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Review

The endocannabinoid system in

inﬂammatory bowel diseases: from pathophysiology to therapeutic

opportunity

Mireille Alhouayek and Giulio G. Muccioli

Bioanalysis and Pharmacology of Bioactive Lipids Research Group, Louvain Drug Research Institute, Universite´ catholique de

Louvain, Av. E. Mounier, 72, B1.72.01, 1200 Bruxelles, Belgium

Crohn’s disease and ulcerative colitis are two major forms considered the primary enzymes controlling anandamide

of inﬂammatory bowel diseases (IBD), which are chronic and 2-arachidonoylglycerol levels, respectively, it is impor-

inﬂammatory disorders of the gastrointestinal tract. tant to consider that under certain conditions (e.g., inﬂam-

These pathologies are currently under investigation to mation) or in speciﬁc cell types (e.g., macrophages as

both unravel their etiology and ﬁnd novel treatments. compared to endothelial cells), NAAA or ABHD6 may have

Anandamide and 2-arachidonoylglycerol are endogenous

bioactive lipids that bind to and activate the cannabinoid

receptors, and together with the enzymes responsible for

their biosynthesis and degradation [fatty acid amide hy- Glossary

drolase (FAAH) and monoacylglycerol lipase (MAGL)]

b.i.d.: abbreviation for bis in die, the administration of a drug twice a day.

constitute the endocannabinoid system (ECS). The ECS Bioactive lipids: lipids for which changes in their levels result in functional

consequences. Usually requires the trilogy: lipid, enzymes producing and

is implicated in gut homeostasis, modulating gastroin-

degrading the lipid, and downstream targets for the bioactive lipid.

testinal motility, visceral sensation, and inﬂammation, as

Ethylenediaminetetraacetate (EDTA): EDTA is a metal chelator with high

well as being recently implicated in IBD pathogenesis. affinity for calcium. When an epithelial (or endothelial) cell monolayer is

incubated in the presence of EDTA, the calcium depletion results in disruption

Numerous subsequent studies investigating the effects of

of tight junctions and opening of the paracellular route.

cannabinoid agonists and endocannabinoid degradation

Fluorescein isothiocyanate-dextran: a high molecular weight polymer of D-

inhibitors in rodent models of IBD have identiﬁed a po- glucose conjugated to the fluorophore fluorescein. Its molecular weight

prevents it from crossing epithelia through the paracellular route. Increased

tential therapeutic role for the ECS.

permeability of the epithelial barrier results in fluorescein isothiocyanate–

dextran crossing to the bloodstream. Thus, its presence in plasma indicates

The ECS in a nutshell increased epithelial permeability.

Hapten: small molecule that becomes immunogenic only once bound to a

Endocannabinoids are lipid mediators that exert most of

carrier (a bigger molecule such as a protein).

their functions by binding and activating two G protein-

Lipopolysaccharide: a component of Gram-negative bacteria made of a

coupled receptors, cannabinoid receptor 1 (CB1) and 2 (CB2), hydrophobic domain known as lipid A (or endotoxin), an oligosaccharidic

core, and a distal polysaccharide (or O-antigen). Its binding to toll-like receptor

that are both expressed throughout the gastrointestinal

4 (TLR4) induces potent inflammatory responses. A normal epithelial barrier

tract at various levels depending on the tissue and condi- prevents translocation of luminal LPS to the bloodstream, but during obesity,

tions. The two most thoroughly studied endocannabinoids increased epithelial permeability leads to increased plasma LPS levels

(significantly lower levels than during an infection or sepsis).

are arachidonic acid derivatives, the N-acylethanolamine

Myeloperoxidase: a lysosomal peroxidase most abundantly expressed in

(NAE) N-arachidonoylethanolamine (anandamide) and the neutrophils and involved in the respiratory burst. Increased myeloperoxidase

monoacylglycerol 2-arachidonoylglycerol. They are found at activity in tissues mirrors increased infiltration of these tissues by neutrophils.

Oil of mustard (OM): is a small nerve fiber stimulant and a potent inflammatory

various levels throughout the gastrointestinal tract, with 2-

irritant, usually administered intrarectally to provoke allodynia (pain response

arachidonoylglycerol being more abundant than ananda- to a usually painless stimulus) and visceral hyperalgesia (an exaggerated pain

response). It has also recently been used for colitis induction.

mide. Their production from cell membrane lipid precursors

Tight junctions: complexes of proteins that mediate epithelial cell-to-cell

is activity-dependent, and their actions are terminated via

adhesion. By closing the space between two adjacent epithelial cells, tight

hydrolysis by speciﬁc lipases (Figure 1) [1]. Anandamide junctions prevent molecules from crossing an epithelium by the paracellular

route. Several of these proteins are transmembrane proteins (claudins,

signaling is terminated by two amidases, FAAH and N-

occludins, and junctional adhesion molecules or JAM), whereas others are

acylethanolamine-hydrolyzing acid amidase (NAAA),

cytoplasmic (zonula occludens proteins).

whereas MAGL and a/b-hydrolase domain 6 (ABHD6) Trans-epithelial electric resistance (TEER): a measure of the resistance that an

epithelial (or endothelial) confluent cell monolayer opposes to the passage of a

are two esterases involved in regulating 2-arachidonoylgly-

current applied between two electrodes separated by the cell monolayer. It is

cerol signaling [2]. Although FAAH and MAGL are

used to determine the presence of functional tight junctions and thus the

integrity of a confluent monolayer of epithelial or endothelial cells.

Corresponding author: Muccioli, G.G. ([email protected]) q.d.: abbreviation for quaque die, the administration of a drug once a day.

Keywords: N-arachidonoylethanolamine; endocannabinoids; inflammatory bowel q.a.d.: abbreviation for quaque alternis die, the administration of a drug every

disease; anandamide; 2-arachidonoylglycerol; palmitoylethanolamide; other day.

monoacylglycerol lipase; fatty acid amide hydrolase.

