Manipulation of the endocannabinoid system in colitis: A comprehensive review

Abstract

Background

Inflammatory bowel disease is a lifelong disease of the gastrointestinal tract whose annual incidence and prevalence is on the rise. Current immunosuppressive therapies available for treatment of inflammatory bowel disease offer limited benefits and lose effectiveness, exposing a significant need for the development of novel therapies. In the clinical setting, cannabis has been shown to provide patients with inflammatory bowel disease symptomatic relief, although the underlying mechanisms of their anti-inflammatory effects remains unclear.

Methods

This review reflects our current understanding of how targeting the endocannabinoid system, including cannabinoid receptors 1 and 2, endogenous cannabinoids anandamide and 2-arachidonoylglycerol, atypical cannabinoids, and degrading enzymes including fatty acid amide hydrolase and monoacylglycerol lipase, impacts murine colitis. In addition, the impact of cannabinoids on the human immune system is summarized.

Results

Cannabinoid receptors 1 and 2, endogenous cannabinoids, and atypical cannabinoids are upregulated in inflammation, and their presence and stimulation attenuates murine colitis, while cannabinoid receptor antagonism and cannabinoid receptor deficient models reverse these anti-inflammatory effects. In addition, inhibition of endocannabinoid degradation via monoacylglycerol lipase and fatty acid amide hydrolase blockade can also attenuate colitis development, and is closely linked to cannabinoid receptor expression.

Conclusions

While manipulation of the endocannabinoid system in murine colitis has proven to be largely beneficial in attenuating inflammation, there is a paucity of human study data. Further research is essential to clearly elucidate the specific mechanisms driving this anti-inflammatory effect for the development of therapeutics to target inflammatory disease such as inflammatory bowel disease.

Introduction

Inflammatory bowel disease (IBD) is a complex, multifactorial, life-long disease of the gastrointestinal (GI) tract that is divided into 2 predominant phenotypes: Crohn’s disease (CD) and Ulcerative colitis (UC). IBD is characterized by chronic inflammation of the GI tract and causes symptoms of abdominal pain, bloody diarrhea, and weight loss, as well as symptoms specific to pediatric-onset IBD including growth failure, impaired bone mineralization and pubertal delay. With an estimated annual disease-attributable direct cost in the United States in excess of $6.3 billion¹ and an estimated 1.6 million affected people in the United States², IBD is one of the most prevalent and costly GI disorders whose annual incidence and prevalence is on the rise. IBD places a significant burden on affected individuals because of the significant effects on quality of life, growth and development, and risk of malignancy later in life³. The cause of IBD and its flare-ups is thought to involve dysregulation of the immune response, and the conventional treatment includes immunosuppression to induce and maintain remission. Current immunosuppressive therapies available for the treatment of IBD offer limited benefits, lose effectiveness rapidly, and carry multiple long-term risks including malignancy, infection, and low bone density⁴. These faults of the available therapies as well as their known complications expose a significant need for the development of highly novel therapies for the treatment of IBD.

One pathway that may offer a novel approach to enhance the impaired host regulatory system in IBD patients is the endogenous cannabinoid (endocannabinoid) pathway. While cannabinoids are traditionally characterized as being synonymous with plant-derived Cannabis or marijuana (MJ), there is an innate, mammalian endocannabinoid system that includes endogenous ligands termed endocannabinoids, their cannabinoid receptors, and the proteins involved in endocannabinoid biosynthesis and degradation. Several physiological effects and pathophysiological roles have been proposed for the endocannabinoid system in the GI tract, including effects on epithelial growth and regeneration, immune function, motor function, appetite control, and secretion⁵. Anecdotal and limited scientific evidence suggests that MJ use may have a positive impact on IBD patients due to its analgesic and anti-inflammatory effects⁶. Cannabinomimetics can provide IBD patients symptomatic relief by improving appetite, stimulating weight gain, reducing abdominal pain and decreasing intestinal motility but their anti-inflammatory function remains unclear⁷. Due to legalization of medical MJ and its increased use in the setting of inflammatory diseases, this uncertainty has sparked a focus on basic science research to further elucidate the biologic effects of manipulation of the endocannabinoid system. In this review, we will focus on defining the endocannabinoid system, and highlight the current data from targeting of the cannabinoid receptors 1 and 2, endogenous cannabinoids, atypical cannabinoids, and the enzymes of degradation in murine colitis.

