental autoimmune encephalomyelitis, and ends with identification of knowledge gaps.

Conclusion: Overall, the data overwhelmingly support the notion that CBD is immune suppressive and that the mechanisms involve direct suppression of activation of various immune cell types, induction of apoptosis, and promotion of regulatory cells, which, in turn, control other immune cell targets.

Cannabidiol History and Therapeutic Uses

Cannabidiol (CBD) is a plant-derived cannabinoid that has structural similarity to the primary psychotropic congener in cannabis, Δ⁹-tetrahydrocannabinol (THC). While CBD was initially isolated in the 1940s, its structure was not elucidated until the 1960s.^1,2 Unlike THC, CBD is bicyclic, comprised a terpene and an aromatic ring, and is a pentyl side chain.¹ It exists as two enantiomers, and it is (−)CBD³ that is one of the major constituents found in Cannabis sp., and will be the focus of this review. For many years, THC and CBD were designated as psychoactive and nonpsychoactive, respectively, owing to the fact that THC produces the euphoric high associated with cannabis use, while CBD does not. However, since we know that CBD produces biological effects in the central nervous system (CNS), perhaps it is better defined as psychoactive, but not psychotropic, since it is active in the CNS without producing the euphoric high.

Perhaps it was the association of the euphoric high with THC that provided the initial focus on THC as opposed to CBD for potential medical use, since THC was originally identified as the active component of the plant.⁴ However, in recent years, researchers have begun to explore CBD more as a therapeutic addition or alternative to THC. In the United States, oral THC (dronabinol, Marinol^®) was first approved in 1985 by the Food and Drug Administration (FDA) to treat nausea and vomiting associated with chemotherapy. In 1992, dronabinol was also approved to treat cachexia in AIDS patients.⁵ The next major advancement in cannabinoid pharmaceuticals was not until the mid-2000s when Sativex^® (nabiximols), a combination of THC and CBD as an oromucosal spray, was approved in Canada and the EU for neuropathic pain in multiple sclerosis (MS) and intractable cancer pain.⁶ There are several reasons why combining THC and CBD in a single therapeutic could have value.⁶ First, additional therapeutic benefit might be gained from hitting multiple targets; for example, if THC alleviates pain and CBD alleviates anxiety,^7–16 the combination therapy could be quite effective for chronic pain sufferers. Second, for disease states in which both THC and CBD are efficacious, a combination might allow for lower doses of THC, thereby potentially decreasing the psychotropic effects of THC. Third, there are some studies suggesting pharmacokinetic interactions between CBD and THC in which CBD treatment increases THC levels,^17–20 thereby allowing longer duration of effects of THC. Sativex^® has been evaluated in several clinical trials for spasticity associated with MS, neuropathic pain, and other conditions.^21–37

The latest approved cannabinoid pharmaceutical in the United States is CBD as Epidiolex^®. It was approved by the U.S. FDA in 2018 for epilepsy in children, in particular, for Dravet Syndrome and Lennox-Gastaut Syndrome.^38–42 CBD is also being investigated for its effectiveness in other diseases, including Tuberous Sclerosis, a genetic condition that causes growth of benign tumors all over the body,^43,44 schizophrenia,⁴⁵ and refractory epileptic encephalopathy.⁴⁶

In addition to the federally approved uses of CBD as Epidolex^®, CBD, usually as CBD oil, is widely used for putative medical benefit in several states, and is certainly used in states in which cannabis has been decriminalized, or legalized, for recreational use.⁴⁷ There are reports that CBD and other cannabinoids are beneficial for sleep, anxiety, pain, post-traumatic stress disorder, schizophrenia, neurodegenerative disorders, and immune-mediated diseases.⁴⁸ Often these conditions are self-diagnosed and self-treated, so there can be issues with dosing, other drug interactions, and characterization of CBD safety and efficacy.

Overall, it is clear that exposures to CBD are increasing.^47,49–51 It is also clear that CBD possesses therapeutic benefit, and in some cases, the beneficial effects of CBD are for diseases for which other available treatments have not been efficacious.⁵² Together, these observations demonstrate the critical need to continue research on CBD, and therefore the goal of this review is to provide a summary of the effects and mechanisms by which CBD alters immune function. The review will include an evaluation of the role for various receptors through which CBD acts in the immune system. There will also be a description of CBD effects in animal and human immune responses, a characterization of mechanisms by which CBD mediates immune effects, and identification of knowledge gaps regarding CBD's actions in the immune system.

Identification of CBD Receptors and Other Targets

Upon identification of the cannabinoid receptors, CBD was determined to exhibit low affinity for CB₁⁵³ and CB₂ receptors.⁵⁴ Consistent with this, we showed CBD-induced suppression of cytokine production in mouse splenocytes in both wild-type and double cannabinoid receptor knockout mice (Cnr1^−/−/Cnr2^−/− mice).⁵⁵ Another study demonstrated that ophthalmic administration of CBD following corneal inflammation reduced neutrophils in both wild-type and CB₂ receptor knockout mice.⁵⁶ CBD-mediated suppression of anti-CD3-mediated proliferation of T cells also occurred in both wild-type and CB₂ receptor knockout splenocytes.⁵⁷ However, there are a few reports using inflammatory stimuli in which CBD's actions have been attributed to either CB₁ or CB₂ receptors (Table 1). In a sepsis model induced with bacterial lipopolysaccharide (LPS), CBD-mediated inhibition of gastric emptying was reversed with the CB₁ receptor antagonist, AM251.⁵⁸ Similarly, CBD inhibited interleukin (IL)-1 in a hypoxia-ischemia brain insult model and this effect was reversed with the CB₂ receptor antagonist, AM630.⁵⁹ Use of ovalbumin to induce an asthma-like disease in mice demonstrated that some cytokines and chemokines induced in the lungs of mice that were suppressed by CBD (IL-4, IL-5, IL-13, and eotaxin) were differentially regulated by CB receptors.⁶⁰ Specifically, CBD-induced suppression of IL-5 was reversed in the presence of the CB₂ receptor antagonist in bronchoalveolar lavage fluid and lung tissue, but there was no clear receptor dependence identified for CBD's suppression of IL-4, IL-13, or eotaxin.⁶⁰ Thus, several studies do suggest a possible role for cannabinoid receptors in CBD-mediated suppression of inflammatory effects. It should also be noted that there are several reports suggesting that CBD acts as an allosteric modulator of CB₁ or CB₂ receptors,^61–64 although the role for CB₁ or CB₂ receptor allosteric modulation by CBD in immune function has not yet been determined.

Table 1.

Receptors Identified in Mediating Cannabidiol Immune Effects

Receptor	Activity	References
CB1	Agonist	58
CB2	Agonist	59,60
FAAH	Inhibition	58,65–67,84,157,160
TRPV1	Agonist	65,66,74,82–88,105,148,194
Adenosine A2A	Agonist	89–91,125,164
PPAR-γ	Activation	92–94,96–98,136
5-HT1a	Agonist	59
GPR55	Antagonist	109,110

FAAH, fatty acid amide hydrolase; PPAR-γ, peroxisome proliferator-activated receptor gamma; TRPV1, transient receptor potential vanilloid 1.