Author's personal copy

Review Trends in Molecular Medicine October 2012, Vol. 18, No. 10

Ethanolamine Glycerol

FA PPAR

AA FAAH NAAA MAGL ABHD6

AEA PEA OEA 2-AG

GPR119 GPR55 CB CB1 2 TRPV1

OEA AEA 2-AG PEA

Phospholipids Phospholipids NAPE-PLD Lyso-PL DAGL

TRENDS in Molecular Medicine

Figure 1. The endocannabinoid system in a nutshell. The N-acylethanolamines (NAE) – anandamide (AEA), N-palmitoylethanolamine (PEA) and N-oleoylethanolamine

(OEA) – are synthesized from membrane N-acylphosphatidylethanolamine (NAPE) precursors either directly through a NAPE-selective phospholipase D or through the

serial action of lipases (phospholipase C and then a phosphatase or phospholipase A2 and then lysophospholipase D). 2-arachidonoylglycerol (2-AG) is primarily produced

from phospholipid-derived diacylglycerols by diacylglycerol lipases a and b (DAGL). These bioactive lipids can activate the following: (i) G protein-coupled receptors such

as the CB1 and CB2 cannabinoid receptors (AEA, 2-AG), GPR119 (OEA), or GPR55 (putatively by PEA); (ii) the channel receptor TRPV1 (AEA); and (iii) the peroxisome

proliferator-activated receptors (PPARs) (AEA, OEA, PEA, 2-AG). These lipids are hydrolyzed inside the cell (a putative and elusive transporter facilitating their recapture is

not represented in this cartoon) by several lipases. The two prime enzymes hydrolyzing NAEs are the serine amidase FAAH and the cysteine amidase NAAA. 2-AG is

hydrolyzed by two serine hydrolases, MAGL and ABHD6.

a more predominant role. This is also true when considering (IBD) (Box 1). The receptors mediating the effects of endo-

the biosynthetic pathways of these mediators. cannabinoids and the enzymes controlling their levels

In addition to generating anandamide and 2-arachido- represent prime pharmacological targets to interfere with

noylglycerol, the enzymes responsible for their biosynthe- the intestinal endocannabinoid signaling and study its

sis produce other bioactive lipids (see Glossary). These physiopathological relevance. Over the years, selective

other lipids do not always bind to cannabinoid receptors pharmacological tools have been developed to selectively

but they do share their metabolic pathways and some probe each element of the ECS, and some of these are listed

molecular targets with the endocannabinoids. For in- in Box 2.

stance, N-palmitoylethanolamine and N-oleoylethanola-

mine, differing from anandamide by their acyl chain, do Role of the ECS in gut homeostasis

not bind to cannabinoid receptors but to the nuclear recep- Cannabinoids have numerous effects on the gastrointesti-

tors PPAR and to G protein-coupled receptors such as nal tract ranging from modulating food intake and feeding

GPR119, which is highly expressed in the gut [3]. 2-Oleoyl- behavior [6] to gastroprotection and inhibition of nausea or

glycerol, a 2-arachidonoylglycerol analogue, also lacks sig- vomiting [5]. Several ECS effects could play a role in IBD,

niﬁcant afﬁnity for the cannabinoid receptors but binds including modulation of gastrointestinal motility, visceral

and activates GPR119 [4]. Finally, GPR55 and the receptor sensation, inﬂammation, and intestinal permeability.

channel TRPV1 (activated, for instance, by anandamide) The inhibition of intestinal transit and colonic propul-

are additional targets related to the ECS. sion is of particular interest when considering inﬂamma-

Endocannabinoids and related molecules such as N- tory states of the bowel, where dysmotility is a clinically

palmitoylethanolamine have pleiotropic effects in the or- signiﬁcant component of the disease. Cannabinoids and

ganism, including in the gastrointestinal tract [5]. In this endocannabinoid degradation inhibitors decrease intesti-

review, we focus on the role of the ECS in gut homeostasis nal motility, peristalsis, and colonic propulsion in rodents,

and its potential beneﬁts in inﬂammatory bowel diseases primarily in a CB1-dependent manner [7–12]. Accordingly,

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Box 1. Inflammatory bowel diseases

IBDs are chronic relapsing-remitting or progressive inflammatory b UC: less clearly associated with Th2

conditions of the gastrointestinal tract [68,69]. b Th17 also implicated in IBD

Symptoms: abdominal pain, diarrhea, rectal bleeding, weight loss, b T cell activation leads to a ﬁnal common pathway of inﬂamma-

fever and fatigue. tion involving cytokines such as TNF-a, IL-1b or IL-6 that further

Two major forms differing mostly by the localization and the nature amplify and sustain the inﬂammatory response

of the inflammatory lesions. Regulatory T cells (Treg) producing anti-inﬂammatory cytokines

Crohn’s disease: such as IL-10 and/or TGF-b

b affects any portion of the digestive tract (mostly ileum and colon) The transition from a normal state of tolerance to an

b discontinuous lesions active immune response could be due to either/or a combination

b inflammation involves the full thickness of the bowel wall of [70,73]:

b complications: granulomas, obstructive strictures or fistulae defective effector T cells overreacting to usual microbial antigens

defective mucosal Treg cells that do not modulate otherwise

Ulcerative colitis:

normal helper T cells

b restricted to the rectum and colon

Conventional treatments [75]:

b uninterrupted pattern

5-aminosalycilic acid derivatives

b inﬂammation conﬁned to the mucosa

Corticosteroids

b complications less common

Immunomodulators

Etiology is believed to be a combination of genetic and environmen-

Biologicals (e.g., anti-TNF-a antibodies)

tal factors leading to an aberrant immune response [43,69,70].