The Endocannabinoid System (ECS)

The endocannabinoid system is comprised of endogenous ligands termed endocannabinoids, their cannabinoid receptors, and the proteins involved in endocannabinoid biosynthesis and degradation. The primary endocannabinoids are arachidonoyl ethanolamines, also known as anandamide (AEA), and 2-arachidonoylglycerol (2-AG). These endogenous ligands are lipid mediators that, in contrast to classic neurotransmitters which are stored in vesicles, are synthesized on-demand from membrane precursors and phospholipids⁸ including N-acyl-phosphatidylethanolamine-selective phospholipase D (NAPE-PLD), then released from cells immediately after production⁵. They activate receptors to elicit a biologic response, then become inactivated through reuptake by carrier proteins in the cell membrane named the endocannabinoid membrane transporters (EMT), followed by enzymatic degradation. AEA is degraded by fatty acid amide hydrolase (FAAH) in to arachidonic acid and ethanolamine and 2-AG is degraded by monoacylglycerol lipase (MAGL) in to arachidonic acid and glycerol⁵.

The endocannabinoids AEA and 2-AG act primarily on two heterotrimeric G-protein coupled receptors, the cannabinoid 1 (CB₁) receptor and cannabinoid 2 (CB₂) receptor. CB₁ receptors are located primarily on central and peripheral neurons, and more specifically in the enteric nervous system, in the intrinsic neurons, extrinsic neurons such as the cell bodies of sensory neurons in the dorsal root ganglia and nodose ganglion, and vagal efferent nerves, as well as on epithelial cells⁸. In neurons, endocannabinoids act mainly presynaptically, modulating the transmission of other neurotransmitters including γ-aminobutyric acid, glutamate and acetylcholine⁸. CB₂ receptors are mainly expressed on immune cells, specifically, neutrophils, activated macrophages, and subsets of T and B cells, as well as on epithelial cells⁹. CB₁ receptors modulate neurotransmitter release, while CB₂ receptors are mainly associated with immune functions⁵. In addition to signaling through CB₁ and CB₂ receptors, endocannabinoids also activate the transient receptor potential vanilloid type 1 (TRPV1), which is mainly expressed by primary afferent neurons, as well as the orphan G protein-coupled receptor GPR55⁵ (Figure 1).

Figure 1. Colonic distribution of classical cannabinoid receptors CB₁ and CB₂.

CB₁ receptors (CB₁R) are located in the intrinsic neurons, extrinsic neurons such as the cell bodies of sensory neurons in the dorsal root ganglia and nodose ganglion, and vagal efferent nerves within the enteric nervous system, as well as on epithelial cells. CB₂ receptors (CB₂R) are expressed on epithelial cells and immune cells including neutrophils, activated macrophages, and subsets of T and B cells.

Other endogenous molecules exhibit "cannabinoid-like" effects and may be termed atypical endocannabinoids, including 2-arachidonoylglycerol ether (noladin ether), N-arachidonoyl dopamine (NADA), virodhamine, N-homo-γ-linolenoyl-ethanolamine (HEA) and N-docosatetraenoyl-ethanolamine (DEA), palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). These additional molecules do not seem to bind to cannabinoid receptors but rather to non-CB₁ or CB₂ receptors or to a specific isozyme belonging to a class of nuclear receptors/transcription factors with three subsets, known as the alpha, beta (delta), or gamma peroxisome proliferator-activated receptors (PPARs). However, these atypical endocannabinoids may augment the effect of AEA by competitive inhibition of FAAH, and/or through allosteric effects on other receptors such as ion channel TRPV1, and thus play an important, although less defined role in the endocannabinoid system¹⁰.