Another mechanism by which CBD acts is through inhibition of fatty acid amide hydrolase (FAAH),^65–67 suggesting that some of CBD's effects are mediated by anandamide elevation since FAAH is responsible for the breakdown of anandamide.^65,66 Anandamide is an endogenous cannabinoid that exhibits affinity for CB₁ and CB₂ receptors.^68,69 A recent study suggested that the mechanism by which CBD elevates anandamide involves CBD interaction with fatty acid binding proteins, which prevents anandamide binding to these proteins to block anandamide transport to FAAH.⁶⁷ Since anandamide exhibits affinity for CB₁ and CB₂ receptors, and oxidation products of anandamide through cyclooxygenase or cytochrome P450 enzymes produce metabolites that also exhibit affinity for CB₁ and CB₂ receptors,^70,71 anandamide or its metabolites could account for some of the reports that CBD acts through CB₁ and/or CB₂ receptors.^{58,61–64,72–84}

Actions of CBD in immune function might also be mediated by the transient receptor potential V1, known as the vanilloid receptor (TRPV1), which was found to be activated by CBD.⁶⁵ Specifically, CBD was found to increase intracellular calcium in HEK cells transfected with TRPV1, and the CBD-induced increase in calcium was blocked by the TRPV1 antagonist, capsazepine.^65,66 Follow-up studies demonstrated that CBD desensitizes TRPV1 following activation.⁸⁵ Other studies have suggested that CBD acts through TRPV1 in the immune system (Table 1). CBD can induce myeloid-derived suppressor cells (MDSCs), a type of regulatory cell, in the liver, and this effect is lost in TRPV1 knockout mice.⁸⁶ Specifically, regarding inflammation, CBD attenuated thermal hyperalgesia in response to carrageenan injections or in a neuropathic pain model in a capsazepine-dependent manner.^87,88 CBD suppression of cytokines in inflamed primary human colonic tissue was attenuated by the TRPV1 antagonist, SB366791.⁸² SB366791 was also effective in reversing CBD's suppression of rolling and adherent leukocytes in the sodium monoiodoacetate model of osteoarthritis in rats.⁸³ Together, these data suggest that TRPV1 is a critical receptor through which CBD acts in the immune system.

There have been several critical articles in which adenosine A_2A receptors have been shown to mediate CBD's effects in the immune system.^89–91 CBD was shown to inhibit microglial cell proliferation, which was associated with inhibition of adenosine uptake into cells.⁸⁹ The studies also demonstrated that CBD suppression of tumor necrosis factor-alpha (TNF-α) could be reversed using an adenosine A_2A receptor antagonist, and CBD-induced suppression of LPS-stimulated TNF-α was not observed in adenosine A_2A receptor knockout mice.⁸⁹ The role for adenosine A_2A receptor in CBD-mediated neuroprotection or suppression of neuroinflammation was demonstrated in a model of hypoxia-ischemia in newborn mouse brains.⁹⁰ CBD inhibited adenosine uptake into rat microglial cells and CBD enhanced adenosine's ability to inhibit TNF-α, which was prevented by the adenosine A_2A receptor antagonist, ZM241385.⁹¹ These studies show that CBD acts through the adenosine A_2A receptor, especially in microglial cells.

CBD's effects have also been shown to be mediated by peroxisome proliferator-activated receptor gamma (PPAR-γ) using PPAR-γ antagonists in models of β amyloid neuroinflammation,⁹² apoptosis,^93,94 dinitrobenzene sulfonic acid (DNBS)-induced colitis,⁹⁵ human ulcerative colitis,⁹⁶ LPS activation of microglial cells,⁹⁷ and hypoxia-ischemia model of neuroinflammation.⁹⁸

There are several reports that CBD acts through the serotonin 5-HT1a receptor (Table 1). Although most of the evidence for the involvement of this receptor comes from the attenuation of CBD's effects using the 5-HT1a antagonist, WAY100635, early studies demonstrated that CBD displaced binding of the 5-HT1a agonist, 8-OH-DPAT, in membranes from CHO cells expressing the human 5-HT1a receptor.⁹⁹ Few of the CBD-mediated effects acting through the serotonin 5-HT1a receptor have been reported in immune cells, but immune cells do express 5-HT1a.^100–103 One study showed that IL-1 produced in the brain in response to hypoxia-ischemia insult was inhibited by CBD, and reversed with the 5-HT1a receptor antagonist, WAY100635.⁵⁹

Studies have suggested that CBD might act through other receptors, including other TRP receptors,^{66,85,104–107} or opioid receptors.¹⁰⁸ There is also evidence that CBD acts through blockade of GPR55,¹⁰⁹ and specifically that CBD modestly antagonized proinflammatory effects in human innate cells following GPR55 activation.¹¹⁰ Thus, together, the current data support that immune effects of CBD are mediated through activation of CB₁, CB₂, TRPV1, adenosine A2A, and PPAR-γ receptors, blockade of GPR55 receptors, and FAAH inhibition.

CBD Immune System Effects and Mechanisms

Immunity is maintained through various cell types acting together to provide protection against foreign invaders, and simultaneously avoid reactions against self-proteins. Thus, an appropriate immune response requires a regulated balance between robust reactions against non-self, but limited or no reactions against self. Cell types include neutrophils, macrophages, and other myeloid cells comprising the innate immune system, which reacts quickly to destroy pathogens. In the event that an innate response is insufficient, certain innate cells can activate the adaptive immune response, comprised predominantly of T and B cells. T cells can then provide signals that recruit and activate other immune cells, or directly lyse or induce apoptosis of infected cells. T cells can also help stimulate B cells, which produce antibodies to neutralize pathogens and/or enhance destruction of the pathogens. Communication between the various cell types, and therefore the innate and adaptive immune responses, is mediated by expressed or secreted proteins called cytokines or chemokines. Inflammation is the process commonly associated with the innate immune response since pathogen destruction can also cause tissue damage, although T cells certainly are proinflammatory as well. In fact, many cell types, regardless of whether they are immune cells, produce proinflammatory cytokines in response to inflammation.

The effects of CBD on immune responses can involve innate or adaptive responses. In assessing these responses, various cell types and their functions have been examined. For instance, a common end-point to examine regardless of cell type is cytokine or chemokine production. Typical proinflammatory cytokines include IL-1α, IL-1β, IL-6, TNF-α and IL-17A, while IL-10 is considered anti-inflammatory. Some cytokines are produced by specific T cell subsets; for instance, the Th1 subset produces interferon-gamma (IFN-γ) and promotes cell-mediated cytotoxicity, while the Th2 subset produces IL-4 and promotes B cell responses. Other end-points that might provide clues of disruption of immune competence are nitric oxide or myeloperoxidase (MPO) production from innate cells, as these are often released during pathogen destruction. Thus, the effects of CBD on immune function are presented by cell type, outlining known mechanisms by which CBD alters various end-points. Tables 2–4 include the studies described in the text (and others) and are organized by experimental approach. As indicated above, inflammation can induce proinflammatory cytokine production in nonimmune cells, so there are also a few of those examples included in the tables.

Table 2.

Cannabidiol-Induced Immune Suppression by Cell Type in Human Cells In Vitro

Cell type	End-point(s)	References
PBMCs	↓rosette formation	138a
PBMCs	↓cytokines	111,112a
Human cell linesb	↓cytokines	186
HL-60b	↑apoptosis	113
Jurkat and MOLT-4 T cellsb	↑apoptosis	80a
Human coronary artery endothelial cells	↓adhesion molecules, migration, transcription factors, nitrative stress	119a
Jurkat T cellsb	↓cytokines, transcription factors	55a
Human neutrophils	↓migration	195
PBMCs	↓indoleamine-2,3-dioxygenase (IDO), ↓cytokines	142
THP-1 cellsb	↓IDO	142
PBMCs	↑apoptosis	114a
Human intestine	↓proteins and nitric oxide	96
Human liver sinusoidal endothelial cells	↓adhesion molecules	118
Human gingival mesenchymal stem cells	↓inflammatory genes	79
Caco-2 cellsb	↓phosphoproteins	82a
Primary colonic explants	↓cytokines	82a
Human neutrophils	↓ROS	185
Human PBMCs	↓proliferation and cytokines	146a
HaCaT human keratinocytesb	↓cytokines	84
Human monocytes	↑apoptosis	115a
Human plasmacytoid dendritic cells	↓CD83 expression in HIV+ dendritic cells	134a

^aDiscussed in review.

^bCell line.

ROS, reactive oxygen species.

Table 3.