Novel therapeutic targets for IBD (reviewed in [76]):

Gut microﬂora, a major environmental player in IBD [43,68,71]:

reducing the T cell mediated aberrant immune response by acting at

altered immune response to otherwise ‘normal’ mucosal ﬂora in IBD

different stages of the initiation and maintenance of inﬂammation:

altered microﬂora in some cases of IBD: contributor to the disease,

b inhibiting major effector cytokines (such as TNF-a)

or a consequence of inﬂammation?

b inﬂuencing T cell differentiation or subsets

Major players in IBD [43,69,70,72–74]:

Innate immunity implicated in initiating the inﬂammatory cascade, reinforcing the innate immune system to prevent, or control, the T

and subsequent adaptive immune responses cell response

T cell driven processes dominated by mucosal CD4 lymphocytes initiating intestinal repair since defects in the intestinal barrier

(for a comprehensive review of T cell differentiation and regulation contribute to the persistent activation of the immune system

in IBD, see [73]): using probiotics to re-establish a balanced microﬂora.

b CD: classically Th1-mediated

pharmacological blockade of CB1, or its genetic deletion, decrease in reabsorption) accompanied by reduced colon

result in prokinetic effects in the gastrointestinal tract contractility. It is noteworthy, however, that small intesti-

(enhanced motility) [8,9,13,14]. It is likely that cannabi- nal contractility appears to be enhanced, leading, in ﬁne, to

noids inhibit contractility by reducing acetylcholine re- decreased orocecal transit time [23]. Cannabinoids can

lease from enteric nerves because they do not inhibit decrease intestinal transit time and hypersecretion. They

contractions produced by exogenous administration of ace- inhibit cholera toxin-induced hypersecretion in the mouse

tylcholine but reduce electrically-evoked acetylcholine re- small intestine and delay the onset of castor oil-induced

lease from myenteric nerves [7,15,16]. Additionally, diarrhea in the rat, via CB1 receptor activation [24,25].

cannabinoids have an inhibitory effect on the non-adren- However, cannabinoids have mostly been evaluated in

ergic, non-cholinergic excitatory transmission in the intes- acute inﬂammation and should therefore be evaluated

tine [7,17,18]. Under normal conditions, CB1 mediates the for transit time and diarrhea reduction during colitis.

effects of cannabinoids on motility; however, in inﬂamma- Moreover, some patients might experience gastroparesis

tory states both CB1 and CB2 receptor activation may and a delay in small intestinal motility, in which cases,

reduce inﬂammation-induced hypermotility. For instance, cannabinoids (if considered a potential treatment) should

in lipopolysaccharide (LPS)-induced intestinal inﬂamma- be used at doses that do not affect gastric emptying or

tion, hypermotility was normalized following CB2 (but not intestinal motility.

CB1) activation [19,20], whereas in croton oil-induced Feelings of discomfort, nausea, and pain are commonly

hypermotility CB2 antagonism did not reverse the effect associated with inﬂammatory disorders of the gastrointes-

of the cannabinoid agonist CP55,940 [8]. In another study tinal tract. Cannabinoid receptors have been implicated in

of LPS-induced inﬂammation, the FAAH inhibitor pain processing in various neuropathic and inﬂammatory

AM3506 normalized hypermotility in a CB1- and CB2- states, mainly in somatic pain [26]. Putative actions of

dependent manner, whereas colonic propulsion was CB1- cannabinoids on visceral sensation have been proposed,

dependent [21]. Interestingly, the effect of cannabinoids in and the effects of centrally and peripherally restricted

cases of inﬂammatory hypermotility occurred at lower cannabinoids have been recently investigated, implicating

doses than in control states [8,22]. This could be due to primarily CB1 [27–29] but also CB2 [27,30]. In one inter-

the increased receptor expression during inﬂammation, to esting study, the CB1/CB2 agonist WIN55,212-2 and the

increased coupling of the receptor to effectors proteins, or CB2 agonist JWH-015 (both at doses of 10 mg/kg) attenu-

both. Therefore, in an acute inﬂammatory setting, the ated the degree of visceral sensitivity under baseline con-

cannabinoid-induced normalization of hypermotility ditions. When assessed versus the heightened sensitivity

should be beneﬁcial. It is nevertheless important to con- due to colonic inﬂammation of trinitrobenzene sulfonic acid

sider the whole picture in chronic IBD where dysmotility is (TNBS)-induced colitis, both agonists were effective at a

predominantly altered towards diarrhea, mainly due to much lower dose (0.1 mg/kg) that had no effect under

secretory colon disturbances (increase in secretion and/or baseline conditions [27].

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Box 2. The ECS toolbox

Potent and selective pharmacological tools are needed to dissect the role of the ECS in IBD. The cannabinoid receptor ligands as well as the

inhibitors of endocannabinoid degradation used in the studies described in this review are listed in Table I [3,59].

Table I. Pharmacological modulators of the ECS

CANNABINOID RECEPTORS LIGANDS

Drug CB1 Ki (nM) CB2 Ki (nM) Functionality

CB1/CB2 receptors agonists

HU210 0.06–0.7 0.1–0.5 Full agonist at both receptors

CP55,940 0.5–5 0.69–2.8 Full agonist at both receptors

WIN55,212-2 1.9–123 0.3–16.2 When assayed in the same study, slightly more afﬁnity for CB2

receptors. Full agonist at both receptors

9 9

D -THC 5–80 3–75 D -Tetrahydrocannabinol is a nonselective partial agonist

SAB378 15 98 Peripherally restricted CB1-CB2 agonist

Noladin ether 9.1 480 Has higher afﬁnity for CB1 receptors, full agonist at both receptors

CB1 receptors selective agonists

ACEA 1.4 >2000 Arachidonyl-2 -chloroethylamide is a CB1 receptor agonist

CB2 receptors selective agonists

JWH-133 677 3.4 Full agonist at CB2 receptors

AM1241 280 3.4 Protean agonist at CB2 receptors

JWH-015 383 13.8 Full agonist at CB2 receptors

CB1 receptors selective antagonists

SR141716A 1.8–12.3 >1000 Rimonabant behaves as an inverse agonist at CB1 receptors