Cannabinoid 1 Receptor

CB₁ receptor expression is significantly increased in models of inflammation, with increased expression demonstrated by Western Blot analysis in croton oil-induced inflammation of the jejunum¹¹, by increased mRNA expression in dinitrobenzene sulfonic acid (DNBS)-induced colitis¹², dextran sulphate sodium (DSS) induced murine colitis¹³ and in lipopolysaccharide (LPS) stimulated peritoneal macrophages¹⁴, and was shown by immunostaining of colonic sections to be increased in myenteric ganglia in both oil of mustard (OM) and DSS-induced colitis, as well as in the endothelium of OM-induced colitis¹⁵. In addition, mRNA expression of CB₁ receptor is upregulated in both DNBS-induced colitis and control mice when treated with the atypical cannabinoid PEA¹⁶. Based on this link of upregulated expression of the CB₁ receptor in inflammation, many studies have sought to demonstrate the key role of the CB₁ receptor in attenuation of murine colitis. CB₁ receptor agonist ACEA attenuated inflammation in OM and DSS-induced colitis models¹⁵. Massa et al showed that treatment with CB₁ receptor agonist R(-)-7-hydroxy-Δ6-tetra-hydrocannabinol-dimethylheptyl (HU210) in DNBS-induced colitis consistently resulted in protection against colitis¹². Furthermore, WIN55, 212-2, a weakly selective CB₁ receptor agonist, protects against TNBS-induced colitis¹⁷ in one particular study; however, this model does not reflect the inflammatory cascade found in human disease. WIN 55, 212-2 was also found to be protective against DSS-induced colitis in WT mice^17,18, and this protective effect is further enhanced in C57BL Mk2^−/− mice, demonstrating an amplified benefit with simultaneous activation of the CB₁ pathway while blocking p38/Mk2 pro-inflammatory pathway¹⁸.

Genetically engineered knockout mouse models have been instrumental in elucidating the CB₁ receptor pathways of the endocannabinoid system. Both 2,4,6-trinitrobenzene sulfonic acid (TNBS) and DSS treatment induced more severe colitis in CB₁^−/− mice than in wild type (WT) littermates, and treatment with the specific CB₁ antagonist N-(piperidino-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-pyrazole-3- carboxamide (SR141716A) mimicked the phenotype of CB₁^−/− mice, showing an acute requirement of CB₁ receptors for protection from inflammation¹². Engel et al reaffirmed this finding several years later by showing that TNBS-induced colitis in CB₁^−/− mice had a higher stool score, increased colon weight, a shorter colon, higher macroscopic and histologic scores, and increased mRNA expression of IL-1β and TNF-α, indicating a more severe colitis in the absence of the CB₁ receptor¹⁹.

Many compounds act via the endocannabinoid system in a broader sense and only recently have the pathways via CB₁ receptor been more clearly explained. One such study demonstrated that oral treatment with α,β-amyrin, a pentacyclic triterpene, reduced leukocyte infiltration and pro-inflammatory cytokine production, and reduced inhibition of adhesion molecule expression¹³. These changes indicated that α,β-amyrin attenuated DSS-induced colitis, and this protective and therapeutic effect was partially reversed with the use of CB₁ receptor antagonist AM251, while CB₂ antagonist aminoalkylindole 6-iodo-pravadoline (AM630) failed to reduce the anti-inflammatory benefit of α,β-amyrin¹³. In addition, the enzymatic degradation pathway of the compound PF-3845, a potent and selective FAAH inhibitor, is mediated by the CB₁ receptor pathway, as administration of CB₁ receptor antagonist AM251 blocked the anti-inflammatory effect of PF-3845 in TNBS-induced colitis while CB₂ blockade with CB₂ selective antagonist AM630 had no effect on inflammation²⁰.

Overall, while these described studies provided no strong consensus of the mechanism of CB₁-mediated attenuation of intestinal inflammation, in vitro studies describing a CB₁-selective improvement in intestinal epithelial barrier restitution following inflammatory insult suggest that this may represent one mechanism through which CB₁ receptors are protective²¹.

Cannabinoid 2 Receptor

Mirroring the upregulation of CB₁ receptors in inflammation, CB₂ receptor mRNA expression is increased in chemically-induced colitis²², and CB₂ receptors are highly expressed on infiltrated immune cells in colonic sections in OM-induced colitis and to a lesser extent, in DSS-colitis as demonstrated by immunostaining¹⁵. However, in vitro LPS stimulation of peritoneal macrophages led to decreased mRNA expression of CB₂ receptors in one study¹⁴.