Cannabidiol-Induced Immune Suppression by Animal Cell Type In Vitro

Cell type	End-point(s)	References
B6C3F1 female splenocytes	↓IL-2	196
EL-4 T cellsa	↑apoptosis	80b
Mouse EOC-20 microglial cellsa	↓proliferation	89b
BALB/c male splenocytes	↓IL-4 and IFN-γ	140b
B6C3F1 female splenocytes	↓IL-2 and IFN-γ	55b
BALB/c male thymocytes and EL-4 T cellsa	↑apoptosis	150b
BALB/c male splenocytes	↑apoptosis	151b
Sprague-Dawley rat microglial cellsc	↓adenosine uptake,	91b
	↓TNF-α
BV-2 cellsa	↓cytokines, ↓NF-κB activation	147b
Mouse brain slicesc	↓cytokines	90b
Rat male astroglial cells	↓gliosis	92b
C57BL/6 male Kupffer cells	↓TNF-α	118
BALB/c microglial cellsc	↑apoptosis	156b
BV-2 cellsa	↓oxidative stress, ↓Ccl2	159
MOG-specific female T cells	↓IL-17A and IL-6	144b
Mouse brain endothelial cellsa	↓VCAM-1 and leukocyte adhesion	164b
Rat astrocytesc	↓Ccl2	164b
RAW cellsa	↓TNF-α	148
MOG-specific female T cells	↓cytokines	143b
Rat male splenocytes and mesenteric lymph nodes	↓proliferation and cytokines	146b
Primary mouse male and female microglial cells	↓activation	97b
BV-2 cellsa	Alteration of circadian rhythm-associated genes	197
BV-2 cellsa	alteration of miRNAs	161b
C57BL/6 or BALB/c female splenocytes	↓proliferation and cytokines	57

^aCell line.

^bDiscussed in review.

^cSex not stated for cells derived from animals (or in the case of primary microglial cell isolates, not determined in newborn animals).

IFN-γ, interferon-gamma; IL, interleukin; miRNA, microRNA; MOG, myelin oligodendrocyte glycoprotein; NF-κB, nuclear factor-κB; TNF-α, tumor necrosis factor-alpha; VCAM-1, vascular cell adhesion molecule-1.

Table 4.

Cannabidiol-Induced Immune Suppression in Animals In Vivo

Model	Disease model	Route, dose range, and duration/frequencya	Major effects	Reference
Male CD-1 mice	sRBC	i.p.	Modest ↓antibody production	155
		25 mg/kg
		4 days
Male DBA/2 mice	Collagen-induced arthritis	i.p. or oral	↓disease, ↓TNF-α and IFN-γ	139b
		2.5–20 mg/kg for i.p.
		5–50 mg/kg for oral
		10 days
Male ICR mice	Carrageenan-induced inflammation	ethosome (CBD in ethosomal gel)	↓inflammation	198
		100 mg of ethosomal CBD (3%)
Male Wistar rats	Carrageenan-induced inflammation	Oral	↓disease, ↓prostaglandin (PGE2)	199
		5–40 mg/kg
		3 days
Female NOD mice	Diabetes	i.p.	↓disease incidence, ↓IL-12, TNF-α and IFN-γ, ↑IL-4	123
		5 mg/kg/day
		10–20 injections
Female C57BL/6 mice	EL-4 leukemia growth	i.p.	↑apoptosis of tumor cells	80b
		12.5 or 25 mg/kg once
Male Wistar rats	Sciatic nerve pain or CFA-induced inflammation	Oral	↓pain, ↓TNF-α, ↓prostaglandin (PGE2)	88
		2.5–20 mg/kg
		7 days
Male Sprague-Dawley rats	Ischemia-reperfusion injury (myocardial)	i.p.	Modest ↓infarct size, ↓TNF-α	200
		5 mg/kg
		twice
C57BL/6J micec	Aβ inflammation	i.p.	↓IL-1β, ↓iNOS	117
		2.5 or 10 mg/kg
		7 days
Male BALB/c mice	Ovalbumin (asthma)	i.p.	↓serum antibodies, ↓IL-2, IL-4, and IFN-γ	140b
		5–20 mg/kg
		once
Male ddY mice	Focal cerebral ischemia	i.p.	↓infarct size, ↓neutrophil MPO activity	129b
		3 mg/kg
		various times surrounding occlusion
Female NOD mice	Diabetes	i.p.	↓disease incidence, ↓IL-6 and IL-12, ↑IL-4 and IL-10	124b
		5 mg/kg/day
		5 injections per week for 4 weeks
Female B6C3F1 mice	sRBC	Oral	Modest ↓antibody production	55b
		25–100 mg/kg/day
		5 days
Male ICR mice	DNBS colitis	i.p.	↓inflammation, ↓colon weight:length ratio, ↓iNOS, IL-1β, ↑IL-10	95b
		1–10 mg/kg
		6 days
Male Wistar rats	None	i.p.	↓blood leukocytes and lymphocytes, ↓B, T and CTL cells, ↑NK and NKT cells	201
		2.5 or 5 mg/kg
		14 days
Male CD-1 mice	Diabetes	i.p. or i.n.	↓diabetic pain, ↓density of microglial cells	81b
		0.1–2 mg/kg i.n.
		1–20 mg/kg i.p.
		3 months
Male C57BL/6 mice	Streptozotocin-induced diabetes	i.p.	↓disease, ↓TNF-α, NF-κB activity, ICAM-1, VCAM-1, iNOS, p-p38, p-JNK, ↑p-AKT	120b
		1–20 mg/kg
		11 weeks
Male Wistar rats	TNBS colitis	i.p.	Modest ↓disease, ↓colonic contractions, ↓neutrophil MPO activity	130
		5–20 mg/kg
		once
Male Wistar rats	Cecal ligation and puncture	i.p.	↑disease survival	184
		2.5–10 mg/kg
		once or up to 9 days
Female Sabra mice	Hepatic encephalopathy (bile duct ligation)	i.p.	Improved disease-associated cognitive impairments, ↓TNF-α	202
		5 mg/kg
		4 weeks
Male BALB/c mice	Ovalbumin (footpad)	i.p.	↓footpad swelling, ↓TNF-α and IFN-γ, ↑IL-10	188
		1–10 mg/kg
		5 days
Male Swiss OFI mice	LPS i.p.	i.p.	↓mast cell infiltration, macrophage activation marker, ↓TNF-α	96
		10 mg/kg
		twice
Female C57BL/6 mice	Experimental autoimmune hepatitis	i.p.	↓hepatic inflammation, ↓IL-2, TNF-α, IFN-γ, IL-6, IL-17A, IL-12, MCP-1 (CCL-2), and eotaxin, ↑MDSCs	86b
		10–50 mg/kg
		once
Male C57BL/6 mice	Ischemia reperfusion injury (liver)	i.p.	↓hepatic inflammation, ↓MIP-1α, ICAM, MIP-2, TNF-α, NF-κB activity, ICAM-1, iNOS, p-p38, p-JNK	118
		3 or 10 mg/kg
		once
C57BL/6 micec	LPS i.v.	i.v.	↓vasodilation, leukocyte margination, and extravasation, ↓COX-2, TNF-α, and iNOS	121
		1 or 3 mg/kg
		once
Male C57BL/6 mice	LPS-induced pulmonary inflammation	i.p.	↓BALF lymphocytes, macrophages, and neutrophils, ↓TNF-α, IL-6, MCP-1 (CCL-2), and MIP-2	125b
		0.3–80 mg/kg
		once
Male Wistar rats	Meningitis (Streptococcus pneumoniae)	i.p.	Improved disease-associated cognitive impairments, ↓TNF-α	203
		2.5–10 mg/kg
		once or up to 9 days
C57BL/6 micec	Cerulein (pancreatitis)	i.p.	↓disease, ↓TNF-α and IL-6, ↓neutrophil MPO	128b
		0.5 mg/kg
		twice
Newborn pigsc	Hypoxia-ischemic brain injury	i.v.	neuroprotection, ↓IL-1	59b
		1 mg/kg
		once
Male Wistar rats	Ovalbumin (asthma)	i.p.	↓TNF-α, IL-6, IL-4, IL-5, and IL-13	127b
		5 mg/kg
		twice
Male C57BL/6 mice	LPS-induced pulmonary inflammation	i.p.	↓inflammation, ↓BALF lymphocytes, macrophages, and neutrophils, ↓TNF-α, IL-6, MCP-1 (CCL-2), and MIP-2	132
		20–80 mg/kg
		once
Female C57BL/6 mice	None	i.p.	↑MDSCs	136b
		20 mg/kg
		once
Female C57BL/6 mice	Malaria (Plasmodium berghei)	i.p.	↓IL-6 and TNF-α	204
		30 mg/kg
		3–5 days
Male Sprague Dawley rats	Freund's Adjuvant (osteoarthritis)	Transdermal	↓inflammation, ↓TNF-α	205
		0.6–63.2 mg/day
		4 days
Male ICR mice	DNBS Colitis	i.p. or orald	↓colon weight:length ratio, ↓neutrophil MPO	131
		5–30 mg/kg for i.p. 10–60 mg/kg oral
		3 days
Female NOD mice	Type 1 diabetes	i.p.	↓disease	206
		5 mg/kg
		5 injections/week for 10 weeks
Male A/J mice	Experimental autoimmune myocarditis	i.p.	↓disease, ↓lymphocyte populations in heart, ↓IL-6, IFN-γ, IL-1β, and MCP-1 (CCL-2)	126b
		10 mg/kg
		46 days
Male Wistar rats	Middle cerebral artery occlusion	i.c.v.	↓infarct size	149
		50–200 ng/rat
		5 days
Male Wistar rats	Middle cerebral artery occlusion	i.c.v.	↓infarct size, ↓TNF-α	207
		50–200 ng/rat
		5 days
Male Wistar rats	Sodium monoiodoacetate (osteoarthritis)	Intra-arterial	↓pain, ↓rolling and adherent leukocytes, ↓joint nerve demyelination	83b
		100–300 μg/rat
		multiple doses
Female C57BL/6 mice	Alcoholic liver disease	i.p.	↓liver damage, ↓neutrophils, ↓TNF-α, MIP-1, IFN-γ, IL-1β, and MCP-1 (CCL-2)	185
		5 or 10 mg/kg
		11 days
Male and female dogs	Osteoarthritis	Orale	↓pain	208
		2 and 8 mg/kg
		every 12 h for 4 weeks
Male Wistar rats	Ulcerative tongue lesion	i.p.	↓inflammation	209
		5 or 10 mg/kg
		3 or 7 days
Female C57BL/6 mice	Spinal cord contusion	i.p.	↓spinal cord CD4 T cells, ↓IL-23A, IL-23R, IFN-γ, CXCL9, CLCL11, NOS2, and IL-10	189
		1.5 mg/kg
		1 and 24 h after injury, on day 3, then twice/week up to 10 weeks
Male Sprague-Dawley rats	Carrageenan-induced inflammation	Oral	↓hyperalgesia	210
		100 or 10,000 μg/kg
		once
Male Swiss mice	Haloperidol-induced inflammation	i.p	↓IL-1β and TNF-α, ↑IL-10	97b
		60 mg/kg
		twice/day up to 21 days
Male BALB/c mice	Corneal inflammation	Topical (ophthalmic)	↓pain, ↓neutrophils	56b
		3% or 5%
Male ICR mice	Ischemia-reperfusion injury (kidney)	i.p.	↓kidney injury, ↓TH17 cells, ↑Tregs and Treg17 cells	152b
		10 mg/kg
		once
Female C57BL/6 and BALB/c mice	Syngeneic or allogeneic bone marrow transplant	i.p.	↓lymphocyte recovery	57
		5 mg/kg
		every other day for 2 weeks
BALB/c mice	Ovalbumin (asthma)	i.p.	↓airway resistance; ↓IL-4, IL-5, IL-13, and eotaxin	60b
		5 or 10 mg/kg
		three times at time of ovalbumin challenge