AM251 7.5 2300 Inverse agonist at CB1 receptors

CB2 receptors selective antagonists

AM630 5152 31.2 Inverse agonist at CB2 receptors

INHIBITORS OF ENDOCANNABINOID DEGRADATION

Drug FAAH IC50 (nM) MAGL IC50 (nM) Mode of action

Fatty acid amide hydrolase inhibitors

URB597 4.6 >30 000 Irreversible carbamate-type inhibitor

AM3506 48 11 000 Sulfonylﬂuoride-type covalent inhibitor

39 88 >30 000 Putatively reversible FAAH inhibitor

AA-5HT 5600 / Arachidonoylserotonin is a FAAH inhibitor also able to antagonize

TRPV1 receptor

VDM11 2000–11000 / Also inhibits the putative anandamide transporter

(IC50=9000–23 000 nM)

Monoacylglycerol lipase inhibitors

JZL184 4690 6 Irreversible carbamate-type inhibitor

An intriguing observation is the modulation of cannabi- roles for both cannabinoid receptors in modulating inﬂam-

noid receptors expression by gut microﬂora [31,32]. For matory processes, including suppression of the activation

instance, administration of Lactobacillus acidophilus of macrophages and mast cells, secretion of proinﬂamma-

increases CB2 expression, resulting in analgesic functions tory cytokines, and modulation of T helper lymphocytes by

in the gut, a response reversed by the CB2 receptor antag- reducing the activated T cell population, inducing their

onist AM630 [32]. It is well accepted that the microﬂora apoptosis, and inhibiting their proliferation [33,35–40].

plays a role in IBD by initiating or maintaining the im- The intestinal epithelium is a selective barrier that

mune response, however, the microﬂora also seems to plays an important role in gut homeostasis. It is permeable

inﬂuence visceral pain sensation through cannabinoid re- to nutrients and essential macromolecules but constitutes

ceptor modulation. A point to consider, however, is that an obstacle for luminal antigens, intestinal bacteria,

cannabinoid agonists differentially modulated afferent in- and harmful macromolecules. Increased epithelial

testinal nerve sensitivity depending on the stimuli used permeability is implicated in IBD pathogenesis. Indeed,

[29]. Thus, the understanding of the underlying mecha- a leaky intestinal barrier allows access to the underlying

nisms of visceral sensitivity is important to effectively mucosal tissue that becomes increasingly and permanently

target visceral pain with cannabinoids. exposed to antigens of the microﬂora. This results in a

Cannabinoids exert pharmacological actions on epithe- change from immune tolerance to immune activation

lial cells as well as immune cells, resulting in protective [41–43]. Pro-inﬂammatory cytokines are known to alter

and immunomodulatory effects [33]. Indeed, cannabinoids the barrier function [39,40], thus forming a vicious circle

such as anandamide, noladin ether, and arachidonoylcy- where increased inﬂammation leads to a further increase

clopropylamide (ACPA) induce wound-closure in human in intestinal permeability. In monolayers of Caco-2 cells, a

colonic epithelial cell lines, suggesting that delayed wound model of the intestinal epithelial barrier, application of

healing, a feature of IBD lesions, might be reversed by EDTA, pro-inﬂammatory cytokines, or LPS increases per-

cannabinoids [34]. Additionally, in vitro studies suggest meability as measured by a decrease in trans-epithelial

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electric resistance (TEER) [31,44,45]. The endocannabi- The ECS is off-balance in IBD, evidence from human and

noids anandamide and 2-arachidonoylglycerol increased animal studies

permeability when applied on the apical surface of Caco-2 Due to its implication in the homeostasis and functionality

monolayers [44], and they enhanced the EDTA- or cyto- of the gastrointestinal tract, the ECS seems positioned to

kine-induced increase in permeability [44,45]. These exert a protective role in many of the points where homeo-

effects were blocked by antagonism of CB1, but not CB2, stasis breaks in IBD. The ﬁrst evidence for this was

implicating CB1 receptors in the control of intestinal provided by studies in knockout mice that revealed an

permeability [31,44,45]. Actually, the CB1 antagonist increased susceptibility to dinitrobenzene sulfonic acid

AM251 decreased EDTA- and cytokines-induced altera- (DNBS)-induced and dextran sulfate sodium (DSS)-in-

tions of epithelial permeability [44,45]. The in vivo rele- duced colitis in CB1 mice when compared with their

vance of CB1 receptors in controlling gut permeability was wild type counterparts [50] (see Box 3 for an overview of the

demonstrated in a study looking at the disrupted epithe- rodent models of IBD discussed in this review), indicating a

lial barrier found in obese mice [31]. Indeed, obesity is protective role for the CB1 receptor in IBD. More recently,

characterized by altered gut permeability leading to in- increased susceptibility to colitis was conﬁrmed not only

/ /

creased plasma LPS levels, that is, metabolic endotoxemia for CB1 mice but also for CB2 mice [51], suggesting

[46]. Treatment with the antagonist SR141716A reduced that both cannabinoid receptors play complementary roles

plasma LPS levels in obese mice, mirroring an improve- in intestinal homeostasis because the absence of one or the

ment of the gut barrier function [31]. In another experi- other cannabinoid receptor results in an exaggerated in-

ment, administration of the CB1/CB2 agonist HU210 to ﬂammatory response. Interestingly, the genetic disruption

lean mice increased plasma LPS levels. HU210 adminis- of both the CB1 and CB2 receptors did not result in further

tration also increased plasma levels of ﬂuorescein isothio- worsening of colitis, when compared with either CB1 or

cyanate-dextran administered by oral gavage, conﬁrming CB2 alone [51]. It is possible that in double knockout

in vivo the cannabinoids-induced increase in gut perme- mice compensatory mechanisms, or the action of endocan-

ability [31]. However, the anti-inﬂammatory effects of nabinoids on other receptors, account for the lack of an

cannabinoids could have a beneficial impact on inflamma- even more exaggerated inflammatory response. Together,

tion-induced alterations of the intestinal epithelial barrier these studies suggest that cannabinoid receptors play a

(see below). Indeed, available treatments for IBD, such predominant role in suppressing gut inﬂammation.

as anti-TNF-a antibodies or corticosteroids, indirectly The initial studies in knockout mice prompted investi-

restore barrier function by reducing inﬂammation gations of the endocannabinoid signaling pathways in