Furthermore, CB₂ receptors have also been shown to play a critical role in the attenuation of murine colitis. Both plant derived Δ9-tetrahydrocannabinol (THC) and endogenous cannabinoid AEA demonstrated an anti-inflammatory effect in in vitro cell assays of antibody formation, and these effects were blocked by CB₂ receptor antagonist SR144528 but not by CB₁ receptor antagonist SR141716²³. Moreover, CB₂ agonist JWH-133 has been shown to inhibit LPS/IFN-γ induced pro-inflammatory cytokine IL-12p40 in vitro²⁴, and induce apoptosis in activated T cells both in vivo and in vitro²⁵. Additionally, CB₂ activation with JWH-133 also ameliorates inflammation in IL10^−/− colitic mice²⁵, as well as in chemically-induced colitis^15,25. These protective effects of JWH-133 in DSS-colitis were reversed in the presence of CB₂ receptor antagonist AM630²⁵. Similar findings were demonstrated in TNBS-induced colitis; CB₂ agonists JWH-133 and AM1241 significantly reduced murine colitis, and CB antagonist AM630 exacerbated colitis and abolished the anti-inflammatory effect of JWH-133 when given as a pre-treatment in the colitis model²². Genetically engineered CB₂ receptor knockout mice further confirmed the importance of the CB₂ pathway in attenuation of murine colitis. In the previously described study, neither JWH-133 nor AM1241 maintained their anti-inflammatory effects on TNBS-induced colitis in CB₂^−/− mice²². Engel et al’s 2010 study reiterated the importance of the CB₂ receptor pathway by showing that TNBS-induced colitis in CB₂^−/− mice had increased mRNA expression of IL-1β and TNF-α and a more severe colitis in the absence of the CB₂ receptor¹⁹.

The CB₂ receptor pathway also modulates the effects of several atypical cannabinoids. In the DNBS-induced colitis model, the beneficial effects of treatment with atypical cannabinoid PEA were abolished by CB₂ antagonist AM630 (as well as by GPR55 and PPAR antagonists), but the CB₁ antagonist rimonabant did not have any effect¹⁶. The CB₂ receptor pathway was also found to modulate the favorable effects of cannabigerol (CBG), a non-psychotropic cannabinoid capable of reducing nitric oxide production in macrophages and attenuating murine DNBS-induced colitis in both a preventive (pretreatment) model and therapeutic (established colitis) model²⁶. In these models, treatment with CB₂ antagonist SR144528 further increased the inhibitory effect of CBG on nitric oxide production²⁶. Finally, the plant metabolite β-caryophyllene (BCP) ameliorates DSS- and oxazolone-induced colitis in a dose dependent manner, and this effect was reversed in the presence of CB₂ antagonist AM630 and in the presence of PPAR antagonist GW9662, suggesting the anti-inflammatory effect of BCP not only involves CB₂ receptor pathway, but also the PPAR pathway²⁷.

Based on evidence that CB₂ receptor agonism demonstrates promising therapeutic benefits in murine colitis, several novel, highly selective CB₂ receptor agonists were developed, of which two of these compounds had promising in vivo anti-inflammatory effects in the DSS-induced colitis model²⁸. Furthermore, El Bakali et al developed constrained analogues from 4-oxo-1,4-dihydroquinoline-3-carboxamides with improved affinity for the CB₂ receptor and increased selectivity over the CB₁ receptor and tested their effects on prevention and treatment of TNBS-induced colitis; the highly CB₂ receptor selective agonistic compound 26 (ALICB459) exerted a strong protective effect when given orally to mice with TNBS-induced colitis²⁹ (Table 1).

Table 1. CB₁ and CB₂ targeted compounds have varying effects on colitis.

Multiple compounds specifically target CB₁ or CB₂ receptors. CB₁ and CB₂ receptor agonism is largely anti-inflammatory while CB₁ and CB₂ receptor antagonism is pro-inflammatory.