^aMaximum duration or frequency noted; some studies in the article might have been shorter.

^bDiscussed in review.

^cSex not stated.

^dCBD or CBD BDS (botanical drug substance).

^eCBD oil.

CBD, Cannabidiol; DNBS, dinitrobenzene sulfonic acid; iNOS, inducible nitric oxide synthase; i.n. intranasal; i.p., intraperitoneal; JNK, c-jun N-terminal kinase; LPS, lipopolysaccharide; MDSCs, myeloid-derived suppressor cells; MPO, myeloperoxidase; sRBC, sheep red blood cell; TNBS, 2,4,6-trinitrobenzene sulfonic acid; Treg, regulatory T cell.

CBD effects and mechanisms of immune suppression in innate cells

One of earliest effects reported with CBD was in human mononuclear cells,^111,112 in which TNF-α, IFN-γ, and IL-1α were all suppressed (0.01–20 μg/mL CBD or 0.03–64 μM CBD). Later studies focused on human monocytic cells revealed that CBD can induce apoptosis in either HL-60 (1–8 μg/mL CBD or 3.2–26 μM CBD)¹¹³ or primary human monocytic cells (1–16 μM CBD).^114,115 Macrophages are also targets, although they have been studied more commonly in animal models. Peritoneal macrophages were used early on to demonstrate that CBD (3 μg/mL or 10 μM) targets nitric oxide,¹¹⁶ and this has also been a well-studied target of suppression by CBD in many tissues and cell types. The mechanism by which CBD suppressed nitric oxide involves suppression of endothelial⁸⁷ or inducible nitric oxide synthase (iNOS)^{58,95,117–121} in response to various inflammatory stimuli. iNOS is known to be regulated by the transcription factor nuclear factor-κB (NF-κB),¹²² which is comprised of p65 and other proteins, and becomes active after degradation of the inhibitory protein, IκB. Decreased expression of iNOS by CBD correlated with stimulation of the inhibitory IκBα protein and inhibition of NF-κB p65 protein expression.^119,120 Using peritoneal macrophages from diabetic mice stimulated ex vivo with LPS revealed that macrophages isolated from CBD-treated mice did not produce as much TNF-α or IL-6 as macrophages isolated from vehicle-treated mice.^123,124 A direct effect of CBD decreasing macrophage numbers in the bronchoalveolar lavage fluid was shown following intranasal LPS administration to induce pulmonary inflammation.¹²⁵ There was also decreased expression of F4/80 (a marker of macrophages) mRNA expression by CBD in heart tissue in experimental autoimmune myocarditis.¹²⁶ Although this study identified CBD only affecting F4/80 mRNA expression as opposed to F4/80 cell surface staining, it does suggest a novel target (i.e., heart tissue) of CBD in a relatively understudied autoimmune model.

IL-6 is a proinflammatory cytokine produced by many cell types, predominantly innate cells. Many studies have shown that circulating IL-6 is readily inhibited by CBD in inflammatory models, including diabetes,¹²⁴ asthma,¹²⁷ pancreatitis,¹²⁸ and hepatitis.⁸⁶ CBD treatment in vivo resulted in lower IL-6 production in peritoneal macrophages stimulated ex vivo with LPS,¹²⁴ in the pancreas in acute pancreatitis,¹²⁸ and in bronchoalveolar lavage fluid in LPS-induced pulmonary inflammation.¹²⁵

There have been some reports that CBD alters neutrophil function. Compromised MPO activity by CBD has been studied in several tissues, including brain,¹²⁹ colon,^130,131 lung,^125,128,132 and pancreas.¹²⁸ Interestingly, in the pulmonary inflammation studies with LPS, neutrophil cell counts in the bronchoalveolar lavage fluid were also decreased by CBD compared to LPS.^125,132 Together, the results suggest that CBD's mechanism for neutrophil suppression involves both decreased numbers of neutrophils and compromised MPO activity.

There are two recent studies focused on CpG stimulation of IFN-α production from human plasmacytoid dendritic cells.^133,134 While these studies are focused primarily on THC and other CB₂ agonists, CBD was also used (1–10 μM) and did not affect IFN-α production.^133,134 It was interesting, however, that CBD suppressed the CD83 dendritic cell activation marker on dendritic cells derived from HIV⁺, but not healthy, individuals.¹³⁴ Reduction in dendritic cell CD83 signaling can compromise T cell function,¹³⁵ although additional studies using CBD in human dendritic cells and T cells are needed to establish the consequences of CBD-induced reduction in CD83 on HIV⁺ dendritic cells.