[42,47–49]. human IBD and revealed an unbalanced ECS. Indeed,

In summary, the ECS seems well positioned to be a anandamide and N-palmitoylethanolamine levels are al-

regulatory system under inﬂammatory conditions because tered in colon mucosal biopsies of patients with ulcerative

the effects of cannabinoids were more evident in situations colitis (UC) or Crohn’s disease (CD), especially during

where increased gastrointestinal motility and visceral acute ﬂares, with no change in 2-arachidonoylglycerol

sensation were induced by an inﬂammatory stimulus. levels [35,52,53] (changes in the ECS in human IBD are

Moreover, the CB2 receptor, although not always relevant summarized in Table 1). NAE levels were reported to be

in a normal setting, seems to play an important role during either increased or decreased depending on the study.

gut inﬂammation, as is also the case in other organs or However, a closer look at anandamide-metabolizing

systems [e.g., the central nervous system (CNS)]. enzymes in acute colitis seems to support the notion of

Box 3. Overview of mouse models of acute IBD

Numerous rodent models of mucosal inflammation (chemically- chemical agent such as DSS, DNBS, TNBS, or oxazolone. These models

induced, genetically-manipulated, and immune-mediated) are avail- mimic the acute phase of the disease, such as epithelial response to

able to unravel the mechanisms implicated in IBD pathogenesis and injury or neutrophil infiltration, and thus are not relevant to address

investigate novel therapeutic strategies [77]. Although these models changes occurring in the chronic phases of colitis. They are never-

differ from one another, primarily in the method or mechanisms of theless useful for studying the pathogenic or protective roles of specific

colitis induction, the resulting inflammation is generally channeled systems and for proposing potential therapeutic strategies [77,81].

into a final common pathway, mediated by an excessive Th1 T cell DSS, administered in drinking water for 5 days, disrupts the

response (associated with secretion of IL-12, IFN-g, and TNF-a) as is epithelial barrier and promotes increased cellular exposure to gut

the case with most models, or an excessive Th2 response (associated microflora. This leads to activation of mucosal phagocytes by luminal

with IL-4 and IL-5). Inflammation is driven by antigens of the normal flora and, consequently, a nonspecific release of proinflammatory

mucosal microflora, such as LPS, and one factor tipping the balance cytokines. In an intact immune system, a T cell-mediated response is

between a Th1- or Th2-mediated response could be the nature of the superimposed on the initial macrophage-mediated inflammation

antigen driving the inflammation or the specificity of the T cell leading to lymphocyte activation and a Th1 response. Colitis is

receptor on reactive cells [78,79]. assessed on day 7 [78,81].

The majority of genetic models of IBD are gene knockouts, such as TNBS and oxazolone are haptenating agents, where inflammation

IL-10 mice, where colitis develops spontaneously around 12 weeks results from an immune response to a specific antigen (a hapten).

of age in mice kept in a conventional environment. Although these Both agents are administered intrarectally with ethanol (to disrupt the

models allow researchers to study the role of specific cytokines, their mucosal barrier), however, they lead to different immune responses:

utility is limited for understanding the underlying causative factors in whereas TNBS leads to a Th1-mediated response, oxazolone leads to

IBD [77,78,80]. Th2-mediated inflammation. These models are more aggressive than

To characterize the role of ECS in IBD, mostly chemically-induced DSS; colon alterations develop in less than 24 h and colitis is assessed

models are used where colitis induction requires administration of a 4 days later [78,81].

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decreased anandamide levels in IBD. Indeed, one study

reported decreased expression of N-acylphosphatidyletha-

nolamine selective phospholipase D (NAPE-PLD) with no

Protein increased Protein unchanged

change in FAAH expression [54], which was conﬁrmed in a

second study showing a decrease in NAPE-PLD activity

and an increase in FAAH activity during acute ﬂares of UC

or CD [35]; one could infer that this leads to decreased

anandamide levels. Thus, although there appears to be a mRNA decreased mRNA unchanged

deregulated anandamide tone, the whole picture of anan-

damide metabolism in the human colon during IBD

remains to be developed.

Data concerning the levels of endocannabinoids in ro- Protein increased Protein increased

dent models of IBD is also scarce. Anandamide levels have

been shown to be increased in the inﬂamed colons of

DNBS-mice and the submucosa of TNBS-rats, with no

change in 2-arachidonoylglycerol levels [52]. Interestingly,

increased unchanged 35/10 35/14

anandamide levels in the mucosa of the same TNBS-rats

remained unchanged [52]. This points to a differential

regulation of anandamide levels in the various colon struc-

tures and might explain the differences in anandamide

levels observed between human studies. In several rodent

Activity unchanged Activity unchanged 2–13/ 9–15

models of colitis, decreased or unchanged FAAH mRNA

expression was reported at different time points during the

course of the disease in the inﬂamed distal colon [55–57].

However, this decrease in mRNA expression was not ac-

Activity increased Activity decreased 2–13/ 9–15

companied by a decrease in FAAH activity [57], raising the

question of whether the observed increase in anandamide

levels is actually due to a decrease of its catabolism by

FAAH or to increased synthesis. FAAH mice chal-

lenged with DSS or DNBS exhibit less colonic inﬂamma-

increased decreased 9–15

tion, thus strengthening the potential role of anandamide

in IBD [50]. We must keep in mind, however, that along

with anandamide, FAAH hydrolyzes other NAEs, such as

N-palmitoylethanolamine, as well as other bioactive lipids,

the levels of which are also increased in FAAH mice

[58,59] and could be implicated in the observed reduction of

inﬂammation.