Compound	Cannabinoid Receptor & Mechanism	Effect
ACEA	CB1 agonist	Anti-inflammatory
HU210	CB1 agonist	Anti-inflammatory
WIN55, 212-2	Weak CB1 agonist	Anti-inflammatory
SR141716A	CB1 antagonist	Pro-inflammatory
α,β-amyrin	CB1 agonist	Anti-inflammatory
AM251	CB1 antagonist	Pro-inflammatory
SR144528	CB2 antagonist	Pro-inflammatory
JWH-133	CB2 agonist	Anti-inflammatory
AM630	CB1,2 antagonist	Pro-inflammatory
AM1241	CB2 agonist	Anti-inflammatory
β-caryophyllene	CB2 agonist	Anti-inflammatory
ALICB459	CB2 agonist	Anti-inflammatory
AM841	CB1,2 agonist	Anti-inflammatory
JWH-015	CB2 agonist	Anti-inflammatory
O-1966	CB2 agonist	Anti-inflammatory

Endogenous Cannabinoids

Endogenous cannabinoids AEA and 2-AG signal via cannabinoid receptors and are also fundamental in the pathway to attenuate murine colitis. AEA is upregulated in LPS stimulated peritoneal macrophages¹⁴, and levels of endogenous cannabinoids AEA and 2-AG are elevated via investigation by isotope dilution liquid chromatography-mass spectrometry in DNBS-induced colitis, while treatment with plant-derived, anti-inflammatory cannabinoid cannabidiol (CBD) significantly counteracted these changes³⁰. Additionally, intraperitoneal administration of AEA ameliorates macroscopic and microscopic colonic inflammation and suppresses the expression of pro-inflammatory cytokines in AKR mice with TNBS-induced colitis³¹. Furthermore, selective MAGL inhibitor JZL184 prevents degradation of 2-AG and thus allows 2-AG to activate cannabinoid receptors, leading to decreased levels of pro-inflammatory cytokines and attenuation of inflammation in TNBS-induced murine colitis³².

Atypical Cannabinoids

Little is known about the mechanistic pathways of atypical cannabinoids in murine colitis, but they appear to play a dynamic role in attenuation of inflammation. Atypical cannabinoid and GPR55 agonist O-1602 attenuates inflammation in both DSS-induced and TNBS-induced colitis in wild type mice, and this effect is preserved when tested in CB₁^−/− and CB₂^−/− mice and in GPR55^−/− mice, indicating that this therapeutic effect may have been achieved by off-target effect or by alternate pathways³³. Conversely, GPR55 antagonist CID16020046 attenuated inflammation in DSS-induced colitis, and GPR55^−/− mice showed reduced inflammation scores as compared to wild type mice in the DSS model, both suggesting that GPR55 activates a pro-inflammatory signal in intestinal inflammation³⁴.

Atypical cannabinoid PEA, which has been shown to act via several targets, including CB₁ receptors, CB₂ receptors, TRPV1 channels, PPARs, and GPR55, was found to be increased 2.5 fold in DNBS-treated colonic tissue, and led to attenuation of inflammation in this murine model of colitis¹⁶. In addition, PEA counteracted DNBS-induced downregulation of GPR55 and TRPV1, however, GPR55 antagonist ML-191 and PPARα antagonist GW6471 reduced the anti-inflammatory effects of PEA while TRPV1 channel antagonist capsazepine had a synergistic anti-inflammatory effect¹⁶.

Treatment of LPS activated peritoneal macrophages with non-psychotropic cannabinoid cannabichromene (CBC), a transient receptor potential ankyrin-type 1 (TRPA1) agonist, weak MAGL inhibitor, and inhibitor of endocannabinoid activation, significantly increased OEA levels^14,26. CBC treatment also reduced pro-inflammatory cytokines and nitric oxide production, and this anti-inflammatory effect was mimicked by cannabinoid receptor and TRPA1 agonists carvacrol and cinnamaldehyde, further enhanced by CB₁ receptor antagonists rimonabant and AM251, with no change on CBC effect by CB₂ antagonist SR144528^14,26. Finally, CBC treatment attenuated DNBS-induced colitis in ICR mice, although the exact mechanistic pathway of these effects is somewhat unknown^14,26.

MAGL and FAAH

As the enzymes responsible for degradation of endocannabinoids 2-AG and AEA, respectively, MAGL and FAAH are vital components in the ECS during the inflammatory cascade. A significant increase in FAAH activity was observed in croton oil treated mice¹¹, but in contrast, FAAH mRNA expression is significantly reduced in early stages of inflammation and returned to normal levels over time in multiple colitis models^30,35. In addition to the previously reviewed effects of α,β-amyrin as an anti-inflammatory agent via the CB₁ receptor pathway in DSS-induced colitis, preventive treatment with α,β-amyrin in DSS-induced colitis caused a marked decrease in both monoglyceride lipase 1 (MGL1) and FAAH mRNA expression, suggesting that the α,β-amyrin beneficial effects could be related to the modulation of endocannabinoid hydrolase expression¹³.