Another mechanism by which CBD controls immune function is induction of regulatory cells. MDSC are innate, myeloid cells that possess the ability to control immune responses. Hegde et al. demonstrated that CBD induced CD11b⁺Gr-1⁺ MDSCs in the liver in a mouse hepatitis model.⁸⁶ Importantly, the isolated MDSCs were functional, that is, they suppressed proliferation of responder T cells ex vivo and improved liver function when administered before hepatitis induction.⁸⁶ CBD-induced MDSCs from the peritoneal cavity were able to attenuate inflammation in response to LPS.¹³⁶ In the experimental autoimmune encephalomyelitis (EAE) model, CBD induced MDSCs in the peritoneal cavity, but decreased the infiltration of MDSCs in the spinal cord and brain.¹³⁷ CBD-induced MDSCs from the peritoneal cavity were able to attenuate responder T cell proliferation ex vivo and attenuate EAE disease when administered in vivo.¹³⁷

CBD effects and mechanisms of immune suppression in lymphocytes

The area in which most of the effects of CBD in the immune system have been studied is T cells. Early studies examining rosette formation in response to sheep red blood cells (sRBCs) (generally considered to be a T cell response) revealed that CBD (1 and 100 μM) reduced this response.¹³⁸ Phytohemagglutinin (PHA)-stimulated IFN-γ production in T cells has also been shown to be inhibited by CBD (0.01–20 μg/mL or 0.03–64 μM).^111,112 Other studies have provided further evidence that T cell-produced IFN-γ is a critical target of CBD suppression. CBD inhibited IFN-γ production from lymph node cells isolated from arthritic mice stimulated ex vivo with collagen,¹³⁹ and from splenocytes isolated from NOD mice stimulated ex vivo with ConA.^123,124 IFN-γ production from splenocytes isolated from untreated mice was suppressed by CBD following ex vivo stimulation with phorbol 12-myristate 13-acetate/ionomycin (PMA/Io).¹⁴⁰ In the latter study, a 1-h exposure of CBD to the mice was meant to mimic the time for CBD distribution before receiving antigen sensitization with ovalbumin to induce asthma-like disease.¹⁴⁰ Thus, CBD's ability to compromise various cytokines at the time of antigen sensitization might suggest that CBD affects primary activation of T cells, as has been suggested as part of the mechanism for other cannabinoids, such as THC.¹⁴¹ Indeed, we have shown that a 30-min pre-treatment with CBD (0.1–20 μM) suppressed IFN-γ production in mouse splenocytes in response to PMA/Io or anti-CD3/CD28.⁵⁵ In these studies, it was shown that the mechanism by which CBD suppressed IFN-γ occurred at the level of transcription and that two important transcription factors for IFN-γ, activator protein-1 (AP-1) and nuclear factor of activated T cells (NFAT), were inhibited by CBD, suggesting a transcriptional mechanism for suppression.⁵⁵ CBD-induced suppression (0.1–10 μg/mL or 0.3–32 μM) of Ifng mRNA expression was shown using PHA-stimulated human PBMCs.¹⁴² Given the many reports that IFN-γ seems to be a sensitive target of suppression by CBD, it was surprising that Ifng mRNA was not affected by CBD (5 μM) using encephalitogenic T cells stimulated by antigen-presenting cells (APCs) and myelin oligodendrocyte glycoprotein peptide (MOG_35–55) in vitro.¹⁴³ However, CBD did inhibit expression of IFN-γ receptor 1 and CBD increased several IFN-γ-responsive genes known to attenuate T cell proliferation.¹⁴³ Overall, the data reveal that an important part of CBD's action in the immune system is its ability to affect IFN-γ in multiple ways. Not only did CBD directly suppress IFN-γ production through a transcriptional mechanism under several conditions^55,142 but also suppressed IFN-γ receptor expression, and increased IFN-γ-induced genes that subsequently attenuate other immune targets.¹⁴³

A few other T cell-derived cytokines have been shown to be targets of CBD. As noted above, IL-6 is a critical target of CBD in many cells and tissues,^{82,84,86,97,125–128,132} many of which are innate cells. However, IL-6 was also suppressed by CBD (5 μM) using encephalitogenic T cells stimulated by APCs and MOG_35–55 in vitro,¹⁴⁴ and “IL-6 signaling” as a critical pathway suppressed by CBD.¹⁴³ Interestingly, “IL-17 signaling” was also identified as a critical pathway suppressed by CBD (5 μM) in T cells in vitro.¹⁴³ It should be noted that IL-6 promotes the differentiation of TH17 cells,¹⁴⁵ so the simultaneous suppression of IL-6 and IL-17A by CBD is consistent with CBD suppressing TH17 cell differentiation. Indeed, CBD (1–20 μg/mL or 3.2–64 μM) suppressed IL-17A production in human CD3⁺ T cells (derived from healthy patients or patients with MS or nonseminomatous germ cell tumors) stimulated ex vivo with PMA/Io.¹⁴⁶ Taken together with the data described in innate cells above, it is clear that CBD's action in inflammation and immune function involves suppression of cytokine production from many different cell types.

The ability of CBD to suppress transcription factors such as NFAT, AP-1, and NF-κB likely accounts for its widespread suppression of many cytokines.^{74,82,118–120,147–149} Some of the studies suggest that CBD increased, or perhaps stabilized, expression of IκB as part of the mechanism by which it suppresses NF-κB.^119,120,147 CBD (4 μM) stimulated IκB-α expression in high glucose-treated human coronary artery endothelial cells.¹¹⁹ CBD induced expression of IκB-α in heart tissue from diabetic mice in vivo¹²⁰ and in LPS-stimulated microglial cells in vitro (CBD 1–10 μM).¹⁴⁷ It is interesting that NF-κB activity has not yet been identified as a target in T cells, suggesting that CBD-mediated suppression of NF-κB plays a bigger role in mediating anti-inflammatory effects in non-T cells.

Certainly, some of the dysregulation of these transcription factors is the result of suppression of various kinases upstream of their activation. Extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), and p38 MAPKs have all been identified as targets of suppression by CBD in various cell types.^{74,80–82,118,120} Of these reports, one was conducted in human T cells.⁸⁰ In these studies, CBD (5 μM) was shown to suppress expression of total and phosphorylated p38 at the 16-h timepoint following CBD treatment. The authors also showed that the CBD-mediated inhibition of phosphorylated p38 was reversed by SR1445328 or tocopherol, suggesting that CBD acts through CB₂ and that the mechanism of suppression involves reactive oxygen species (ROS) production.⁸⁰

Although well studied in cancer cell lines and primary tumor tissue, CBD-mediated apoptosis is also a contributor to the immune suppressive mechanism. Initially CBD-induced apoptosis in T cells was described in Jurkat and MOLT4 human T cells.⁸⁰ In the same study, McKallip et al. observed increased apoptosis of mouse lymphoma cells injected into, and then recovered from, the peritoneal cavity of mice that were treated with CBD.⁸⁰ Since then, there has been a series of studies characterizing the mechanisms by which CBD induced apoptosis in mouse immune cells. CBD (1–16 μM) was shown to induce apoptosis in mouse thymocytes and EL-4 T cells.¹⁵⁰ The same group demonstrated that CBD (1–16 μM) induced apoptosis in mouse splenocytes, including assessment of CBD-induced apoptosis by cell type (B220⁺ B cells and CD4⁺ and CD8⁺ T cells).¹⁵¹ In both studies, CBD increased ROS, and CBD-mediated apoptosis was attenuated by N-acetylcysteine.^150,151 Wu et al. further demonstrated that the CBD increased ROS-activated caspase-8 to mediate apoptosis.¹⁵¹ In follow-up studies in human monocytes, Wu et al. noted that CBD (1–16 μM) readily induced apoptosis, but that the effect of CBD on apoptosis was lost if the monocytes were pre-cultured for 72 h.¹¹⁴ The authors suggest that the differential responsiveness to CBD was due to an increase in antioxidant capacity in cultured cells, which is a thought consistent with the mechanism by which CBD induced apoptosis in mouse lymphocytes.^150,151 CBD-induced apoptosis (1–16 μM CBD) in human monocytes was due to a cascade of intracellular events, including opening of the mitochondrial permeability transition pore, depolarization of the mitochondrial membrane potential, oxidation of a lipid in the mitochondrial inner membrane, and mitochondrial ROS generation, leading to cytochrome C release.¹¹⁵ Thus, this latest study demonstrates a critical role of the mitochondria in CBD-induced apoptosis.