Interestingly, in the case of 2-arachidonoylglycerol, ex-

pression of both diacylglycerol lipase (DAGL)-a and MAGL

Increased Increased IncreasedUnchanged Unchanged Unchanged Unchanged Unchanged Activity Decreased Unchanged Activity Unchanged DecreasedIncreased Increased Unchanged Increased Unchanged Unchanged Unchanged Unchanged mRNA mRNA

was increased in colon mucosal biopsies of patients with

UC, which implies an increased 2-arachidonoylglycerol

turnover during inﬂammation [54]. Note that maintenance

of the DAGL-a/MAGL ratio [54] could explain the unal-

tered 2-arachidonoylglycerol levels observed both in

Unchanged Unchanged Unchanged Unchanged

humans and mice [35,52,57]. The question remains, how-

ever, whether the differences in expression of MAGL and

DAGL-a translate into differences in enzymatic activity.

Indeed, in TNBS-induced colitis decreased MAGL mRNA

Increased IncreasedIncreased Decreased Decreased Unchanged Unchanged Unchanged Unchanged [53]UCAcute [52]8/8 UC Acute 8/8 [54] Acute UC 22/24 Quiescent Inflamed Inflamed [35] Uninflamed CD Acute 2–13/ UC Acute CD and UC CD [60] UC

in the distal colon was not accompanied by decreased

MAGL activity [57].

a b

So far, data from human studies seem insufﬁcient to

1 2

provide a conclusion concerning the ECS tone in IBD,

CB MAGL DAGL- DAGL- CB AEA 2-AG PEA

mainly due to the complexity in obtaining large enough

cohorts of subjects and to a lack of sub-classiﬁcation of

these diseases in published studies. In general, one of the

cannabinoid receptors appears to be increased in CD or

Hydrolysis FAAH Synthesis NAPE-PLD

UC, with the other increased or unchanged (Table 1)

[35,54,60]. A sub-classiﬁcation of acute UC according to

clinical score (mild, moderate, or severe) identiﬁed various

changes in cannabinoid receptors and enzymes expression Endocannabinoid levels were measured by HPLC-MS.

Receptors Metabolic enzymes EC levels Regarding the enzymes and receptors, mRNA expression was measured, except where mentioned otherwise. IBD form Disease stage Patient number: CTL/IBD Table 1. Summary ofRefs the ECS tone in human IBD a

according to the clinical score [54]. For instance, whereas

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Review Trends in Molecular Medicine October 2012, Vol. 18, No. 10

expression of DAGL-a is unchanged during mild colitis, it colonic wall. Usual ways to assess colitis and the effect

is signiﬁcantly increased during moderate colitis. Interest- of treatments include evaluation of macroscopic and mi-

ingly, fewer differences were observed in severe colitis croscopic (histological) signs of colon inﬂammation, mea-

when compared with healthy controls, suggesting a dimin- surement of colon weight and length, and additional

ished ECS response to the inflammatory insult at that measures of colon inflammation such as inflammatory

stage [54]. A second interesting fact to consider is the cytokines expression or myeloperoxidase activity. Treat-

modiﬁed ECS tone in quiescent phases of the diseases ment schemes for the TNBS- or DNBS-induced models

[54] reﬂecting either a role for cannabinoid receptors in generally consist of drug administration once prior to

acute inﬂammation or a diminished colonic functionality in colitis induction and then once or twice daily for 3 days.

chronic stages, given that cannabinoid receptors, aside In the DSS model, drugs are administered concomitantly

from modulating inﬂammation, have other functional roles with the start of DSS or 4 days later.

in the colon. Various cannabinoid agonists have been used to evalu-

The discrepancies, between human studies, in the in- ate the impact of ECS activation on colitis. Nonselective

ﬂammation-induced variation of expression of cannabinoid agonists such as D -THC (10 mg/kg or 20 mg/kg, q.d.) [62],

receptors (Table 1) could be explained by varying degrees of HU210 (0.05 mg/kg, q.d.) [50], WIN55,212-2 (2 mg/kg,

inﬁltration and mucosal remodeling or alteration as well as b.i.d.) [12], or anandamide (5 mg/kg, q.d.) [63] were beneﬁ-

an analysis of different zones of the inﬂamed colon (ulcera- cial in murine models of colitis and reduced colon inﬂam-

tions or at a distance from ulcerations). Indeed, in normal mation. ACEA, a CB1-selective agonist, was also effective

colonic tissue, CB1 immunoreactivity is most evident on in alleviating inﬂammation in oil of mustard (OM)-induced

the microvillus border of the apical surface of the colonic colitis (2.5 mg/kg, q.d.) and DSS-induced colitis (10 mg/kg,

epithelium, as well as the smooth muscle and the submu- b.i.d.) [64].

cosal nerve bundles [34,54,60]. By contrast, CB2 expression Because the use of CB1 receptor agonists comes with

was more evident in subepithelial macrophages and plas- central side-effects owing to activation of CB1 receptors in

mocytes characteristically present within the lamina pro- the CNS, targeting solely CB2 receptors might be a sound

pria of normal bowel, with no evident staining in the strategy for treating colitis, seeing as CB2 activation is not

colonic epithelial border [34,54,60]. In acute phase IBD, associated with such side-effects. JWH-133, a CB2-selec-

CB1 immunostaining was evident in the same structures tive agonist, effectively reduces inﬂammation at the stated

as normal tissue, in contrast to CB2 immunoreactivity doses in DSS-induced colitis (10 mg/kg or 20 mg/kg, b.i.d.)

which was also evident in the microvillus border. This [36,64], TNBS-induced colitis (20 mg/kg, q.d. or b.i.d.) [61],

shift towards an upregulation of CB2 expression in epithe- OM-induced colitis (2.5 mg/kg, b.i.d.) [64], and spontaneous

lial cells in IBD was consistent in all the studies, even when colitis in IL-10 mice (2.5 mg/kg, q.a.d.) [36]. In all of

total CB2 protein or mRNA was unchanged [54,60]. these models, CB2 activation led to improved macroscopic

Unfortunately, animal studies do not shed light on these as well as histological colon alterations and reduced cellu-

discrepancies in expression. Indeed, CB1 expression was lar inﬁltrates. In the IL-10 model of colitis, inﬂamma-

increased in the myenteric plexuses of DNBS-mice, owing tion is primarily mediated by Th1 cells, and in this model,

to an increase in cells expressing CB1, with no change in JWH-133 not only inhibited neutrophil inﬁltration in the

the overall neuronal population [50], whereas no difference lamina propria (LP) and mesenteric lymph nodes (MLN)

was observed in CB1 mRNA expression in the colon of but also reduced the number of activated T cells in the