In addition, MAGL and FAAH blockade are important to attenuation of colitis, and are closely linked to CB receptors in the ECS. While FAAH inhibition was shown to ameliorate DSS-induced colitis in one study³⁶, it failed to treat colitis using the same model in another study³⁷. The FAAH inhibitor PF-3845 successfully attenuated TNBS-induced colitis³⁷. Potent MAGL inhibitor JZL184 attenuated TNBS-induced murine colitis while preserving the expression of MAGL and altering function only³². The MAGL inhibitory pathway also involves the CB₁ and CB₂ receptors, as administration of the MAGL inhibitor JZL184 in the presence of CB₁ receptor antagonist SR141716A and CB₂ receptor antagonist AM630 completely abolished the protective effect of MAGL inhibition³². Furthermore, FAAH inhibitor URB597 attenuated TNBS-induced colitis, and this anti-inflammatory effect was abolished in both CB₁^−/− and CB₂^−/− mice³⁵. Lastly, FAAH^−/− mice are impaired in their ability to degrade AEA and display protection from DNBS-induced colitis as compared to the more severe inflammation seen in their FAAH^+/+ littermates¹² (Table 2).

Table 2. Atypical cannabinoid pathway and FAAH and MAGL targeted compounds have varying effects on colitis.

Multiple compounds specifically act on atypical cannabinoid pathways TRPA1 or GPR55 and on enzymatic degradation pathways FAAH and MAGL. GPR55 pathway is pro-inflammatory, as antagonism has an anti-inflammatory effect. TRPA1 agonism, as well as FAAH and MAGL inhibition have anti-inflammatory effects on experimental colitis.

Compound	Receptor & Mechanism	Effect
CID16020046	GPR55 antagonist	Anti-inflammatory
cannabichromene	TRPA1 agonist & weak MAGL antagonist	Anti-inflammatory
carvacrol	TRPA1 agonist	Anti-inflammatory
cinnamaldehyde	TRPA1 agonist	Anti-inflammatory
PF-3845	FAAH antagonist	Anti-inflammatory
JZL184	MAGL antagonist	Anti-inflammatory
URB597	FAAH antagonist	Anti-inflammatory

Impact of Cannabinoids in Human Disease

Several studies have looked at the endocannabinoid system and its effects on human immune cell function. Anandamide has been shown to suppress proliferation and cytokine release from human T-lymphocytes via the CB₂ receptor³⁸. Also, human neutrophil transmigration in vitro is impaired by treatment with synthetic cannabinoid receptor agonist WIN 55,212-2, though the mechanism does not appear to be mediated via the CB₁ or CB₂ receptor³⁹, suggesting an off-target effect may be responsible.

In addition, two plant cannabinoids, THC and CBD, have received greatest interest for their therapeutic potential in humans. THC, the partial CB_1/2 receptor agonist which was the first identified active ingredient in cannabis with potent psychotropic effects⁴⁰, can suppress production of IFNγ by human CD4⁺ T cells in vitro albeit at relatively high concentrations⁴¹. THC has also been shown to enhance TGFβ production⁴² in unfractionated human peripheral blood mononuclear cells and reduces both cyclic AMP⁴³ and IL-10 production⁴⁴ by human lymphocytes via the CB₂ receptor.

CBD first gained interest when it was shown to provide significant improvement in a phase II placebo-controlled clinical trial for the treatment of epilepsy⁴⁵. CBD has been reported to suppress TNFα, IL-1β and IFNγ production by human peripheral blood mononuclear cells⁴⁶. In contrast to THC, CBD appears to have little or no CB₁ receptor or CB₂ receptor affinity⁴⁷ and a number of possible alternative methods have been postulated for its mechanism of action, including prolonging of the effect of anandamide. In a study looking at CBD in schizophrenia, CBD enhanced AEA signaling⁴⁸ potentially by acting as a false substrate for FAAH. CBD can also enhance adenosine signaling by inhibiting its uptake via equilibrative nucleoside transporter 1⁴⁹. CBD, however, has also been reported to strongly inhibit IL-10 production by a human HUT-78 T cell line⁴⁴. Interestingly, CBD increases TGFβ production⁵⁰ at low doses, which is important for expansion of both Treg and Th17 cells, but does the reverse at high CBD concentrations suggesting that there may be a significant dose effect in patients.