Another important mechanism by which CBD acts to control immune responses is through regulatory T cell (Treg) induction. In the ConA model of hepatitis, CBD modestly enhanced Tregs in the liver as quantified by CD4⁺Foxp3⁺ cells.⁸⁶ A confirmation of in vivo induced Tregs by CBD was noted in an ischemia-reperfusion injury model in the kidney, in which CBD returned the disease-induced reduction in CD3⁺Foxp3⁺ cells to baseline.¹⁵² Interestingly, in the ischemia-reperfusion kidney model, CBD also induced “TReg17 cells,” which were defined as CD3⁺Foxp3⁺CCR6⁺STAT3⁺.¹⁵² It has been suggested that Treg17 cells help control a TH17 response. In vitro, CBD (5 μM) induced a CD69⁺LAG⁺ population in CD4⁺CD25⁻ cells, which were identified as one type of regulatory cell, and induced Il10 mRNA expression.¹⁵³ We showed in vitro that CBD (1–15 μM) induced functional CD4⁺CD25⁺Foxp3⁺ T cells under conditions of suboptimal stimulation and that Il10 mRNA expression was induced.¹⁵⁴

There are only a few studies in which B cells are identified as targets of CBD. CBD given at 25 mg/kg by intraperitoneal (i.p.) injection modestly reduced the sRBC-induced plaque-forming cells, which is a measure of antibody production.¹⁵⁵ We conducted a similar study using oral administration of CBD and also found modest inhibition of antibody production.⁵⁵ Other studies have shown that CBD robustly inhibited the sRBC-induced antibody production in vitro,⁵⁵ suppressed ovalbumin-induced IgM, IgG1, and IgG2a in an in vivo asthma model,¹⁴⁰ and reduced expression of activation markers such as major histocompatibility complex II, CD25, and CD69, on B cells.¹⁵³ CBD has also been shown to induce apoptosis in B cells.¹⁵¹ Overall, the results suggest that B cells can be targets of suppression by CBD.

CBD-induced neuroprotection by suppression of microglial cell activation

There is no doubt that many of the mechanisms already identified for innate cells and lymphocytes also account for CBD's ability to decrease microglial cell activation. CBD (1–16 μM) induced apoptosis in microglial cells,¹⁵⁶ which was dependent on activation of caspase 8 and 9, and was reversed in the presence of an agent that depletes cholesterol and disrupts lipid rafts.¹⁵⁶ These results suggest that CBD-induced apoptosis is dependent on lipid raft formation,¹⁵⁶ and indeed, this observation was confirmed by another group in BV-2 microglial cells.¹⁵⁷

BV-2 microglial cells have been used as a model in several articles, in which detailed transcriptional effects of CBD have been evaluated.^{147,157–159} The mechanisms contributing to CBD (10 μM)-mediated suppression of LPS-stimulated cytokine production in microglial cells includes decreased activation of the Toll/IL-1 receptor domain-containing adapter-inducing IFN-β (TRIF)/IFN-β/signal transducer and activator of transcription (STAT) signaling pathway.¹⁴⁷ CBD suppressed LPS-stimulated NF-κB activation, and induced LPS-stimulated STAT3 activation, which has been shown to suppress NF-κB activation.¹⁴⁷ CBD (10 μM) was shown to affect several genes involved in lipid metabolism in unstimulated BV-2 cells,¹⁵⁷ which might account for CBD's ability to increase anandamide^{58,65–67,84,157,160} or could account for CBD's dependence on lipid raft formation to induce apoptosis^156,157 Follow-up studies examining CBD's effects in unstimulated BV-2 cells demonstrated that CBD (10 μM) alters zinc homeostasis, oxidative stress, and glutathione levels in microglial cells.^158,159 A recent study demonstrated that CBD alters microRNA (miRNA) expression,¹⁶¹ and two of the CBD miRNA targets identified are discussed. First, CBD downregulated miR146-a, which acts as a negative regulator of inflammation, in both resting and LPS-stimulated cells, thereby contributing to CBD's ability to downregulate proinflammatory cytokines.¹⁶¹ Second, CBD upregulated miR-34a, which has several roles in cell survival, such as cell cycle, apoptosis, and differentiation.¹⁶¹ These results show that CBD-induced alterations in miRNA expression are involved in the mechanism by which CBD suppresses immune function.

In vivo, CBD has been shown to decrease microglial accumulation in the spinal cord in diabetic mice,⁸¹ which might contribute to attenuation of neuropathic pain, and CBD decreased haloperidol-induced activation of reactive microglial cells.⁹⁷ CBD's suppression of TNF-α production from microglial cells in vitro was mediated by A_2A adenosine receptors in EOC-20 mouse microglial cells (0.5–5 μm)⁸⁹ or rat retinal microglial cells (1 μM).⁹¹

CBD Effects in Autoimmune Disease Models

EAE and MS

The immunosuppressive and neuroprotective mechanisms of CBD make it an ideal therapeutic candidate for MS, a neurodegenerative autoimmune disease of the CNS that affects ∼2.5 million people worldwide. The average age of onset is around 30 years, and symptoms can vary greatly for each patient based on the lesion locations within the CNS.¹⁶² Two models frequently used in the laboratory environment to study MS are the EAE and Theiler's murine encephalomyelitis virus (TMEV) models, and an increasing number of studies have shown promising results with CBD using these models (Table 5). In 2011, Kozela et al. successfully demonstrated that CBD (5 mg/kg i.p.) administered at the onset of disease attenuated clinical disease, microglial activation, and T cell infiltration into the CNS in EAE, and that CBD reduced T cell proliferation in vitro.¹⁶³ CBD showed similar effects in the TMEV model, in which Mecha et al. demonstrated that CBD (5 mg/kg i.p.) administered for the first 10 days following disease onset reduced clinical disease and neuroinflammation by decreasing microglial activation and immune cell trafficking signals in the CNS.¹⁶⁴ Use of MOG_35–55-specific T cells isolated from EAE mice in vitro has also been extremely vital to determining how CBD might be affecting T cells in these and other disease models. As outlined above, in the T cell section, in vitro CBD treatment of MOG_35–55-specific T cells co-cultured with APCs with CBD suppressed IL-17A and IL-6 production, suggesting CBD suppressed TH17 development; however, production of Il10 mRNA was potentiated with CBD treatment, suggesting that CBD may have multiple suppressive mechanisms.¹⁴⁴ In vitro treatment of MOG_35–55-specific T cells with CBD induced a Treg with a CD4⁺CD25⁻LAG3⁺CD69⁺ phenotype, promoted upregulation of anergy-associated genes, such as Lag3, Erg2, and Il10, and altered the balance between STAT3 and STAT5 activation.¹⁵³ In another study, CBD administered during disease onset increased the number of functional MDSCs present within the peritoneal cavity, decreased neuroinflammation, and reduced IL-17A and IFN-γ in the serum.¹³⁷ When splenocytes from these mice were restimulated ex vivo, the CBD-treated mice had significantly decreased levels of IL-17A and IFN-γ, and increased levels of IL-10 in the supernatants.¹³⁷ Finally, a recent study using an adoptive transfer EAE model showed a reduction in neuroinflammation, demyelination, and axonal damage with CBD treatment during disease onset.¹⁶⁵ Adoptive transfer EAE is a variation of the EAE model induced by transfer of encephalitogenic T cells into naive mice, which allows experiments performed with this model to focus more on the T cell-specific mechanisms of pathogenesis in the EAE model. From the accumulation of data, it is obvious that multiple immune cell types, proinflammatory and anti-inflammatory, within the EAE model are modulated by CBD, but overall, CBD appears to downregulate proinflammatory pathways and upregulate anti-inflammatory pathways in the EAE model.

Table 5.