TNBS-mice [57]. CB2 expression, by contrast, was in- colon, probably due to increased apoptosis. The reduction

creased in one study of DSS-induced colitis and TNBS in cellular inﬁltrates in JWH-133-treated mice was further

mice [61] but unchanged in a second study of TNBS mice conﬁrmed with isolated cells from the LP and MLN of DSS-

[57]. mice. CD11b cells were increased in the LP and MLN of

untreated mice – consistent with the macrophage-induced

The ECS as a therapeutic target in IBD inﬂammation characteristic of DSS – and reduced follow-

Overall, data from animal and human studies suggest an ing JWH-133 treatment [36]. A similar effect was observed

up-regulation of the ECS during bowel inﬂammation, ei- for INF-g cells [36], consistent with the fact that INF-g

ther by increased receptor expression and/or increased plays a critical role in colitis induction and progression.

endocannabinoids levels. Moreover, data from knockout Interestingly, JWH-133 was more effective in OM-in-

mice underline a protective role of the ECS in the patho- duced colitis when administered therapeutically (24 h af-

genesis of colitis. Thus, its pharmacological modulation ter colitis induction) versus prophylactically (24 h prior to

could be a therapeutic strategy for IBD. colitis induction) [64]. This observation might offer insight

Regardless of the model, colitis in rodents generally into the mechanism of action of CB2 agonists in colitis; CB2

translates into colon shrinkage and thickening, inﬂamma- signaling might be related to a time-dependent recruit-

tory lesions such as tissue damage to epithelial and smooth ment of CB2-expressing cells or to their activation state.

muscle architecture and invasion by monocytic and gran- Indeed, CB2 is not highly expressed in the normal colon,

ulocytic cells, as well as bowel dysmotility and diarrhea. whereas under inﬂammatory conditions its expression is

Colon shrinkage is the result of neuromuscular-induced increased. In this setting, CB2 agonists might act primarily

contractions of the colonic smooth muscle, which in a to inhibit inﬂammation by acting on CB2-expressing cells,

clinical context is the correlate of acute abdominal pain. instead of preventing their recruitment. Additionally, this

Colon thickening is indicative of glandular hypersecretion, observation implies that cannabinoid signaling can be

muscular hypertrophy, and edematous swelling of the effective in correcting colitis even after its initiation and

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not only at induction of disease. However, seeing as most FAAH inhibitor, URB597 (5 mg/kg, b.i.d.) was used in

studies to date used a pre-treatment scheme, further stud- TNBS-mice at a dose known to increase anandamide levels

ies in a therapeutic setting are warranted to conﬁrm, or in vivo [66] and resulted in a signiﬁcant attenuation of

disprove, this effect. inﬂammation [55]. Interestingly, combining VDM11 and

AM1241 (10 mg/kg or 20 mg/kg, b.i.d.), a CB2 agonist, URB597 was not more effective at reducing colitis than

was used in the context of TNBS-induced colitis with the either drug given alone [55] suggesting that the individual

same outcome as JWH-133 [61]. The effects of both ago- doses used allowed for a maximal increase of anandamide

nists were conﬁrmed to be CB2-mediated because they levels. The beneﬁcial effects of increased anandamide

were reversed in the presence of AM630 (10 mg/kg or levels are CB1- and CB2-dependent because URB597

/ / /

20 mg/kg, q.d.) and absent in CB2 mice [36,61]. In and VDM11 had no effect in CB1 or CB2 mice

one study, AM630 seemed to exacerbate TNBS-induced [55]. In addition, a newly described FAAH inhibitor, com-

colitis [61], but this was not consistent over all measured pound (39) in [67] (10 mg/kg, q.d.), also reduced inﬂamma-

parameters and was not conﬁrmed by two other studies tion in TNBS-colitis, although its effects on anandamide

using AM630 in studies of TNBS-induced and DSS-in- levels or FAAH activity in vivo were not reported [67].

duced colitis [57,65]. The potentially increased turnover of 2-arachidonoyl-

A potential way to avoid the central side-effects of CB1 glycerol under conditions of inﬂammation is also an inter-

receptor activation is to use peripherally-restricted canna- esting fact to consider. Administration of JZL184 (16 mg/

binoid receptor agonists. SAB378, a CB1/CB2 peripherally kg, b.i.d.) in TNBS-induced colitis effectively inhibited

restricted agonist has been studied for treatment of DSS- MAGL activity, led to increased 2-arachidonoylglycerol

induced colitis (0.1 mg/kg or 1 mg/kg, b.i.d.) and TNBS- levels, and subsequently to a decrease in inﬂammatory

induced colitis (0.1 mg/kg or 1 mg/kg, 1 h pre-TNBS and markers [57], conﬁrming the therapeutic potential of 2-

then 8 h and 24 h later), and its effect was compared to that arachidonoylglycerol. An interesting observation is that

of the brain-penetrating CB1/CB2 agonist WIN55,212-2 colitis-induced alterations of the intestinal barrier led to

[12]. SAB378 treatment reduced inﬂammation in TNBS- increased intestinal permeability, as is the case in IBD,

induced colitis by an amount that was not statistically and subsequently to endotoxemia and systemic as well as

significant, and was ineffective in DSS-colitis [12]. This central inflammation. Decreasing colon inflammation

study suggests that central cannabinoid receptor activa- improved epithelial barrier function and resulted in

tion might be required to achieve the anti-inﬂammatory a decrease of colitis-derived systemic and central

effect of cannabinoid agonists on colitis; however, the doses inﬂammation. The use of antagonists selective for CB1

used were low compared with other cannabinoid agonists (SR141716A; 3 mg/kg, q.d.) and CB2 (AM630; 10 mg/kg,

used in the same models or to the doses of WIN55,212-2 q.d.) established that the effects of 2-arachidonoylglycerol

used in the same study. Higher doses of SAB378 were were mediated by both receptors. In fact, blocking each

suggested (but not tested) to be potentially efﬁcient in receptor completely abrogated the beneﬁcial effects of 2-

reducing colitis, with the concern that they might exert arachidonoylglycerol on macroscopic and microscopic colon

inhibitory actions on gastrointestinal transit [12]. How- inﬂammation markers, suggesting that both cannabinoid

ever, inhibition of gastrointestinal transit could prove receptors are needed to maintain colon integrity [57]. This

beneﬁcial in acute colitis where bowel dysmotility and is consistent with previously reported data from knockout

diarrhea are often observed. mice, where in each knockout strain the remaining canna-