Three observational studies focused on MJ use in adults with IBD report subjective improvement in symptoms with MJ use^51,52,53, but the study by Storr et al in 2014 also demonstrated increased surgical procedures in those with use of MJ for greater than 6 months⁵³. A prospective trial using THC versus placebo for the treatment of Crohn’s disease in 21 patients found statistically significant reductions in Crohn’s Disease Activity Index scores and increased quality of life in the THC treatment group; however, there was no change in serum markers of inflammation in either the THC treatment or the placebo group⁶. Lastly, a clinical trial sponsored by Bial and performed by Biotrial studied the effect of a fatty acid amide hydrolase (FAAH) inhibitor as an analgesic in human subjects, but the study was aborted after 5 of 6 subjects developed significant neurologic side effects, including one death⁵⁴. Subsequent investigations coupled with studies on other FAAH inhibitors, strongly suggest that this is highly unlikely to represent a class effect.

Discussion

The ECS and its components, including CB₁ and CB₂ receptors, endocannabinoids AEA and 2-AG, atypical cannabinoids, and endocannabinoid hydrolases FAAH and MAGL, are vastly interrelated but incompletely understood pathways that highly contribute to attenuation of murine colitis. Plant cannabinoids THC and CBD proved beneficial in DNBS-induced colitis in a bell-shaped dose-related response, but more importantly, the effects of the phytocannabinoids were additive, as CBD increased an ineffective THC dose to the level of an effective one⁵⁵. As previously discussed, TNBS induced a more severe colitis in CB₁^−/− mice, CB₂^−/− mice, as well as in CB_1/2 double knockout mice as compared to WT littermates, however, there was no difference in the severity of colitis between CB₁^−/−, CB₂^−/− or the CB_1/2 double knockout mice, indicating the importance of the presence of both cannabinoid receptor pathways in the attenuation of inflammation¹⁹. Moreover, covalent cannabinoid agonist AM841 attenuated both DSS and TNBS-induced colitis when administered both before induction of colitis as well as a treatment for existing inflammation, and this anti-inflammatory effect was abolished in the setting of CB₁^−/− mice, CB₂^−/− mice, as well as in CB_1/2 double knockout mice, which provides further evidence to support the vital role of both cannabinoid pathways in attenuation of murine colitis⁵⁶. Finally, MAGL and FAAH blockade, as well as blockade of atypical cannabinoid receptor TRPV1⁵⁷, are important to attenuation of colitis, and studies demonstrate a close link of these endocannabinoid hydrolases to CB receptors in the ECS (Figure 2).

Figure 2. Therapeutic targeting of the endocannabinoid system in murine colitis.

A schematic overview of cannabinoid receptors, the endogenous cannabinoids that act on them, pathway of enzymatic degradation of endocannabinoids, and how manipulation of this pathway impacts colitis.

Investigators have produced similar results from manipulation of the endocannabinoid system in other disease models of inflammation. In the Mixed Lymphocyte Reaction (MLR), an in vitro correlate of organ graft rejection, selective CB₂ agonists JWH-015 and O-1966, as well as THC, significantly suppressed the MLR (thus inflammation) in a dose dependent fashion, and this effect was reversed in the setting of CB₂ antagonist SR144528 but not in the presence of CB₁ antagonist SR141716A⁵⁸, indicating that the anti-inflammatory effect was CB₂ receptor mediated. In the same MLR model, selective CB₂ agonist O-1966 increased percentage of T regulatory cells (Treg), decreased active markers of inflammation including transcription factors on T cells and decreased pro-inflammatory cytokines, but these anti-inflammatory effects were abolished in CB₂^−/− mice, and the increased Treg percentage was reversed with administration of anti-IL10⁵⁹. In Theiler’s virus (TMEV)-activated macrophages, a viral model of multiple sclerosis, CB₂ agonist JWH-133 inhibited inflammation and this effect was reversed in the presence of CB₂ antagonist SR144528²⁴. Thus, the mammalian ECS likely plays an important role in a multitude of inflammatory diseases, and with time, novel drug therapies targeting the ECS may prove critical for the advancement of therapeutics in inflammatory disease, including IBD.