Cannabidiol Effects in Experimental Autoimmune Encephalomyelitis

Model	Approach	Dosage/concentration	Effects	Reference
EAE in ABH	In vivo	In vivo: 0.5–25 mg/kg i.p.	No effects	211
EAE in C57BL/6	In vivo and in vitro	In vivo: 5 mg/kg i.p. in vitro: 1, 5, and 10 μM	in vivo: ↓disease severity, ↓T cell infiltration into the CNS, ↓microglial activation, ↓axonal damage in vitro: ↓T cell proliferation	163a
TMEV in SJL/J	In vivo and in vitro	In vivo: 5 mg/kg i.p. in vitro: 1 and 5 μM	in vivo:↓disease severity, ↓leukocyte infiltration into the CNS, ↓microglial activation, ↓CCL2 (MCP-1), ↓CCL5, ↓IL-1β ↓TNF-α in vitro: ↓sVCAM-1 production from endothelial cells, ↓leukocyte adhesion, ↓CCL2 (MCP-1)	164a
MOG35–55-specific T cells from EAE mice	In vitro	In vitro: 0.1, 1, and 5 μM	in vitro: ↓IL-17A, ↓IL-6, ↑IL-10	144a
MOG35–55-specific T cells from EAE mice	In vitro	In vitro: 5 μM	in vitro:↓IL-17A, ↓IL-6, ↑IL-10, ↑EGR2, ↑CD4+CD25−CD69+LAG3+ phenotype, ↑STAT5/↓STAT3, ↓B cell activity, ↑Nfatc1, ↑Casp4, ↑Cdkn1a, ↑Icos, ↑Fas	153a
EAE in C57BL/6	In vivo	In vivo: 5 mg/kg i.p.	in vivo: ↓disease severity, ↓leukocyte invasion, ↓demyelination, ↓TNF-α, ↓IFN-γ, ↓IL-17A	212
EAE in C57BL/6	In vivo	In vivo: 10 mg/kg i.p.	in vivo: ↓disease severity, ↓FAS ligand, ↓ERK phosphorylation, ↓Caspase-3 activity, ↓Bax/↑Bcl-2, ↓p53-p21 activation, ↓apobody formation	166a
MOG35–55-specific T cells from EAE mice	In vitro	In vitro: 5 μM	in vitro: ↓IL-1β, ↓IL-3, ↓Xcl1 mRNA, ↓IL-12a mRNA, ↑Dusp6 mRNA, ↑Btla mRNA, ↑Lag3 mRNA, ↑Irf4 mRNA, ↑IL-10 mRNA	143a,b
EAE in C57BL/6	In vivo	In vivo: 10 mg/kg i.p.	in vivo: ↓disease severity, ↓leukocyte infiltration, ↑PI3k/Akt/mTOR phosphorylation, ↑S6k phosphorylation, ↑BDNF expression, ↑PPAR-γ, ↓IFN-γ, ↓IL-17A, ↓JNK activity, ↓p38 MAP kinase activity	167a
Adoptive Transfer EAE in C57BL/6	In vivo and in vitro	In vivo: 5–50 mg/kg i.p in vitro: 1, 5 & 10 μM	in vivo: ↓disease severity, ↓leukocyte invasion, ↓demyelination, ↓axonal damage, ↓microglial activation, ↓CB2 receptor expression in CNS, ↓GPR55 receptor expression in CNS in vitro: ↓Cell viability, ↓IL-6, ↑apoptosis, ↑ROS	165a
EAE in C57BL/6	In vivo	In vivo: 20 mg/kg i.p	in vivo: ↓disease severity, ↓leukocyte invasion, ↓IL-17A, ↓IFN-γ, ↓RORγT, ↓T-bet, ↑IL-10, ↑MDSC ex vivo: ↓IL-17A, ↓IFN-γ, ↑IL-10	137a

^aDiscussed in review.

^bSee¹⁴⁰ for a full description of the microarray results.

CNS, central nervous system; EAE, experimental autoimmune encephalomyelitis; ERK, extracellular signal-regulated kinase; STAT, signal transducer and activator of transcription; TMEV, Theiler's murine encephalomyelitis virus.

In addition to its immunomodulatory effects, CBD's neuroprotective properties in the EAE model also indicate its therapeutic potential in MS. CBD has been shown to decrease the activation of proapoptotic proteins, such as caspase-3 and Bax,¹⁶⁶ and to counteract the effects of EAE on the PI3K/Akt/mTOR pathway, JNK, and p38 MAP kinases in the CNS of EAE mice.¹⁶⁷ Interestingly, the study from Giacoppo et al. found the PI3k/Akt/mTOR pathway was upregulated in neural tissues when EAE mice were treated with CBD.¹⁶⁷ However, Kozela et al.¹⁵³ observed a reduction in the activation of Akt in vitro in MOG_35–55-reactive T cells, which might suggest a differential role for CBD's effects on the PI3K/Akt/mTOR pathway in various cell types.

Despite the growing number of studies involving the neuroprotective and immunosuppressive effects of CBD, the majority of human studies involving cannabinoids and MS have been focused on the use of THC:CBD mixtures, with a particular focus on Sativex. Clinical studies that have been performed have shown that Sativex has beneficial effects on spasticity, mobility, bladder function, and pain in MS patients, and is well tolerated^{22,25,28,31,168–175}; however, there has been little focus on the neuroprotective and immunosuppressive effects of THC:CBD mixtures in MS, and so it is difficult to say at this point if the successful results observed with CBD in the animal models of MS will be observed in MS patients. For a more complete review on the effects of Sativex in MS, see Zettl et al.¹⁷⁶

Other autoimmune disease states

CBD has been shown to attenuate experimental autoimmune hepatitis,⁸⁶ experimental autoimmune myocarditis,¹²⁶ and autoimmune diabetes^123,124 in mice. There are few studies done with CBD only in human autoimmune diseases. In human patients, CBD at 20 mg/kg did not reduce clinical Crohn's disease.¹⁷⁷ However, CBD is effective at attenuating intestinal inflammation in other models of human inflammatory bowel disease,^82,96 so it is possible that CBD could be effective at higher doses. Indeed, CBD as Epidolex for epilepsy in children is being used as high as 20 mg/kg, but CBD doses as high as 300 mg/kg have been evaluated, and have not exhibited significant adverse effects.¹⁷⁸

CBD Immune Enhancement Effects

Much of the data support the fact that CBD is immune suppressive and anti-inflammatory; however, there have been a few reports over the years that CBD has produced some immune enhancing effects (Table 6). The potential for CBD, and other cannabinoids, to produce immune enhancing effects has been attributed to differences in hormetic (i.e., biphasic) responses depending on CBD concentration/dose, cell culture conditions, including serum presence and/or percent, immune stimulant, and magnitude of cellular activation in response to the immune stimulant. Indeed, studies from our laboratory and others have shown that CBD either enhanced or suppressed cytokine production (IL-2 and IFN-γ) in response to relatively low or high degree of immune stimulation, respectively.^154,179,180 The mechanism for the differential responsiveness likely involves alterations in intracellular calcium, as CBD increases intracellular calcium in mouse splenocytes regardless of the increase of intracellular calcium produced by the immune stimulant.¹⁷⁹ In addition, the differential cytokine production was correlated with nuclear expression of the NFAT transcription factor,¹⁷⁹ which is calcium responsive. Interestingly, CBD's ability to increase intracellular calcium also likely accounts for some of the other enhancing effects, including stimulation of neutrophil degranulation,¹⁸¹ chemotaxis,¹⁸² and mast cell/basophil activation.¹⁸³

Table 6.

Immune Enhancement by Cannabidiol

Cell type/model	In vivo	Effect	Reference
Male human subjects	X	↑antibody response	213
Rabbit neutrophilsa		↑neutrophil degranulation	181b
Female Hartley guinea pigs	X	↑skin sensitization	214
Female B6C3F1 mouse splenocytes		↑IL-2 production	180
Mouse BV-2 microglial cellsc		↑chemotaxis	182b
Male Swiss mouse peritoneal macrophages		↑IL-12 production	187
Male Swiss mouse peritoneal macrophages	X	↑IL-12 production (stimulated ex vivo)	187
Rat RBL-2H3 mast cellsc		↑mast cell/basophil activation	183b
Female B6C3F1 and C57BL/6 mouse splenocytes		↑IL-2 and IFN-γ production	179b
Female C57BL/6 mice	X	↑LPS-induced pulmonary inflammation	191b
Mouse BV-2 microglial cellsc, mouse RAW264.7 macrophage cellsc, rat HAPI microglial cellsc, male C57BL/6 microglial cells		↑phagocytosis	105
Female C57BL/6 splenocytes		↑IL-2 production	154b

^aSex not stated.