Several studies established a role for CB2 receptors in binoid receptor was not sufﬁcient to suppress colonic in-

alleviating colitis, shying away from CB1 because of its ﬂammation, showing either an increased susceptibility to

potential central side-effects. Nevertheless, targeting both colitis or a complete abrogation of the beneﬁcial effects of

receptors could have added beneﬁts versus a receptor- inhibitors of endocannabinoid degradation [51,55].

speciﬁc approach. Along this line, another strategy to

explore is inhibiting endocannabinoid degradation to ben- Concluding remarks

eﬁt from the natural turnover of endocannabinoids in Since the ﬁrst evidence implicating the ECS in IBD, much

inﬂamed tissues. additional work has been done. The effects in animal

Anandamide was the ﬁrst studied endocannabinoid models of IBD of modulating the four main targets of

based on its altered levels in human IBD and rodent the ECS – the CB1 and CB2 receptors, FAAH, and MAGL

models and the availability of several selective FAAH – are now well described. Collectively, these studies show

inhibitors. VDM11 increases anandamide tone by decreas- that increasing the ECS tone results in decreased inﬂam-

ing its cellular uptake (the ﬁrst step in endocannabinoid mation and improved colon integrity. However, there are

degradation) and by inhibiting FAAH. VDM11 adminis- still numerous questions to be answered. One pertinent

tration resulted in a signiﬁcant improvement of colon question would be determining which, if not both, canna-

inﬂammation in TNBS-induced colitis (5 mg/kg, b.i.d.) binoid receptor to target because both seem to play a role in

[55] and DNBS-mice (5 mg/kg, q.d.) [52] and increased colon homeostasis. However, there are no reports of a study

anandamide levels in the colon of DNBS-mice [52]. Two comparing potent and selective CB1 and CB2 agonists,

inhibitors of FAAH were also assayed. AA-5-HT (10 mg/kg, alone or in combination, in the same experimental setting.

q.d.) decreased inﬂammation in DNBS-mice, although to a One characteristic of the ECS is the large number of

lesser extent than VDM11 in the same study [52], possibly enzymes and molecular targets that regulate the

due to the absence of a signiﬁcant increase in colon anan- levels of endocannabinoids and mediate their effects,

damide levels with AA-5-HT treatment [52]. Another respectively. Thus, another direction for future research

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Box 4. Non-psychotropic cannabinoids in IBD Box 5. Outstanding questions

Although anecdotal reports of Cannabis sativa preparations being Which of the two cannabinoid receptors is the most effective

used to treat IBD offer a potential therapeutic pathway for target for counteracting colitis?

investigation, the psychoactive side-effects of D -THC constitute a Are CB2 agonists the best method to avoid central side effects and

major obstacle to widespread use. Thus, components of the decrease motility only when hypermotility is induced by inflam-

cannabis plant that do not bind and activate classical cannabinoid mation?

receptors, or synthetic analogues, have been tested as treatments Is the protection conferred by FAAH blockade the result of

for IBD. Cannabidiol (CBD), present in Cannabis sativa, has elevated levels of one bioactive lipid (e.g., anandamide) or of

antioxidant, anti-inflammatory, and immunomodulatory effects several of the bioactive lipids regulated by FAAH?

and is devoid of psychoactive properties [82]. It has very low affinity Beyond CB1 and CB2, what is the relevance of other targets of

for CB1 and CB2 receptors and could exert part of its actions through endocannabinoids and related compounds?

a PPARg pathway [3,83]. CBD effectively reduces colon alterations in Regardless of the target, would a treatment initiated post-colitis

TNBS- and DNBS-induced colitis [56,84]. Moreover, in DNBS- induction (i.e., more closely mimicking a therapeutic setting) work

induced colitis, CBD counteracted colitis-induced alterations in in reducing colitis?

cytokine and endocannabinoid levels in the colon; CBD reduced IL- What is the exact state of the ECS during the development of IBD

1b and increased IL-10, thus regulating the aberrant immune in humans?

response, and diminished the colitis-induced increase in endocan- What are the underlying mechanisms of the ECS effects in IBD?

nabinoid levels [56]. Beneficial effects of CBD in IBD could extend How relevant are the translational studies in humans? To date,

further than its immunomodulatory properties. Indeed, CBD was they are mostly performed with D -THC, a partial agonist; are they

shown not to affect colonic motility in vivo in normal mice, while always analogous to what we have learned from animal models?

inhibiting inflammation-induced hypermotility [62,85]. Furthermore, Where does modulation of the ECS stand in comparison to

on Caco-2 cells, a model of the intestinal epithelial barrier, CBD available therapies (from efficacy and safety standpoints)?

reduced the inflammation-induced increase in epithelial permeabil- Would targeting the ECS be a valuable therapeutic strategy in all

ity [45]. Moreover, CBD reduced nitrite production in vivo and forms of IBD?

oxidative stress in vitro in Caco-2 cells and in human colonic

cultures derived from UC patients. This could be beneficial in this

setting because oxidative stress is a tissue-destructive factor playing

notion of the ECS as a potentially valuable therapeutic

a role in IBD pathogenesis [56,83]. In another experiment, CBD

potentiated the beneficial effects of D -THC on colitis, significantly target also in IBD.

improving the effect of the same dose of D -THC alone [62]. This

could prove interesting if it holds true with other cannabinoids. References

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