Multiple studies have struggled to elucidate specific mechanistic pathways in attenuation of inflammation, but nonetheless, their findings highlight the importance of manipulation of the ECS to produce biologic effects. Two groups investigated intraperitoneal administration of peripherally-restricted CB agonists and found that they had no effect on murine colitis, suggesting an inclusive role of CNS receptors^56,17. One group went a step further and demonstrated that intracerebroventricular (i.c.v.) administration of peripherally-restricted CB agonists attenuated colitis, imparting credibility to the concept that both central and peripheral CB receptors are responsible for the protective and therapeutic effects of cannabinoids⁵⁶, and that the lack of efficacy in peripheral restriction could be overcome by systemic administration.

Furthermore, upregulation in expression of the components of the ECS in the setting of murine colitis may be location and timing dependent. In one study, there was marked upregulation of CB₂ expression in the proximal colon within one day of TNBS instillation which progressed to the distal colon by day three, consistent with the aboral direction of disease progression; these findings were mirrored in the DSS-induced colitis model as well²². An additional study notes that method of administration may be critical in attenuating inflammation, as intraperitoneal (i.p.) and rectal administration of CBD attenuated TNBS-induced colitis but oral administration made no effect on inflammation⁶⁰. These studies highlight the complicated nature of the ECS, and suggest that further research to clearly elucidate the specific mechanistic pathways of the ECS will be crucial to the development of therapeutics to target inflammation.

Conclusion

There is a significant need for the development of highly novel therapies for the treatment of IBD, as IBD is one of the most prevalent and costly GI disorders whose annual incidence and prevalence is on the rise. Manipulation of multiple pathways of the ECS in murine colitis has proven to be beneficial in attenuating inflammation, and the ECS pathway may offer an innovative approach to enhance the impaired host regulatory system in IBD patients. However, studies on the impact of manipulation the endocannabinoid system in intestinal inflammation are lacking, as the few human trials on MJ use in IBD have evaluated subjective, although positive, results only and failed to evaluate objective histologic data. This review highlights the complicated nature of the ECS, its critical role in a multitude of inflammatory diseases, and emphasizes that further research to clearly elucidate the specific mechanistic pathways of the ECS is essential to the development of therapeutics to target inflammation.

Nonstandard Abbreviations

IBD

Inflammatory bowel disease

FAAH

Fatty acid amide hydrolase

MAGL

Monoacylglycerol lipase

GI

Gastrointestinal

UC

Ulcerative Colitis

CD

Crohn’s disease

MJ

marijuana

ECS

Endocannabinoid System

AEA

anandamide

2-AG

2-arachidonoylglycerol

NAPE-PLD

N-acyl-phosphatidylethanolamine-selective phospholipase D

EMT

endocannabinoid membrane transporters

CB₁

Cannabinoid 1

CB₂

Cannabinoid 2

TRPV1

transient receptor potential vanilloid type 1

NADA

N-arachidonoyl dopamine

HEA

N-homo-γ-linolenoyl-ethanolamine

DEA

N-docosatetraenoyl-ethanolamine

PEA

palmitoylethanolamide

OEA

oleoylethanolamide

PPARs

peroxisome proliferator-activated receptors

DNBS

dinitrobenzene sulfonic acid

DSS

dextran sulphate sodium

LPS

lipopolysaccharide

OM

oil of mustard

TNBS

2,4,6-trinitrobenzene sulfonic acid

WT

wild type

THC

Δ9-tetrahydrocannabinol

CBG

cannabigerol

BCP

b-caryophyllene

CBD

cannabidiol

CBC

cannabichromene

TRPA1

transient receptor potential ankyrin-type 1

MGL1

monoglyceride lipase 1

MLR

Mixed Lymphocyte Reaction

Treg

Regulatory T cell

TMEV

Theiler’s virus

i.c.v.

intracerebrovascular

i.p.

intraperitoneal

CB₁R

CB₁ receptor

CB₂R

CB₂ receptor