^bDiscussed in review.

^cCell line.

In addition to the ones listed in Table 6, there are a few well-studied end-points for which CBD treatment has produced opposing effects, one of which is apoptosis. As described in detail above, part of the mechanism by which CBD produces immune suppression is induction of apoptosis; however, there are a few studies in which CBD has inhibited inflammation-induced apoptosis.^118,120 Interestingly, the reports of CBD on oxidative stress are different across studies, with some articles identifying CBD as an antioxidant,^{59,118,184,185} and others reporting that CBD induces oxidative stress.^{114,115,150,151} CBD-mediated effects on IL-10 production also revealed opposing effects when compared across several studies.^{95,123,124,127,148,186–189} Effects of CBD on IL-10 could be tied to regulatory cell production (i.e., Tregs or MDSCs) or changes in T cell subpopulations.

Although it is not entirely clear why CBD produces opposing effects for many end-points, a critical part in understanding the mechanisms of CBD involves investigation of the consequences of all the changes. Let us consider two examples, the first of which was introduced in the section on cytokines (IFN-γ). We know that IFN-γ is a critical target of suppression by CBD,^{55,111,112,123,124,139,140,142,143} but there are some conditions under which CBD had no effect¹⁴³ or enhanced it.¹⁷⁹ Perhaps under some conditions, the CBD-induced enhancement of IFN-γ would increase the IFN-γ-responsive genes that attenuate T cell proliferation, as suggested by Kozela et al.¹⁴³ Thus, although CBD increased a “proinflammatory” cytokine, its consequence could be immune suppression. The second example is IL-2, which was enhanced under conditions of low-level T cell stimulation.^154,179,180 We recently showed that CBD, by producing IL-2 under some conditions, contributed to the appropriate milieu to drive Treg induction,¹⁵⁴ again demonstrating that enhancement of seemingly proinflammatory cytokines by CBD still resulted in immune suppression.

Conclusions, Challenges, and Knowledge Gaps

Considering all the studies conducted on immune responses and inflammation, the data overwhelmingly demonstrate that CBD is immune suppressive and anti-inflammatory (Fig. 1). Critical targets of suppression include cytokines such as TNF-α, IFN-γ, IL-6, IL-1β, IL-2, IL-17A, and chemokines, such as CCL-2. The overall mechanism of CBD involves direct suppression of target cells, such as effector T cells and microglial cells, through suppression of kinase cascades and various transcription factors. An example of this is CBD-induced suppression of phosphorylated p38, leading to compromised AP-1 or NF-κB activity. Direct suppression of target cells also includes induction of IκB, which could contribute to decreased NF-κB activity. The involvement of regulatory cell induction by CBD is also a major part of the mechanism by which CBD controls immune responses, and CBD has been shown to induce Tregs and MDSCs. Finally, CBD-induced apoptosis is likely an important mechanism in many target cells.

FIG. 1.

Summary of CBD's mechanisms of immune suppression. Overall, CBD's immune system suppression is mediated by direct inhibition of various cell types (microglial, innate, and T cells) and induction of apoptosis and regulatory cells (Tregs and MDSCs). CBD, Cannabidiol; MDSCs, myeloid-derived suppressor cells; Treg, regulatory T cell.

It is often argued that the concentrations/doses at which CBD acts in vitro/in vivo are high. However, it should be noted that CBD is highly lipophilic and subject to first-pass metabolism after oral dosing.¹⁹⁰ In fact, we have shown that in mice at 6 h after oral CBD at 75 mg/kg/day for 3 days resulted in plasma CBD levels of ∼40 ng/mL and were not detectable by 24 h.¹⁹¹ This is ∼0.12 μM CBD, which is on the lower end of concentrations typically used in vitro to evaluate the effects of CBD as detailed in this review. On the other hand, recent data obtained in one human clinical trial that was used to support the indication of CBD as Epidiolex in epilepsy studies showed that plasma CBD levels were as high as 400 ng/mL following 20 mg/kg/day dosing for 22 days.¹⁹² This concentration is ∼1.2 μM CBD, which is a more common concentration used in vitro at which CBD effects are observed. These two studies in mice and humans^191,192 suggest that the doses and concentrations of CBD used in many of the studies in this review are appropriate. There are still limitations on our knowledge about CBD dosing and plasma levels and how those relate to immune modulation. Some of these limitations might be clarified with many of the planned clinical trials with CBD in the coming years. Specifically related to immune effects of CBD, there is a planned randomized, open-label interventional study assessing CBD and THC on immune cell activation in HIV⁺ patients.¹⁹³ Importantly, this trial will evaluate dose escalation of relatively high CBD doses compared to THC; the dose escalation for CBD will go from 45–225 mg/kg/day over a 5-week period and then maintain the highest dose for an additional 7 weeks.¹⁹³

In addition to the need for more data on CBD dosing and pharmacokinetics, this broad summary of immune and inflammatory effects of CBD revealed a number of data gaps that should be addressed. First, identification of the receptor(s) through which CBD acts in the immune system remains a critical question. An important part of this question is whether CBD-induced FAAH inhibition generates anandamide metabolites that bind to various receptors to mediate some of the immune suppressive or anti-inflammatory effects of CBD. Coupled with the observation that some of the effects of CBD can be attenuated with PPAR-γ antagonists,^92–98 the possibility exists that CBD-mediated anandamide production drives the subsequent production of (yet unidentified) metabolites that activate PPAR-γ. Another critical determination needed for many of the receptor studies is identification of the cell type(s) on which the receptors are expressed, which are mediating the CBD effects. Second, although combined cannabinoids were not a major focus of this review, it will be critical to determine the CBD contribution to immune function compromise in cannabis and/or combined pharmaceuticals such as Sativex. Third, there are still several cell types for which little data exist, notably B cells and dendritic cells. Even in the rich CBD-T cell literature, several well-established targets have not been extensively studied in T cells. In fact, there are limited data examining CBD's effects on various T cell subsets. Fourth, increasing our understanding of CBD's effects in response to a variety of immune stimuli and degrees of immune stimulation will help in interpreting effects of CBD in humans and other outbred species that are naturally exposed to a variety of pathogens. Thus, the last identified knowledge gap is the need for increased studies on the effects of CBD in human and veterinary immune responses. These include well-controlled studies considering differences with administration routes, dose, and pharmacokinetics.

Abbreviations Used

AP-1

activator protein-1

APC

antigen-presenting cell

CBD

Cannabidiol

CNS

central nervous system

DNBS

dinitrobenzene sulfonic acid

EAE

experimental autoimmune encephalomyelitis

ERK

extracellular signal-regulated kinase

FAAH

fatty acid amide hydrolase

IFN-γ

interferon-gamma

IL

interleukin

i.n.

intranasal

iNOS

inducible nitric oxide synthase

i.p.

intraperitoneal

JNK

c-jun N-terminal kinase

LPS

lipopolysaccharide

MDSCs

myeloid-derived suppressor cells

miRNA

microRNA

MOG

myelin oligodendrocyte glycoprotein

MPO

myeloperoxidase

MS

multiple sclerosis

NFAT

nuclear factor of activated T cells

NF-κB

nuclear factor-κB

PHA

phytohemagglutinin

PMA/Io

phorbol 12-myristate 13-acetate/ionomycin

PPAR-γ

peroxisome proliferator-activated receptor gamma

ROS

reactive oxygen species

sRBC

sheep red blood cell

STAT

signal transducer and activator of transcription

THC

Δ⁹-tetrahydrocannabinol

TMEV

Theiler's murine encephalomyelitis virus

TNBS

2,4,6-trinitrobenzene sulfonic acid

TNF-α

tumor necrosis factor-alpha

Treg

regulatory T cell

TRPV1

transient receptor potential vanilloid 1

VCAM-1

vascular cell adhesion molecule-1

Author Disclosure Statement

No competing financial interests exist.

Funding Information

Funding provided by Mississippi State University College of Veterinary Medicine.

Cite this article as: Nichols JM, and Kaplan BLF (2020) Immune responses regulated by cannabidiol, Cannabis and Cannabinoid Research 5:1, 12–31, DOI: 10.1089/can.2018.0073.